the genetics of nephrotic syndrome in pakistani...

I

THE GENETICS OF NEPHROTIC SYNDROME IN

PAKISTANI CHILDREN

Saba Shahid

Centre of Human Genetics and Molecular Medicine

Sindh Institute of Medical Sciences

Sindh Institute of Urology and Transplantation (SIUT)

Karachi Pakistan

2013

II

THE GENETICS OF NEPHROTIC SYNDROME IN PAKISTANI

CHILDREN

Thesis submitted to the Sindh Institute of Medical Sciences

for the degree of Doctor of Philosophy

BY

Saba Shahid




Karachi Pakistan

2013

III

IV

Table of Contents

page

Acknowledgments i

List of abbreviations iii

Publications v

List of Tables vi

List of Figures viii

Summary ix

1 Literature review on nephrotic syndrome (NS) 1

11 The Kidney 2

111 Structure of the kidney 2

112 Glomerular filtration barrier 4

113 Fenestrated endothelial cells 4

114 Glomerular basement membrane 6

115 Podocyte 6

12 Glomerular diseases of the filtration system 7

121 Nephrotic syndrome 9

122 Definition 9

123 Classification 9

13 Genetics of nephrotic syndrome 13

131 Autosomal recessive mode of steroid resistant NS 14

132 Congenital NS caused by the NPHS1 gene (nephrin) 14

133 NS caused by NPHS2 gene (podocin) 18

134 NS caused by LAMB2 gene (laminin) 21

135 NS caused by PLCE1 gene (phospholipase C epsilon 1) 23

V

136 NS caused by PTPRO gene (protein tyrosine phosphatase

receptor-type O) 24

14 Autosomal dominant mode of steroid resistant NS 24

141 NS caused by ACTN4 gene (α-actinin 4) 24

142 NS caused by WT1 gene (Wilmrsquos tumor) 26

143 NS caused by CD2AP gene (CD2 associated protein) 27

144 NS caused by TRPC6 gene (transient receptor potential

canonical channel 6) 29

145 NS caused by INF2 gene (inverted formin-2) 30

15 Nephrotic syndrome caused by other genetic factors 31

151 Angiotensin converting enzyme (ACE) gene

insertiondeletion polymorphism 31

152 Methyltetrahydrofolate reductase enzyme

(MTHFR) gene polymorphism 32

16 References 33

2 Materials and Methods 48

21 Sample collection 49

22 Extraction of DNA from blood samples 49

221 Quantification of DNA 50

23 Polymerase chain reaction (PCR) 51

24 Agarose gel electrophoreses 52

25 Automated fluorescence DNA sequencing 53

251 Precipitation for sequencing reaction 53

252 Sequencing reaction 53

26 Polyacrylamide gel electrophoresis (PAGE) 54

27 Restriction fragment length polymorphism (RFLP) 55

28 Statistical analysis 57

29 References 58

VI

3 A spectrum of novel NPHS1 and NPHS2 gene mutations in pediatric

nephrotic syndrome patients from Pakistan 59

31 Introduction 60

32 Materials and methods 62

321 Patient recruitment and data collection 62

322 Mutation analysis 63

33 Results 67

331 Clinical characteristics of patients 67

332 Mutations in the NPHS1 gene 67


34 Discussion 86

35 References 90

4 Association of the ACE-II genotype with the risk of nephrotic

syndrome in Pakistani children 94

41 Introduction 95

42 Subjects and Methods 96


422 Genotyping 97


43 Results 99

44 Discussion 103

45 References 106

VII

5 Association of the MTHFR gene polymorphisms

(C677T amp A1298C) with the nephrotic syndrome in Pakistani

children 109

51 Introduction 110


521 Genotyping 113

53 Results 115

54 Discussion 118

55 References 122

6 General Discussion 125

61 Genetic screening and counseling 129

62 Therapeutic options 131

63 Future perspectives 133

64 Conclusion 135

65 References 136

i

Acknowledgments

All praise for Allah the most compassionate and the most merciful

I would like to express my sincerest gratitude to my mentor Dr Syed Qasim Mehdi

HI SI (Centre for Human Genetics and Molecular Medicine) for his guidance

advice and for provision of excellent laboratory facilities for doing scientific work

I gratefully acknowledge my supervisor Dr Aiysha Abid for her support and

valuable suggestions throughout this research work

I admire Dr Shagufta Khaliq (Co-supervisor) for her dedicated attitude towards

research and her encouragement and advice that has been a great source of

inspiration for me

I am thankful to my senior lab colleague Dr Sadaf Firast for her help and

cooperation

I thank all my lab colleagues for their help Miss Sadia Ajaz who helped me in

statistical analysis Mr Ali Raza for his help in DNA extraction and also great

ldquofightsrdquo with him that makes the environment lively Mr Hajan Shah for his

support and friendship

I am grateful to Dr Ali Lanewala and his team of the pediatric nephrology

department SIUT who provided samples and did clinical analysis of all the

nephrotic syndrome patients I am also very grateful to all the patients who

participated in this study

I thank our lab attendant Mr Mohammad Imran Baig for his support and hard

work

ii

I am grateful to my best friend Sajida Batool (Nottinghum University UK) for her

constant love and support at every step in my life and especially for sharing

valuable research articles that were not available in Pakistan

It has been a privilege for me to work at the Sindh Institute of Urology and

Transplantation (SIUT) the worldrsquos largest kidney transplant centre I am

especially thankful to Dr Adeeb-ul-Hassan Rizvi HI SI Director SIUT for his kind

guidance laboratory facilities and funding for my research work

I acknowledge the love and support of my parents and family without which the

completion of this work would have not been possible

iii

List of abbreviations

ACD Acid Citrate Dextrose

ACE Angiotensin Converting Enzyme

ACEI Angiotensin Converting Enzyme Inhibitor

ACTN4 α-Actinin 4

AD Autosomal Dominant

Ang-I Angiotensin I

Ang-II Angiotensin II

APS Ammonium Persulphate

ARB Angiotensin Receptor Blocker

CBEC Centre for Biomedical Ethics and Culture

CD2AP CD2 Associated Protein

CNF Nephrotic Syndrome of Finnish Type

CNS Congenital Nephrotic Syndrome

CRF Chronic Renal Failure

CsA Cyclosporine

DAG Diacylglyecerol

DDS Denys-Drash Syndrome

DMS Diffuse Mesengial Sclerosis

DNA Deoxyribonucleic Acid

eGFR Estimated Glomerular Filtration Rate

EDTA Ethylenediaminetetraacetic Acid

ESRD End Stage Renal Disease

FECs Fenestrated Endothelial Cells

FS Frasier Syndrome

FSGS Focal Segmental Glomerulosclerosis

GBM Glomerular Basement Membrane

GFB Glomerular Filtration Barrier

GLEP1 Glomerular Epithelial Protein 1

Hcy Homocysteine

HSPG Heparin Sulfate Proteoglycans

HWE Hardy-Weinberg Equilibrium

ID InsertionDeletion Polymorphism

Ig Immunoglobulin

INF2 Inverted Formin 2

IP3 Inositol 1 4 5-Triphosphate

IRB Institutional Review Board

iv

LAMB2 Laminin Beta 2

MCD Minimal Change Disease

MCGN Mesengio Capillary Glomerulonephritis

MesPGN Mesengial Proliferative Glomerular Nephropathy

MGN Membranous Glomerulonephritis

MTHFR Methylenetetrahydrofolate Reductase

NPHS1 Nephrotic Syndrome Type 1


NS Nephrotic Syndrome

OD Optical Density

PAGE Polyacrylamide Gel Electrophoresis

4-PBA Sodium 4-Phenylbutyrate

PLC Phospholipase C

PLCE1 Phospholipase C Epsilon 1

PTPRO Protein Tyrosine Phosphatase

RAAS Renin-Angiotensin-Aldosterone System

RCLB Red Cell Lysis Buffer

RFLP Restriction Fragment Length Polymorphism

RTx Renal Transplantation

SD Slit Diaphragm

SDS Sodium Dodecyl Sulfate

SIUT Sindh Institute of Urology and Transplantation

SNPs Single Nucleotide Polymorphisms

SPSS Statistical Package for Social Sciences

SRNS Steroid Resistant Nephrotic Syndrome

SSNS Steroid Sensitive Nephrotic Syndrome

TBE Tris Boric Acid EDTA Buffer

TEMED N N N N Tetramethylethylenediamine

TRP Transient Receptor Potential

TRPC-6 Transient Receptor Potential Canonical Channel 6

WT1 Wilmrsquos Tumor

v

Publications

Saba Shahid Aiysha Abid S Qasim Mehdi Sadaf Firasat Ali Lanewala

S Ali Anwar Naqvi S Adeebul Hasan Rizvi Shagufta Khaliq (2012)

Association of the ACE-II genotype with the risk of nephrotic syndrome in

Pakistani children Gene 493 165-168 Erratum in Gene 2012 495 93

Aiysha Abid Shagufta Khaliq Saba Shahid Ali Lanewala Mohammad

Mubarak Seema Hashmi Javed Kazi Tahir Masood Farkhanda Hafeez S

Ali Anwar Naqvi S Adeebul Hasan Rizvi S Qasim Mehdi (2012) A

spectrum of novel NPHS1 and NPHS2 gene mutations in pediatric nephrotic

syndrome patients from Pakistan Gene 502 133-137

vi

List of Tables

Table Title

Page

11 Summary of genes that cause inherited NS

13

31 Primer pairs and PCR conditions for mutation screening of the

NPHS1 gene

65


NPHS2 gene

66

33 Clinical characteristics of children with idiopathic nephrotic

syndrome

68

34 Clinical characteristics of all 145 patients examined

69

35 List of homozygouscompound heterozygous mutations

identified in the NPHS1 gene

81

36 List of heterozygous mutationsvariants identified in the

NPHS1 gene

82

37 List of mutations identified in the NPHS2 gene

85

41 The clinical parameters of NS patients

99

42 Genotypic and allelic frequencies of the ACE ID

polymorphism and their distribution in terms of II ID and

IIDD genotypes with respect to DD genotype in NS patients

and controls

101

43 Frequency distribution of the ACE ID polymorphism in

SRNSSSNS FSGSnon-FSGS and MCDnon-MCD patients

102


113

52 Genotypic and allelic frequencies of the MTHFR C667T

polymorphism and their distribution in terms of CC CT and

vii

CCCT genotypes with respect to TT genotype in NS patients

and controls

116

53 Frequency distribution of the MTHFR C677T polymorphism

in SRNSSSNS FSGSnon-FSGS and MCDnon-MCD

patients

117

54 Genotypic and allelic frequencies of the MTHFR A1298C

polymorphism and their distribution in terms of CC CA and

CCCA genotypes with respect to AA genotype in NS patients

and controls

119

55 Frequency distribution of the MTHFR A1298C polymorphism


patients

120

viii

List of Figures

Figure Title

Page

11 Systemic diagram of the kidney and nephron structure

3

12 The glomerular filtration barrier comprises of the glomerular

basement membrane fenestrated endothelial cells and

podocyte

5

13 Diagrammatic representation of the podocyte structure and SD

composed of nephrin podocin α-actinin 4 TRPC6 CD2AP

and PLCE1

8

14 Protein leakage through the GFB in nephrotic syndrome

10

15 Diagrammatic structure of the NPHS1 protein

15

16 An illustration of the membranous localization of podocin

protein

19

31 Illustration of the identified mutations in the NPHS1 gene and

their respective locations in the gene and protein domains

80


their locations

84

41 ACE gene ID polymorphism genotyping on agarose gel

98

51 Dysregulation of MTHFR leads to the accumulation of

homocysteine

112

52 MTHFR gene C677T polymorphism genotyping on agarose

gel

114

53 MTHFR gene A1298C polymorphism genotyping on agarose

gel

114

ix

SUMMARY

x

SUMMARY

The kidneys play a central role in removing water soluble metabolic waste

products from the organism Many acquired and inherited renal diseases in humans

lead to kidney dysfunctions such as nephrotic syndrome (NS) It is a common

pediatric kidney disease associated with heavy proteinuria The underlying causes

of hereditary NS are the presence of defects in the podocyte architecture and

function Recent genetic studies on hereditary NS have identified mutations in a

number of genes encoding podocyte proteins In the work presented here genetic

screening of nephrotic syndrome was carried out for the first time in a cohort of

paediatric Pakistani patients The analyses conducted are (1) Mutation screening of

the nephrotic syndrome type 1 (NPHS1) and type 2 (NPHS2) genes (2) The

association studies of NS with insertiondeletion (ID) polymorphism of the

angiotensin converting enzyme (ACE) gene and (3) The C677T and A1298C

polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene

All the studies described in this thesis were approved by the Institutional

Ethical Review Committee and were according to the tenets of the Declaration of

Helsinki Informed consent was obtained from all the participants

1- A spectrum of novel NPHS1 and NPHS2 gene mutations in pediatric

nephrotic syndrome (NS) patients from Pakistan

This study was designed to screen the disease causing mutations in the

NPHS1 and NPHS2 genes in a Pakistani steroid resistant nephrotic syndrome

(SRNS) cohort For this study 145 cases of early onset and familial SRNS were

collected from the pediatric nephrology department at the Sindh Institute of

xi

Urology and Transplantation (SIUT) Mutation analysis was performed by direct

DNA sequencing of all exons of the NPHS1 and NPHS2 genes This study has

identified six novel homozygous mutations in the NPHS1 gene and four in the

NPHS2 gene The main findings of this work are mutations in the NPHS1 gene that

accounted for around 20 of the cases and the NPHS2 gene for 55 of the cases

with early onset NS Another important finding is the absence of disease-causing

mutations in the NPHS2 gene in the familial SRNS and congenital nephrotic

syndrome (CNS) cases These novel findings of a low mutation rate in the NPHS1

and NPHS2 genes are in contrast to the higher mutation rate reported from Europe

and America (39-55 and 10-28 respectively) and suggest that other genetic

causes of the disease remain to be identified

2- Association of the angiotensin converting enzyme (ACE) - II genotype with

the risk of nephrotic syndrome in Pakistani children

This study examined the association of insertiondeletion (ID)

polymorphism of the angiotensin converting enzyme (ACE) gene with nephrotic

syndrome in Pakistani children A total of 268 blood samples from NS patients and

223 samples from control subjects were used The genotyping of ACE gene

polymorphism was performed by the PCR method The results show a significant

association of the II genotype and the I allele of the ACE gene with NS in the

Pakistani children (OR=6755 CI= 3-149) These results suggest that the analysis

of ACE polymorphism should be performed for the early diagnosis of NS patients

in South Asian patients

xii

3- Association of methylenetetrahydrofolate reductase (MTHFR) gene

polymorphisms (C677T and A1298C) with nephrotic syndrome in Pakistani

children

The associations of methylenetetrahydrofolate reductase (MTHFR) gene

polymorphisms (C677T and A1298C) with NS were also examined in this study

Blood samples were obtained from 318 children with NS and 200 normal controls

and were analyzed using the polymerase chain reaction (PCR) and restriction

fragment length polymorphism (RFLP) methods A positive association between

NS and the C677T and A1298C polymorphisms of the MTHFR gene were not

observed in this study This too is in contrast to the higher incidence of the TT

genotype found to be associated with the early development of childhood focal

segmental glomerulosclerosis (FSGS) in Japanese children

In view of the results presented in this thesis genetic testing of the NPHS1

and NPHS2 genes following the diagnosis of NS may have important applications

regarding possible response to steroid treatment The low prevalence of mutations

in these genes in the Pakistani cohort compared to that in other populations of

Europe and the United States suggest the need of finding other genetic markers that

may be involved in disease pathogenesis

1

1 LITERATURE REVIEW ON NEPHROTIC

SYNDROME

2

11 THE KIDNEY

The kidney plays a central role in the regulation of blood pressure acid base

balance and the excretion of metabolic waste products from the blood In addition

the kidneys produce and secrete the hormones renin erythropoietin and 1 25-

dihydroxy vitamin D3 that play an important role in the regulation of the bodyrsquos

calcium and phosphate balance (Greenberg et al 2009)

111 STRUCTURE OF THE KIDNEY

Kidneys are bean shaped organs located in the retroperitoneal space They

exist in pairs each weighing about 150gm In adult humans 180 liters of blood is

filtered through the kidneys every 24 hours producing 1-15 liters of urine The

functional unit of the kidney is the nephron and each kidney has approximately 1

million of them Each nephron consists of a glomerular tuft and a long tubule that is

segmented into different parts the proximal tubule loop of Henle the distal tubule

and the collecting duct (Figure-11) The main filtration unit of the nephron is the

glomerulus It is composed of parietal epithelial cells of the Bowmanrsquos capsule

endothelial cells podocyte (visceral epithelial cells) and mesangial cells The blood

enters the glomerulus through an afferent blood vessel which branches into a

capillary tuft These capillaries form the glomerular filtration barrier (GFB)

responsible for the filtration of blood and the formation of urine The filtrate passes

through the GFB and is collected in the Bowmanrsquos capsule It is finally processed

in the tubular system of the kidney (Greenberg et al 2009)

3

Figure- 11 Systemic diagram of the kidney and nephron structure

(httpwwwpfizercozaruntimepopcontentrunaspxpageidref=2551)

4

112 GLOMERULAR FILTRATION BARRIER (GFB)

The glomerular filtration barrier (GFB) regulates the outflow of solutes

from the blood capillaries to the urinary space (Caulfield and Farquhar 1974) It

selectively permits the ultra filtration of water and solutes and prevents leakage of

large molecules (MW gt 40KDa) such as albumin and clotting factors etc

(Ruotsalainen et al 1999) GFB comprises of fenestrated endothelium glomerular

basement membrane (GBM) and podocyte foot process (Ballermann and Stun

2007 and see Figure-12) The integrity of each of these structural elements is

important for the maintenance of normal ultrafiltration The components of the

GFB are described in detail below

113 FENESTRATED ENDOTHELIAL CELLS (FECs)

The glomerular capillary endothelial cells form the inner lining of the

GBM They contain numerous pores (fenestrae) with a width of up to 100 nm

These pores are large enough to allow nearly anything smaller than a red blood cell

to pass through (Deen and Lazzara 2001) They are composed of negatively

charged proteoglycans and sialoproteins (Weinbaum et al 2007) These charged

molecules have been reported to restrict the filtration of albumin and other plasma

proteins They play an important role in the filtration of blood through the

glomeruli The dysregulation of the endothelial cells may be associated with

proteinuria as well as renal failure (Satchell and Braet 2009)

5

Figure-12 The glomerular filtration barrier comprises of the glomerular

basement membrane fenestrated endothelial cells and podocytes

(httpwwwbiodavidsoneducoursesimmunologyStudentsspring2000carterrest

rictedpaperhtml)

6

114 GLOMERULAR BASEMENT MEMBRANE (GBM)

The glomerular basement membrane (GBM) is a 300-350 nm thick

extracellular matrix It is located between the podocyte and the endothelial cell

layers It is made up of a meshwork of collagen type IV laminin nidogenentactin

and heparin sulfate proteoglycans (HSPG Gubler 2008) The laminin-collagen IV

and nidogen network provides structural support to the GBM and is involved in cell

adhesion and differentiation The HSPG consists of anionic perlecan and agrin

moieties This network forms an electric barrier for plasma protein (Groffen et al

1999) The GBM was initially thought to have a central role in macromolecular

filtration in a size and charge-selective manner (Caulfield and Farquhar 1974)

However recent studies have suggested their major role as a support structure for

the attachment of endothelial cells and podocyte (Goldberg et al 2009)

115 PODOCYTE

The podocytes are specialized epithelial cells that cover the outer surface of

the GBM They play an important role in the size and charge selective

permeability They are also involved in the synthesis and maintenance of the GBM

(Patrakka and Tryggvason 2009) The podocyte is composed of the cell body

which contains a nucleus golgi apparatus mitochondria and rough and smooth

endoplasmic reticulum (Pavenstadt et al 2003) It has several foot processes that

are interconnected with each other and coated with negatively charged molecules

called glycocalyx Glycocalyx is an anti-adhesive protein that is important for the

preservation of normal podocyte architecture and for limiting albumin leakage

(Doyonnas et al 2001) Foot processes are functionally defined by three

7

membrane domains the apical membrane domain the slit diaphragm (SD) and the

basal membrane domain associated with the GBM (Faul 2007) The SD bridges

the space between the adjacent podocyte foot processes It forms a zipper-like

structure with a constant width of 300-450 A and acts as a major size barrier to

prevent protein leakage (Rodewald and Karnovsky 1974) The slit diaphragm is

formed by several proteins including nephrin podocin ά-actinin 4 CD2-associated

protein transient receptor potential 6 channel protein etc (Hinkes et al 2006

Buumlscher and Weber 2012) These proteins play key roles in maintaining the

structural and functional integrity of the podocyte as shown in Figure-13 (Buumlscher

and Weber 2012) Several studies have suggested that the dysfunction of the SDndash

associated molecules cause proteinuria in nephrotic syndrome and some other

glomerular diseases (Shih et al 2001 Reiser et al 2005 Winn et al 2005)

12 GLOMERULAR DISEASES OF THE FILTRATION SYSTEM

Glomerular disorders are a major cause of kidney diseases Renal

dysfunction may be due to genetic factors infections or exposure to toxins Recent

studies have indicated that inherited impairment in the structure and function of the

glomerular filtration barrier ultimately leads to nephrotic syndrome (Clark and

Baratt 1999)

8

Figure- 13 Diagrammatic representation of podocyte structure and slit

diaphragm composed of nephrin podocin α-actinin 4 TRPC6 CD2AP and

PLCE1 (Buumlscher and Weber 2012)

9

121 NEPHROTIC SYNDRME (NS)

122 DEFINITION

Nephrotic syndrome (NS) is a set of symptoms associated with kidney

dysfunction It can be caused by several different defects that affect the kidneys It

is characterized by heavy proteinuria hypoalbuminemia hypercholesterolemia and

edema (Tune and Mendoza 1997) In humans nephrotic range proteinuria is

generally defined as the excretion of more than 35 gm of protein per 24 hours The

decrease in serum albumin level is secondary to the loss of protein in the urine The

underlying mechanism in the majority of patients with NS is permeability defect in

the GFB that allows the loss of proteins from the plasma into the urine (Clark and

Barrat 1999 see Figure-14)

NS is the most common glomerular disease in children (Braden et al

2000) The estimated incidence of pediatric NS is 20 to 27 per 100000 in the

USA with a cumulative frequency of 16 per 100000 Geographic or ethnic

differences have also been reported to contribute towards the incidence of NS with

a 6-fold higher incidence in the Asian than European populations (Sharples et al

1985)

123 CLASSIFICATIONS

NS can be clinically classified on the basis of the age of disease onset as

congenital (CNS) infantile and childhood CNS appears in utero or during the first

three months of life Infantile and childhood onset NS are diagnosed during and

after the first year of life respectively (Eddy and Symons 2003)

10

Figure-14 Protein leakage through the GFB in nephrotic syndrome

(httpwwwunckidneycenterorgkidneyhealthlibrarynephroticsyndromehtml)

11

NS in children is generally divided into steroid resistant (SRNS) and steroid

sensitive nephrotic syndrome (SSNS) depending on the patientrsquos response toward

steroid therapy 80-90 patients with sporadic NS respond well to steroid therapy

However approximately 10-20 children and 40 adults fail to do so and hence

are at a higher risk of developing end stage renal disease (ESRD Ruf et al 2004)

NS can also be categorized histologically into minimal change disease

(MCD) and focal segmental glomerosclerosis (FSGS Obedova et al 2006) MCD

is the most common cause of NS affecting 77 of children followed by FSGS

(8 International Study of Kidney Diseases in Children 1978) However recent

studies have shown a rise in the incidence of FSGS in the NS patients According

to the data available in Pakistan MCD and its variants are the leading cause of NS

in children (43 of cases) followed by FSGS (38 Mubarak et al 2009) Patients

with MCD usually respond to steroid treatment but are accompanied by more or

less frequent relapses FSGS is a histological finding that appears as focal (some of

the glomeruli) and segmental (part of an entire glomerulus) sclerosis of the

glomerular capillary tuft and manifests in proteinuria This histological finding has

been typically shown in steroid resistant NS patients The less frequent lesions are

diffuse mesangial sclerosis (DMS) mesengial membranoproliferative

glomerulonephritis (MesPGN) and membrane glomerulopathy (MG McTaggart

2005)

Most of the children with NS have been found to have a genetic

predisposition for developing this disease NS can occur sporadically but large

numbers of familial cases have also been reported (Eddy and Symons 2003) and

their mode of inheritance can either be autosomal dominant or recessive (Boute et

12

al 2002 Pollak et al 2007) Recent studies on NS have lead to the discovery of

several novel genes that encode proteins that are crucial for the establishment and

maintenance for podocyte Mutations found in different forms of NS are in the

NPHS1 (nephrin) NPHS2 (podocin) LAMB2 (laminin β2) PLCE1 (phospholipase

Cέ1) and PTPRO genes (protein tyrosine phosphatase) in the autosomal recessive

mode of inheritance The ACTN4 (alpha-actinin 4) WT1 (Wilmrsquos tumor) CD2AP

(CD2-associated protein) TRPC6 (transient receptor potential 6) and INF2 genes

(inverted formin-2) are involved in disease etiology are inherited in the autosomal

dominant mode (Buumlscher and Weber 2012)

Mutations in the NPHS1 and NPHS2 genes mainly cause a severe form of

NS in children with congenital and childhood onset The WT1 and LAMB2 genes

have been involved in syndromic forms of NS with other external manifestations

(Hinkes et al 2007) Mutations in the ACTN CD2AP and TRPC6 genes have been

involved in alterating the structure and function of podocyte (Patrie et al 2002

Reiser et al 2005 Winn et al 2005) Recently mutations in the PLCE1 INF2

PTPRO and MYO1E have been reported in the childhood familial cases of NS

(Hinkes et al 2006 Brown et al 2010 Mele et al 2011 Ozaltin et al 2011)

13

13 GENETICS OF NEPHROTIC SYNDROME

A brief overview of the different forms of NS caused by mutations in various genes (Table-11)

Tabe-11 Summary of genes that cause inherited NS

Inheritance Gene Protein Chromosome

Location Age of onset Pathology References

Autosomal

recessive

(AR)

NPHS1 Nephrin 19q131 Congenital

Childhood MCDFSGS

Kestila et al

1998

NPHS2 Podocin 1q25-q31 Childhood

Adulthood FSGSMCD

Boute et al

2000

LAMB2 Laminin 2 3p21 Congenital

Childhood DMSFSGS

Hinkes et al

2007

PLCE1 Phospholipase C epsilon 1 10q23 Childhood DMSFSGS Hinkes et al

2006

PTPRO Protein tyrosine

phosphatase 12p123 Childhood FSGSMCD

Ozaltin et

al 2011

Autosomal

dominant

(AD)

ACTN4 -actinin 4 19q13 Adulthood FSGS Kaplan et

al 2000

WT1 Wilmsrsquo tumor 1 11p13 Congenital

Childhood DMSFSGS

Mucha et al

2006

CD2AP CD2 associated protein 6p123 Adulthood FSGS Lowik et al

2007

TRPC6 Transient receptor

potential channel 6 11q21-22 Adulthood FSGS Winn et al

2005

INF2 Inverted formin-2 14q32 Adulthood FSGS Brown et al

2010

14

131 AUTOSOMAL RECESSIVE INHERITANCE OF STEROID

RESISTANT NEPHROTIC SYDROME (SRNS)

132 CONGENITAL NEPHROTIC SYNDROME CAUSED BY THE NPHS1

GENE (NEPHRIN)

Congenital nephrotic syndrome (CNS) appears in utero or during the first

three months of life (Jalanko 2009) The most common form of CNS first

described by Hallman and colleagues (1956) was congenital nephrotic syndrome of

the Finnish type (CNF) It is characterized by massive proteinuria and nephrosis

which starts in utero (Hallman et al 1973) It rapidly progresses toward ESRD by

the age of 2 to 3 years (Heeringa et al 2008) The resulting phenotype includes

FSGS MCD and DMS (Koziell et al 2002 Lahdenkari et al 2004 Schultheiss et

al 2004)

