genotypic phenotypic correlations€¦ · dr. r saxena professor & head, department of...

Thalassemia Intermedia in India:

genotypic phenotypic correlations

Dr. R Saxena

Professor & Head, Department of Haematology

All India Institute of Medical Sciences, New Delhi, India

Beta Thalassemia -Distribution

Thalassemia Intermedia

A state of thalassemia syndrome:

Manifests later in life (> 2 years)

Less severe than thalassemia major

May require BT occasionally under stress

Patient may develop complications of anemia

Clinical phenotype Number (%)

Thalassemia major 293(76.3)

Thalassemia intermedia 91 (23.7)

Prevalence of Thalassemia Intermedia:

AIIMS (n=384)

Genotypes of Thalassemia Intermedia in

India (n=251)

• Clinico-Haematological Profile of Thalassemia Intermedia Patients. S Tyagi1, M. Kabra, N Tandon, R Saxena, H. P. Pati and V. P.

Choudhry Int J Hum Genet, 3(4): 251-258 (2003)

• Molecular Characterization Of Thalassemia Intermedia In in India . I. Panigrahi, S. Agarwal M, Pradhan, DR Choudhry, VP

Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.

Genotypes of TI

Beta Thalassemia

HbE Beta Thal

HbS Beta Thal

HbD Pb Beta Thal

HbE Lepore

HPFH Beta

Delta Beta Beta Thal

Phenotype in Thalassemia Intermedia

Phenotype in Beta Thalassemia

Depends on Chain imbalance of Beta and Alpha globin

chains

Excess alpha chain cause manifestations of Beta

Thalassaemia

Phenotypic heterogeneity within the various groups

Genotype Phenotype Correlation

What causes heterogeneity of phenotype of

thalassemia intermedia?

Primary Modulators: Mutations which affect β globin

gene

Secondary Modulators: Genetic Alteration that affect α

and γ globin genes

Tertiary Modulators: Genetic Alterations that don’t

affect globin gene directly

Taher A. Blood Cells Mol Dis. 2006;37:12.

Thalassemia intermedia: Beta thalassemia

Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S Agarwal, M. Pradhan, DR Choudhry, VP

Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.

0

20

40

60

80

Beta Thalassemia

75.9

24.1 Homozygous

Heterozygous

Pre

val

ence

(%

)

Types of beta thalassemia

Beta Thalassaemia Beta Zero Beta Plus

Beta globin Chain Synthesis Absent Partial

HbA Absent 10-30%

HbF 95-98% 70-90%

HbA2 2-5% 2-5%

phenotype severe mild

S Europe

Cd 39 C/T

IVS1-110 G/A

IVS1-6 T/C

IVS1-1 G/A

W. Africa

-29 A/G

-88 C/T

Others

S.E. Asia

Cd 41/42

IVS 1-5 G/C

Cd 17 A/T

-28 A/G

S Asia

IVS1-5 G/C

IVS1-1 G/T

Cd 8/9

Cd41/42

619 bp del

Most common beta-thalassemia

mutations in different countries

Beta thalassemia mutations in TI in India

Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S Agarwal, M. Pradhan, DR Choudhry, VP

Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.

Mutations in beta thalassemia

Severe phenotype

IVS-I-5 (G->T) beta+/beta0 (severe)

IVS-I-1 (G->T); beta0

Codons 8/9 (+G); beta0

Codons 41/42 (-TTCT); TT beta0

619 bp deletion beta0

Mild Phenotype:

Capsite +1 ( A-C): 5 (6.7%)

-88 (C-T): 5 (6.7%) Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S Agarwal, M. Pradhan, DR Choudhry, VP

Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.

Hb E

Epidemiology

Hemoglobin E

disease

HbE syndromes

M 1 2 3

1,2 are Hom. 3 is Het.