Mutations in the nephrin gene (NPHS1 OMIM-602716) have been shown

to cause autosomal recessive SRNS worldwide but in Finland the incidence is

approximately 1 in 10000 newborns (Holmberg et al 1995) NPHS1 was

identified in 1998 by the positional cloning method It is localized on chromosome

19q131 and contains 29 exons (Kestila et al 1998) It encodes the multifunctional

protein nephrin which has a molecular weight of 180 KDa It belongs to the

immunoglobulin (Ig) family (Wartiovaara et al 2004) It contains eight

extracellular IgG like motifs a fibronectin III-like domain and a cytosolic C-

terminal tail (Figure-15 Koziell et al 2002 Tryggvason et al 2006)

15

Figure-15 Diagrammatic structure of the NPHS1 protein (Koziell et al

2002)

16

Nephrin is one of the most important structural protein of the podocyte

(Hinkes et al 2006) It is exclusively expressed in the kidney podocyte and is a

key functional component of the SD (Patrakka et al 2001) It plays an important

role in signaling between adjacent podocytes by interacting with podocin and

CD2AP (Khoshnoodi et al 2003 Sellin et al 2003) In the nephrin knockout

mice model the effacement of the podocyte foot processes caused deleterious

proteinuria and neonatal death (Putaala et al 2001) Thus nephrin is essential for

the development and function of the normal GFB

NPHS1 has been identified as the major gene involved in CNF The two

most important mutations found are Fin major (the deletion of nucleotides 121 and

122 leading to a frame shift mutation or stop codon) and Fin minor (nonsense

mutation encoding a truncated protein of 90 and 1109 amino acids Kestila et al

1998) These two mutations account for 95 of the CNF cases in the Finnish

population but are uncommon in other ethnic groups However in other studies on

European North American and Turkish children mutations in the NPHS1 gene

account for 39-55 cases of childhood NS and 40 of all cases of CNS (Lenkkeri

et al 1999 Hinkes et al 2007 Heeringa et al 2008) To date more than 173

different mutations have been identified in the NPHS1 gene including deletions

insertions nonsense and missense mutations (Beltcheva et al 2001 Benoit et al

2010 Ovunc et al 2012)

The homozygous pR1160X mutation in the NPHS1 gene also leads to the

production of a truncated protein causing severe CNS in the first three months

(Koziell et al 2002) It is also reported to develop partial or complete remission in

17

adult hood with a milder phenotype in some patients (Koziell et al 2002) In

recent studies mutations in the NPHS1 gene have been identified in patients with

the age of disease onset ranging from 6 months to 8 years (Philippe et al 2008)

Another study in a Spanish cohort identified more disease causing mutations in the

NPHS1 than in the NPHS2 gene in patients with childhood onset diseases Further

compound heterozygous mutations (pR827X pR979S) were identified in patients

with childhood and adulthood glomerular disorder that also enhanced the clinical

severity in NS (Santin et al 2009)

The variability in disease onset is explained by functional and

computational studies Philippe and colleagues classified the nephrin mutations into

ldquosevererdquo or ldquomildrdquo mutations The severe mutations include nonsense truncated

frame shift splice-site (c609ndash2ArarrC) and missense (pL832P) mutations These

mutations cause a defect in the intracellular transport so that the mutant protein is

retained in the endoplasmic reticulum instead of being transported to the cell

surface This results in the loss of nephrin function which causes severe and early

onset NS On the other hand the milder mutations include missense mutations

(pLp96V pA107T pP575Q pR460Q and pR976S) that allow the mutant

protein to be targeted to the cell surface and to maintain partial protein function

Another splice site mutation (c2072ndash6CrarrG) allows some correct splicing and is

therefore considered a mild mutation This also explains the later onset of disease

in such cases (Philippe et al 2008) Mutation analysis in 15 families of Japanese

and Korean origin excluded the involvement of NPHS1 and NPHS2 in SRNS

(Kitamura et al 2006) This suggests an ethnic diversity in the involvement of

these genes in Asian SRNS patients

18

NS patients with the NPHS1 gene mutations generally show resistance to

steroid therapy (Jalanko 2009) However heterozygous mutations have been found

to respond to therapy and may therefore have a better long-term survival compared

to patients with compound heterozygous and homozygous mutations (Caridi et al

2004) Steroid therapy does not induce remission and the only treatment of choice

is kidney transplantation (Holmberg et al 1995) The recurrence of CNS may

account for 20ndash25 of the patients after renal transplantation (Patrakka et al

2002) However recently it has been reported that gt20 of CNS patients including

patients with NPHS1 mutations may respond to antiproteinuric treatment (Schoeb

et al 2010) Angiotensin-converting enzyme inhibitors are also beneficial in

reducing protein excretion (Sredharan and Bockenhauer 2005 Copelovitch et al

2007) Mutations identified in this gene provide greater insight in understanding of

the clinical manifestation and pathology of NS

133 NEPHROTIC SYNDROME CAUSED BY NPHS2 GENE (PODOCIN)

Mutations in the podocin gene (NPHS2 OMIM-604766) have been shown

to cause autosomal recessive SRNS This gene was identified in year 2000 by

positional cloning It is localized on chromosome 1q25-31 and comprises of 8

exons (Boute et al 2000) It encodes the integral membrane protein podocin (MW

42 KDa) that belongs to the stomatin family It has a single membrane domain

forming a hairpin like structure and both the N and C domains are in the cytosol

(Roselli et al 2002 Figure-16)

19

Figure-16 An illustration of the membranous localization of the

podocin protein (Rellel et al 2011)

20

It is specifically expressed in the podocyte at the foot processes It closely

interacts with nephrin CD2-associated protein and NEPH1 (Huber et al 2003

Roselli et al 2004) Mice lacking podocin develop proteinuria and die after a few

days of life due to fused foot processes and loss of SD that suggests their crucial

role in glomerular filtration (Roselli et al 2004)

Mutations in the podocin gene were originally found in infancy or

childhood but have also been reported in adult onset NS (Caridi et al 2001)

These NPHS2 gene mutations have generally been found with childhood onset

diseases but have also been reported in 51 of CNS cases of European origin

(Heringa et al 2008) These patients show characteristic NS presentation from

birth to 6 years of age and progress to ESRD before the end of the first decade of

life (Berdeli et al 2007 Hinkes et al 2007) Renal biopsies show either MCD or

FSGS and patients are generally steroid resistant (Ruf et al 2004)

Mutations are found in a high proportion in nephrotic syndrome patients

both in familial and sporadic cases (Weber et al 2004) They represent 45-55 of

familial cases and 8-20 of sporadic cases More than 100 pathogenic mutations

have been reported that include missense nonsense and deletion mutations (Caridi

et al 2004 Ruf et al 2004 Benoit et al 2010) Patients with frame shift or

truncation mutations have an early onset whereas patients with missense mutations

have a late onset nephropathy (Huber et al 2003 Roselli et al 2004) The most

frequent pathogenic mutation (pR138Q) has been found to cause earlier onset of

the disease (Weber et al 2004 Hinkes et al 2008) The mutant protein thus

produced is retained in the endoplasmic reticulum and fails to recruit nephrin to the

lipid raft (Huber et al 2003 Roselli et al 2004)

21

An NPHS2 gene variant (pR229Q) has been shown to cause late-onset NS

when found in association with another pathogenic NPHS2 mutation (Machuca et

al 2010 Santin et al 2011) This variant has been found commonly as a

nonsynonymous NPHS2 variant in Caucasians and is particularly common among

Europeans with an observed frequency of heterozygotes that ranges from 003-

013 (Pareira et al 2004 Franceschini et al 2006 Kottgen et al 2008) The

variability in disease severity suggests that some other non genetic or

environmental factors may also influence the disease presentation

The incidence of mutations in familial SRNS cases were found to be 40 in

European and American children 29 in Turkish 76 in Tunisian Libyan and

Moroccan families (Hinkes et al 2008 Ismaili et al 2009 Mbarek et al 2011)

The prevalence of mutations in the SRNS patients is higher in the Europeans and

Turks than in Asian children (Maruyama et al 2003)

Patients with homozygous or compound heterozygous mutations in the

NPHS2 gene do not respond to standard steroid therapy for NS Therefore genetic

testing for the NPHS2 gene mutations is recommended for every child upon

diseases presentation (Ruf et al 2004 Weber et al 2004) Thus podocin may be a

major contributor to the genetic heterogeneity of NS

134 NEPHROTIC SYNDROME CAUSED BY LAMB2 GENE (LAMININ

BETA 2)

Mutations in the laminin gene (LAMB2 OMIM-150325) have been shown

to cause autosomal recessive NS with or without ocular and neurological sclerosis

(Zenker et al 2004) In 1963 Pierson first described the association of glomerular

22

kidney disease with ocular abnormalities (Pierson et al 1963) The characteristic

clinical ophthalmic sign is microcoria or the fixed narrowing of the pupils (Zenker

et al 2004) The LAMB2 gene is localized on chromosome 3p21 and comprises of

32 exons It encodes the basement membrane protein laminin 2 (Tunggal et al

2000)

LAMB2 gene mutations are common in patients with NS manifesting in

their first year of life (Hinkes et al 2007) The histology showed characteristic

patterns of DMS and FSGS The disease causing nonsense and splices site

mutations lead to the formation of truncated protein and complete loss of laminin

β2 expression in patients with Pierson syndrome (Zenker et al 2004) Milder

phenotype of the disease has been shown in some cases of infantile NS with

homozygous or compound heterozygous mutations (Hasselbacher et al 2006

Matejas et al 2006 Choi et al 2008 Kagan et al 2008 Chen et al 2011) This

syndrome shows early progression to ESRD during the first 3 months of life and

the only treatment of choice is kidney transplantation The recurrence of DMS has

not been observed in transplanted patients (Matejas et al 2010) In animal models

of the Pierson syndrome the laminin knockout mice present a disorganized GBM

with proteinuria whereas podocyte foot processes and SD are normal (Noakes et

al 1995) These studies strongly suggest that laminin β2 has an important role in

maintaining the structural and functional integrity of the GFB

23

135 NEPHROTIC SYNDROME CAUSE BY PLCE1 GENE

(PHOSPHOLIPASE C EPSILON-1)

Mutations in the phospholipase C epsilon-1 gene (PLCE1 OMIM-608414)

have been shown to cause childhood onset recessive form of NS with DMS andor

FSGS as histological presentations It is localized on chromosome 10q23 and

comprises of 35 exons (Hinkes et al 2006) It encodes the phospholipase C (PLC)

enzyme that catalyzes the hydrolysis of phosphatidylinositides to the second

messenger inositol 1 4 5-triphosphate (IP3) and diacylglyecerol (DAG) The

second messenger IP3 is involved in intracellular signaling that is important for cell

growth and differentiation (Wing et al 2003) In the kidney PLCE1 is expressed

in the podocyte (Hinkes et al 2006) Mutations in the PLCE1 gene have been

identified in 286 of 35 famillies that showed a histological pattern of DMS in a

worldwide cohort (Gbadegesin et al 2008) Recent studies have found

homozygous mutations in phenotypically normal adults and have suggested that

some other factors could also be involved in disease presentation (Gilbert et al

2009 Boyer et al 2010) Hinkes and colleagues have reported that some patients

carrying the PLCE1 gene mutation respond to steroid therapy (Hinkes et al 2006)

NS caused by mutations in the PLCE1 gene is the only type that can be treated by

steroid therapy thus providing the clinicians an opportunity to treat hereditary NS

(Weins and Pollak 2008)

24

136 NEPHROTIC SYNDROME CAUSED BY PTPRO GENE (PROTEIN

TYROSINE PHOSPHATASE RECEPTOR-TYPE O)

Mutations in the protein tyrosine phosphatase receptor-type O gene

(PTPRO OMIM-600579) have been shown to cause autosomal recessive NS It is

localized on chromosome 12p123 and contains 26 exons It encodes a receptor-like

membrane protein tyrosine phosphatase that is also known as glomerular epithelial

protein 1 (GLEPP1) It is expressed at the apical membrane of the podocyte foot

processes in the kidney (Ozaltin et al 2011) The splice site mutations in the

PTPRO gene were identified in familial cases of Turkish origin with childhood

onset of disease (Ozaltin et al 2011) The Ptpro null mice showed altered

podocyte structure and low glomerular filtration rate This study has suggested its

role in the regulation of podocyte structure and function (Wharram et al 2000)

14 AUTOSOMAL DOMINANT INHERITANCE OF STEROID


141 NEPHROTIC SYNDROME CAUSED BY ACTN4 GENE ( -

ACTININ- 4)

Mutations in the α-actinin 4 gene (ACTN-4 OMIM-604638) have been

reported to cause the familial form of infantile or adult onset NS with an autosomal

dominant (AD) mode of inheritance (Kaplan et al 2000 Pollak et al 2007) It is

localized on chromosome 19q13 and contains 21 exons (Kaplan et al 2000) It

encodes ά-actinin 4 a 100 KDa homodimeric cytoskeletal protein It is an actin

25

binding and cross linking protein that is essential for the podocyte cytoskeleton and

for motility (Weins et al 2007) It is highly expressed in the podocyte in the

glomeruli and interacts with the β integren protein cell adhesion molecules and

signaling proteins (Otey and Carpen 2004) The ά-actinin 4 is responsible for the

interaction between the actin cytoskeleton and the cellular membrane of podocyte

(Honda et al 1998) Actinin knockout mice develop proteinuria and die after 10

weeks with progressive glomerulosclerosis (Kos et al 2003) suggesting their role

in glomerular disease (Yau et al 2004)

Mutations in the ACTN4 gene are less frequent than in the NPHS1 and

NPHS2 genes in associated nephropathies (Obedova et al 2006) The ACTN4 gene

mutations (pI149del pW59R pV801M pR348Q pR837Q pR310Q pK228E

pT232I and pS235P) have been identified in five different families with an AD

mode of inheritance These mutations cause mild proteinuria in teen ages of the

patients and slow progression to ESRD in later life (Kaplan et al 2000 Weins et

al 2005) Most of the mutations in this gene are missense with increased affinity

towards F-actin that alters the mechanical characteristics of the podocyte (Kaplan et

al 2000) However a novel de novo mutation (pS262F) has also been identified

in familial cases of the age of 3-5 years with rapid progression toward ESRD (Choi

et al 2008) Recent studies have also reported a positive association of the

promoter region SNPs in this gene with idiopathic FSGS (Dai et al 2009 2010)

The recurrence of FSGS was not observed after renal transplantation in ACTN4

associated disease

26

142 NEPHROTIC SYNDROME CAUSED BY WT1 GENE (WILMrsquos

TUMOR)

Mutations in the Wilmrsquos tumor gene (WT1 OMIM-607102) have been

reported to cause AD form of SRNS (Mucha et al 2006) WT1 is a zinc finger

tumor suppressor gene and was identified in 1990 The WT1 gene spans

approximately 50 kb on chromosome 11p13 and encodes a 52-54 KDa transcription

factor (Call et al 1990) It contains 10 exons (Haber and Buckler 1992) Exons 1ndash

6 of the gene encode a prolineglutamine rich transcriptional regulatory region

whereas exons 7ndash10 encode the four zinc fingers of the DNA-binding domain

(Reddy and Licht 1996) WT1 expression is critically involved in the normal

development of the kidney and gonads In the kidney it is specifically expressed in

podocyte (Pritchard-Jones et al 1990) Mutations in this gene cause idiopathic

SRNS kidney tumor and glomerular nephropathy in children (Denamur et al

2000 Mucha et al 2006)

The WT1 gene mutations have been identified in patients with Wilmrsquos

tumor Denys-Drash syndrome (DDS OMIM-194080) and Frasier syndrome (FS

OMIM-136680 McTaggart et al 2001) In DDS the clinical presentations include

early onset NS rapid progression toward ESRD urogenital abnormalities XY

pseudohermaphrodism (female phenotype and male genotype) and Wilmrsquos tumor

DDS usually starts within the first year of life with a characteristic histology of

DMS (Habib et al 1985 Mueller 1994) In this gene deletion insertion nonsense

and frame shift mutations have been identified (Little et al 2005) Approximately

95 of the reported mutations are missense and are mainly found in exons 8 and 9

that code for the zinc finger domains 2 and 3 respectively (Jeanpierre et al 1998

27

Koziell et al 1999 Orloff et al 2005) The most common mutation found in this

syndrome is (pR394W) that affects the zinc finger domain 3 resulting in the loss or

alteration of its DNA binding ability (Hastie 1992)

Frasier syndrome is characterized by male pseudohermaphrodism

progressive glomerulopathy with FSGS and late onset ESRD Patients usually

present normal female external genitalia streak gonads and XY karyotype (Niaudet

and Gubler 2006) The knockout mice model showed the absence of both kidneys

and gonads suggesting a crucial role of the WT1 gene in the development of the

genitourinary tract (Patek et al 2003) The splice site mutations in WT1 gene

specifically insertion or deletion of a three amino acids lysine threonine and serine

(KTS) region seems important for normal glomerulogenesis and sex determination

(Barbaux et al 1997 Hammes et al 2001 Lahiri et al 2006) This splice site

mutation has been found in 12 young females with SRNS (Aucella et al 2006)

Several single nucleotide polymorphisms (SNPs) in the WT1 gene have been shown

to be associated with FSGS in the high-risk group of African Americans (Orloff et

al 2005) However further studies are needed to confirm the association of these

SNPs with the pathogenesis of NS by altering the WT1 function

143 NEPHROTIC SYNDROME CAUSED BY CD2AP GENE (CD2

ASSOCIATED PROTEIN)

Mutations in the CD2AP gene (CD2AP OMIM-604241) have been

reported to cause adult onset NS with FSGS CD2AP gene is localized on

chromosome 6p123 and comprises of 18 exons It encodes a multifunctional

adaptor protein of 80 KDa and is presents in the cytoplasm membrane ruffles and

28

leading edges of cells (Kirsch et al 1999) It was initially identified as a ligand

molecule for the T cells adhesion protein CD2 (Dustin et al 1998 Shih et al

1999) It is expressed primarily in podocyte at the site of SD The CD2 associated

protein specifically interacts with nephrin and plays an important role in the

maintenance of the podocyte structure (Shih et al 1999) The specificity of

nephrin and CD2 associated protein interaction was confirmed by the finding that

the C-terminal domain of CD2AP specifically interacts with the cytoplasmic

domain of nephrin (Dustin et al 1998 Shih et al 2001) CD2AP also acts as a

scaffolding protein in the dynamic regulation of the actin cytoskeleton of the

podocyte (Lowik et al 2007)

Mutations in the CD2AP gene cause pediatric and adult onset FSGS To

date five heterozygous and one homozygous mutations have been identified in the

NS patients Lowik and colleagues have provided the first supportive data of a

direct involvement of CD2AP in NS with the identification of a homozygous

truncating (pR612X) mutation of the CD2AP gene in a 10 months old NS child

(Lowik et al 2008) The splice site heterozygous mutation has also been identified

in two African Americans with FSGS (Kim et al 2003) Recent studies in Italy

have found three heterozygous mutations (pK301M pT374A and pdelG525) in

NS patients (Gigante et al 2009) The CD2 associated protein knockout mice have

been shown to develop proteinuria after 2 weeks and they died of renal failure at 6

weeks of age indicating the role of CD2AP in the pathogenesis of NS (Shih et al

1999) Thus further studies are required for confirming the true association with

CD2AP in NS pathogenesis

29

144 NEPHROTIC SYNDROME CAUSED BY TRPC6 GENE (TRANSIENT

RECEPTOR POTENTIAL CANONICAL CHANNEL 6)

Mutations in the transient receptor potential canonical channel 6 gene

(TRPC6 OMIM-603652) have been reported to cause adult onset FSGS with an

AD mode of inheritance (Reiser et al 2005 Winn et al 2005) It is localized on

chromosome 11q21-22 and comprises of 13 exons (Drsquo Esposito et al 1998) It

encodes the transient receptor potential canonical channel 6 (TRPC6) a member of

the transient receptor potential (TRP) ions channels that regulates the amount of

calcium pumped inside the cells It is expressed in the tubules and the glomeruli of

the kidney including podocyte and glomerular endothelial cells It interacts with

nephrin signaling molecules and cytoskeleton elements to regulate SD and

podocyte (Reiser et al 2005) The increased expression of TRPC6 in glomerular

podocyte causes a verity of glomerular diseases including MCD FSGS and MG

(Moller et al 2007) Mutations in the TRPC6 gene were first identified in a family

from Newzeland with an AD form of FSGS A missense (pP112Q) mutation

causes higher calcium influx in response to stimulation by Ang II The increased

signaling of calcium is responsible for podocyte injury and foot processes

effacement Mutation in the TRPC6 gene causes a later onset of diseases and milder

phenotype (Winn et al 2005)

Reiser and colleagues (2005) have reported mutations in the TRPC6 gene

(pN143S pS270T pR895C pE897K and pK874X) in five unrelated families of

Western European African and Hispanic ancestries The recent studies also

reported novel mutations in children and in adults with sporadic cases of FSGS

(Heeringa et al 2009 Santin et al 2009 Mir et al 2011) Zhu and colleagues

30

(2009) have found a novel mutation (pQ889K) in Asians that is associated with

FSGS (Zhu et al 2009) Mutation analysis studies have shown that TRPC6

mutations alter podocyte function control of cytoskeleton and foot process

architecture (Reiser et al 2005) Thus mutations in the TRPC6 gene are

responsible for massive proteinuria and ultimately lead to kidney failure in FSGS

145 NEPHROTIC SYNDROME CAUSED BY INF2 GENE (INVERTED

FORMIN-2)

Mutations in the inverted formin-2 gene (INF2 OMIM-610982) have been

reported to cause the familial AD form of FSGS (OMIM-603278) It is localized on

chromosome 14q3233 and comprises of 22 exons (Brown et al 2010) It encodes

a member of the formin family of actin regulating proteins that plays an important

role in actin filament assembly (Faix and Grosse 2006) The INF2 protein has the

distinctive ability to accelerate both polymerization and depolarization of actin It is

highly expressed in the glomerular podocyte It plays a key role in the regulation of

podocyte structure and function (Faul et al 2007)

Mutations in the INF2 gene have been found in families showing moderate

proteinuria and FSGS lesion in early adolescence or adulthood (Boyer et al 2011)

They account for about 12-17 of familial dominant FSGS cases The disease

often progresses to ESRD All of the mutations identified todate effect the N-

terminal end of the protein suggesting a critical role of this domain in INF2

function (Brown et al 2011) Thus mutation screening provides additional insight

into the pathophysiologic mechanism connecting the formin protein to podocyte

dysfunction and FSGS

31

15 NEPHROTIC SYNDROME CAUSED BY OTHER GENETIC

FACTORS

151 ANGIOTENSIN CONVERTING ENZYME (ACE) GENE

INSERTIONDELETION POLYMORPHISM

The angiotensin converting enzyme (ACE) gene insertiondeletion (ID)

polymorphisms have been extensively investigated in the pathogenesis of NS

(Luther et al 2003) The insertion or deletion of a 287 bp Alu repeat sequence in

intron 16 of the ACE gene is defined as an ID polymorphism (Rigat et al 1990)

ACE catalyzes the conversion of an inactive angiotensin I (AngndashI) into a

vasoactive and aldosterone-stimulating peptide angiotensin II (Ang-II Oktem et

al 2004) The deletion allele (D) has been associated with the higher

concentration of plasma ACE and AngndashII levels (Rigat et al 1990) An increased

ACE level has deleterious effects on renal hemodynamics and enhances

proteinuria (Oktem et al 2004) The use of ACE inhibitors reduces proteinuria in

patients with NS The reduction of proteinuria in these patients has suggested the

involvement of ACE inhibitors in the pathogenesis of NS (White et al 2003)

Therefore this study was carried out to determine the association of this

polymorphism with the risk of NS in Pakistani children The present study also

evaluates the effect of this polymorphism on the response to steroid therapy and

histological findings for FSGS and MCD in these patients

32

152 METHYLTETRAHYDROFOLATE REDUCTASE ENZYME

(MTHFR) GENE POLYMORPHISMS

The methyltetrahydrofolate reductase (MTHFR) enzyme plays an important

role in homocysteine and folate metabolism It catalyzes the NADPH-linked

reduction of 5 10 methyltetrahydrofolate to 5-methyltatrahydrofolate (Goyette et

al 1994) The two most common single nucleotide polymorphisms (SNPs C677T

and A1298C) in the MTHFR gene are known to cause elevated homocysteine levels

in the blood (Weisberg et al 1998 Lucock 2000) Hyperhomocysteinemia is an

independent risk factor for thrombosis atherosclerosis cardiovascular and renal

diseases etc (Buyukcelik et al 2008 Ferechide and Radulescu 2009 Kniazewska

et al 2009 Ciaccio and Bellia 2010) and similar complications are also associated

with the nephrotic syndrome (Louis et al 2003 Kniazewska et al 2009) These

observations emphasize the role of homocysteine metabolism in the NS patients

The present study investigated the role of these polymorphisms for the first time in

Pakistani NS children

For the population based studies described here the Hardy-Weinberg

Equlibrium (HWE) was examined The HW law is an algebraic expression for

genotypic frequencies in a population If the population is in HWE the allele

frequencies in a population will not change generation after generation The allele

frequencies in this population are given by p and q then p + q = 1

Genotype frequencies are given as p + q = 1rarr p2 + 2pq + q

2 = 1

33

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Weins A Pollak MR Inherited Nephrosis In Molecular and genetic basis of renal

disease 1st Edition Mount DM Pollak MR Saunders Elsevier Philadelphia PA

2008 142-145

Weins A Schlondorff JS Nakamura F Denker BM Hartwig JH Stossel TP

Pollak MR (2007) Disease-associated mutant alphaactinin-4 reveals a mechanism

for regulating its F-actin-binding affinity Proc Natl Acad Sci USA 104 16080-

16085

Weisberg I Tran P Christensen B Sibani S Rozen R (1998) A second genetic

polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with

decreased enzyme activity Mol Genet Metab 64 169-172

Wharram BL Goyal M Gillespie PJ Wiggins JE Kershaw DB Holzman LB

Dysko RC Saunders TL Samuelson LC Wiggins RC (2000) Altered podocyte

47

structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low

glomerular filtration rate J Clin Invest 106 1281-1290

White CT Macpherson CF Hurley RM Matsell DG (2003) Antiproteinuric

effects of enalapril and losartan a pilot study Pediatr Nephrol18 1038-1043

Winn MP Conlon PJ Lynn KL Farrington MK Creazzo T Hawkins AF

Daskalakis N Kwan SY Ebersviller S Burchette JL Pericak-Vance MA Howell

DN Vance JM Rosenberg PB (2005) A mutation in the TRPC6 cation channel

causes familial focal segmental glomerulosclerosis Science 308 1801-1804

Wing MR Bourdon DM Harden TK (2003) PLC-epsilon a shared effector

protein in Ras- Rho- and G alpha beta gamma-mediated signaling Mol Interv 3

273-280

Yao J Le TC Kos CH Henderson JM Allen PG Denker BM Pollak MR (2004)

Alpha-actinin-4-mediated FSGS an inherited kidney disease caused by an

aggregated and rapidly degraded cytoskeletal protein PLoS Biol 2 167

Zenker M Aigner T Wendler O Tralau T Muntefering H Fenski R Pitz S

Schumacher V Royer-Pokora B Wuhl E Cochat P Bouvier R Kraus C Mark K

Madlon H Doumltsch J Rascher W Maruniak-Chudek I Lennert T Neumann LM

Reis A (2004) Human laminin beta-2 deficiency causes congenital nephrosis with

mesangial sclerosis and distinct eye abnormalities Hum Mol Genet 13 2625-2632

Zhu B Chen N Wang ZH Pan XX Ren H Zhang W Wang WM (2009)

Identification and functional analysis of a novel TRPC6 mutation associated with

late onset familial focal segmental glomerulosclerosis in Chinese patients Mut Res

664 84-90

48

2 MATERIALS AND METHODS

49

21 SAMPLES COLLECTION

Blood samples of patients and controls were obtained from the pediatric

nephrology OPD at the Sindh Institute of Urology and Transplantation (SIUT)

with their informed consent or that of their parents The blood samples were

collected in 4 ml ethylenediaminetetraacetic acid (EDTA) treated vacutainers

(Beckton Dickinson) All the studies reported in this thesis were approved by the

Institutional Review Board (IRB) Centre for Biomedical Ethics and Culture

(CBEC) SIUT and conformed to the tenets of the Declaration of Helsinki

22 EXTRACTION OF DNA FROM FRESH BLOOD

Isolation of the genomic deoxyribonucleic acid (DNA) was carried out by

using a modified organic extraction protocol (Sambrook amp Russell 2001) The

blood samples were mixed with thrice the volumes of red cell lysis buffer (RCLB

001 M potassium bicarbonate 015 M ammonium chloride and 05 M EDTA pH-

74) and then kept on ice for 30 minutes The samples were centrifuged in an

AllegraTM

25R (Beckman Coulter USA) centrifuge at 1200 rpm for 10 minutes at

4˚C The pellets were then washed with 10 ml of RCLB and resuspended in 475 ml

saline TrisndashEDTA (STE pH-80) 250 microl of 10 sodium dodecyl sulfate (SDS)

was slowly added drop wise with vortexing followed by 5 microl proteinase K (20

mgml) The tubes were then incubated overnight in a rotary water bath at 55˚C

The next day equal volumes of Tris-equilibrated phenol (pH 80) was

added (Maniatis et al 1982) mixed gently for 10 minutes and kept on ice for 10

minutes After centrifugation at 3200 rpm for 30 minutes at 4oC the aqueous layer

was carefully removed with the help of 1ml micropipette tips The samples were

50

then extracted a second time with equal volumes of chloroform-isoamyl alcohol

(241 vv) The samples were mixed gently for 10 minutes placed on ice for 10

minutes and then centrifuged at 3200 rpm for 30 minutes at 4oC The aqueous layer

was again collected in another tube DNA was precipitated by adding one tenth

volume of 10 M ammonium acetate followed by two volumes of absolute ethanol

(or an equal volume of isopropanol) and stored overnight at -20oC The precipitated

DNA was centrifuged at 3200 rpm for 60 minutes at 4oC The DNA pellet was then

washed with 70 ethanol and centrifuged again at 3200 rpm for 40 minutes The

pellet was air dried or vacuum dried for 10 minutes to remove traces of ethanol

The purified DNA was resuspended in 500 microl of TrisndashEDTA (pH 80) and placed in

a shaking water bath at 55oC

221 QUANTIFICATION OF DNA

The optical density (OD) was measured at 260 and 280 nm using a USVIS

spectrometer (Lambda Ez201 Perkin Elmer)

The concentration of DNA in the sample was calculated using the formula

Absorbance at 260 nm X dilution factor X 50 = ngmicrol DNA

(Where 50 is the correction factor for double stranded DNA)

If the ratio OD260OD280 was found to be 17ndash20 the DNA was considered

pure and free of contaminating phenol or protein The samples were then

transferred to an appropriately labeled Eppendorf tube and stored at 4oC

51

23 POLYMERASE CHAIN REACTION (PCR)

Polymerase chain reaction was first described by the efforts of Saiki et al

(1985) and this method was widely used in this thesis to amplify the fragments of

interest from genomic DNA

The polymerase chain reaction was performed with GoTaqreg Flexi DNA

Polymerase kit from Promegareg (Madison WI USA) Briefly the PCR master mix

containing 1X PCR buffer 15 mM magnesium chloride 01 mM dNTPs

(Promega) 025 units of GoTaqTM

DNA polymerase 04 microM of each primer

(MEG Operon) and 60 ng of the genomic DNA were added in a total PCR reaction

volume of 25 microl A negative (master mix only) and a positive control (master mix +

successfully amplified DNA containing target sequence) were set up for each

experiment

The amplification reactions were carried out in the Veriti 96 well thermal

cycler (Applied Biosystemsreg California

reg USA) using the following PCR program

initial denaturation at 95˚C for 5 minutes followed by 35 cycles of denaturation at

95˚C for 1 minute annealing at 55˚C for 1 minute and extension at 72˚C for 1

minute The final extension was at 72˚C for 10 minutes The PCR products were

kept at 4˚C for electrophoresis

A number of precautions were taken to minimize the possibility of

obtaining non-specific PCR products eg varying the concentration of MgCl2 or

annealing temperature etc as described in this thesis where necessary In some

instances where required a lsquohot-startrsquo PCR method was used that involves the

addition of Taq polymerase after the first denaturation step

52

24 AGAROSE GEL ELECTROPHORESIS

A 1-2 solution of agarose (LE analytical grade Promegareg

) was

prepared in TBE electrophoresis buffer (06 M trizma base 09 M boric acid 0024

M EDTA pH 80) The solution was heated in a loosely stoppered bottle to

dissolve the agarose in a microwave oven After mixing the solution and cooling to

about 55oC ethidium bromide was added to the solution at a concentration of 05

microgml and poured onto the casting platform of a horizontal gel electrophoresis

apparatus An appropriate gel comb was inserted at one end The bottom tip of the

comb was kept 05ndash10 mm above the base of the gel After the gel had hardened

the gel comb was withdrawn Sufficient electrophoresis buffer was added to cover

the gel to a depth of approximately 1 mm Each DNA sample in an appropriate

amount of loading dye (0125 Orange G 20 ficoll and 100 mM EDTA) was

then loaded into a well with a micro-pipettor Appropriate DNA molecular weight

markers (100 bp DNA ladder Promega) were included in each run Electrophoresis

was carried out at 100 volts for 30ndash40 minutes The gel was visualized and

recorded using a gel documentation system (Bio Rad system)

On occasions when a particular DNA fragment was required to be isolated

the appropriate band was cut out using a sterile blade or scalpel DNA was

recovered from the agarose gel band using the QIA quick gel extraction kit

(QIAGEN Germany)

53

25 AUTOMATED FLUORESCENT DNA SEQUENCING

Automated DNA sequencing (di-deoxy terminator cycle sequencing

chemistry) method was carried out using a 3100 genetic analyzer (ABI) and the

BigDye terminator cycle sequencing kit (version 31) DNA was first amplified by

polymerase chain reaction in a 25 microl reaction volume The PCR reaction and

thermal cycler conditions were described earlier in the PCR method

251 PRECIPITATION FOR SEQUENCING REACTION

Amplified PCR products were checked on a 2 agarose gel and then

precipitated with 14 volumes of 75 of isopropanol (analytical grade Scharlau)

Samples were washed with 250 microl of 75 isopropanol and the pellets were

resuspended in autoclaved deionized water as required The PCR products were

also purified with the Wizard SV gel and PCR clean-up system (Promegareg)

according to the manufacturerrsquos instructions

252 SEQUENCING REACTION

The following sequencing reaction conditions were used

Autoclaved deionized water 4microl

10X sequencing buffer 1microl

Big Dye Terminator ready reaction mix

labeled dye terminators buffer and dNTPrsquos

2microl

Forward or reverse sequence specific primer 1microl

Template DNA 2microl

Total reaction volume 10microl

54

PCR was performed using a Gene Amp PCR System 9700 thermal cycler

(Applied Biosystem) for 25 cycles as follows 95oC for 10 seconds 50

oC for 5

seconds and 60oC for 4 minutes

After amplification the products were precipitated with 40 microl of 75

isopropanol washed with 125 microl of 75 isopropanol and air or vacuum dried The

pellets were resuspended in 10 microl of Hi-Di Formamide (ABI) denatured at 95oC

for 5 minutes and then loaded into the 96-well plate for sequencing using the ABI

3100 Genetic Analyzer

26 POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

A 10 polyacrylamide gel solution was prepared by adding 62 ml of 40

acrylamide stock solution (391 acrylamide bisacrylamide) to 25 ml of 10 X TBE

buffer (pH-80) and volume was adjusted to 250 ml with deionized water The

casting base seal of electrophoresis cell (Sequi Gen GT nucleic acid electrophoresis

system Bio Rad) was prepared by pouring the 50 ml from 10 acrylamide added

with 300 microl of 25 ammonium persulphate (APS) and 150 microl of N N N N

tetramethylethylenediamine (TEMED) and allowed the gel to polymerize for 10

minutes

The glass plates and spacers were washed and cleaned with 70 ethanol

and treated with siliconizing fluid (Sigma coat Sigma) Spacers (075 mm) were

placed between the front and rear plates that were then tightly clamped and placed

in a tilted position on the table The gel solution was prepared by adding 200 ml of

10 acrylamide solution with 850 microl of 25 APS solution and 150 microl of TEMED

55

mixed thoroughly and carefully poured into the plates without any bubble

formation The comb was inserted between the plates and the gel was allowed to

polymerize for at least 2 hours at room temperature

After polymerization the gel unit was assembled with upper and lower

reservoirs filled with 2 L of 1 X TBE buffer The gel unit was pre-run for 15

minutes at 100 Watts constant power (Bio Rad HV Power Pac) and the comb was

removed carefully Each sample was prepared by adding 6 microl of gel loading dye

(025 bromophenol blue 025 xylenecyanol and 30 ficoll) to each amplified

product and loaded in the appropriate well The molecular weight marker (100 bp)

was added into the first lane The gel was run at 100 Watts for ~4hours After

complete migration of the samples the gel was removed from the casting plates

with care and cut according to expected product sizes The gel was stained with

ethidium bromide for a few minutes and analyzed using the gel documentation

system (Bio Rad)

27 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

Restriction fragment length polymorphism (RFLP) PCR is based on the

principle that a base change results in the creation or abolition of a restriction site

PCR primers are designed from sequences flanking the restriction site to produce a

100-500 base pair product The amplified product is subsequently digested with the

appropriate restriction enzyme and fragments are analyzed by PAGE

The master mix for PCR is as follows 1X PCR buffer 25 mM magnesium

chloride 02 mM dNTPs (Promega) 1 U of Taq polymerase 035 microM of each

primer (MEG Operon) and 64 ng of the genomic DNA were prepared in a total

56

reaction volume of 25 microl The amplification reaction was carried out in a Bio Rad

C-1000 thermal cycler using the following PCR cycling parameters initial

denaturation at 92˚C for 2 minutes followed by 35 cycles of denaturation at 92˚C

for 1 minute annealing at 62˚C for 1 minute and extension at 72˚C for 30 seconds

and a final extension at 72˚C for 7 minutes

RFLP analyses of methylenetetrahydrofolate reductase (MTHFR)

polymorphisms ldquoC6777Trdquo and ldquoA1298Crdquo were performed according to Skibola et

al 1999 The fragment digestion of the amplified product was carried out with

HinfI and MboII restriction enzymes 20 microl of the PCR products were digested with

10 U of HinfI enzyme for C6777T and 25 U of MboII enzyme for A1298C

polymorphisms with 20 μl of the recommended buffer at 37degC overnight

After complete digestion the samples were run on an adjustable PAGE

electrophoresis apparatus 10 acrylamide gel was prepared by adding 62 ml of a

40 polyacrylamide stock solution to 25 ml of 10X TBE buffer and the volume

was adjusted to 25 ml with deionized water The solution was mixed thoroughly

and 85 ul of 25 ammonium persulfate (APS) and 27 ul of TEMED were added

The gel plates (165 cmtimes145 cm) were cleaned with 70 ethanol and adjusted

with 1 mm thick spacer and sealing gaskets The gel solution was poured into the

plates and a 1 mm thick comb was inserted between the plates The gel was

allowed to polymerize for 20 minutes

After polymerization the comb and sealing gaskets were removed and the

plates were placed in the electrophoresis apparatus (adjustable height dual gel unit

Sigma-Aldrich) TBE buffer (1X pH-80) was added to the upper and lower

chambers of the apparatus Initially the gels were pre-run at 200 volts for 15

57

minutes The samples for loading were prepared by adding 6 microl loading dye (see

page 54) into the digested products The gel was run at 200 volts for 1hour and 30

minutes depending on the product size The gel was stained with 05 microgml

ethidium bromide solution for 5 minutes and was analyzed on the gel

documentation system

28 STATISTICAL ANALYSIS

Statistical analyses were carried out using Statistical Package for Social

Sciences (SPSSreg) version 17 for Windows

reg Cochran-Armitage trend test was

carried out with χLSTATreg The associations between polymorphism and clinical

outcomes were analyzed by χsup2 test of independence and odds ratios For all the

statistical analyses p-values less than 005 were considered to be significant

Odds Ratio

An odds ratio (OR) is defined as the ratio of the odds of an event occurring

in one group (disease) to the odds of it occurring in another group (controls) The

OR greater than one means significant association and less than one show no

association between groups

Chi-square test

Chi-square is a statistical test commonly used to compare observed data

with data we would expect to obtain according to a specific hypothesis

The formula for calculating chi-square ( χ2) is

χ

2= sum (o-e)

2e

That is chi-square is the sum of the squared difference between observed

(o) and the expected (e) data (or the deviation d) divided by the expected data in

all possible categories

58

29 REFERENCES

Boyam A (1968) Separation of lymphocytes and erythrocytes by centrifugation

Scand J Clin Lab Invest 21 (Supplement 97) 91

Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual

Cold Spring Harbor laboratory Cold Spring Harbor New York 1982

Mullis KB Faloona FA (1987) Specific synthesis of DNA in vitro via a

polymerase-catalyzed chain reaction Methods Enzymol 155 335-350

Sambrook J Russell DW Molecular Cloning A laboratory manual 3rd

Edition

Cold Spring Harbor Laboratory Press Cold Spring Harbor New York 2001

Saiki RK Scharf S Faloona F Mullis KB Horn GT Erlich HA Arnheim N

(1985) Enzymatic amplification of beta-globin genomic sequences and restriction

site analysis for diagnosis of sickle cell anemia Science 230 1350-1354

Skibola CF Smith MT Kane E Roman E Rollinson S Cartwright RA Morgan

G (1999) Polymorphisms in the methylenetetrahydrofolate reductase gene are

associated with susceptibility to acute leukemia in adults Proc Natl Acad Sci USA

96 12810-12815

59

3 A SPECTRUM OF NOVEL NPHS1 AND NPHS2 GENE

MUTATIONS IN PEDIATRIC NEPHROTIC SYNDROME

PATIENTS FROM PAKISTAN

60

31 INTRODUCTION

Nephrotic syndrome (NS) in children is characterized by proteinuria

edema hypoalbuminaemia and hyperlipidemia Clinically pediatric NS can be

classified as congenital (CNS) infantile and childhood onset CNS appears in utero

or during the first three months of life Infantile and childhood onset NS are

diagnosed during and after the first year of life respectively The majority of early

onset NS cases have a genetic origin with a widespread age of onset that ranges

from fetal life to several years (Avni et al 2011) Most patients respond to steroid

therapy and show a favorable long term outcome However 10-20 of the patients

show resistance to the therapy and are classified as a steroid resistant nephrotic

syndrome (SRNS) These patients tend to progress to end stage renal disease

(ESRD) due to the progressive damage of the glomerular filtration barrier (GFB

Yu et al 2005)

Glomerular pathology in NS mostly appears as minimal change disease

(MCD) focal segmental glomerulosclerosis (FSGS) or diffuse mesengial sclerosis

(DMS) According to ldquoThe International Study of Kidney Diseases in Childrenrdquo

(1978) the most common histological manifestation of childhood NS is sporadic

MCD affecting 77 of the children followed by FSGS (8) According to the data

available in Pakistan MCD is the leading cause of idiopathic NS in children (43

of cases) followed by FSGS (38 of cases) The FSGS is the predominant

pathology in SRNS and adolescent NS (Mubarak et al 2009)

Mutations in several genes that are highly expressed in the GFB and

podocytes have been reported to cause pediatric NS In a study of a large cohort of

patients with isolated sporadic NS occurring within the first year of life two third

61

of the cases were due to mutations in the NPHS1 NPHS2 WT1 and LAMB2 genes

(Hinkes et al 2007) The NPHS1 and NPHS2 genes together share a large

proportion of mutations that cause NS in children The other two genes WT1 and

LAMB2 have also been associated with syndromic or complex forms (Lowik et al

2009 Zenker et al 2009) The TRPC6 PLCE1 CD2AP ACTN4 genes are also

involved in the etiology of NS (Kaplan et al 2000 Santin et al 2009 Benoit et

al 2010 Boyer et al 2010) Recently mutations in the IFN2 MYOE1 and

PTPRO genes have been reported in NS and in childhood familial FSGS cases

(Brown et al 2010 Mele et al 2011 Ozaltin et al 2011)

Mutations in the NPHS1 gene were initially described as the cause of the

lsquoFinnish typersquo of nephrotic syndrome (CNF) In Finland two mutations Finmajor

(c121delCT pLeu41fs) and Finminor (c3325CgtT pArg1109Ter) were found in

78 and 16 of the cases respectively (Kestila et al 1998) These two mutations

have rarely been observed outside Finland However in studies on European North

American and Turkish NS patients mutations in the NPHS1 gene account for 39-

55 cases of childhood NS and 40 of all cases of CNS (Lenkkeri et al 1999

Kestila et al 2007 Heeringa et al 2008) Other reports have observed NPHS1

gene mutations in NS patients that are more than three months of age (Philippe et

al 2008) It has also been suggested that NS caused by NPHS1 gene mutations

consistently show resistance to steroid therapy (Hinkes et al 2007 Heeringa et al

2008 Jalanko 2009) However recently it has been reported that gt20 of CNS

patients including patients with NPHS1 gene mutations may respond to

antiproteinuric treatment (Schoeb et al 2010)

62

Mutations in the NPHS2 gene cause an autosomal recessive form of SRNS

with an early onset of the disease and renal histology of FSGS (Boute et al 2000)

The NPHS2 gene mutations have also been identified in 51 of CNS cases of

European origin and also in adult onset form of FSGS (Tsukaguchi et al 2002

Hinkes et al 2007) The incidence of NPHS2 gene mutations in familial SRNS

was found to be 40 in European and American children 29 in Turkish and 0

in Japanese and Korean children (Lowik et al 2009)

Idiopathic NS is one of the major glomerular diseases in Pakistani children

and approximately 30 of the NS cases show resistance to steroid therapy

(Mubarak et al 2009) This is in contrast to the other parts of the world where 10-

20 of the NS cases show steroid resistance (Ruf et al 2004 Weber et al 2004)

This study was therefore carried out to find the frequency of disease causing

mutations in the NPHS1 and NPHS2 genes in Pakistani children suffering from

congenital early and childhood onset NS To our knowledge this is the first

comprehensive screening of NPHS1 and NPHS2 gene mutations in pediatric NS

cases from South Asia


321 PATIENTS RECRUITMENT AND DATA COLLECTION

A total of 145 NS patients were recruited from the pediatric nephrology

department of the Sindh Institute of Urology and Transplantation Karachi and

pediatric nephrology department of the Children Hospital Lahore The research

protocol was approved by the Institutional Review Board and conformed to the

63

tenets of the Declaration of Helsinki Written informed consent was obtained from

the parents of all the subjects

Patients with CNS infantile and childhood onset NS including familial and

sporadic cases that are younger than 16 years of age were recruited in this study

All the children were resistant to standard steroid therapy NS patients with

extrarenal abnormalities were excluded from this study

NS was diagnosed by the presence of edema urinary protein excretion

equal to or greater than 40 mgm2hr and serum albumin below 25 gl Renal

failure was designated when estimated glomerular filtration rate (eGFR) was less

than 90 mlmin by the Schwartz formula (Schwartz and Work 2009) All the

patients received standard steroid therapy on initial presentation The clinical

response to steroid therapy was classified as described earlier (Mubarak et al

2009) (1) steroid sensitive ie complete remission of proteinuria during the steroid

therapy persisting for at least 12 weeks after therapy (2) steroid dependent ie

remission of proteinuria during therapy but recurrence when the dosage was

reduced below a critical level or relapse of proteinuria within the first three months

after the end of therapy and (3) resistant ie no remission of proteinuria during 4

consecutive weeks of daily steroid therapy

322 MUTATION ANALYSIS

Blood samples were collected in acid citrate dextrose (ACD) vacutainer

tubes Genomic DNA was extracted using the standard phenol-chloroform

extraction procedure as described earlier Mutation analysis was performed by

direct DNA sequencing of all the 29 exons of the NPHS1 gene and the 8 exons of

64

the NPHS2 gene Genomic sequences of the two genes were obtained from the

Ensembl genome browser (Ensembl ID ENSG00000161270 and

ENSG00000116218 respectively) and exon-specific intronic primers were designed

in the forward and reverse directions and were obtained commercially (Eurofins

MWG Operon Germany) The primer sequence and PCR conditions for screening

NPHS1 and NPHS2 gene are described in the Table- 31 and 32 Each exon was

individually amplified by PCR in a 25 microl reaction volume using 1microg of genomic

DNA under standard PCR conditions as described in Materials and Methods

section Amplified PCR products were purified using the PCR clean-up kit

(Promega Wizardreg Promega Corporation Madison WI USA) The sequencing

reaction was performed using the BigDye terminator cycle sequencing kit V31

(Applied Biosystemsreg California USA) Sequencing products were purified using

the Centri-Sep spin columns (Princeton Separationreg) and were analyzed on an

automated DNA analyzer (ABI 3100) Each mutation was confirmed by repeat

sequencing in both the forward and reverse orientations To differentiate between

mutations and polymorphisms 100 healthy controls were also analyzed using direct

DNA sequencing To assess the damaging effects of missense mutations in silico

the online database PolyPhen-2 (Polymorphism Phenotyping v2

httpgeneticsbwhharvardedupph2indexshtml) was used (Adzhubei et al

2010)

65

Table- 31 Primer pairs and PCR conditions for mutation screening of the

NPHS1 gene

EXON PRIMER SEQUENCE (5rsquo gt 3rsquo) PCR product

size (bp)

PCR conditions

1F AGAGGGGAAGAGGAAAACGA 400 bp 52ordmC X 15mMMg+2

1R CACCACCGTCAGGTTTTCAG 400 bp 52ordmC X 15mMMg+2

2F TGCTGACTGAAGGTGAGTGG 463bp 62ordmC X 3mMMg+2

2R CTCATACTCCGCGTCATCG 463bp 62ordmC X 3mMMg+2

3F CCCAGGATCCCAGGCTTC 401bp 65ordmC X 15mMMg+2

3R GGGTAAGCTTCCAGCACTGA 401bp 65ordmC X 15mMMg+2

4F ACCCATGAGTCTGGGCTTC 394bp 63ordmC X 15mMMg+2

4R CCCAGGGATGACATCTTTTC 394bp 63ordmC X 15mMMg+2

5F GGCCCTTTTCCTCTAGAACG 377bp 54ordmC X 15mMMg+2

5R ATGAGCCACCACCTCTGTTC 377bp 54ordmC X 15mMMg+2

6F CTGGATCCCAGAGGAGATCA 354bp 58ordmC X 15mMMg+2

6R GAACCCCCATGTTTCTCTGA 354bp 58ordmC X 15mMMg+2

7F GGGATCACAGGGATTATGGA 388bp 61ordmC X 1mMMg+2

7R GCCTGGGTGTGCTCTGTG 388bp 61ordmC X 1mMMg+2

8F GGGGTAATCCCTTAGCCACA 424bp 59ordmC X 15mMMg+2

8R CCAGACAGAACAGGACTGGAG 424bp 59ordmC X 15mMMg+2

9F GTGTGCCCCCAAATTATGC 398bp 55ordmC X 15mMMg+2

9R CCATGGTCCTCAAGGAGAAA 398bp 55ordmC X 15mMMg+2

10F ATGTCTCCTGTGTCCCTGCT 382bp 63ordmC X 2mMMg+2

10R GAGCTTCTGGCCCTCTGG 382bp 63ordmC X 2mMMg+2

11F TGTCCAACCTGACATTCCTG 480bp 62ordmC X 1mMMg+2

11R CTGATTCCCTGCCAAACCT 480bp 62ordmC X 1mMMg+2

12F TGGTGCTGATGAGAGTGCTT 527bp 60ordmC X 15mMMg+2

12R GTTGGAGGAGCGAGACTCAG 527bp 60ordmC X 15mMMg+2

13F GAGGGACAGAGCCAGGTG 341bp 60ordmC X 15mMMg+2

13R AGCCTTTGAATGGGGCTCT 341bp 60ordmC X 15mMMg+2

14F GACAAGGAAGGGGAGAGGTG 495bp 63ordmC X 15mMMg+2

14R GCTCAGGAGTTGGAGACTGC 495bp 63ordmC X 15mMMg+2

15amp16F ACAACCTTAAACCCCGTCGT 595bp 63ordmC X 3mMMg+2

15amp16R GTTCCAGGATGGGTGGCTAT 595bp 63ordmC X 3mMMg+2

17F GAGGGTGGAGACAACCTCAC 472bp 62ordmC X 3mMMg+2

17R CATTCATTTTGCCACCAACA 472bp 62ordmC X 3mMMg+2

18F AGATGGATGACAGGAGAATTTTT 470bp 60ordmC X 15mMMg+2

18R CAGCTGCAGCCACCTTAGTT 470bp 60ordmC X 15mMMg+2

19F GATTCACCATGCCAAACTGG 469bp 62ordmC X 1mMMg+2

19R CACTCATTCCTCCACCCATT 469bp 62ordmC X 1mMMg+2

20F GGATGAATGGATAGATAGGCAGA 399bp 55ordmC X 1mMMg+2

20R AGGCAAAAACTCCATCCTCA 399bp 55ordmC X 1mMMg+2

21F GTTTGCCAGAGCAGTGTTCA 390bp 50ordmC X 3mMMg+2

66

21R CCACATAGTGGAACCCTGGA 390bp 50ordmC X 3mMMg+2

22F TGACCCTCCATCAGGATTAAA 499bp 56ordmC X 15mMMg+2

22R TGTGACCTTGGACAATTTGC 499bp 56ordmC X 15mMMg+2

23F TCAGCAATTTCTAGCTCTCTTTGA 323bp 56ordmC X 15mMMg+2

23R GCTTGGCCAGAACTAAGTCG 323bp 56ordmC X 15mMMg+2

24amp25F GTCTTGCTGAGGGTGAGGAG 489bp 65ordmC X 3mMMg+2

24amp25R AACAAAGCCCTTTCCATCCT 489bp 65ordmC X 3mMMg+2

26amp27F CAGGTTGATCATTGCCCTTC 495bp 56ordmC X 15mMMg+2

26amp27R CATGGTCAGGCCTCTTTGT 495bp 56ordmC X 15mMMg+2

28F CATGGGGTTCATCATAAGCA 440bp 60ordmC X 3mMMg+2

28R CCTCTCCTGACACCAAGTCC 440bp 60ordmC X 3mMMg+2


NPHS2 gene


size (bp)

PCR conditions

1F ACCCGACGGTCTTTAGGG 514bp 55ordmC X 15mMg+2

1R AGCATCCAGCAATCTGCTCT 514bp 55ordmC X 15mMg+2

2F CAGGCCCTGTGAACTCTGAC 400bp 63ordmC X 3mMg+2

2R GAAGGTGAGTCTGGGGTGAG 400bp 63ordmC X 3mMg+2

3F TTTTTCCTGGTTCTCAAAACAAA 396bp 61ordmC X 2mMg+2

3R CCAATTCTCTCTCTTGGCTACC 396bp 61ordmC X 2mMg+2

4F GATGGGCCAATGGTCTGTAA 391bp 62ordmC X 3mMg+2

4R TCCCTAGATTGCCTTTGCAC 391bp 62ordmC X 3mMg+2

5F GGGTAGGCCAACTCCATTTT 455bp 55ordmC X 15mMg+2

5R TATGAGCTCCCAAAGGGATG 455bp 55ordmC X 15mMg+2

6F CTCTTTGCAAGGCACTGTGA 372bp 55ordmC X 15mMg+2

6R TGGCTGTAAGATATTAGGTGATTTG 372bp 55ordmC X 15mMg+2

7F AGGAATGGCACACTCTGGTC 343bp 58ordmC X 2mMg+2

7R GTTGTAAGGGCCCAAGACAG 343bp 58ordmC X 2mMg+2

8F CTGTCTCCCCAGCTCAAGAC 596bp 61ordmC X 08mMg+2

8R TGGATGGTGCATTGTGACTT 596bp 61ordmC X 08mMg+2

67

33 RESULTS

331 CLINICAL CHARACTERISTICS OF PATIENTS

In this study a total of 145 patients including 36 early-onset and 109

childhood-onset NS were screened for disease-causing mutations in the NPHS1 and

NPHS2 genes Early-onset cases include children with congenital and infantile

onset of NS Among these 106 patients were sporadic cases whereas 39 patients

belonged to 30 different families The clinical characteristics of the patients are

given in Table- 33 Clinical data were obtained for all the cases (Table- 34) Renal

failure was established in 22 patients One patient had undergone kidney

transplantation with no recurrence over a period of 2 years of follow up Renal

biopsy results were available for 99 cases mostly representing FSGS (48 cases) and

MCD (27 cases)

332 MUTATIONS IN THE NPHS1 GENE

A total of 7 homozygous mutations were identified in 8 patients in the

NPHS1 gene (Figure- 31 Table- 35) Among these 6 mutations were novel while

only one known mutation was found in three patients All these mutations were

identified in either CNS or infantile cases only These mutations were not present

in the 100 normal controls

Three patients (NS145 NS300 and NS310) who had severe proteinuria at

birth or in early infancy were identified to have a homozygous pR1160X mutation

that resulted in the premature termination of the nephrin protein This mutation has

been reported to be associated with both severe and mild CNF cases (Koziell et al

2002) All the children had a normal renal outcome at the ages of 6 months 15

years and 25 years respectively

68

Table- 33 Clinical characteristics of children with idiopathic nephrotic

syndrome

Total number of children n 145

Age of onset since birth ndash 14 years

Males () 88 (607)

Females () 57 (393)

Male to female ratio 151

Classification of NS

Congenital infantile NS () 36 (25)

Childhood NS () 109 (75)

Renal biopsy findings n=99

FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)

Negative () 106 (73)

Outcome

ESRDg CRF

h 14 (96)

Lost to follow-up 9 (62)

Expired 8 (55)

a focal segmental glomerular sclerosis

bminimal change disease

cIgM nephropathy

dmesengial proliferative glomerulonephritis

emembranous glomerulonephritis

fmesengio capillary glomerulonephritis

gend stage renal disease

hchronic renal

failure

69

Table- 34 Clinical characteristics of all 145 patients examined

S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid

response Response to therapy Patient outcome

1 NS001 No 14 M bIgMN a

SRNS q- d

ESRD ndash eTx

2 NS003 No 1 F fMCD SRNS No response Lost to follow up

3 NS008 No 5 M - SRNS Complete remission to

CyA -

4 NS015A Yes 10 M MCD SRNS Partial remission to CyA -

5 NS015B Yes 11 M gFSGS SRNS Partial remission to CyA -

6 NS021 Yes 25 F FSGS SRNS - ESRD Expired

7 NS030 Yes 7 M - SRNS - Lost to follow up

8 NS032 Yes 10 F FSGS SRNS Partial remission to CyA -


10 NS034 No 04 F iMesPGN SRNS Partial remission to CyA -

11 NS037 No 12 F jMGN SRNS Maintained on

kACEI +

lARB

-

12 NS039A Yes 5 M MCD SRNS Maintained on ACEI

+ARB -

13 NS039B Yes 85 F - SRNS Maintained on ACEI

+ARB -

70

14 NS044 No 8 M FSGS SRNS No remission -


16 NS049B Yes 25 F - SRNS No response -

17 NS050 No 12 M FSGS SRNS Partial remission to CyA -

18 NS052 No 07 M MCD SRNS Complete remission to

CyA

19 NS060 No 11 F MCD SRNS - Lost to follow up

20 NS061 No 11 F MCD SRNS - Expired

21 NS064 Yes 4 F - - In remission -

22 NS065 Yes 1 F IgMN - Partial remission to CyA mCRF

23 NS084 No 5 M C1q

Nephropathy SRNS Partial remission to CyA -

24 NS088 No 8 F FSGS SRNS Complete remission to

CyA -


26 NS104 No 105 M MesPGN SRNS Partial remission to CyA CRF

27 NS110 No 9 F FSGS SRNS - Expired

28 NS113 No 07 F - SRNS No remission -

29 NS118 No 22 M FSGS SRNS Complete remission to

CyA -

30 NS122 Yes 13 F FSGS SRNS Maintained on ACEI

+ARB -


71

32 NS124 No 125 M IgMN SRNS Complete remission to

CyA -

33 NS125 No 3 F FSGS SRNS Partial remission to CyA ESRD

34 NS128 No 7 F MCD SRNS Partial remission to CyA -

35 NS129 No 1 M MCD SRNS Partial remission to CyA ESRD

36 NS130 No 5 M FSGS SRNS Maintained on ACEI

+ARB -


nCyP

-


CyA -

39 NS135 No 7 F - - No remission -

40 NS136 No 85 M - - No remission -


42 NS138 Yes 8 M FSGS SRNS Partial remission to CyA -

43 NS139 No 4 F MCD oSDNS On ACEI +ARB -

44 NS140 No 35 M - SDNS - -

45 NS141 No 7 M - SNS Partial remission to ACEI -


47 NS145 No 01 F FSGS SRNS Maintained on ACEI

+ARB -

48 NS146A Yes 11 M FSGS SRNS Partial remission to CyA -

49 NS146C Yes 10 M FSGS SRNS Complete remission to

CyA -

72

50 NS146D Yes 115 F FSGS SRNS - -

51 NS147 No 35 M MCD SRNS No response to CyA Tac CRF

52 NS148 No 4 M - - No response -

53 NS152 No 1 M - SRNS - Lost to follow up

54 NS153 No 5 F - - No response -

55 NS154 No 11 F IgMN SRNS Complete remission to

CyA -

56 NS155 No 3 M - SRNS In remission -



CyA -


60 NS162 No 9 M pMCGN SRNS Maintained on ACEI +

ARB CRF

61 NS165 No 7 M MCD SRNS Maintained on ACEI

+ARB -

62 NS167 Yes 9 M - - - -

63 NS169 Yes 3 M FSGS SRNS Complete remission to

CyA -


65 NS175 No 11 M FSGS SRNS Partial remission to CyA ESRD

66 NS176 No 55 M IgMN SRNS Partial remission to CyA -

67 NS180 No 4 F - SRNS - Lost to follow up

73

68 NS181A Yes 7 M - SSNS Being treated for first

relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -


72 NS187 No 4 F MCD SRNS Complete remission to

CyA -


Tac -


75 NS193 Yes 65 F FSGS SRNS Complete remission to

CyP -


CyP -

77 NS196 No 3 F FSGS SRNS - ESRD

78 NS197 No 4 F MCD SRNS Partial remission CyA -

79 NS200 No 4 M FSGS SRNS Partial remission CyA -


81 NS202A Yes 3 M FSGS SRNS Partial remission CyA -

82 NS202C Yes 5 F FSGS SRNS Partial remission CyA -

83 NS203 No 11 M - - - -


85 NS206 No 95 F FSGS SRNS Partial remission to Tac -

74

86 NS207 No 3 M MesPGN SRNS - -

87 NS209 No 25 M MesPGN SRNS Maintained on ACEI

+ARB -

88 NS211 No 2 M MCD SRNS Partial response to Tac -

89 NS213 Yes 5 M FSGS - No response -

90 NS214 Yes 6 M FSGS - - -


CyP -


93 NS217 No 6 M - - - Expired

94 NS218 No 25 F FSGS SRNS Partial remission to CyA -

95 NS220 No 5 M FSGS SRNS - ESRD

96 NS221 Yes 1 M - - - -

97 NS222 No 3 F FSGS SRNS Partial remission to Taq -

98 NS223 No 85 M MCD SRNS - -

99 NS228 No 1 M MesPGN SRNS No response to CyA -

100 NS230 No 9 M MGN SRNS Maintained on ACEI

+ARB -

101 NS231 No 4 M MesPGN SRNS Complete remission to

CyP -


CyA -


75

104 NS234 No 03 F - SRNS Maintained on ACEI

+ARB -

105 NS235 No 115 M pMCGN SRNS Maintained on ACEI

+ARB -

106 NS236 No 14 M FSGS SRNS Partial response to CyA -

107 NS239 Yes 11 F - SRNS - ESRD


CyP -

109 NS245 No 18 F FSGS SRNS -

110 NS248 No 2 F MGN SRNS Maintained on ACEI

+ARB -



Tac -

113 NS251 No 5 M MesPGN SRNS Complete remission -



116 NS255 No 95 M FSGS SRNS - Lost to follow up


CyP -

118 NS257 Yes 3 F - SNS - Lost to follow up

119 NS267 Yes 01 M - SRNS No remission -

120 NS268 No 24 M MesPGN SRNS Partal response to CyA ESRD

121 NS269 No 8 F SRNS - Expired

76

122 NS270 No 04 M SRNS - ESRD

123 NS275 No 3 F - SRNS - ESRD

124 NS276 No 5 M MCD SRNS In complete remission to

CyA -

125 NS278 No 1 M - CNS Maintained on ACEI

+ARB -

126 NS279 Yes 25 M MCD SDNS Partial response to CyP -

127 NS281 No 10 M SRNS - -


129 NS288 No 1 M IgMN SRNS Partial response to CyA

Tac -


CyA -


CyA -


133 NS292 No 45 M MCD SRNS Response to CyA -


CyA -

135 NS295 Yes 03 F - CNS Maintained on ACEI

+ARB -

136 NS300 No 09 M - SRNS Maintained on ACEI

+ARB

137 NS301 Yes 01 M - CNS Maintained on ACEI

+ARB -

138 NS302 Yes 12 M - - - Expired

77

139 NS303 Yes 3 M - SRNS - -


141 NS305 No 02 M - Maintained on ACEI

+ARB -

142 NS306 No 25 M SRNS - -

143 NS308 Yes 2 M FSGS SRNS No response -


+ARB -

145 NS310 No 01 F - CNS Maintained on ACEI

+ARB -

aSteroid resistant nephrotic syndrome

bIgM nephropathy

ccyclosporine

dend stage renal disease

etransplantation

fminimal change

disease gfocal segmental glomerular sclerosis

htacrolimus

imesengial proliferative glomerulonephritis

jmembranous

glomerulonephritis kangiotensin converting enzyme inhibitor

langiotensin receptor blocker

mchronic renal failure

ncyclophosphamide

oSteroid dependant nephrotic syndrome

pmesengio capillary glomerulonephritis

q (-)

78

A novel pG1020V mutation was present in patient NS228 who had

infantile NS This change was predicted to be damaging since it had a PolyPhen-2

score of 10 The biopsy report showed that this patient had a unique presentation

of mesengial proliferative glomerular nephropathy (MesPGN) Another novel

homozygous pT1182A mutation was identified in patient NS254 who had biopsy

proven FSGS with a typical clinical presentation This child died at the age of 15

years because of ESRD Another child (NS309) who had congenital NS at the age

of two months had a novel homozygous pG867P mutation which is probably

damaging according to the Polyphen-2 analysis His parents were first cousins and

were segregating the mutation in a heterozygous state One infantile NS case was

found to have compound heterozygous mutations (pL237P and pA912T) and had

inherited one mutation from each parent A novel homozygous 2 bp duplication

(c267dupCA) was found in a child who had severe NS since birth His elder sister

died of NS at the age of two months His parents were first cousin and analysis

revealed that both were carriers of the mutation

Besides these homozygous mutations identified in the NPHS1 gene 12

patients carried heterozygous mutations (Table- 36) Among these the pR408Q

mutation was identified in 3 patients This mutation has previously been reported in

a compound heterozygous condition in patients with CNS (Lenkkeri et al 1999)

while in the present study patients carrying the heterozygous pR408Q mutation

had a late onset of the disease with NS symptoms appearing at the ages of 4-10

years Along with the pR408Q mutation in the NPHS1 gene one patient (NS130)

also had a heterozygous missense mutation (pP341S) in the NPHS2 gene (Tablendash

36 and 37) Kidney biopsy results of the two patients that only had the pR408Q

79

mutation showed MCD while patient NS130 who had both gene mutations showed

FSGS

A GgtA substitution (pE117K rs3814995) was found in a homozygous

condition in six patients and in a heterozygous condition in 21 patients However

this was considered to be a common variant since it was found in both homozygous

and heterozygous states in normal individuals (Lenkkeri et al 1999)

80

Figure- 31 Illustration of identified mutations in the NPHS1 gene and their respective locations in the gene and protein

domains

81

Table- 35 List of homozygouscompound heterozygous mutations identified in the NPHS1 gene

Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228

M no Infantile cMesPGN c3059G-T pG1020V Partial remission

to dCyA

Normal 15yrs 100

NS254

F no CNS FSGS c3426A-G pT1182A Expired 15yrs 000

NS291

M no Infantile c710T-C

c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099

afocal segmental glomerular sclerosis

b angiotensin converting enzyme inhibitor

c mesengial proliferative glomerular nephropathy

dcyclosporine

82

Table- 36 List of heterozygous mutationsvariants identified in the NPHS1 gene

aMinimal change disease

b cyclosporine

cfocal segmental glomerular sclerosis

dangiotensin converting enzyme inhibitor

eangiotensin receptor blocker

fmesengial proliferative glomerular nephropathy


Mutation in the NPHS2 gene also

Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to Therapy Renal

Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I

Partial remission to bCyA

Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q

Maintained on dACEI+

eARB

Maintained on ACEI+ ARB

Complete remission to CyA

Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W

Partial remission to ACEI Normal 100

NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I

Partial remission to CyA


Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206

F No 11 FSGS c881C-T pT294I Partial remission to

Tacrolimus

Normal 000

NS049 M yes Infantile MCD c791C-G pP264R

Partial remission to CyA Normal 002

NS267 M yes CNS _ c3047G-A pS1016N 7mo

follow up

019

83


The NPHS2 gene was sequenced in 145 NS patients and 4 mutations were

identified (Figure- 32 Table- 37) The pP341S mutation was identified in patient

NS130 in a heterozygous state who also carried the pR408Q mutation in the

NPHS1 gene in a heterozygous condition (Table- 36 and 37) This patient was

diagnosed with FSGS at the age of 5 years As observed by others patients

carrying mutations in the NPHS2 gene initially showed complete remission of

proteinuria but developed secondary resistance to steroid therapy (Caridi et al

2001) Two previously known homozygous pK126N and pV260E mutations were

identified in two infantile NS cases while no NPHS2 gene mutation was found in

the CNS cases in our Pakistani cohort Similarly no mutation was identified in any

of the familial SRNS cases

A homozygous pR229Q mutation was found in two patients aged 25 and 3

years This change causes a decrease in the binding of the podocin protein to the

nephrin protein and in association with a second NPHS2 mutation enhances

susceptibility to develop FSGS (Tsukaguchi et al 2002) One of these children

(NS125) developed end stage renal disease at the age of 14 years

84

Figure- 32 Illustration of the identified mutations in the NPHS2 gene and their locations

85

Table- 37 List of Mutations identified in the NPHS2 gene

Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q

Partial remission to

Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130

M no 5 FSGS c1090C-T pP341S Maintained on dACEI and

eARB

Normal 10yrs 0998

NS278

M no Infantile

c378G-C pK126N Maintained on dACEI and

eARB

Normal 3yrs 100

NS288

M no Infantile

c779T-A pV260E Partial remission to

Tacrolimus

Normal 3yrs 0998

a

Focal segmental glomerular sclerosis b end stage renal disease

cminimal change disease

dangiotensin converting

enzyme inhibitor eangiotensin receptor blocker


86

34 DISCUSSION

This study describes the identification of 6 novel mutations out of 7 in the

NPHS1 and 4 mutations in the NPHS2 gene The primary findings of this study

show that as opposed to Europe mutations in the NPHS1 and NPHS2 genes are not

the frequent causes of paediatric NS in Pakistan Another important finding is the

absence of disease-causing mutation in the NPHS2 gene in the familial SRNS and

CNS cases By contrast homozygous mutations in the NPHS2 gene have been

reported to account for 42 of the autosomal recessive SRNS families and 39-51

of CNS cases of European origin (Weber et al 2004 Hinkes et al 2007)

Reports of the European populations have shown that in children up to three

months of age mutations in the NPHS1 gene account for 39ndash82 of the NS cases

and that most of the mutations are homozygous (Caridi et al 2001 Koziell et al

2002 Philippe et al 2008 Schoeb et al 2010) Consequently these mutations

have been associated with the earliest and most severe type with the onset of NS in

utero or within the first three months of life (Hinkes et al 2007) However we

have observed that in our cohort the mutations are in children who have NS since

birth but up to a longer period of one year of life

Although the exact role of heterozygous NPHS1 mutations in disease

progression is not established in the current screening it was found that

homozygous NPHS1 mutations caused a severe and early disease type while

heterozygous mutations caused milder NS that manifested relatively later in life

(Table- 35 and 36) In patients with the heterozygous NPHS1 gene mutations we

also examined the possible disease-causing involvement of some other genes

87

However no mutation was found in the NPHS2 WT1 and LAMB2 genes that are

known to cause early onset NS

Several previous studies have shown that children with the NPHS1 gene

mutations progressed to ESRD very rapidly within one to three years of age

(Hinkes et al 2007 Machuca et al 2010) However in our study children with

the NPHS1 gene mutations retained some renal function up to 25 years of age

(Table- 35 and 36)

Koziell et al (2002) have reported digenic inheritance of NPHS1 and

NPHS2 gene mutations In one of our patients a heterozygous pR408Q mutation

was observed in the NPHS1 gene and a second heterozygous pP321S mutation in

the NPHS2 gene (Table- 36 and 37) The child was diagnosed with FSGS at the

age of 5 years In silico analysis with the PolyPhen 2 program suggested that both

the mutations are damaging

Weber et al (2004) have shown that 42 of the familial SRNS cases and

10 of the sporadic cases are due to the mutations in the NPHS2 gene (Weber et

al 2004) By contrast in our cohort no mutation was found in the familial SRNS

cases and only 34 of all the NS cases have mutations in the NPHS2 gene

An NPHS2 gene variant pR229Q has been found to occur with at least one

pathogenic mutation and it was therefore suggested that it has no functional effects

(Machuca et al 2010 Santin et al 2011) However in vitro studies of Tsukaguchi

et al (2002) have shown that this variant decreases the binding of the podocin-

nephrin complex and hence its function In our study two children aged 25 and 3

years carried this variant in the homozygous state with no other mutation in both

these genes Our observation supports that of Tsukaguchi that this variant may be

88

the cause of NS in these children In the world population the pR229Q allele is

more frequent in the Europeans and South American (4-7) than in the African

African American and Asian populations (0-15 Santin et al 2011) In our

population only one out of 100 control samples was found to have this variant

allele in a heterozygous state (001 allele frequency)

Mutations in the NPHS1 gene account for ~20 and NPHS2 gene account

for 55 of the patients with early onset NS in our cohort This observation is in

marked contrast to the studies from Europe and US where the prevalence of the

NPHS1 gene mutations ranges from 39-55 and the NPHS2 gene mutations ranges

from 10-28 (Koziell et al 2002 Lahdenkari et al 2004 Philippe et al 2008

Schoeb et al 2010) Studies from Japan and China also report a low prevalence of

the two genes in their NS patients (Sako et al 2005 Mao et al 2007) Although

the NPHS1 and NPHS2 genes together make a significant contribution to the

spectrum of disease causing mutations there are a number of other genes including

WT1 LAMB2 PLCE1 TRPC6 CD2AP ACTN and INF2 that are known to cause

NS in children (Hinkes et al 2007) In view of this observation all the early onset

NS patients with no NPHS1 and NPHS2 gene mutations are being screened for the

WT1 LAMB2 and PLCE1 gene mutations

Population genetic analysis has shown in a study of heart failure the South

Asian populations are strikingly different compared to the Europeans in disease

susceptibility (Dahandapany et al 2009) Our results therefore reaffirm that the

genetic factors causing NS are different in Asian and European populations and

that other genes that may contribute to the etiology of the NS need to be identified

89

Thus low prevalence of disease-causing mutations in our population may reflect the

geographic and ethnic genetic diversity of NS in the world populations

90

35 REFERENCES

Adzhubei IA Schmidt S Peshkin L Ramensky VE Gerasimova A Bork P

Kondrashov AS Sunyaev SR (2010) A method and server for predicting missense

mutations Nat Methods 7 248-249

Avni EF Vandenhoute K Devriendt A Ismaili K Hackx M Janssen F Hall M

(2011) Update on congenital nephrotic syndromes and the contribution of US

Pediatr Radiol 41 76-81

Benoit G Machuca E Heidet L Antignac C (2010) Analysis of recessive CD2AP

and ACTN4 mutations in steroid-resistant nephrotic syndrome Pediatr Nephrol 25

445-451


Gubler MC Niaudet P Antignac C (2000) NPHS2 encoding the glomerular

protein podocin is mutated in autosomal recessive steroid-resistant nephrotic

syndrome Nat Genet 24 349-354







cause focal segmental glomerulosclerosis Nat Genet 42 72-76




mutations in steroid resistant nonfamilial focal segmental glomerulosclerosis J Am


Dhandapany PS Sadayappan S Xue Y Powell GT Rani DS Nallari P Rai TS

Khullar M Soares P Bahl A Tharkan JM Vaideeswar P Rathinavel A

Narasimhan C Ayapati DR Ayub Q Mehdi SQ Oppenheimer S Richards MB

Price AL Patterson N Reich D Singh L Tyler-Smith C Thangaraj K (2009) A

common MYBPC3 (cardiac myosin binding protein C) variant associated with

Cardiomyopathies in South Asia Nat Genet 41 187-191





91


D Ozaltin F Zenker M Hildebrandt FArbeitsgemeinschaft fuumlr (2007) Nephrotic

syndrome in the first year of life Two thirds of cases are caused by mutations in 4

genes (NPHS1 NPHS2 WT1 and LAMB2) Paediatrics 119 e907-e919





2128



ACTN4 encoding alpha-actinin 4 cause familial focal segmental

glomerulosclerosis Nat Genet 24 251-256





1 575-582








Lenkkeri U Ma nnikko M McCready P Lamerdin J Gribouval O Niaudet P







1198-1203

Machuca E Benoit G Nevo F Tecircte MJ Gribouval O Pawtowski A Brandstroumlm

P Loirat C Niaudet P Gubler MC Antignac C (2010) Genotype-phenotype

correlations in non-Finnish congenital nephrotic syndrome J Am Soc Nephrol 21

1209-1217

92

Mao J Zhang Y Du L Dai Y Gu W Liu A Shang S Liang L (2007) NPHS1

and NPHS2 gene mutations in Chinese children with sporadic nephrotic syndrome

Pediatr Res 61 117-122









Nephrol 13 589-593














Sako M Nakanishi K Obana M Yata N Hoshii S Takahashi S Wada N

Takahashi Y Kaku Y Satomura K Ikeda M Honda M Iijima K Yoshikawa N

(2005) Analysis of NPHS1 NPHS2 ACTN4 and WT1 in Japanese patients with

congenital nephrotic syndrome Kidney Int 67 1248-1255












93




1148







Schwartz GJ Work DF (2009) Measurement and estimation of GFR in children

and adolescents Clin J Am Soc Nephrol 4 1832-1843

Tsukaguchi H Sudhakar A Le TC Nguyen T Yao J Schwimmer JA Schachter

AD Poch E Abreu PF Appel GB Pereira AB Kalluri R Pollak MR (2002)

NPHS2 mutations in late-onset focal segmental glomerulosclerosis R229Q is a

common disease-associated allele J Clin Invest 110 1659-1666




Int 66 571-579

Yu Z Ding J Huang J Yao Y Xiao H Zhang J Liu J Yang J (2005) Mutations

in NPHS2 in sporadic steroid resistant nephrotic syndrome in Chinese children






mesangial sclerosis and distinct eye abnormalities Hum Molec Genet 13 2625-

2632

94

4 ASSOCIATION OF THE ACE ndash II GENOTYPE WITH

THE RISK OF NEPHROTIC SYNDROME IN

PAKISTANI CHILDREN

95

41 INTRODUCTION

Nephrotic Syndrome (NS) is the most common glomerular disease in

children (Braden et al 2000) The estimated incidence of pediatric NS in the USA

is 20 to 27 per 100000 populations with a cumulative frequency of 16 per 100000

(Eddy and Symons 2003) It is characterized by heavy proteinuria

hypoalbuminemia hypercholesterolemia and edema The primary variants of NS

are focal segmental glomerulosclerosis (FSGS) minimal change disease (MCD)

and membranous glomerulopathy (MGN Obeidova et al 2006) The majority of

patients with sporadic NS respond well to steroid therapy However approximately

10-20 fail to do so and hence are at a higher risk of developing end stage renal

disease (ESRD Ruf et al 2004) Geographic as well as ethnic differences have

been reported to contribute towards the incidence of NS with a 6-fold higher

incidence in the Asians compared to the European populations (Sharlpes et al

1985)

The gene for angiotensin-converting enzyme (ACE) is located on

chromosome 17q23 It is an important enzyme in the renin-angiotensin-aldosterone

system (RAAS) It is responsible for converting an inactive angiotensin I (Ang-I)

into a vasoactive and aldosterone-stimulating peptide angiotensin II (Ang-II Oktem

et al 2004) The insertion or deletion of a 287 bp Alu repeat sequence in intron 16

of the ACE gene is defined by the ID polymorphism The deletion allele (D) has

been associated with the higher concentration of plasma ACE and AngndashII levels

(Rigat et al 1990) The increased concentration of Ang-II stimulates the expression

of several different growth factors and nuclear transcription factors that cause

96

deleterious effects on renal hemodynamics and may result in the manifestation of

NS (Serdaroglu et al 2005)

This study was carried out to determine the association of the ACE ID

polymorphism with the risk of NS in Pakistani children and to further evaluate the

relation between this polymorphism and the risk of developing steroid resistant and


42 SUBJECTS AND METHODS


Blood samples were collected from 268 NS patients from the pediatric

nephrology department SIUT with their informed consent or that of their parents

A panel of 223 control samples was also included in the study The controls

consisted of unrelated healthy individuals with no history of kidney disease or

hypertension The criteria for the inclusion of patients in the study were the clinical

presentation of NS and an age less than 16 years The diagnosis of NS was based

upon the presence of edema urinary protein excretion ge 40mgm2hr and serum

albumin below 25gml All the patients received standard steroid therapy and were

classified into two categories on the basis of their responses towards steroids the

steroid sensitive nephrotic syndrome (SSNS) and steroid resistant nephrotic

syndrome (SRNS) The renal biopsy results were available for 105 cases

97

422 GENOTYPING

Genomic DNA was prepared using the standard phenol-chloroform

extraction procedure (Sambrook and Russell 2006) The forward and reverse

primer sequences for ACE ID polymorphism were

5rsquoCTGGAGACCACTCCCATCCTTTCT3rsquo and 5rsquoGATGTGGCCATCACATTGG

TCAGAT3rsquo(Eurofins MWG Operon Germany) respectively The polymerase chain

reaction was performed in a total reaction volume of 10 microl as decribed priviousely

in the Materials and Methods section with some modifications such as 1X PCR

buffer (GoTaqreg

Flexi DNA polymerase Promega USA) 15 mM magnesium

chloride 02 mM dNTPs (Gene Ampreg

dNTP Applied Biosystems USA) 01 units

of GoTaq DNA polymerase and 20ng of the genomic DNA The reaction mixture

was amplified for 30 cycles with denaturation at 94˚C for 1min annealing at 58˚C

for 1 min and extension at 72˚C for 2 min using a Gene Ampreg PCR System 9700

(Applied Biosystems USA) The PCR products were electrophoresed on 2

agarose gel A PCR product of 490 bp represents a homozygous insertion genotype

(II) a 190 bp fragment of homozygous deletion genotype (DD) and the presence of

both the fragments revealed heterozygosity (ID) as shown in Figure- 41

98

Figure- 41 ACE gene ID polymorphism genotyping on 2 agarose gel

M

ACE gene ID polymorphism genotyping on 2 agarose gel stained with

ethidium bromide and photographed with automated gel documentation system

The I allele was detected as a 490 bp band (upper band) the D allele was detected

as a 190 bp band (lower band) while heterozygotes showed both the bands The lane

on the right shows the 100 bp molecular weight marker

99


The statistical analysis was carried out using the Statistical Package for

Social Sciences (SPSS version 17) Chi-Square and OR tests were used to analyze

the distribution of the genotypic and allelic frequencies of the ACE ID

polymorphism in the NS cases and controls as well as steroid therapy response and

histological features A p-value less than 005 was considered to be significant

43 RESULTS

A total of 268 children with NS were selected for this study Of these 164

were males and 104 were females with the ages ranging between 2 months to 15

years Steroid resistance was established in 105 patients whereas 163 patients were

classified as SSNS End stage renal disease (ESRD) was developed in 12 patients

The clinical parameters of NS patients are shown in Table- 41

Table- 41 The clinical parameters of NS patients

Steroid response

SRNS

N=105

SSNS

N=163

Malefemale 6047 10457

Age of onset 02-15 yrs 1-10 yrs

Family history 24 6

ESRD 12 No

Biopsy 105 No

Proteinuria (gdl) 0-4 0-4

Serum albumin (gl) 0-36 0-35

Serum creatinine (mgdl) 0-92 0-162

Hematuria 0-3 No

100

The genotyping of the ACE ID polymorphism in NS and control samples

showed that the incidence of II ID and DD genotypes were 82 (306) 128

(478) and 58 (216) in the NS patients and 9 (40) 171 (767) and 43

(193) in the control samples respectively The frequency distribution of I and D

alleles were 292 (545) and 244 (455) in the NS group and 189 (42) and 257

(58) in the control samples respectively The difference between the two groups

was statistically significant (plt0001 χ2

=142) having an OR of 16 (95 CI =13-

20) as shown in Table- 42 The NS samples were in Hardy-Weinberg equilibrium

(HWE) with p=085 However the control samples deviated from HWE (plt0001)

The frequency distribution of II and DD genotypes were 82 (59) and 58

(41) in the NS group and 9 (17) and 43 (83) in the control samples

respectively This showed a statistically significant association of the II genotype

with NS (plt0001 χ2

=258) having an OR of 67 (95 CI=3-149) The I-carrier

genotypes (II and ID) were evaluated in the NS group and no significant difference

was found with the control samples as shown in Table- 42

The frequency distribution of II ID and DD genotypes were 35 (33) 47

(45) and 23 (22) in the SRNS group and 47 (29) 82 (50) and 34 (42) in

the SSNS group No significant association was found with steroid response in the

NS patients (pgt005) as shown in Table- 43

The biopsies of 105 SRNS patients were available in which 48 patients had

FSGS and 25 had MCD The frequency distribution of II and DD genotypes and ID

alleles were not significantly associated with FSGS or MCD in our NS population

as shown in Table- 43

101

Table- 42 Genotypic and allelic frequencies of the ACE ID polymorphism

and their distribution in terms of II ID and IIDD genotypes with respect to

DD genotype in NS patients and controls

NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)

Cochran-Armitage trend test = 37 plt0001

ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102

Table- 43 Frequency distribution of the ACE ID polymorphism in SRNS

SSNS FSGS non-FSGS and MCD non-MCD patients

II genotype ID genotype DD genotype Total P value

SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION

ACE is an important component of RAAS that plays an important role in the

renal and cardiovascular pathophysiology by regulating blood pressure fluid-

electrolyte and acid-base balance (Seikaly et al 1990) ACE (ID) polymorphism

has been studied in different diseases like hypertension myocardial infarction and

IgA nephropathy (Bantis et al 2004 Ismail et al 2004) Similarly an association

between the ACE ID polymorphism and the etiology of NS has been investigated

in several epidemiologic studies However conflicting results have been reported

from different parts of the world

The present study was carried out to determine the association of ID

polymorphism in the ACE gene with pediatric NS in Pakistan We found a

significant association of II genotype and the I allele with NS as compare to the

normal controls Our results are in agreement with a study from India where the II

genotype was more frequent in SSNS patients as compared to the controls (Patil et

al 2005) However another study from India has reported that the frequency

distribution of the DD genotype was significantly higher in the SRNS group

compared to the control subjects (Prasun et al 2011) Similarly the II genotype

was found at higher frequency among the Malays (Jayapalan et al 2008) By

contrast the association of the DD genotype with NS has been reported from

Taiwan Egypt and Turkey (Serdaroglu et al 2005 Tsai et al 2006 Fahmy et al

2008) On the other hand no association of ACE gene polymorphism was found in

the Swiss children (Sasse et al 2006) In a recently published meta-analysis Zhou

et al (2011) have concluded that the DD genotype or D allele was not associated

104

with SRNS susceptibility in Asians and Caucasian children but the D allele was

associated with SRNS onset for African children

The NS samples were in HWE (p=085) whereas control samples deviated

from HWE (plt0001) due to the presence of a larger number of heterozygotes than

expected Deviation from HWE indicates that one or more model assumptions for

HWE have been violated The first source for deviation is genotyping error To

exclude the possibility of genotyping errors the genotypes of randomly selected

samples were confirmed by sequencing The Pakistani population is genetically

heterogeneous and the samples used in this study are of mixed ethnicity Another

source of the observed deviation from HWE in these samples could be due to

population stratification However population stratification always leads to a deficit

of heterozygotes (Ziegler et al 2011) which was not the case in this study It has

been suggested that in the case of observed deviation from HWE with no

attributable phenomena a test for trend such as Cochran-Armitage trend test should

be used in order to reduce the chances of false positive association (Zheng et al

2006) Therefore the Cochran-Armitage trend test was performed and the results

confirm the allelic association (plt0001 Table- 42)

The II and DD genotypes showed no significant differences in the SRNS

and SSNS patients in the Pakistani children (Table- 43) However the sample size

(SSNS=163 and SRNS=105) is rather small to conclude any significant role of ACE

polymorphism with response to standard steroid therapy Similarly the D allele

frequency was not found to be associated with steroid sensitivity in NS patients in

the Egyptian and Indonesian populations (Sasongko et al 2005 Saber-Ayad et al

2010)

105

The MCD and FSGS are common histological variants of NS found in our

population (Mubarak et al 2009) As also reported by others (Serdaroglu et al

2005 Saber-Ayad et al 2010) the ID polymorphism showed no association with

FSGS and MCD in our NS population (Table- 43) By contrast the DD genotype

was associated with FSGS in the Kuwaiti Arab and Korean patients (Lee et al

1997 Al-Eisa et al 2001)

In conclusion NS is associated with a higher incidence of the II genotype in

the ACE gene in Pakistani children No significant association of allele and

genotype frequencies with steroid sensitivity and histological patterns are found in

these children

106

45 REFERENCES

Al-Eisa A Haider MZ Srivastva BS (2001) Angiotensin converting enzyme gene

insertiondeletion polymorphism in idiopathic nephrotic syndrome in Kuwaiti Arab

children Scand J Urol Nephrol 35 239-242

Bantis C Ivens K Kreusser W Koch M Klein-Vehne N Grabensee B Heering P

(2004) Influence of genetic polymorphism of the rennin-angiotensin system on IgA

nephrotpathy Am J Nephrol 24 258-267

Braden GL Mulhern JG OrsquoShea MH Nash SV Ucci AA Germain MJ (2000)

Changing incidence of Glomerular diseases in adults Am J Kidney Dis 35 878-

883


629-639

Fahmy ME Fattouh AM Hegazy RA Essawi ML (2008) ACE gene

polymorphism in Egyptian children with idiopathic nephrotic syndrome Bratisl Lek

Listy 109 298-301

Hussain R Bittles AH (2004) Assessment of association between consanguinity

and fertility in Asian populations J Health Popul Nutr 22 1-12

Ismail M Akhtar N Nasir M Firasat S Ayub Q Khaliq S (2004) Association

between the angiotensin-converting enzyme gene insertiondeletion polymorphism

and essential hypertension in young Pakistani patients J Biochem Mol Biol 3 552-

555

Jayapalan JJ Muniandy S Chan SP (2008) Angiotensin-1 converting enzyme

ID gene polymorphism Scenario in Malaysia Southeast Asian J Trop Med Public

Health 39 917-921

Lee DY Kim W Kang SK Koh GY Park SK (1997) Angiotensin-converting

enzyme gene polymorphism in patients with minimal-change nephrotic syndrome

and focal segmental glomerulosclerosis Nephron 77 471-473

Mubarak M Lanewala A Kazi JI Akhter F Sher A Fayyaz A Bhatti S (2009)


Nephrol 13 589-593

Obeidova H Merta M Reiterova J Maixnerova D Stekrova J Rysava R Tesar V




Nephrol 19 384-389

107

Patil SJ Gulati S Khan F Tripathi m Ahmed M Agrawal S (2005) Angiotensin

converting enzyme gene polymorphism in Indian children with steroid sensitive

nephrotic syndrome Indian J Med Sci 59 431-435




1346



Bakkaloglu A Hildebrandt F Arbeitsgemeinschaft Fuumlr Padiatrische Nephrologie

Study Group (2004) Patients with mutations in NPHS2 (podocin) do not respond

to standard steroid treatment of nephrotic syndrome J Am Soc Nephrol 15 722-

732

Saber-Ayad M Sabry S Abdel-Latif I Nabil H El-Azm SA Abdel-Shafy S

(2010) Effect of angiotensin-converting enzyme gene insertiondeletion

polymorphism on steroid resistance in Egyptian children with idiopathic nephrotic

syndrome Renin Angiotensin Aldosterone Syst 11 111-118

Sambrook J Russell DW The condensed protocol From molecular cloning a

laboratory manual Coldspring Harbour Laboratory Press Coldspring Harbour

New York 2006 241-243

Sasongko T Sadewa AH Kusuma PA Damanik MP Lee MJ Ayaki H Nozu K

Goto A Matsuo M Nishio H (2005) ACE gene polymorphism in children with

nephrotic syndrome in the Indonesian population Kobe J Med Sci 51 41-47

Sasse B Hailemariam S Wuthrich RP Kemper MJ Neuhaus TJ (2006)

Angiotensin converting enzyme gene polymorphisms do not predict the course of

idiopathic nephrotic syndrome in Swiss children Nephrology 11 538-5341

Seikaly MG Arant BS Seney FD (1990) Endogenous angiotensin concentrations

in specific intrarenal fluid compartments in the rat J Clin Invest 86 1352-1357

Serdaroglu E Mir S Berdeli A Aksu N Bak M (2005) ACE gene insertiondele-

tion polymorphism in childhood idiopathic nephrotic syndrome Pediatr Nephrol

20 1738-1743



Tsai LJ Yang YH Lin Wu VC Tsau YK Hsieh FJ (2006) Angiotensin-

converting enzyme gene polymorphism in children with idiopathic nephrotic

syndrome Am J Nephrol 26 157-162

108

Zheng G Freidlin B Gastwirth JL (2006) Robust genomic control for association

studies Am J Hum Genet 78 350-356

Zhou TB Qin YH Su LN Lei FY Huang WF Zhao YJ Pang YS (2011)

Insertiondeletion (ID) polymorphism of angiotensin-converting enzyme gene in

steroid-resistant nephrotic syndrome for children A genetic association study and

Meta-analysis Renal Failure 33 741-748

109

5 ASSOCIATION OF MTHFR GENE

POLYMORPHISMS (C677T AND A1298C) WITH

NEPHROTIC SYNDROME IN PAKISTANI

CHILDREN

110

51 INTRODUCTION

The gene for the enzyme methyltetrahydrofolate reductase (MTHFR

OMIM-607093) is localized on chromosome 1p363 (Gaughan et al 2000) This

enzyme catalyzes the NADPH-linked reduction of 5 10 methyltetrahydrofolate to

5-methyltatrahydrofolate which serves as an important cofactor in the methylation

of homocysteine (Hcy) to methionine as shown in Figure-51 (Goyette et al 1994)

Mutations in the MTHFR gene have been suggested to be responsible for increased

homocysteine levels in the blood (Lucock 2000)

The two most common single nucleotide polymorphisms (SNPs) in the

MTHFR gene are C677T (dbSNP I rs1801133) a missense mutation that results in

an alanine to valine substitution at codon 222 and A1298C (dbSNP ID rs1801131)

a point mutation that leads to change from a glutamine to alanine at codon 429 of

the gene (Weisberg et al 1998) The C677T polymorphism is localized in the

catalytic N-terminal domain of the enzyme while A1298C is localized in the

regulatory domain of the enzyme (Friso et al 2002)

The C677T polymorphism is associated with a 30 decrease in the activity

of the enzyme in the CT heterozygous state and a 60 decrease in the TT

homozygous state (Frosst et al 1995) This polymorphism is known to cause mild

hyperhomocysteinemia particularly in homozygotes and also in compound

heterozygotes along with the A1298C polymorphism (Weisberg et al 1998

Andreassi et al 2003) The frequency of TT homozygotes among healthy

individuals ranges from 0 to 1 in African Americans 25 in Hispanic

111

Americans and 10 to 15 in Canadians Americans Europeans Asians and

Australian populations (Rozen 2001)

Hyperhomocysteinemia is a commonly recognized risk factor for several

multifactorial disorders associated with thrombotic complications atherosclerosis

cardiovascular and renal diseases etc (Buumlyuumlkccedilelik et al 2008 Ferechide and

Radulescu 2009 Kniazewska et al 2009 Ciaccio and Bellia 2010) Nephrotic

syndrome has also been associated with a higher risk of infections thrombotic

complications early atherosclerosis and cardiovascular diseases (Louis et al 2003

Kniazewska et al 2009)

In the healthy individuals 75 of the total Hcy is bound to albumin and

only a small amount is available in the free form (Hortin et al 2006) However in

the NS patients heavy proteinuria is supposed to cause a decrease in the plasma

Hcy concentration and an increase in urinary Hcy excretion (Refsum et al 1985

Sengupta et al 2001) The change in the plasma Hcy concentration affects its

metabolism and may suggests a role for MTHFR polymorphisms in NS

This study was carried out to determine the association of MTHFR gene

polymorphisms (C677T and A1298C) with the progression of NS in Pakistani

children and to further evaluate the relationship between these polymorphisms and

the outcome of steroid therapy and histological findings in these patients

112

Figure- 51 Dysregulation of MTHFR leads to the accumulation of

homocysteine (Kremer 2006)

113



nephrology department SIUT with their informed consent A panel of 200 normal

control samples was also included in the study The diagnosis of patients and their

inclusion for the study has been discussed earlier The NS patients were classified

into 166 SRNS and 152 SSNS patients (Table-51)

Table-51 The clinical parameters of NS patients

SRNS

N=166

SSNS

N=152


Age of onset 02mo-15 yrs 1-10 yrs

Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING

Genotyping for the MTHFR gene polymorphisms was performed using

polymerase chain reaction (PCR) and restriction fragment length polymorphism

(RFLP) techniques as described earlier The presence of C677T and A1298C

polymorphisms in the MTHFR gene were analyzed by HinfI and MobII restriction

enzymes digestion respectively according to Skibola et al 1999 (Figure- 52 and

53)

114

Figure- 52 MTHFR gene C677T polymorphism genotyping

MTHFR gene polymorphism genotyping on a 2 agarose gel stained with


The C allele of C677T polymorphism was detected as a single 198 bp band (upper

band) the T allele was detected as a 175 and 23 bp bands (lower band) while

heterozygotes showed both the bands The lane on the left (M) shows the 100 bp

molecular weight marker

Figure- 53 MTHFR gene A1298C polymorphism genotyping

115

The C and A alleles of the MTHFR A1298C polymorphism were detected as a

major visible band of 84 bp (upper band) and 56 bp (lower band) respectively while

heterozygotes showed both the bands

53 RESULTS


were males and 137 were females with ages ranging between 2 months to 15 years

The genotyping of the MTHFR C667T polymorphism in the NS and control

samples showed that the incidence of CC CT and TT genotypes were 236 (74)

70 (22) and 12 (4) in the NS patients and 140 (70) 52 (26) and 8 (4) in

the control samples respectively The frequency distribution of C and T alleles were

542 (85) and 94 (15) in the NS group and 332 (83) and 68 (17) in the

control samples respectively The difference between the two groups was not

statistically significant (χ2=0917 pgt005) having an OR of 1181 (95 CI= 0840-

1660) as shown in Table- 52 The controls samples were in Hardy-Weinberg

equilibrium (HWE) with (χ2=124 pgt005) However the NS samples deviated

from HWE (plt005)

The frequency distribution of CC and TT genotypes were 236 (74) and 12


respectively There was no statistically significant difference in the frequencies of

the CC and TT genotypes in the two groups (χ2=0062 pgt005) having an OR of

1124 (95 CI= 0448-2816) as shown in Table- 52 The T-carrier genotypes (CT

and TT) were evaluated in the NS group but no significant difference (pgt005) was

found in the NS and control samples as shown in Table- 52

116

Table- 52 Genotypic and allelic frequencies of the MTHFR C667T

polymorphism and their distribution in terms of CC CT and CCCT

genotypes with respect to TT genotype in NS patients and controls

Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value

MTHFR C667T genotype

CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117

The frequency distribution of CC CT and TT genotypes of C677T

polymorphism were 124 (75) 37 (22) and 5 (3) in the SRNS group and 112

(74) 33 (22) and 7 (4) in the SSNS group No significant association was

found with steroid response in the NS patients (pgt005) as shown in Table- 53


FSGS and 30 had MCD The frequency distribution of CC and TT genotypes and

CT alleles were not significantly associated with FSGS or MCD in our NS

population as shown in Table- 53

Table- 53 Frequency distribution of the MTHFR C677T polymorphism in

SRNS SSNS FSGS non-FSGS and MCD non-MCD patients

CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137

The genotyping of the MTHFR A1298C polymorphism in the NS and

control samples showed that the incidence of CC CA and AA genotypes were 52

(16) 152 (48) and 114 (36) in the NS patients and 37 (185) 93 (465)

and 70 (35) in the control samples respectively The frequency distribution of C

and A alleles were 256 (40) and 380 (60) in the NS group and 167 (42) and

118

233 (58) in the control samples respectively The difference between the two

groups was not statistically significant (χ2=0191 pgt005) having an OR of 0945

(95 CI=0733-1218) as shown in Table- 54 The NS and control samples were

in Hardy-Weinberg equilibrium with (χ2

=001 and 039 pgt005)

The frequency distribution of CC and AA genotypes were 52 (16) and

114 (36) in the NS group and 37 (185) and 70 (35) in the control samples

respectively There was no statistically significant association of A1298C

polymorphism with NS (χ2=0314 pgt005) having an OR of 0863 (95

CI=0515-1446) as shown in Table- 54

The frequency distribution of CC CA and AA genotypes were 32 (193)

72 (434) and 62 (373) in the SRNS group and 23 (15) 77 (51) and 52

(34) in the SSNS group No significant association was found with steroid

response in the NS patients (pgt005) The frequency distribution of CC and AA

genotypes and CA alleles were not significantly associated with FSGS or MCD in

our NS population as shown in Table- 55

54 DISCUSSION

MTHFR gene polymorphisms have been studied in different diseases like

atherosclerosis vascular and thrombotic diseases neural birth defect and cancers

etc (Buumlyuumlkccedilelik et al 2008 Ferechide and Radulescu 2009 Kniazewska et al

2009 Taioli E et al 2009 Ciaccio and Bellia 2010 Deb et al 2011) However

only a few studies have been reported on the association of the MTHFR gene

polymorphism with NS (Zou et al 2002 Prikhodina et al 2010) The present

study was carried out to determine the association of C667T and A1298C

polymorphisms in the MTHFR gene with pediatric NS patients in Pakistan

119

Table- 54 Genotypic and allelic frequencies of the MTHFR A1298C

polymorphism and their distribution in terms of CC CA and CCCA

genotypes with respect to AA genotype in NS patients and controls

Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value

MTHFR A1298C genotype

CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)

MTHFR A1298Cgenotype

CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120

Table- 55 Frequency distribution of the MTHFR A1298C polymorphism in


The MTHFR enzyme regulates homocysteine metabolism Mutations in the

MTHFR gene are associated with increased plasma homocysteine levels Similar to

that of hyperhomocysteinemia the NS patients have a higher risk of infections

thrombotic complications and arthrosclerosis These observations give insight into

the role of homocysteine metabolism in the NS patients However some studies

have reported decreased plasma Hcy levels in the NS patients (Arnadottir et al

2001 Tkaczyk et al 2009) while other have shown normal (Dogra et al 2001)

and increased levels as compared to healthy controls (Joven et al 2000 Podda et

al 2007) Since contradictory results were observed in the NS patients these

studies have suggested that plasma Hcy concentration is not a predictable marker

In agreement with Prikhodina et al (2010) the association between C677T

and A1298C polymorphisms of the MTHFR gene with NS was not observed in this

study However Zou et al (2002) have reported that the frequency distribution of

CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121

the TT genotype was significantly higher with the early development and

progression of childhood FSGS

The NS samples for C667T polymorphism were not in HWE whereas the

control samples were The possible explanation of HWE deviation in the Pakistani

population has been discussed previously in Chapter 4 On the other hand the NS

patients and healthy controls for A1298C polymorphism were in HWE To exclude

the possibility of genotyping errors the genotypes of randomly selected samples

were confirmed by sequencing

The C677T and A1298C genotypes showed no significant differences in the

SRNS and SSNS patients in the Pakistani children (Table- 53 and 55) As also

reported by (Prikhodina et al 2006) the MTHFR gene polymorphisms showed no

association with steroid therapy (Table- 53) The common histological variants of

NS found in our patient population are MCD and FSGS (Mubarak et al 2009)

However the MTHFR polymorphisms showed no association with FSGS and MCD

in our NS population (Table- 53 and 55)

In conclusion the genotypic and allelic frequencies of C677T and A1298C

polymorphisms were not associated with the progression of NS in Pakistani

children By contrast the TT genotype was significantly higher with the early

development of childhood FSGS in the Japanese patients No significant

association of allele and genotype frequencies was found with steroid sensitivity

and histological patterns of these children

122

55 REFERENCES

Andreassi MG Botto N Battaglia D Antonioli E Masetti S Manfredi S

Colombo MG Biagini A Clerico A (2003) Methylenetetrahydrofolate reductase

gene C677T polymorphism homocysteine vitamin B12 and DNA damage in

coronary artery disease Hum Genet 112 171-177

Arnadottir M Hultberg B Berg AL (2001) Plasma total homocysteine

concentration in nephrotic patients with idiopathic membranous nephropathy





494



Deb R Arora J Meitei SY Gupta S Verma V Saraswathy KN Saran S Kalla

AK (2011) Folate supplementation MTHFR gene polymorphism and neural tube

defects a community based case control study in North India Metab Brain Dis 26

241-246

Dogra G Irish AB Watts GF (2001) Homocysteine and nephrotic syndrome



Med Life 2 53-59

Friso S Choi SW Girelli D Mason JB Dolnikowski GG Bagley PJ Olivieri O

Jacques PF Rosenberg IH Corrocher R Selhub J (2002) A common mutation in

the 5 10-methylenetetrahydrofolate reductase gene affects genomic DNA

methylation through an interaction with folate status Proc Natl Acad Sci USA 99

5606-5611

Frosst P Blom HJ Milos R Goyette P Sheppard CA Matthews RG Boers GJ

den Heijer M Kluijtmans LA van den Heuvel LP Rozen R (1995) A candidate

genetic risk factor for vascular disease a common mutation in

methylenetetrahydrofolate reductase Nat Genet 10 111-113

Gaughan DJ Barbaux S Kluijtmans LA Whitehead AS (2000) The human and

mouse methylenetetrahydrofolate reductase (MTHFR) genes genomic

organization mRNA structure and linkage to the CLCN6 gene Gene 257 279-

289

123




Hortin GL Seam N Hoehn GT (2006) Bound homocysteine cysteine and

cysteinylglycine distribution between albumin and globulins Clin Chem 52 2258-

2264

Joven J Arcelus R Camps J Ordoacutentildeez-Llanos J Vilella E Gonzaacutelez-Sastre F

Blanco-Vaca F (2000) Determinants of plasma homocyst(e)ine in patients with

nephrotic syndrome J Mol Med 78 147-154





Kremer JM (2006) Methotrexate pharmacogenomics Ann Rheum Dis 65 1121-

1123



1300





Nephrol 13 589-593

Podda GM Lussana F Moroni G Faioni EM Lombardi R Fontana G Ponticelli

C Maioli C Cattaneo M (2007) Abnormalities of homocysteine and B vitamins in

the nephrotic syndrome Thromb Res 120 647-652

Prikhodina L Vinogradova T Poltavets N Polykov A Dlin V (2010)

Hyperhomocysteinaemia and mthfr c677t gene polymorphism in

children with steroid-resistant nephrotic syndrome In The 15th

Congress

of the IPNA (August 29-September 2 2010) New York USA Pediatric

Nephrology 25 1881 pp 432

Prikhodina L Poltavets N Zaklyazminskaya E Galeeva N Tverskay S Polykov

A Dlin V Ignatova M (2006) Methylentetrahydrofolate reductase (mthfr) 677c-t

gene polymorphism and progression of steroid-resistant nephrotic syndrome in

children Pediatr Nephrol 21 ОР 43 c1517

124

Refsum H Helland S Ueland PM (1985) Radioenzymic determination of

homocysteine in plasma and urine Clin Chem 31 624-628

Rozen R Polymorphisms of folate and cobalamin metabolism In Homocysteine

in Health and Disease Edited by Carmel R Jacobsen DW UK Cambridge

University Press 2001 259-270

Sengupta S Wehbe C Majors AK Ketterer ME DiBello PM Jacobsen DW

(2001) Relative roles of albumin and ceruloplasmin in the formation of

homocystine homocysteine-cysteine-mixed disulfide and cystine in circulation J

Biol Chem 276 46896-46904







96 12810-12815

Taioli E Garza MA Ahn YO Bishop DT Bost J Budai B Chen K Gemignani F

Keku T Lima CS Le Marchand L Matsuo K Moreno V Plaschke J Pufulete M

Thomas SB Toffoli G Wolf CR Moore CG Little J (2009) Meta- and pooled

analyses of the methylenetetrahydrofolate reductase (MTHFR) C677T

polymorphism and colorectal cancer a HuGE-GSEC review Am J Epidemiol 170

1207-1221

Tkaczyk M Czupryniak A Nowicki M Chwatko G Bald E (2009)

Homocysteine and glutathione metabolism in steroid-treated relapse of idiopathic

nephrotic syndrome Pol Merkur Lekarski 26 294-297 Polish




Zou C Tsukahara H Hiraoka M Mizu J Todoroki Y Ohshima Y Kimura H

Tsuzuki K Mayumi M (2002) Methylenetetrahydrofolate reductase

polymorphism in childhood primary focal segmental glomerulosclerosis Nephron

92 449-451

125

6 GENERAL DISCUSSION

126

Single gene defects have been shown to cause a number of kidney diseases

eg nephrotic syndrome Nail-Patella syndrome Alport syndrome etc The disease

causing mutation in a single gene is sufficient to cause monogenic diseases

(Hildebrandt 2010) The present work on ldquoGenetics of nephrotic syndrome in

Pakistani childrenrdquo is such an example of monogenic disorders and is carried out to

find the genetic causes of steroid resistant nephrotic syndrome in pediatric

Pakistani population

It is well established that the glomerular filtration barrier consists of a

dynamic network of proteins that are involved in maintaining its function and

structural integrity (Hinkes et al 2007) The identification of disease-causing

mutations in the genes encoding these proteins helps in understanding the diseases

pathophysiology prognosis and treatments

A large number of Pakistani children suffer from NS and a significant

proportion of these become steroid resistant In the first year of life two thirds of

the cases of SRNS are reported to be caused by mutations in one of the four genes

NPHS1 (nephrin) NPHS2 (podocin) WT1 (Wilmrsquos tumor) and LAMB2 (laminin

beta 2 Hinkes et al 2007) Recently the panel of genes that are involved in the

pathogenesis of SRNS has expanded These genes include NPHS1 NPHS2

LAMB2 PLCE1 PTPRO ACTN4 WT1 CD2AP TRPC6 and INF2 (Weins and

Pollak 2008 Sinha and Bagga 2012) However the NPHS1 and NPHS2 genes

constitute a major spectrum of disease causing mutations Therefore it was of

interest to find the frequencies of disease-causing mutations in these two genes in

the Pakistani pediatric NS patients

127

The present study analyzed 145 cases that included 36 samples of

congenital or infantile onset NS and 39 samples of familial cases from 30 different

families The diagnosis was based on the presence of edema urinary protein

excretion equal to or greater than 40mgm2hr and serum albumin below 25 gl

Detailed clinical analysis was obtained for all the patients

Mutation analysis was performed by direct DNA sequencing of all the 29

exons of the NPHS1 gene and 8 exons of the NPHS2 gene A total of seven

homozygous (six novel) mutations in the NPHS1 gene and four homozygous

mutations in the NPHS2 gene were identified exclusively in the early onset cases

Our results showed a low prevalence of disease causing mutations in the NPHS1

(22 early onset 55 overall) and NPHS2 (33 early onset and 34 overall)

genes in the Pakistani NS children as compared to the European populations No

mutation was found in the familial Pakistani cases contrary to the high frequency of

NPHS2 gene mutations reported for familial SRNS in Europe These observations

suggested that patients that do not have disrupted NPHS1 and NPHS2 genes should

be screened for mutations in other genes encoding the WT1 LAMB2 and PLCE1

genes This is the first comprehensive screening of the NPHS1 and NPHS2 gene

mutations in sporadic and familial NS cases from Pakistan (South Asia)

The identified mutations have important implications in disease progression

but underlying genetic association studies are thought to affect several aspects of

the disease etiology These may include susceptibility for acquiring the disease

treatment responses histological findings and disease progression The genetic

association study of ACE gene polymorphism has been largely investigated in the

nephrotic syndrome patients and therefore the present studies were designed to

128

determine the association of the ACE and MTHFR gene polymorphisms with

pediatric NS in Pakistan

The ACE gene insertiondeletion (ID) polymorphism is a putative genetic

risk factor for NS This study analyzed 268 NS and 223 control samples by a PCR-

based method The results showed that the frequency distribution of the II ID and

DD genotypes were 82 (306) 128 (478) and 58 (216) in the NS patients

and 9 (40) 171 (767) and 43 (193) in the control samples respectively The

II genotypic and allelic frequencies were found to be significantly associated with

the disease in the Pakistani pediatric NS population (OR=67 CI=3-149) No

significant association was found between this polymorphism and the response to

standard steroid therapy Thus in contrast to reports from other parts of the world

the II genotype was found to be significantly associated with NS in the Pakistani

population This is similar to reports of the Indian and Malay populations (Patil et

al 2005 Jayapalan et al 2008) To our knowledge this is the first report from

Pakistan describing the association of the ACE ID polymorphism with pediatric

NS On the basis of these results it is suggested that analysis of the ACE (ID)

polymorphism should be performed for early diagnosis in the high risk NS patients

in South Asia

MTHFR gene polymorphisms cause elevated homocysteine levels

Hyperhomocysteinemia is an independent risk factor for thrombosis hypertension

arthrosclerosis and renal diseases etc and these similar complications are also

associated with the nephrotic syndrome (Kniazewska et al 2009 Ciaccio and

Bellia 2010) The MTHFR gene polymorphisms (C677T and A1298C) were also

analyzed in the nephrotic syndrome patients in this study A total of 318 children

129

with NS were ascertained and a panel of 200 healthy control samples was also

included Genotypes of the MTHFR polymorphisms (C677T and A1298C) were

analyzed using the PCR and RFLP techniques The frequencies for all three

possible genotypes of MTHFR C667T polymorphism ie CC CT and TT

genotypes were 74 22 and 4 in the NS patients and 70 26 and 4 in the

control samples respectively

The frequencies of CC CA and AA genotypes of MTHFR A1298C

polymorphism were 16 48 and 36 in the NS patients and 185 465 and

35 in the control samples respectively The genotypic and allelic frequencies of

C677T and A1298C polymorphisms were not associated with NS in Pakistani

children (OR=1181 0945 respectively) By contrast the TT genotype of the

MTHFR C667T polymorphism was associated with the early development and

progression of childhood FSGS in the Japanese patients (Zou et al 2002)

61 GENETIC SCREENING AND COUNSELING

The genetic screening guidelines for SRNS patients were described by

Santin et al (2011) It has been recommended that genetic screening should be

carried out for all SRNS children under the age of 13 years It is a non invasive

technique and is suggested to be performed before renal biopsies of SRNS patients

This precise testing approach depends on the age of the patient In congenital neph-

rotic syndrome the NPHS1 gene should be screened first whereas in cases of

infantile and childhood-onset NS the NPHS2 gene should be screened first (Santin

et al 2011) Other studies have also recommended the screening of the NPHS1

NPHS2 and WT1 genes for childhood onset SRNS (Hinkes et al 2007) If SRNS

130

patients are associated with renal histology of DMS the screening of PLCE1 and

LAMB2 genes should be carried out (Hasselbacher et al 2006 Hinkes et al

2006) In cases of late onset SRNS screening of INF2 TRPC6 and ACTN4 may be

performed in familial cases but no further investigation is recommended for

sporadic cases (Machuca et al 2009 Benoit et al 2010 Brown et al 2010

Boyer et al 2011 Santin et al 2011) This genetic testing guideline is generally

recommended for patients of European Middle Eastern or North African origin

but may not be appropriate for other part of the world as NPHS2 mutations are less

prevalent in Asian and African American children suffering from SRNS (Sako et

al 2005 Mao et al 2007)

There is no guideline available for the South Asian region and therefore the

present study was designed to carry out the screening of the NPHS1 and NPHS2

gene mutations in the pediatric SRNS cases from Pakistan The selection criteria of

patients were according to Santin et al (2011) and the results showed that

mutations in the NPHS1 and NPHS2 genes were not the frequent causes of

pediatric NS in Pakistan These results are in accordance with the studies from

Japan and China that reported a low prevalence of defects of the two genes in their

NS patients (Sako et al 2005 Mao et al 2007) Thus the low prevalence of

disease-causing mutations in the NPHS1 and NPHS2 genes suggests the

contribution of ethnic diversity in world populations Further investigations are

required to identify other novel podocyte genes that may be responsible for disease

in these patients

Genetic counseling is recommended for every patient with hereditary NS

and their families due to a higher risk of disease transmission from parents to

131

progeny The prenatal diagnosis should be accessible to families with a known risk

of CNS NPHS1 gene screening in these cases may help in counseling the families

at early pregnancies and also in future family planning In some patients genotypendash

phenotype correlations may facilitate counseling providing further information for

the NS patients which may modify the clinical course This has been observed in

the NPHS2-associated disease where some mutations have severe early onset of

the disease whereas others have shown to be late onset with a milder phenotype

(Buscher and Weber 2012)

62 THERAPEUTIC OPTIONS

NS patients generally respond to glucocorticoids or immunosuppressant

agents including cyclosporine (CsA) cyclophosphamide azathioprine and

mycophenolate mofetil (Plank et al 2008) Immunosuppressants suppress the

immune response and have beneficial effects directly on podocyte architecture

(Tejani and Ingulli 1995)

Patients with hereditary NS do not respond to standard steroid therapy This

observation suggested that there is no need to give heavy doses of steroids to these

patients However a partial response to and angiotensin converting enzyme (ACE)

inhibitors have been observed in some patients bearing NPHS1 NPHS2 TRPC6 or

WT1 mutations This response may be an effect of the antiproteinuric action of

calcineurin inhibitors or cyclosporine A (Machuca et al 2009 Benoit et al 2010

Buscher et al 2010 Santin et al 2011) Similarly in the current screening the

patients bearing NPHS1 and NPHS2 mutations have shown partial response to

immunosuppressants and ACE inhibitors

132

It has been observed that remission rates after CsA therapy are significantly

lower in patients with a known genetic basis compared with non hereditary SRNS

(17 vs 68 Buscher et al 2010) Intensified immunosuppressive therapy

regimens should not be recommended for hereditary SRNS patients ACE

inhibitors or blockers are also beneficial in reducing protein excretion and have

been found to be a better therapeutic option for SRNS patients (Sredharan and

Bockenhauer 2005 Liebau et al 2006 Copelovitch et al 2007) Further studies

are needed to determine which treatment would be beneficial for hereditary SRNS

patients Genetic screening also spares patients from the side effects associated with

these drugs Thus mutation analysis provides a guideline for long term therapy and

is also helpful in avoiding unnecessary steroid treatment for patients (Ruf et al

2004 Weber et al 2004)

The hereditary SRNS patients generally progress to ESRD and need dialysis

andor renal transplantation (RTx) The SRNS patients with NPHS2 gene mutations

have a lower risk of recurrent FSGS after renal transplantation (Caridi et al 2005

Jungraithmayr et al 2011) However these patients are not completely protected

from post-transplant recurrence of proteinuria Among these patients with a

heterozygous mutation show a higher risk of recurrence as compared to the patients

with homozygous or compound heterozygous mutations Thus a kidney from the

carrier of the mutation (such as parents) is not recommended as a donor for

transplantation due to the higher risk of FSGS recurrence in the recipient (Caridi et

al 2004) Therefore genetic screening of SRNS patients is also valuable in the

selection of the donor Patients with NPHS1 gene mutations have a higher risk of

post-transplant recurrence of NS due to the development of anti-nephrin antibodies

133

Such patients showed partial response to cyclophosphamide (Patrakka et al 2002)

In the dominant form of NS only one parent is the carrier of the causative

mutations In this case genetic testing will help to identify carriers within the family


63 FUTURE PERSPECTIVES

Recent genetic studies are providing exciting knowledge related to NS The

exact roles and functions of the newly discovered genes and proteins have been

under investigation using a combination of in vitro and in vivo approaches

(Woroniecki and Kopp 2007) These approaches have resulted in the development

of animal models of disease which will be helpful in understanding the disease

mechanisms as well as providing important tools to analyze novel therapeutic

strategies The better understanding of the pathophysiology of the NS will

influence future therapies and outcomes in this complicated disease

The use of chemical chaperones such as sodium 4-phenylbutyrate (4-PBA)

may be a potential therapeutic approach for the treatment of mild SRNS caused by

mutations in the NPHS1 and NPHS2 genes or in some patients with a non familial

NS or other similar diseases affecting renal filtration 4-PBA can correct the

cellular trafficking of several mislocalized or misfolded mutant proteins It has been

shown to efficiently rescue many mutated proteins that are abnormally retained in

the ER and allow them to be expressed normally on the cell surface and also

function properly (Burrows et al 2000)

Other important targets are the calcineurin inhibitors or CsA that provide

direct stabilization to the actin cytoskeleton in podocyte Recent advances indicate

134

that calcineurin substrates such as synaptopodin have the potential for the

development of antiproteinuric drugs This novel substrate also helps in avoiding

the severe side effects associated with the extensive use of CsA (Faul et al 2008)

The study presented here reports that mutations in the NPHS1 and NPHS2

genes are not the frequent causes of pediatric NS in Pakistan and no mutation was

found in the familial SRNS cases This study indicates that there are additional

genetic causes of SRNS that remain to be identified Novel genomic approaches

including next generation sequencing (Mardis et al 2008) and copy number

analysis based strategies may lead to the identification of novel genes in the near

future

In this current screening the exact role of heterozygous NPHS1 and NPHS2

mutations in disease progression were not established The newer techniques such

as whole exome screening may facilitate to analyze all the NS genes in a single

array and will be helpful in investigating the role of digenic or multigenic

(heterozygous) mutations These techniques will also aid in the diagnosis of

mutation specific prognosis and therapy

135

64 CONCLUSION

The main finding reported here is the low frequency of causative mutations

in the NPHS1 and NPHS2 genes in the Pakistani NS children These results

emphasize the need for discovery of other novel genes that may be involved in the

pathogenesis of SRNS in the South Asian region For this purpose genetic analysis

of large populations and the use of resequencing techniques will be required to find

other novel genesfactors in the pathogenesis of NS

The work presented here has important clinical relevance Genetic

screening should be done for every child upon disease presentation The

identification of a disease causing mutation would help in avoiding unnecessary

steroidimmunosuppressive drugs Mutation analysis may also encourage living

donor kidney for transplantation and offer prenatal diagnosis to families at risk

136

65 REFERENCES


T Hafeez F Naqvi SA Rizvi SA Mehdi SQ (2012) A spectrum of novel NPHS1


Gene 502 133-137



1214 83-98








Nature Genet 42 361

Buumlscher AK Kranz B Buumlscher R Hildebrandt F Dworniczak B Pennekamp P

Kuwertz-Broumlking E Wingen AM John U Kemper M Monnens L Hoyer PF

Weber S Konrad M (2010) Immunosuppression and renal outcome in congenital

and pediatric steroid-resistant nephrotic syndrome Clin J Am Soc Nephrol 5

2075-2084


Pediatr Eur J Pediatr 171 1151-1160

Burrows JA Willis LK Perlmutter DH (2000) Chemical chaperones mediate

increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z A potential

pharmacological strategy for prevention of liver injury and emphysema in alpha 1-

AT deficiency Proc Natl Acad Sci USA 97 1796-1801


NPHS2 mutations in responsive nephrotic syndrome and the multifactorial origin of

proteinuria Kidney Int 66 1715-1716



61R



137




Faul C Donnelly M Merscher-Gomez S Chang YH Franz S Delfgaauw J

Chang JM Choi HY Campbell KN Kim K Reiser J Mundel P (2008) The actin

cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of

cyclosporine A Nat Med 14 931-938




Hildebrandt F (2006) Recessive missense mutations in LAMB2 expand the clinical

spectrum of LAMB2-associated disorders Kidney Int 70 1008-1012

Hildebrandt F (2010) Genetic kidney diseases Lancet 375 1287-1295

















Health 39 917-921

Jungraithmayr TC Hofer K Cochat P Chernin G Cortina G Fargue S Grimm

P Knueppel T Kowarsch A Neuhaus T Pagel P Pfeiffer KP Schaumlfer F

Schoumlnermarck U Seeman T Toenshoff B Weber S Winn MP Zschocke J

Zimmerhackl LB (2011) Screening for NPHS2 mutations may help predict FSGS

recurrence after transplantation J Am Soc Nephrol 22 579-585





138

Liebau MC Lang D Boumlhm J Endlich N Bek MJ Witherden I Mathieson PW

Saleem MA Pavenstaumldt H Fischer KG (2006) Functional expression of the renin-

angiotensin system in human podocytes Am J Physiol Renal Physiol 290 F710-

719



R185-194




Mardis ER (2008) Next-generation DNA sequencing methods Annu Rev

Genomics Hum Genet 9 387-402

Patil SJ Gulati S Khan F Tripathi M Ahmed M Agrawal S (2005) Angiotensin







Plank C Kalb V Hinkes B Hildebrandt F Gefeller O Rascher W (2008)

Arbeitsgemeinschaft fuumlr Paumldiatrische Nephrologie Cyclosporin A is superior to

cyclophosphamide in children with steroid-resistant nephrotic syndrome-a

randomized controlled multicentre trial by the Arbeitsgemeinschaft fuumlr Paumldiatrische

Nephrologie Pediatr Nephrol 23 1483-1493













1148

139




Sinha A Bagga A (2012) Nephrotic syndrome Indian J Pediatr 79 1045-1055



Tejani A Ingulli E (1995) Cyclosporin in steroid-resistant idiopathic nephrotic

syndrome Contrib Nephrol 114 73-77




Int 66 571-579


disease 1st Edition Mount DM Pollak MR Sundher Elsevier Philadelphia PA

2008 142-145

Woroniecki RP Kopp JB (2007) Genetics of focal segmental glomerulosclerosis





92 449-451

II

THE GENETICS OF NEPHROTIC SYNDROME IN PAKISTANI

CHILDREN

Thesis submitted to the Sindh Institute of Medical Sciences

for the degree of Doctor of Philosophy

BY

Saba Shahid




Karachi Pakistan

2013

III

IV

Table of Contents

page

Acknowledgments i


Publications v

List of Tables vi


Summary ix


11 The Kidney 2





115 Podocyte 6



122 Definition 9








V


receptor-type O) 24













16 References 33













29 References 58

VI



31 Introduction 60




33 Results 67




34 Discussion 86

35 References 90



41 Introduction 95



422 Genotyping 97


43 Results 99

44 Discussion 103

45 References 106

VII



children 109

51 Introduction 110


521 Genotyping 113

53 Results 115

54 Discussion 118

55 References 122





64 Conclusion 135

65 References 136

i

Acknowledgments









inspiration for me


cooperation










work

ii










iii





ACTN4 α-Actinin 4


Ang-I Angiotensin I









CsA Cyclosporine

DAG Diacylglyecerol








FS Frasier Syndrome





Hcy Homocysteine




Ig Immunoglobulin




iv










OD Optical Density



PLC Phospholipase C







SD Slit Diaphragm












v

Publications










vi

List of Tables

Table Title

Page


13


NPHS1 gene

65


NPHS2 gene

66


syndrome

68


69



81


NPHS1 gene

82


85


99




and controls

101



102


113



vii


and controls

116



patients

117




and controls

119



patients

120

viii

List of Figures

Figure Title

Page


3



podocyte

5



and PLCE1

8


10


15


protein

19



80


their locations

84


98


homocysteine

112


gel

114


gel

114

ix

SUMMARY

x

SUMMARY























xi























xii



children
















1


SYNDROME

2

11 THE KIDNEY





















3



4





















5




rictedpaperhtml)

6













115 PODOCYTE











7


















Baratt 1999)

8




9


122 DEFINITION















1985)

123 CLASSIFICATIONS





10



11




















2005)





12


















13






Autosomal

recessive

(AR)


Childhood MCDFSGS

Kestila et al

1998


Adulthood FSGSMCD

Boute et al

2000


Childhood DMSFSGS

Hinkes et al

2007


2006



Ozaltin et

al 2011

Autosomal

dominant

(AD)


al 2000


Childhood DMSFSGS

Mucha et al

2006


2007



2005


2010

14




GENE (NEPHRIN)








al 2004)










15


2002)

16
























17

























18






















19



20

























21




















BETA 2)




22





2000)
















23





















24
















ACTININ- 4)






25






















associated disease

26


TUMOR)























27



















ASSOCIATED PROTEIN)





28
























29

























30







FORMIN-2)

















31


FACTORS



















32






















2 = 1

33

16 REFERENCES




Gene 502 133-137










467-470



373



1214 83-98








24 349-354









34








878-883




Nature Genet 42 361


Pediatr 171 1151-1160




494




Cell 60 509-520











61R




35



Nephrol 22 849-858










Nephrology 4th














281 F860-868








Cell Genet 83 46-47




36




Cell 94 667-677


629-639






Med Life 2 53-59



Genet Med 8 63-75





1297












2044-2051




37



Edition The












Nephrol 24 269-278


11 101-110





















38
















Nephrol 19 365-371



Nephrol 9 87-93















2128




39
















1 575-582













6211-6216








40




























1300




1198-1203





41









21 3283-3286











R185-194











937-941







42



205-209







Nephrol 13 589-593







1653-1660



2 Nature 374 258-262





Nephrol 19 384-389






212-221






c139-146

43








Mol Genet 12 2379-2394



Nephrol 12 289-296





















184-213






44




Genet 10 1-8







739-744



24 1101-1110




1346





J Pathol 160 131-139














369-381

45















1148



F947- 956




















46



Pathol 159 2303-2308
















Int 66 571-579








2008 142-145




16085






47











273-280












664 84-90

48


49















AllegraTM










50





















51













experiment













52



) was


















(QIAGEN Germany)

53




















2microl




54



oC for 5















minutes






55














system (Bio Rad)










56

























57













Odds Ratio





Chi-square test




χ

2= sum (o-e)

2e




58

29 REFERENCES








Edition








96 12810-12815

59




60

31 INTRODUCTION












Yu et al 2005)












61
























62























63
























64




















2010)

65


NPHS1 gene


size (bp)

PCR conditions








































66













NPHS2 gene


size (bp)

PCR conditions

















67

33 RESULTS











MCD (27 cases)













68


syndrome



Males () 88 (607)

Females () 57 (393)






FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)


Outcome

ESRDg CRF

h 14 (96)


Expired 8 (55)



cIgM nephropathy





hchronic renal

failure

69


S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid



SRNS q- d

ESRD ndash eTx



CyA -









kACEI +

lARB

-


+ARB -


+ARB -

70






CyA





23 NS084 No 5 M C1q



CyA -






CyA -


+ARB -


71


CyA -





+ARB -


nCyP

-


CyA -






44 NS140 No 35 M - SDNS - -




+ARB -



CyA -

72







CyA -




CyA -



ARB CRF


+ARB -

62 NS167 Yes 9 M - - - -


CyA -





73


relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -



CyA -


Tac -



CyP -


CyP -







83 NS203 No 11 M - - - -



74



+ARB -



90 NS214 Yes 6 M FSGS - - -


CyP -





96 NS221 Yes 1 M - - - -





+ARB -


CyP -


CyA -


75


+ARB -


+ARB -




CyP -



+ARB -



Tac -






CyP -





76




CyA -


+ARB -


127 NS281 No 10 M SRNS - -



Tac -


CyA -


CyA -




CyA -


+ARB -


+ARB


+ARB -


77

139 NS303 Yes 3 M - SRNS - -



+ARB -

142 NS306 No 25 M SRNS - -



+ARB -


+ARB -


bIgM nephropathy

ccyclosporine


etransplantation

fminimal change


htacrolimus


jmembranous




ncyclophosphamide



q (-)

78

























79


FSGS





80


domains

81


Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228


to dCyA

Normal 15yrs 100

NS254


NS291


c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099




dcyclosporine

82



b cyclosporine







Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change


Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I


Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q


eARB



Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W


NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I



Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206


Tacrolimus

Normal 000




follow up

019

83


















84


85


Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q


Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130


eARB

Normal 10yrs 0998

NS278

M no Infantile


eARB

Normal 3yrs 100

NS288

M no Infantile


Tacrolimus

Normal 3yrs 0998

a






86

34 DISCUSSION























87







(Table- 35 and 36)


















88
























89



90

35 REFERENCES









445-451



























91









2128









1 575-582















1198-1203




1209-1217

92












Nephrol 13 589-593





























93




1148
















Int 66 571-579









2632

94



PAKISTANI CHILDREN

95

41 INTRODUCTION













1985)










96




















97

422 GENOTYPING








buffer (GoTaqreg











98


M






99







43 RESULTS







Steroid response

SRNS

N=105

SSNS

N=163



Family history 24 6

ESRD 12 No

Biopsy 105 No




Hematuria 0-3 No

100








=142) having an OR of 16 (95 CI =13-


















101




NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)


ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102




SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION























104
























2010)

105










these children

106

45 REFERENCES









883


629-639



Listy 109 298-301






555



Health 39 917-921






Nephrol 13 589-593





Nephrol 19 384-389

107







1346






732







New York 2006 241-243











20 1738-1743






108







109




CHILDREN

110

51 INTRODUCTION






















111




















112



113








SRNS

N=166

SSNS

N=152



Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING






53)

114









115




53 RESULTS












from HWE (plt005)








116




Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117











CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137






118





=001 and 039 pgt005)












54 DISCUSSION









119




Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)


CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120
















CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121






















122

55 REFERENCES











494






241-246




Med Life 2 53-59





5606-5611








289

123






2264









1123



1300





Nephrol 13 589-593







Congress







124









Biol Chem 276 46896-46904







96 12810-12815






1207-1221










92 449-451

125


126
























127

























128


















in South Asia







129
























130










al 2005 Mao et al 2007)












in these patients



131
























132

























133
























134










future







135

64 CONCLUSION












136

65 REFERENCES




Gene 502 133-137



1214 83-98








Nature Genet 42 361





2075-2084












61R



137






























Health 39 917-921










138




719



R185-194






























1148

139












Int 66 571-579



2008 142-145






92 449-451

III

IV

Table of Contents

page

Acknowledgments i


Publications v

List of Tables vi


Summary ix


11 The Kidney 2





115 Podocyte 6



122 Definition 9








V


receptor-type O) 24













16 References 33













29 References 58

VI



31 Introduction 60




33 Results 67




34 Discussion 86

35 References 90



41 Introduction 95



422 Genotyping 97


43 Results 99

44 Discussion 103

45 References 106

VII



children 109

51 Introduction 110


521 Genotyping 113

53 Results 115

54 Discussion 118

55 References 122





64 Conclusion 135

65 References 136

i

Acknowledgments









inspiration for me


cooperation










work

ii










iii





ACTN4 α-Actinin 4


Ang-I Angiotensin I









CsA Cyclosporine

DAG Diacylglyecerol








FS Frasier Syndrome





Hcy Homocysteine




Ig Immunoglobulin




iv










OD Optical Density



PLC Phospholipase C







SD Slit Diaphragm












v

Publications










vi

List of Tables

Table Title

Page


13


NPHS1 gene

65


NPHS2 gene

66


syndrome

68


69



81


NPHS1 gene

82


85


99




and controls

101



102


113



vii


and controls

116



patients

117




and controls

119



patients

120

viii

List of Figures

Figure Title

Page


3



podocyte

5



and PLCE1

8


10


15


protein

19



80


their locations

84


98


homocysteine

112


gel

114


gel

114

ix

SUMMARY

x

SUMMARY























xi























xii



children
















1


SYNDROME

2

11 THE KIDNEY





















3



4





















5




rictedpaperhtml)

6













115 PODOCYTE











7


















Baratt 1999)

8




9


122 DEFINITION















1985)

123 CLASSIFICATIONS





10



11




















2005)





12


















13






Autosomal

recessive

(AR)


Childhood MCDFSGS

Kestila et al

1998


Adulthood FSGSMCD

Boute et al

2000


Childhood DMSFSGS

Hinkes et al

2007


2006



Ozaltin et

al 2011

Autosomal

dominant

(AD)


al 2000


Childhood DMSFSGS

Mucha et al

2006


2007



2005


2010

14




GENE (NEPHRIN)








al 2004)










15


2002)

16
























17

























18






















19



20

























21




















BETA 2)




22





2000)
















23





















24
















ACTININ- 4)






25






















associated disease

26


TUMOR)























27



















ASSOCIATED PROTEIN)





28
























29

























30







FORMIN-2)

















31


FACTORS



















32






















2 = 1

33

16 REFERENCES




Gene 502 133-137










467-470



373



1214 83-98








24 349-354









34








878-883




Nature Genet 42 361


Pediatr 171 1151-1160




494




Cell 60 509-520











61R




35



Nephrol 22 849-858










Nephrology 4th














281 F860-868








Cell Genet 83 46-47




36




Cell 94 667-677


629-639






Med Life 2 53-59



Genet Med 8 63-75





1297












2044-2051




37



Edition The












Nephrol 24 269-278


11 101-110





















38
















Nephrol 19 365-371



Nephrol 9 87-93















2128




39
















1 575-582













6211-6216








40




























1300




1198-1203





41









21 3283-3286











R185-194











937-941







42



205-209







Nephrol 13 589-593







1653-1660



2 Nature 374 258-262





Nephrol 19 384-389






212-221






c139-146

43








Mol Genet 12 2379-2394



Nephrol 12 289-296





















184-213






44




Genet 10 1-8







739-744



24 1101-1110




1346





J Pathol 160 131-139














369-381

45















1148



F947- 956




















46



Pathol 159 2303-2308
















Int 66 571-579








2008 142-145




16085






47











273-280












664 84-90

48


49















AllegraTM










50





















51













experiment













52



) was


















(QIAGEN Germany)

53




















2microl




54



oC for 5















minutes






55














system (Bio Rad)










56

























57













Odds Ratio





Chi-square test




χ

2= sum (o-e)

2e




58

29 REFERENCES








Edition








96 12810-12815

59




60

31 INTRODUCTION












Yu et al 2005)












61
























62























63
























64




















2010)

65


NPHS1 gene


size (bp)

PCR conditions








































66













NPHS2 gene


size (bp)

PCR conditions

















67

33 RESULTS











MCD (27 cases)













68


syndrome



Males () 88 (607)

Females () 57 (393)






FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)


Outcome

ESRDg CRF

h 14 (96)


Expired 8 (55)



cIgM nephropathy





hchronic renal

failure

69


S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid



SRNS q- d

ESRD ndash eTx



CyA -









kACEI +

lARB

-


+ARB -


+ARB -

70






CyA





23 NS084 No 5 M C1q



CyA -






CyA -


+ARB -


71


CyA -





+ARB -


nCyP

-


CyA -






44 NS140 No 35 M - SDNS - -




+ARB -



CyA -

72







CyA -




CyA -



ARB CRF


+ARB -

62 NS167 Yes 9 M - - - -


CyA -





73


relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -



CyA -


Tac -



CyP -


CyP -







83 NS203 No 11 M - - - -



74



+ARB -



90 NS214 Yes 6 M FSGS - - -


CyP -





96 NS221 Yes 1 M - - - -





+ARB -


CyP -


CyA -


75


+ARB -


+ARB -




CyP -



+ARB -



Tac -






CyP -





76




CyA -


+ARB -


127 NS281 No 10 M SRNS - -



Tac -


CyA -


CyA -




CyA -


+ARB -


+ARB


+ARB -


77

139 NS303 Yes 3 M - SRNS - -



+ARB -

142 NS306 No 25 M SRNS - -



+ARB -


+ARB -


bIgM nephropathy

ccyclosporine


etransplantation

fminimal change


htacrolimus


jmembranous




ncyclophosphamide



q (-)

78

























79


FSGS





80


domains

81


Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228


to dCyA

Normal 15yrs 100

NS254


NS291


c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099




dcyclosporine

82



b cyclosporine







Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change


Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I


Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q


eARB



Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W


NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I



Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206


Tacrolimus

Normal 000




follow up

019

83


















84


85


Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q


Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130


eARB

Normal 10yrs 0998

NS278

M no Infantile


eARB

Normal 3yrs 100

NS288

M no Infantile


Tacrolimus

Normal 3yrs 0998

a






86

34 DISCUSSION























87







(Table- 35 and 36)


















88
























89



90

35 REFERENCES









445-451



























91









2128









1 575-582















1198-1203




1209-1217

92












Nephrol 13 589-593





























93




1148
















Int 66 571-579









2632

94



PAKISTANI CHILDREN

95

41 INTRODUCTION













1985)










96




















97

422 GENOTYPING








buffer (GoTaqreg











98


M






99







43 RESULTS







Steroid response

SRNS

N=105

SSNS

N=163



Family history 24 6

ESRD 12 No

Biopsy 105 No




Hematuria 0-3 No

100








=142) having an OR of 16 (95 CI =13-


















101




NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)


ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102




SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION























104
























2010)

105










these children

106

45 REFERENCES









883


629-639



Listy 109 298-301






555



Health 39 917-921






Nephrol 13 589-593





Nephrol 19 384-389

107







1346






732







New York 2006 241-243











20 1738-1743






108







109




CHILDREN

110

51 INTRODUCTION






















111




















112



113








SRNS

N=166

SSNS

N=152



Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING






53)

114









115




53 RESULTS












from HWE (plt005)








116




Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117











CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137






118





=001 and 039 pgt005)












54 DISCUSSION









119




Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)


CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120
















CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121






















122

55 REFERENCES











494






241-246




Med Life 2 53-59





5606-5611








289

123






2264









1123



1300





Nephrol 13 589-593







Congress







124









Biol Chem 276 46896-46904







96 12810-12815






1207-1221










92 449-451

125


126
























127

























128


















in South Asia







129
























130










al 2005 Mao et al 2007)












in these patients



131
























132

























133
























134










future







135

64 CONCLUSION












136

65 REFERENCES




Gene 502 133-137



1214 83-98








Nature Genet 42 361





2075-2084












61R



137






























Health 39 917-921










138




719



R185-194






























1148

139












Int 66 571-579



2008 142-145






92 449-451

IV

Table of Contents

page

Acknowledgments i


Publications v

List of Tables vi


Summary ix


11 The Kidney 2





115 Podocyte 6



122 Definition 9








V


receptor-type O) 24













16 References 33













29 References 58

VI



31 Introduction 60




33 Results 67




34 Discussion 86

35 References 90



41 Introduction 95



422 Genotyping 97


43 Results 99

44 Discussion 103

45 References 106

VII



children 109

51 Introduction 110


521 Genotyping 113

53 Results 115

54 Discussion 118

55 References 122





64 Conclusion 135

65 References 136

i

Acknowledgments









inspiration for me


cooperation










work

ii










iii





ACTN4 α-Actinin 4


Ang-I Angiotensin I









CsA Cyclosporine

DAG Diacylglyecerol








FS Frasier Syndrome





Hcy Homocysteine




Ig Immunoglobulin




iv










OD Optical Density



PLC Phospholipase C







SD Slit Diaphragm












v

Publications










vi

List of Tables

Table Title

Page


13


NPHS1 gene

65


NPHS2 gene

66


syndrome

68


69



81


NPHS1 gene

82


85


99




and controls

101



102


113



vii


and controls

116



patients

117




and controls

119



patients

120

viii

List of Figures

Figure Title

Page


3



podocyte

5



and PLCE1

8


10


15


protein

19



80


their locations

84


98


homocysteine

112


gel

114


gel

114

ix

SUMMARY

x

SUMMARY























xi























xii



children
















1


SYNDROME

2

11 THE KIDNEY





















3



4





















5




rictedpaperhtml)

6













115 PODOCYTE











7


















Baratt 1999)

8




9


122 DEFINITION















1985)

123 CLASSIFICATIONS





10



11




















2005)





12


















13






Autosomal

recessive

(AR)


Childhood MCDFSGS

Kestila et al

1998


Adulthood FSGSMCD

Boute et al

2000


Childhood DMSFSGS

Hinkes et al

2007


2006



Ozaltin et

al 2011

Autosomal

dominant

(AD)


al 2000


Childhood DMSFSGS

Mucha et al

2006


2007



2005


2010

14




GENE (NEPHRIN)








al 2004)










15


2002)

16
























17

























18






















19



20

























21




















BETA 2)




22





2000)
















23





















24
















ACTININ- 4)






25






















associated disease

26


TUMOR)























27



















ASSOCIATED PROTEIN)





28
























29

























30







FORMIN-2)

















31


FACTORS



















32






















2 = 1

33

16 REFERENCES




Gene 502 133-137










467-470



373



1214 83-98








24 349-354









34








878-883




Nature Genet 42 361


Pediatr 171 1151-1160




494




Cell 60 509-520











61R




35



Nephrol 22 849-858










Nephrology 4th














281 F860-868








Cell Genet 83 46-47




36




Cell 94 667-677


629-639






Med Life 2 53-59



Genet Med 8 63-75





1297












2044-2051




37



Edition The












Nephrol 24 269-278


11 101-110





















38
















Nephrol 19 365-371



Nephrol 9 87-93















2128




39
















1 575-582













6211-6216








40




























1300




1198-1203





41









21 3283-3286











R185-194











937-941







42



205-209







Nephrol 13 589-593







1653-1660



2 Nature 374 258-262





Nephrol 19 384-389






212-221






c139-146

43








Mol Genet 12 2379-2394



Nephrol 12 289-296





















184-213






44




Genet 10 1-8







739-744



24 1101-1110




1346





J Pathol 160 131-139














369-381

45















1148



F947- 956




















46



Pathol 159 2303-2308
















Int 66 571-579








2008 142-145




16085






47











273-280












664 84-90

48


49















AllegraTM










50





















51













experiment













52



) was


















(QIAGEN Germany)

53




















2microl




54



oC for 5















minutes






55














system (Bio Rad)










56

























57













Odds Ratio





Chi-square test




χ

2= sum (o-e)

2e




58

29 REFERENCES








Edition








96 12810-12815

59




60

31 INTRODUCTION












Yu et al 2005)












61
























62























63
























64




















2010)

65


NPHS1 gene


size (bp)

PCR conditions








































66













NPHS2 gene


size (bp)

PCR conditions

















67

33 RESULTS











MCD (27 cases)













68


syndrome



Males () 88 (607)

Females () 57 (393)






FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)


Outcome

ESRDg CRF

h 14 (96)


Expired 8 (55)



cIgM nephropathy





hchronic renal

failure

69


S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid



SRNS q- d

ESRD ndash eTx



CyA -









kACEI +

lARB

-


+ARB -


+ARB -

70






CyA





23 NS084 No 5 M C1q



CyA -






CyA -


+ARB -


71


CyA -





+ARB -


nCyP

-


CyA -






44 NS140 No 35 M - SDNS - -




+ARB -



CyA -

72







CyA -




CyA -



ARB CRF


+ARB -

62 NS167 Yes 9 M - - - -


CyA -





73


relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -



CyA -


Tac -



CyP -


CyP -







83 NS203 No 11 M - - - -



74



+ARB -



90 NS214 Yes 6 M FSGS - - -


CyP -





96 NS221 Yes 1 M - - - -





+ARB -


CyP -


CyA -


75


+ARB -


+ARB -




CyP -



+ARB -



Tac -






CyP -





76




CyA -


+ARB -


127 NS281 No 10 M SRNS - -



Tac -


CyA -


CyA -




CyA -


+ARB -


+ARB


+ARB -


77

139 NS303 Yes 3 M - SRNS - -



+ARB -

142 NS306 No 25 M SRNS - -



+ARB -


+ARB -


bIgM nephropathy

ccyclosporine


etransplantation

fminimal change


htacrolimus


jmembranous




ncyclophosphamide



q (-)

78

























79


FSGS





80


domains

81


Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228


to dCyA

Normal 15yrs 100

NS254


NS291


c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099




dcyclosporine

82



b cyclosporine







Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change


Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I


Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q


eARB



Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W


NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I



Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206


Tacrolimus

Normal 000




follow up

019

83


















84


85


Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q


Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130


eARB

Normal 10yrs 0998

NS278

M no Infantile


eARB

Normal 3yrs 100

NS288

M no Infantile


Tacrolimus

Normal 3yrs 0998

a






86

34 DISCUSSION























87







(Table- 35 and 36)


















88
























89



90

35 REFERENCES









445-451



























91









2128









1 575-582















1198-1203




1209-1217

92












Nephrol 13 589-593





























93




1148
















Int 66 571-579









2632

94



PAKISTANI CHILDREN

95

41 INTRODUCTION













1985)










96




















97

422 GENOTYPING








buffer (GoTaqreg











98


M






99







43 RESULTS







Steroid response

SRNS

N=105

SSNS

N=163



Family history 24 6

ESRD 12 No

Biopsy 105 No




Hematuria 0-3 No

100








=142) having an OR of 16 (95 CI =13-


















101




NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)


ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102




SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION























104
























2010)

105










these children

106

45 REFERENCES









883


629-639



Listy 109 298-301






555



Health 39 917-921






Nephrol 13 589-593





Nephrol 19 384-389

107







1346






732







New York 2006 241-243











20 1738-1743






108







109




CHILDREN

110

51 INTRODUCTION






















111




















112



113








SRNS

N=166

SSNS

N=152



Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING






53)

114









115




53 RESULTS












from HWE (plt005)








116




Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117











CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137






118





=001 and 039 pgt005)












54 DISCUSSION









119




Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)


CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120
















CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121






















122

55 REFERENCES











494






241-246




Med Life 2 53-59





5606-5611








289

123






2264









1123



1300





Nephrol 13 589-593







Congress







124









Biol Chem 276 46896-46904







96 12810-12815






1207-1221










92 449-451

125


126
























127

























128


















in South Asia







129
























130










al 2005 Mao et al 2007)












in these patients



131
























132

























133
























134










future







135

64 CONCLUSION












136

65 REFERENCES




Gene 502 133-137



1214 83-98








Nature Genet 42 361





2075-2084












61R



137






























Health 39 917-921










138




719



R185-194






























1148

139












Int 66 571-579



2008 142-145






92 449-451

V


receptor-type O) 24













16 References 33













29 References 58

VI



31 Introduction 60




33 Results 67




34 Discussion 86

35 References 90



41 Introduction 95



422 Genotyping 97


43 Results 99

44 Discussion 103

45 References 106

VII



children 109

51 Introduction 110


521 Genotyping 113

53 Results 115

54 Discussion 118

55 References 122





64 Conclusion 135

65 References 136

i

Acknowledgments









inspiration for me


cooperation










work

ii










iii





ACTN4 α-Actinin 4


Ang-I Angiotensin I









CsA Cyclosporine

DAG Diacylglyecerol








FS Frasier Syndrome





Hcy Homocysteine




Ig Immunoglobulin




iv










OD Optical Density



PLC Phospholipase C







SD Slit Diaphragm












v

Publications










vi

List of Tables

Table Title

Page


13


NPHS1 gene

65


NPHS2 gene

66


syndrome

68


69



81


NPHS1 gene

82


85


99




and controls

101



102


113



vii


and controls

116



patients

117




and controls

119



patients

120

viii

List of Figures

Figure Title

Page


3



podocyte

5



and PLCE1

8


10


15


protein

19



80


their locations

84


98


homocysteine

112


gel

114


gel

114

ix

SUMMARY

x

SUMMARY























xi























xii



children
















1


SYNDROME

2

11 THE KIDNEY





















3



4





















5




rictedpaperhtml)

6













115 PODOCYTE











7


















Baratt 1999)

8




9


122 DEFINITION















1985)

123 CLASSIFICATIONS





10



11




















2005)





12


















13






Autosomal

recessive

(AR)


Childhood MCDFSGS

Kestila et al

1998


Adulthood FSGSMCD

Boute et al

2000


Childhood DMSFSGS

Hinkes et al

2007


2006



Ozaltin et

al 2011

Autosomal

dominant

(AD)


al 2000


Childhood DMSFSGS

Mucha et al

2006


2007



2005


2010

14




GENE (NEPHRIN)








al 2004)










15


2002)

16
























17

























18






















19



20

























21




















BETA 2)




22





2000)
















23





















24
















ACTININ- 4)






25






















associated disease

26


TUMOR)























27



















ASSOCIATED PROTEIN)





28
























29

























30







FORMIN-2)

















31


FACTORS



















32






















2 = 1

33

16 REFERENCES




Gene 502 133-137










467-470



373



1214 83-98








24 349-354









34








878-883




Nature Genet 42 361


Pediatr 171 1151-1160




494




Cell 60 509-520











61R




35



Nephrol 22 849-858










Nephrology 4th














281 F860-868








Cell Genet 83 46-47




36




Cell 94 667-677


629-639






Med Life 2 53-59



Genet Med 8 63-75





1297












2044-2051




37



Edition The












Nephrol 24 269-278


11 101-110





















38
















Nephrol 19 365-371



Nephrol 9 87-93















2128




39
















1 575-582













6211-6216








40




























1300




1198-1203





41









21 3283-3286











R185-194











937-941







42



205-209







Nephrol 13 589-593







1653-1660



2 Nature 374 258-262





Nephrol 19 384-389






212-221






c139-146

43








Mol Genet 12 2379-2394



Nephrol 12 289-296





















184-213






44




Genet 10 1-8







739-744



24 1101-1110




1346





J Pathol 160 131-139














369-381

45















1148



F947- 956




















46



Pathol 159 2303-2308
















Int 66 571-579








2008 142-145




16085






47











273-280












664 84-90

48


49















AllegraTM










50





















51













experiment













52



) was


















(QIAGEN Germany)

53




















2microl




54



oC for 5















minutes






55














system (Bio Rad)










56

























57













Odds Ratio





Chi-square test




χ

2= sum (o-e)

2e




58

29 REFERENCES








Edition








96 12810-12815

59




60

31 INTRODUCTION












Yu et al 2005)












61
























62























63
























64




















2010)

65


NPHS1 gene


size (bp)

PCR conditions








































66













NPHS2 gene


size (bp)

PCR conditions

















67

33 RESULTS











MCD (27 cases)













68


syndrome



Males () 88 (607)

Females () 57 (393)






FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)


Outcome

ESRDg CRF

h 14 (96)


Expired 8 (55)



cIgM nephropathy





hchronic renal

failure

69


S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid



SRNS q- d

ESRD ndash eTx



CyA -









kACEI +

lARB

-


+ARB -


+ARB -

70






CyA





23 NS084 No 5 M C1q



CyA -






CyA -


+ARB -


71


CyA -





+ARB -


nCyP

-


CyA -






44 NS140 No 35 M - SDNS - -




+ARB -



CyA -

72







CyA -




CyA -



ARB CRF


+ARB -

62 NS167 Yes 9 M - - - -


CyA -





73


relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -



CyA -


Tac -



CyP -


CyP -







83 NS203 No 11 M - - - -



74



+ARB -



90 NS214 Yes 6 M FSGS - - -


CyP -





96 NS221 Yes 1 M - - - -





+ARB -


CyP -


CyA -


75


+ARB -


+ARB -




CyP -



+ARB -



Tac -






CyP -





76




CyA -


+ARB -


127 NS281 No 10 M SRNS - -



Tac -


CyA -


CyA -




CyA -


+ARB -


+ARB


+ARB -


77

139 NS303 Yes 3 M - SRNS - -



+ARB -

142 NS306 No 25 M SRNS - -



+ARB -


+ARB -


bIgM nephropathy

ccyclosporine


etransplantation

fminimal change


htacrolimus


jmembranous




ncyclophosphamide



q (-)

78

























79


FSGS





80


domains

81


Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228


to dCyA

Normal 15yrs 100

NS254


NS291


c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099




dcyclosporine

82



b cyclosporine







Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change


Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I


Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q


eARB



Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W


NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I



Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206


Tacrolimus

Normal 000




follow up

019

83


















84


85


Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q


Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130


eARB

Normal 10yrs 0998

NS278

M no Infantile


eARB

Normal 3yrs 100

NS288

M no Infantile


Tacrolimus

Normal 3yrs 0998

a






86

34 DISCUSSION























87







(Table- 35 and 36)


















88
























89



90

35 REFERENCES









445-451



























91









2128









1 575-582















1198-1203




1209-1217

92












Nephrol 13 589-593





























93




1148
















Int 66 571-579









2632

94



PAKISTANI CHILDREN

95

41 INTRODUCTION













1985)










96




















97

422 GENOTYPING








buffer (GoTaqreg











98


M






99







43 RESULTS







Steroid response

SRNS

N=105

SSNS

N=163



Family history 24 6

ESRD 12 No

Biopsy 105 No




Hematuria 0-3 No

100








=142) having an OR of 16 (95 CI =13-


















101




NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)


ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102




SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION























104
























2010)

105










these children

106

45 REFERENCES









883


629-639



Listy 109 298-301






555



Health 39 917-921






Nephrol 13 589-593





Nephrol 19 384-389

107







1346






732







New York 2006 241-243











20 1738-1743






108







109




CHILDREN

110

51 INTRODUCTION






















111




















112



113








SRNS

N=166

SSNS

N=152



Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING






53)

114









115




53 RESULTS












from HWE (plt005)








116




Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117











CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137






118





=001 and 039 pgt005)












54 DISCUSSION









119




Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)


CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120
















CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121






















122

55 REFERENCES











494






241-246




Med Life 2 53-59





5606-5611








289

123






2264









1123



1300





Nephrol 13 589-593







Congress







124









Biol Chem 276 46896-46904







96 12810-12815






1207-1221










92 449-451

125


126
























127

























128


















in South Asia







129
























130










al 2005 Mao et al 2007)












in these patients



131
























132

























133
























134










future







135

64 CONCLUSION












136

65 REFERENCES




Gene 502 133-137



1214 83-98








Nature Genet 42 361





2075-2084












61R



137






























Health 39 917-921










138




719



R185-194






























1148

139












Int 66 571-579



2008 142-145






92 449-451

VI



31 Introduction 60




33 Results 67




34 Discussion 86

35 References 90



41 Introduction 95



422 Genotyping 97


43 Results 99

44 Discussion 103

45 References 106

VII



children 109

51 Introduction 110


521 Genotyping 113

53 Results 115

54 Discussion 118

55 References 122





64 Conclusion 135

65 References 136

i

Acknowledgments









inspiration for me


cooperation










work

ii










iii





ACTN4 α-Actinin 4


Ang-I Angiotensin I









CsA Cyclosporine

DAG Diacylglyecerol








FS Frasier Syndrome





Hcy Homocysteine




Ig Immunoglobulin




iv










OD Optical Density



PLC Phospholipase C







SD Slit Diaphragm












v

Publications










vi

List of Tables

Table Title

Page


13


NPHS1 gene

65


NPHS2 gene

66


syndrome

68


69



81


NPHS1 gene

82


85


99




and controls

101



102


113



vii


and controls

116



patients

117




and controls

119



patients

120

viii

List of Figures

Figure Title

Page


3



podocyte

5



and PLCE1

8


10


15


protein

19



80


their locations

84


98


homocysteine

112


gel

114


gel

114

ix

SUMMARY

x

SUMMARY























xi























xii



children
















1


SYNDROME

2

11 THE KIDNEY





















3



4





















5




rictedpaperhtml)

6













115 PODOCYTE











7


















Baratt 1999)

8




9


122 DEFINITION















1985)

123 CLASSIFICATIONS





10



11




















2005)





12


















13






Autosomal

recessive

(AR)


Childhood MCDFSGS

Kestila et al

1998


Adulthood FSGSMCD

Boute et al

2000


Childhood DMSFSGS

Hinkes et al

2007


2006



Ozaltin et

al 2011

Autosomal

dominant

(AD)


al 2000


Childhood DMSFSGS

Mucha et al

2006


2007



2005


2010

14




GENE (NEPHRIN)








al 2004)










15


2002)

16
























17

























18






















19



20

























21




















BETA 2)




22





2000)
















23





















24
















ACTININ- 4)






25






















associated disease

26


TUMOR)























27



















ASSOCIATED PROTEIN)





28
























29

























30







FORMIN-2)

















31


FACTORS



















32






















2 = 1

33

16 REFERENCES




Gene 502 133-137










467-470



373



1214 83-98








24 349-354









34








878-883




Nature Genet 42 361


Pediatr 171 1151-1160




494




Cell 60 509-520











61R




35



Nephrol 22 849-858










Nephrology 4th














281 F860-868








Cell Genet 83 46-47




36




Cell 94 667-677


629-639






Med Life 2 53-59



Genet Med 8 63-75





1297












2044-2051




37



Edition The












Nephrol 24 269-278


11 101-110





















38
















Nephrol 19 365-371



Nephrol 9 87-93















2128




39
















1 575-582













6211-6216








40




























1300




1198-1203





41









21 3283-3286











R185-194











937-941







42



205-209







Nephrol 13 589-593







1653-1660



2 Nature 374 258-262





Nephrol 19 384-389






212-221






c139-146

43








Mol Genet 12 2379-2394



Nephrol 12 289-296





















184-213






44




Genet 10 1-8







739-744



24 1101-1110




1346





J Pathol 160 131-139














369-381

45















1148



F947- 956




















46



Pathol 159 2303-2308
















Int 66 571-579








2008 142-145




16085






47











273-280












664 84-90

48


49















AllegraTM










50





















51













experiment













52



) was


















(QIAGEN Germany)

53




















2microl




54



oC for 5















minutes






55














system (Bio Rad)










56

























57













Odds Ratio





Chi-square test




χ

2= sum (o-e)

2e




58

29 REFERENCES








Edition








96 12810-12815

59




60

31 INTRODUCTION












Yu et al 2005)












61
























62























63
























64




















2010)

65


NPHS1 gene


size (bp)

PCR conditions








































66













NPHS2 gene


size (bp)

PCR conditions

















67

33 RESULTS











MCD (27 cases)













68


syndrome



Males () 88 (607)

Females () 57 (393)






FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)


Outcome

ESRDg CRF

h 14 (96)


Expired 8 (55)



cIgM nephropathy





hchronic renal

failure

69


S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid



SRNS q- d

ESRD ndash eTx



CyA -









kACEI +

lARB

-


+ARB -


+ARB -

70






CyA





23 NS084 No 5 M C1q



CyA -






CyA -


+ARB -


71


CyA -





+ARB -


nCyP

-


CyA -






44 NS140 No 35 M - SDNS - -




+ARB -



CyA -

72







CyA -




CyA -



ARB CRF


+ARB -

62 NS167 Yes 9 M - - - -


CyA -





73


relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -



CyA -


Tac -



CyP -


CyP -







83 NS203 No 11 M - - - -



74



+ARB -



90 NS214 Yes 6 M FSGS - - -


CyP -





96 NS221 Yes 1 M - - - -





+ARB -


CyP -


CyA -


75


+ARB -


+ARB -




CyP -



+ARB -



Tac -






CyP -





76




CyA -


+ARB -


127 NS281 No 10 M SRNS - -



Tac -


CyA -


CyA -




CyA -


+ARB -


+ARB


+ARB -


77

139 NS303 Yes 3 M - SRNS - -



+ARB -

142 NS306 No 25 M SRNS - -



+ARB -


+ARB -


bIgM nephropathy

ccyclosporine


etransplantation

fminimal change


htacrolimus


jmembranous




ncyclophosphamide



q (-)

78

























79


FSGS





80


domains

81


Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228


to dCyA

Normal 15yrs 100

NS254


NS291


c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099




dcyclosporine

82



b cyclosporine







Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change


Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I


Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q


eARB



Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W


NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I



Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206


Tacrolimus

Normal 000




follow up

019

83


















84


85


Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q


Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130


eARB

Normal 10yrs 0998

NS278

M no Infantile


eARB

Normal 3yrs 100

NS288

M no Infantile


Tacrolimus

Normal 3yrs 0998

a






86

34 DISCUSSION























87







(Table- 35 and 36)


















88
























89



90

35 REFERENCES









445-451



























91









2128









1 575-582















1198-1203




1209-1217

92












Nephrol 13 589-593





























93




1148
















Int 66 571-579









2632

94



PAKISTANI CHILDREN

95

41 INTRODUCTION













1985)










96




















97

422 GENOTYPING








buffer (GoTaqreg











98


M






99







43 RESULTS







Steroid response

SRNS

N=105

SSNS

N=163



Family history 24 6

ESRD 12 No

Biopsy 105 No




Hematuria 0-3 No

100








=142) having an OR of 16 (95 CI =13-


















101




NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)


ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102




SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION























104
























2010)

105










these children

106

45 REFERENCES









883


629-639



Listy 109 298-301






555



Health 39 917-921






Nephrol 13 589-593





Nephrol 19 384-389

107







1346






732







New York 2006 241-243











20 1738-1743






108







109




CHILDREN

110

51 INTRODUCTION






















111




















112



113








SRNS

N=166

SSNS

N=152



Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING






53)

114









115




53 RESULTS












from HWE (plt005)








116




Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117











CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137






118





=001 and 039 pgt005)












54 DISCUSSION









119




Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)


CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120
















CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121






















122

55 REFERENCES











494






241-246




Med Life 2 53-59





5606-5611








289

123






2264









1123



1300





Nephrol 13 589-593







Congress







124









Biol Chem 276 46896-46904







96 12810-12815






1207-1221










92 449-451

125


126
























127

























128


















in South Asia







129
























130










al 2005 Mao et al 2007)












in these patients



131
























132

























133
























134










future







135

64 CONCLUSION












136

65 REFERENCES




Gene 502 133-137



1214 83-98








Nature Genet 42 361





2075-2084












61R



137






























Health 39 917-921










138




719



R185-194






























1148

139












Int 66 571-579



2008 142-145






92 449-451

VII



children 109

51 Introduction 110


521 Genotyping 113

53 Results 115

54 Discussion 118

55 References 122





64 Conclusion 135

65 References 136

i

Acknowledgments









inspiration for me


cooperation










work

ii










iii





ACTN4 α-Actinin 4


Ang-I Angiotensin I









CsA Cyclosporine

DAG Diacylglyecerol








FS Frasier Syndrome





Hcy Homocysteine




Ig Immunoglobulin




iv










OD Optical Density



PLC Phospholipase C







SD Slit Diaphragm












v

Publications










vi

List of Tables

Table Title

Page


13


NPHS1 gene

65


NPHS2 gene

66


syndrome

68


69



81


NPHS1 gene

82


85


99




and controls

101



102


113



vii


and controls

116



patients

117




and controls

119



patients

120

viii

List of Figures

Figure Title

Page


3



podocyte

5



and PLCE1

8


10


15


protein

19



80


their locations

84


98


homocysteine

112


gel

114


gel

114

ix

SUMMARY

x

SUMMARY























xi























xii



children
















1


SYNDROME

2

11 THE KIDNEY





















3



4





















5




rictedpaperhtml)

6













115 PODOCYTE











7


















Baratt 1999)

8




9


122 DEFINITION















1985)

123 CLASSIFICATIONS





10



11




















2005)





12


















13






Autosomal

recessive

(AR)


Childhood MCDFSGS

Kestila et al

1998


Adulthood FSGSMCD

Boute et al

2000


Childhood DMSFSGS

Hinkes et al

2007


2006



Ozaltin et

al 2011

Autosomal

dominant

(AD)


al 2000


Childhood DMSFSGS

Mucha et al

2006


2007



2005


2010

14




GENE (NEPHRIN)








al 2004)










15


2002)

16
























17

























18






















19



20

























21




















BETA 2)




22





2000)
















23





















24
















ACTININ- 4)






25






















associated disease

26


TUMOR)























27



















ASSOCIATED PROTEIN)





28
























29

























30







FORMIN-2)

















31


FACTORS



















32






















2 = 1

33

16 REFERENCES




Gene 502 133-137










467-470



373



1214 83-98








24 349-354









34








878-883




Nature Genet 42 361


Pediatr 171 1151-1160




494




Cell 60 509-520











61R




35



Nephrol 22 849-858










Nephrology 4th














281 F860-868








Cell Genet 83 46-47




36




Cell 94 667-677


629-639






Med Life 2 53-59



Genet Med 8 63-75





1297












2044-2051




37



Edition The












Nephrol 24 269-278


11 101-110





















38
















Nephrol 19 365-371



Nephrol 9 87-93















2128




39
















1 575-582













6211-6216








40




























1300




1198-1203





41









21 3283-3286











R185-194











937-941







42



205-209







Nephrol 13 589-593







1653-1660



2 Nature 374 258-262





Nephrol 19 384-389






212-221






c139-146

43








Mol Genet 12 2379-2394



Nephrol 12 289-296





















184-213






44




Genet 10 1-8







739-744



24 1101-1110




1346





J Pathol 160 131-139














369-381

45















1148



F947- 956




















46



Pathol 159 2303-2308
















Int 66 571-579








2008 142-145




16085






47











273-280












664 84-90

48


49















AllegraTM










50





















51













experiment













52



) was


















(QIAGEN Germany)

53




















2microl




54



oC for 5















minutes






55














system (Bio Rad)










56

























57













Odds Ratio





Chi-square test




χ

2= sum (o-e)

2e




58

29 REFERENCES








Edition








96 12810-12815

59




60

31 INTRODUCTION












Yu et al 2005)












61
























62























63
























64




















2010)

65


NPHS1 gene


size (bp)

PCR conditions








































66













NPHS2 gene


size (bp)

PCR conditions

















67

33 RESULTS











MCD (27 cases)













68


syndrome



Males () 88 (607)

Females () 57 (393)






FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)


Outcome

ESRDg CRF

h 14 (96)


Expired 8 (55)



cIgM nephropathy





hchronic renal

failure

69


S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid



SRNS q- d

ESRD ndash eTx



CyA -









kACEI +

lARB

-


+ARB -


+ARB -

70






CyA





23 NS084 No 5 M C1q



CyA -






CyA -


+ARB -


71


CyA -





+ARB -


nCyP

-


CyA -






44 NS140 No 35 M - SDNS - -




+ARB -



CyA -

72







CyA -




CyA -



ARB CRF


+ARB -

62 NS167 Yes 9 M - - - -


CyA -





73


relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -



CyA -


Tac -



CyP -


CyP -







83 NS203 No 11 M - - - -



74



+ARB -



90 NS214 Yes 6 M FSGS - - -


CyP -





96 NS221 Yes 1 M - - - -





+ARB -


CyP -


CyA -


75


+ARB -


+ARB -




CyP -



+ARB -



Tac -






CyP -





76




CyA -


+ARB -


127 NS281 No 10 M SRNS - -



Tac -


CyA -


CyA -




CyA -


+ARB -


+ARB


+ARB -


77

139 NS303 Yes 3 M - SRNS - -



+ARB -

142 NS306 No 25 M SRNS - -



+ARB -


+ARB -


bIgM nephropathy

ccyclosporine


etransplantation

fminimal change


htacrolimus


jmembranous




ncyclophosphamide



q (-)

78

























79


FSGS





80


domains

81


Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228


to dCyA

Normal 15yrs 100

NS254


NS291


c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099




dcyclosporine

82



b cyclosporine







Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change


Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I


Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q


eARB



Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W


NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I



Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206


Tacrolimus

Normal 000




follow up

019

83


















84


85


Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q


Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130


eARB

Normal 10yrs 0998

NS278

M no Infantile


eARB

Normal 3yrs 100

NS288

M no Infantile


Tacrolimus

Normal 3yrs 0998

a






86

34 DISCUSSION























87







(Table- 35 and 36)


















88
























89



90

35 REFERENCES









445-451



























91









2128









1 575-582















1198-1203




1209-1217

92












Nephrol 13 589-593





























93




1148
















Int 66 571-579









2632

94



PAKISTANI CHILDREN

95

41 INTRODUCTION













1985)










96




















97

422 GENOTYPING








buffer (GoTaqreg











98


M






99







43 RESULTS







Steroid response

SRNS

N=105

SSNS

N=163



Family history 24 6

ESRD 12 No

Biopsy 105 No




Hematuria 0-3 No

100








=142) having an OR of 16 (95 CI =13-


















101




NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)


ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102




SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION























104
























2010)

105










these children

106

45 REFERENCES









883


629-639



Listy 109 298-301






555



Health 39 917-921






Nephrol 13 589-593





Nephrol 19 384-389

107







1346






732







New York 2006 241-243











20 1738-1743






108







109




CHILDREN

110

51 INTRODUCTION






















111




















112



113








SRNS

N=166

SSNS

N=152



Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING






53)

114









115




53 RESULTS












from HWE (plt005)








116




Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117











CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137






118





=001 and 039 pgt005)












54 DISCUSSION









119




Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)


CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120
















CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121






















122

55 REFERENCES











494






241-246




Med Life 2 53-59





5606-5611








289

123






2264









1123



1300





Nephrol 13 589-593







Congress







124









Biol Chem 276 46896-46904







96 12810-12815






1207-1221










92 449-451

125


126
























127

























128


















in South Asia







129
























130










al 2005 Mao et al 2007)












in these patients



131
























132

























133
























134










future







135

64 CONCLUSION












136

65 REFERENCES




Gene 502 133-137



1214 83-98








Nature Genet 42 361





2075-2084












61R



137






























Health 39 917-921










138




719



R185-194






























1148

139












Int 66 571-579



2008 142-145






92 449-451

i

Acknowledgments









inspiration for me


cooperation










work

ii










iii





ACTN4 α-Actinin 4


Ang-I Angiotensin I









CsA Cyclosporine

DAG Diacylglyecerol








FS Frasier Syndrome





Hcy Homocysteine




Ig Immunoglobulin




iv










OD Optical Density



PLC Phospholipase C







SD Slit Diaphragm












v

Publications










vi

List of Tables

Table Title

Page


13


NPHS1 gene

65


NPHS2 gene

66


syndrome

68


69



81


NPHS1 gene

82


85


99




and controls

101



102


113



vii


and controls

116



patients

117




and controls

119



patients

120

viii

List of Figures

Figure Title

Page


3



podocyte

5



and PLCE1

8


10


15


protein

19



80


their locations

84


98


homocysteine

112


gel

114


gel

114

ix

SUMMARY

x

SUMMARY























xi























xii



children
















1


SYNDROME

2

11 THE KIDNEY





















3



4





















5




rictedpaperhtml)

6













115 PODOCYTE











7


















Baratt 1999)

8




9


122 DEFINITION















1985)

123 CLASSIFICATIONS





10



11




















2005)





12


















13






Autosomal

recessive

(AR)


Childhood MCDFSGS

Kestila et al

1998


Adulthood FSGSMCD

Boute et al

2000


Childhood DMSFSGS

Hinkes et al

2007


2006



Ozaltin et

al 2011

Autosomal

dominant

(AD)


al 2000


Childhood DMSFSGS

Mucha et al

2006


2007



2005


2010

14




GENE (NEPHRIN)








al 2004)










15


2002)

16
























17

























18






















19



20

























21




















BETA 2)




22





2000)
















23





















24
















ACTININ- 4)






25






















associated disease

26


TUMOR)























27



















ASSOCIATED PROTEIN)





28
























29

























30







FORMIN-2)

















31


FACTORS



















32






















2 = 1

33

16 REFERENCES




Gene 502 133-137










467-470



373



1214 83-98








24 349-354









34








878-883




Nature Genet 42 361


Pediatr 171 1151-1160




494




Cell 60 509-520











61R




35



Nephrol 22 849-858










Nephrology 4th














281 F860-868








Cell Genet 83 46-47




36




Cell 94 667-677


629-639






Med Life 2 53-59



Genet Med 8 63-75





1297












2044-2051




37



Edition The












Nephrol 24 269-278


11 101-110





















38
















Nephrol 19 365-371



Nephrol 9 87-93















2128




39
















1 575-582













6211-6216








40




























1300




1198-1203





41









21 3283-3286











R185-194











937-941







42



205-209







Nephrol 13 589-593







1653-1660



2 Nature 374 258-262





Nephrol 19 384-389






212-221






c139-146

43








Mol Genet 12 2379-2394



Nephrol 12 289-296





















184-213






44




Genet 10 1-8







739-744



24 1101-1110




1346





J Pathol 160 131-139














369-381

45















1148



F947- 956




















46



Pathol 159 2303-2308
















Int 66 571-579








2008 142-145




16085






47











273-280












664 84-90

48


49















AllegraTM










50





















51













experiment













52



) was


















(QIAGEN Germany)

53




















2microl




54



oC for 5















minutes






55














system (Bio Rad)










56

























57













Odds Ratio





Chi-square test




χ

2= sum (o-e)

2e




58

29 REFERENCES








Edition








96 12810-12815

59




60

31 INTRODUCTION












Yu et al 2005)












61
























62























63
























64




















2010)

65


NPHS1 gene


size (bp)

PCR conditions








































66













NPHS2 gene


size (bp)

PCR conditions

















67

33 RESULTS











MCD (27 cases)













68


syndrome



Males () 88 (607)

Females () 57 (393)






FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)


Outcome

ESRDg CRF

h 14 (96)


Expired 8 (55)



cIgM nephropathy





hchronic renal

failure

69


S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid



SRNS q- d

ESRD ndash eTx



CyA -









kACEI +

lARB

-


+ARB -


+ARB -

70






CyA





23 NS084 No 5 M C1q



CyA -






CyA -


+ARB -


71


CyA -





+ARB -


nCyP

-


CyA -






44 NS140 No 35 M - SDNS - -




+ARB -



CyA -

72







CyA -




CyA -



ARB CRF


+ARB -

62 NS167 Yes 9 M - - - -


CyA -





73


relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -



CyA -


Tac -



CyP -


CyP -







83 NS203 No 11 M - - - -



74



+ARB -



90 NS214 Yes 6 M FSGS - - -


CyP -





96 NS221 Yes 1 M - - - -





+ARB -


CyP -


CyA -


75


+ARB -


+ARB -




CyP -



+ARB -



Tac -






CyP -





76




CyA -


+ARB -


127 NS281 No 10 M SRNS - -



Tac -


CyA -


CyA -




CyA -


+ARB -


+ARB


+ARB -


77

139 NS303 Yes 3 M - SRNS - -



+ARB -

142 NS306 No 25 M SRNS - -



+ARB -


+ARB -


bIgM nephropathy

ccyclosporine


etransplantation

fminimal change


htacrolimus


jmembranous




ncyclophosphamide



q (-)

78

























79


FSGS





80


domains

81


Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228


to dCyA

Normal 15yrs 100

NS254


NS291


c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099




dcyclosporine

82



b cyclosporine







Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change


Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I


Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q


eARB



Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W


NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I



Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206


Tacrolimus

Normal 000




follow up

019

83


















84


85


Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q


Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130


eARB

Normal 10yrs 0998

NS278

M no Infantile


eARB

Normal 3yrs 100

NS288

M no Infantile


Tacrolimus

Normal 3yrs 0998

a






86

34 DISCUSSION























87







(Table- 35 and 36)


















88
























89



90

35 REFERENCES









445-451



























91









2128









1 575-582















1198-1203




1209-1217

92












Nephrol 13 589-593





























93




1148
















Int 66 571-579









2632

94



PAKISTANI CHILDREN

95

41 INTRODUCTION













1985)










96




















97

422 GENOTYPING








buffer (GoTaqreg











98


M






99







43 RESULTS







Steroid response

SRNS

N=105

SSNS

N=163



Family history 24 6

ESRD 12 No

Biopsy 105 No




Hematuria 0-3 No

100








=142) having an OR of 16 (95 CI =13-


















101




NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)


ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102




SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION























104
























2010)

105










these children

106

45 REFERENCES









883


629-639



Listy 109 298-301






555



Health 39 917-921






Nephrol 13 589-593





Nephrol 19 384-389

107







1346






732







New York 2006 241-243











20 1738-1743






108







109




CHILDREN

110

51 INTRODUCTION






















111




















112



113








SRNS

N=166

SSNS

N=152



Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING






53)

114









115




53 RESULTS












from HWE (plt005)








116




Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117











CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137






118





=001 and 039 pgt005)












54 DISCUSSION









119




Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)


CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120
















CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121






















122

55 REFERENCES











494






241-246




Med Life 2 53-59





5606-5611








289

123






2264









1123



1300





Nephrol 13 589-593







Congress







124









Biol Chem 276 46896-46904







96 12810-12815






1207-1221










92 449-451

125


126
























127

























128


















in South Asia







129
























130










al 2005 Mao et al 2007)












in these patients



131
























132

























133
























134










future







135

64 CONCLUSION












136

65 REFERENCES




Gene 502 133-137



1214 83-98








Nature Genet 42 361





2075-2084












61R



137






























Health 39 917-921










138




719



R185-194






























1148

139












Int 66 571-579



2008 142-145






92 449-451

ii










iii





ACTN4 α-Actinin 4


Ang-I Angiotensin I









CsA Cyclosporine

DAG Diacylglyecerol








FS Frasier Syndrome





Hcy Homocysteine




Ig Immunoglobulin




iv










OD Optical Density



PLC Phospholipase C







SD Slit Diaphragm












v

Publications










vi

List of Tables

Table Title

Page


13


NPHS1 gene

65


NPHS2 gene

66


syndrome

68


69



81


NPHS1 gene

82


85


99




and controls

101



102


113



vii


and controls

116



patients

117




and controls

119



patients

120

viii

List of Figures

Figure Title

Page


3



podocyte

5



and PLCE1

8


10


15


protein

19



80


their locations

84


98


homocysteine

112


gel

114


gel

114

ix

SUMMARY

x

SUMMARY























xi























xii



children
















1


SYNDROME

2

11 THE KIDNEY





















3



4





















5




rictedpaperhtml)

6













115 PODOCYTE











7


















Baratt 1999)

8




9


122 DEFINITION















1985)

123 CLASSIFICATIONS





10



11




















2005)





12


















13






Autosomal

recessive

(AR)


Childhood MCDFSGS

Kestila et al

1998


Adulthood FSGSMCD

Boute et al

2000


Childhood DMSFSGS

Hinkes et al

2007


2006



Ozaltin et

al 2011

Autosomal

dominant

(AD)


al 2000


Childhood DMSFSGS

Mucha et al

2006


2007



2005


2010

14




GENE (NEPHRIN)








al 2004)










15


2002)

16
























17

























18






















19



20

























21




















BETA 2)




22





2000)
















23





















24
















ACTININ- 4)






25






















associated disease

26


TUMOR)























27



















ASSOCIATED PROTEIN)





28
























29

























30







FORMIN-2)

















31


FACTORS



















32






















2 = 1

33

16 REFERENCES




Gene 502 133-137










467-470



373



1214 83-98








24 349-354









34








878-883




Nature Genet 42 361


Pediatr 171 1151-1160




494




Cell 60 509-520











61R




35



Nephrol 22 849-858










Nephrology 4th














281 F860-868








Cell Genet 83 46-47




36




Cell 94 667-677


629-639






Med Life 2 53-59



Genet Med 8 63-75





1297












2044-2051




37



Edition The












Nephrol 24 269-278


11 101-110





















38
















Nephrol 19 365-371



Nephrol 9 87-93















2128




39
















1 575-582













6211-6216








40




























1300




1198-1203





41









21 3283-3286











R185-194











937-941







42



205-209







Nephrol 13 589-593







1653-1660



2 Nature 374 258-262





Nephrol 19 384-389






212-221






c139-146

43








Mol Genet 12 2379-2394



Nephrol 12 289-296





















184-213






44




Genet 10 1-8







739-744



24 1101-1110




1346





J Pathol 160 131-139














369-381

45















1148



F947- 956




















46



Pathol 159 2303-2308
















Int 66 571-579








2008 142-145




16085






47











273-280












664 84-90

48


49















AllegraTM










50





















51













experiment













52



) was


















(QIAGEN Germany)

53




















2microl




54



oC for 5















minutes






55














system (Bio Rad)










56

























57













Odds Ratio





Chi-square test




χ

2= sum (o-e)

2e




58

29 REFERENCES








Edition








96 12810-12815

59




60

31 INTRODUCTION












Yu et al 2005)












61
























62























63
























64




















2010)

65


NPHS1 gene


size (bp)

PCR conditions








































66













NPHS2 gene


size (bp)

PCR conditions

















67

33 RESULTS











MCD (27 cases)













68


syndrome



Males () 88 (607)

Females () 57 (393)






FSGSa 48

MCDb 27

IgMNc 9

MesPGNd 9

MGNe 3

MCGNf 2

C1q nephropathy 1

Family history

Positive () 39 (27)


Outcome

ESRDg CRF

h 14 (96)


Expired 8 (55)



cIgM nephropathy





hchronic renal

failure

69


S

No Patient

ID Family

history Age of

onset Sex Renal

Biopsy Steroid



SRNS q- d

ESRD ndash eTx



CyA -









kACEI +

lARB

-


+ARB -


+ARB -

70






CyA





23 NS084 No 5 M C1q



CyA -






CyA -


+ARB -


71


CyA -





+ARB -


nCyP

-


CyA -






44 NS140 No 35 M - SDNS - -




+ARB -



CyA -

72







CyA -




CyA -



ARB CRF


+ARB -

62 NS167 Yes 9 M - - - -


CyA -





73


relapse -

69 NS181B Yes 9 M - SSNS - -


CyA -



CyA -


Tac -



CyP -


CyP -







83 NS203 No 11 M - - - -



74



+ARB -



90 NS214 Yes 6 M FSGS - - -


CyP -





96 NS221 Yes 1 M - - - -





+ARB -


CyP -


CyA -


75


+ARB -


+ARB -




CyP -



+ARB -



Tac -






CyP -





76




CyA -


+ARB -


127 NS281 No 10 M SRNS - -



Tac -


CyA -


CyA -




CyA -


+ARB -


+ARB


+ARB -


77

139 NS303 Yes 3 M - SRNS - -



+ARB -

142 NS306 No 25 M SRNS - -



+ARB -


+ARB -


bIgM nephropathy

ccyclosporine


etransplantation

fminimal change


htacrolimus


jmembranous




ncyclophosphamide



q (-)

78

























79


FSGS





80


domains

81


Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow up

Polyphen 2

scores

NS145

NS300

NS310

F

M

F

no

no

no

CNS

Infantile

CNS

FSGS

c3478C-T

c3478C-T

c3478C-T

pR1160X

pR1160X

pR1160X

Maintained on bACEI

Normal

Normal

Normal

25yrs

15yrs

6mo

NS228


to dCyA

Normal 15yrs 100

NS254


NS291


c2734G-A

pL237P

pA912T

Normal 1yr 100

035

NS301

NS309

M

yes

no

CNS

CNS

c2673dupCA

c2600G-A

pG867P

Normal

Normal

6mo

9mo

099




dcyclosporine

82



b cyclosporine







Patient

Sex Family

history

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change


Outcome

Polyphen

2 scores

NS015

M

yes

11

aMCD

c563A-T

pN188I


Normal

015

NS039

NS130

NS187

M

M

F

yes

no

no

5-10

5

4

MCD cFSGS

MCD

c1223G-A

c1223G-A

c1223G-A

pR408Q

pR408Q

pR408Q


eARB



Normal

Normal

Normal

098

NS141

M No 7

_ c766C-T pR256W


NS161

NS104

M

M

yes

no

4

11

FSGS fMesPGN

c1822G-A

c1822G-A

pV608I

pV608I



Normal gESRD

030

NS165

NS223

M

M

no

no

7

9

MCD

MCD

c565G-A

c565G-A

pE189K

pE189K


Normal

Normal

011

NS206


Tacrolimus

Normal 000




follow up

019

83


















84


85


Patient

Sex Family

History

Age at

Onset

(yrs)

Biopsy Nucleotide

Change

Amino

Acid

Change

Response to

Therapy

Renal

Outcome

Time to

follow

up

Polyphen 2

scores

NS125

NS211

F

M

no

no

3

25

aFSGS

cMCD

c755G-A

c755G-A

pR229Q

pR229Q


Tacrolimus

bESRD

Normal

11yrs

15yr

0673

NS130


eARB

Normal 10yrs 0998

NS278

M no Infantile


eARB

Normal 3yrs 100

NS288

M no Infantile


Tacrolimus

Normal 3yrs 0998

a






86

34 DISCUSSION























87







(Table- 35 and 36)


















88
























89



90

35 REFERENCES









445-451



























91









2128









1 575-582















1198-1203




1209-1217

92












Nephrol 13 589-593





























93




1148
















Int 66 571-579









2632

94



PAKISTANI CHILDREN

95

41 INTRODUCTION













1985)










96




















97

422 GENOTYPING








buffer (GoTaqreg











98


M






99







43 RESULTS







Steroid response

SRNS

N=105

SSNS

N=163



Family history 24 6

ESRD 12 No

Biopsy 105 No




Hematuria 0-3 No

100








=142) having an OR of 16 (95 CI =13-


















101




NS patients

N=268

Controls

N=223

Total

N=491

p-value

ACE genotype

II 82 (306) 9 (4) 91

ID 128 (478) 171 (767) 299

DD 58 (216) 43 (193) 101

ACE allele

I 292 (545) 189 (42) 481 lt0001

D 244 (455) 257 (58) 501

χ2=142 df=1 OR=16 (95 CI=12-20)


ACE genotype

II 82 (59) 9 (17) 91 lt0001

DD 58 (41) 43 (83) 101 OR=67 (30-149)

Total 140 52 192

ID 128 (69) 171 (80) 299 0011

DD 58 (31) 43 (20) 101 OR=05 (03-08)

Total 186 214 400

IIID 210 (78) 180 (81) 390

DD 58 (22) 43 (19) 101 gt005

Total 268 223 491

102




SRNS 35 (33) 47 (45) 23 (22) 105 pgt005

SSNS 47 (29) 82 (50) 34 (21) 163

FSGS 14 (29) 20 (42) 14 (29) 48 pgt005

Non-FSGS 21 (37) 27 (47) 9 (16) 57

MCD 8 (32) 14 (56) 3 (12) 25 pgt005

Non-MCD 27 (34) 33 (41) 20 (25) 80

103

44 DISCUSSION























104
























2010)

105










these children

106

45 REFERENCES









883


629-639



Listy 109 298-301






555



Health 39 917-921






Nephrol 13 589-593





Nephrol 19 384-389

107







1346






732







New York 2006 241-243











20 1738-1743






108







109




CHILDREN

110

51 INTRODUCTION






















111




















112



113








SRNS

N=166

SSNS

N=152



Family history 42 7

ESRD 12 No

Biopsy 114 No




Hematuria 0-36 No

521 GENOTYPING






53)

114









115




53 RESULTS












from HWE (plt005)








116




Genotypes

and Alleles

C667T

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 236 (74) 140 (70) 376

CT 70 (22) 52 (26) 122

TT 12 (4) 8 (4) 20

MTHFR C667T allele

C 542 (85) 332 (83) 874 gt005

T 94 (15) 68 (17) 162

χ2=0917 df=1 OR=1181 (95 CI=0840-166)


CC 236 (74) 140 (70) 376 gt005

TT 12 (4) 8 (4) 20 OR=1124

Total 248 148 396

CT 70 (22) 52 (26) 122 gt005

TT 12 (4) 8 (4) 20 OR=0897

Total 82 60 142

CCCT 306 (96) 192 (96) 498 gt005

TT 12 (4) 8 (4) 20 OR=1063

Total 318 200 518

117











CC

genotype

CT

genotype

TT

genoty

pe

Total P value

SRNS 124 (75) 37 (22) 5 (3) 166 pgt005

SSNS 112 (74)

33 (22) 7 (4) 152

FSGS 42 (79) 9 (17) 2 (4) 53 pgt005

Non-

FSGS 82 (73) 27 (24) 3 (3) 112

MCD 19 (63) 11 (37) 0 (0) 30 pgt005

Non-

MCD 105 (77) 27 (20) 5 (3) 137






118





=001 and 039 pgt005)












54 DISCUSSION









119




Genotypes and

Alleles A1298C

NS patients

N=318

Controls

N=200

Total

N=518 p-value


CC 52 (16) 37 (185) 89

CA 152 (48) 93 (465) 245

AA 114 (36) 70 (35) 184

MTHFR A1298C allele

C 256 (40) 167 (42) 423 gt005

A 380 (60) 233 (58) 613

χ2=0191 df=1 OR=0945 (95 CI=0733-1218)


CC 52 (16) 37 (185) 89 gt005

AA 114 (36) 70 (35) 184 OR=0863

Total 166 107 273

CA 152 (48) 93 (465) 245 gt005

AA 114 (36) 70 (35) 184 OR=1004

Total 266 163 429

CCCA 204 (64) 130 (65) 334 gt005

AA 114 (36) 70 (35) 184 OR=0964

Total 318 200 518

120
















CC

genotype

CA

genotype

AA

genotype

Total P

value

SRNS 32(193) 72(434) 62(373) 166 pgt005

SSNS 23(15) 77(51) 52(34)

152

FSGS 7(135) 22(423) 23(442) 52 pgt005

Non-

FSGS

22(19) 50(45) 40(36) 112

MCD 6(19) 17(53) 9(28) 32 pgt005

Non-

MCD

25(18) 57(41) 56(41) 138

121






















122

55 REFERENCES











494






241-246




Med Life 2 53-59





5606-5611








289

123






2264









1123



1300





Nephrol 13 589-593







Congress







124









Biol Chem 276 46896-46904







96 12810-12815






1207-1221










92 449-451

125


126
























127

























128


















in South Asia







129
























130










al 2005 Mao et al 2007)












in these patients



131
























132

























133
























134










future







135

64 CONCLUSION












136

65 REFERENCES




Gene 502 133-137



1214 83-98








Nature Genet 42 361





2075-2084












61R



137






























Health 39 917-921










138




719



R185-194






























1148

139












Int 66 571-579



2008 142-145






92 449-451

the genetics of nephrotic syndrome in pakistani...

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