330 bp

310 bp

Status Occurrence Frequency phenotype

Heterozygous E 52/ 339 15.33% asymptomatic

Homozygous E 41/339 12.1% asymptomatic

E beta 246 /339 72.56 TI

HbE Mutation (PCR-RFLP) variant Hb caused by Glutamic acid to Lysine (G-A) at

26th position of β globin chain

E beta thalassemia:Phenotypes

Clinical Characteristics Non Severe Severe

Age of presentation >3yrs ≤3yrs

Transfusion Requirements None or < 4units/year, frequent

Age of transfusion started at least after three years of age <3yrs

Spleen Size ≤3cm 3—10 cm

Hb >5g/dl 3-5g/dl

Growth & development >5percentile 3-5percentile

β mutations in HbE Beta thalassemia

Genotype Total (240) Severe (132) Non severe (108) P value

β globin gene mutations

IVS1–5 (G C) 102(42.5%) 53(40.1%) 49(45.3%) NS

CD 41/42 (-CTTT) 35 (14.6%) 18(13.6%) 17(15.7%) NS

CD 8/9 (+G) 30 912.5%) 20(15.1%) 10(9.2%) NS

CD 30(G-C) 18 (7.5%) 11(8.3%) 7(6.5%) NS

CD 15 (G-A) 24 (10%) 12(9.1%) 12(11.1%) NS

Cap site +1(A-C) 4(1.7%) 0 4(3.7%) 0.026

619 bp del. 9 (3.75%) 6(4.5%) 3(2.8%) NS

-88(C-T) 3(1.25%) 0 3(2.8%) 0.05

CD 16(-C) 2(0.83%) 2(1.5%) NS

IVS1-1 (G-T) 8(3.3%) 6(4.5%) 2(1.9%) NS

CD 39(C-T) 1(0.42%) 1(.76%) -

IVS-2 position 1(G-A) 1 (0.42% 1(.76) -

IVSII-613(C-T) 1 (0.42%) 1(.76%) -

initiation codon (ATG-ACG) 2 (0.84%) 1(.76%) 1(.92%) NS

Total 240 132 108

HS5 HS4 HS3 HS2 HS1

ε Gγ Aγ θβ δ β 5 4 3 2 1

Promoter Exon 1 IVSI Exon 2 IVSII Exon 3

3’ enhancer region 5’ silencer region

(AT)x(T)y

LCR Locus control region in beta globin gene

Locus control region (LCR)is located upstream of the β globin complex and regulates beta globin

gene.

Polymorphisms in Beta LCR in India (by

sequencing)

Region Status in our Population

HS1 : (CA)x(TA)y Reported Alteration Found

HS2 : (AT)x(N)y(AT)z Novel Alteration found

HS3 : sequencing , no repeats No Alteration found

HS4 : TGGGG(A/G)CCCCA Reported Alteration found

HS5 : sequencing, no repeats No Alteration found

5’Silencer Region: (AT)x (T)y Reported Alteration Found

3’Enhancer Region: sequencing, no repeats No Alteration found

LCR genotypes and phenotypes in Hb E thalassemia Gene variation Non severe (108) Severe (132) P value

HS1 (CA)x(TA)y

(CA)12(TA)6(Wild)

(CA)10(TA)8

(CA)12(TA)7

(CA)11(TA)7

41(37.9%)

24(22.2%)

22(20.3%)

21(19.4%)

85(64.3%)

20(15.1%)

17(12.8%)

10(7.5%)

.001

NS

NS

.01

HS2 (AT)xNy(TA)z

(AT)9N12(AT)10

(AT)10N12(AT)11(Wild)

(AT)9N12(AT)11

(AT)8N12(AT)11

31(28.7%)

30(27.7%)

31(28.7%)

16(14.8%)

23(17.4%)

45(34%)

47(35.6%)

17(12.9%)

.025

NS

NS

NS

5’ Silencer region (AT)x(T)y

(AT)9(T)5

(AT)8(T)4

(AT)8(T)7

(AT)7(T)7(Wild)

(AT)8(T)5

(AT)8(T)6

48(44.4%)

6(5.5%)

12(1.8%)

3(2.8%)

32(29.6%)

7(6.5%)

51(38.6%)

6(4.5%)

21(15.9%)

11(8.3%)

32(24.2%)

11(8.3%)

NS

NS

.001

.06

NS

NS

HS4 (TGGGG(A/G)CCCCA)

A(Wild)

G

79(73.1%)

29(19.4%)

109(82.5%)

23(17.4%)

.001

NS

Hb S

Epidemiology:

Hemoglobin S

disease

S Beta thalassemia

AIIMS

(n=349)

Phenotype West

SA 99 (28.3%) mild 23%

SS 175 (50.2%) severe 65.5%

Sβ

Sβ+

Sβ0

75 (21.5%)

TI 11.5% 29 (39%)

46(61%)

Comparative clinical parameters of HbSβ0-thalassemia and

HbSβ+-thalassemia patients(N=75)

Beta Thalassemia mutations in HbSβ patients (n= 75)

HbSβ0 : Common mut.

HbSβ+ : Cap site, -88 CT

0% 5%

10% 15% 20% 25% 30%

24.1%

13.8% 10.3%

13.0% 8.7% 6.9%

17.2%

26.1%

19.6% 17.4% 17.2%

10.3%

6.5% 8.7%

Hb… Hb…

Secondary modifiers

Alpha Globin Gene defects:

Alpha gene deletions and triplications

Gamma globin gene defects:

Xmn-1 polymorphisms

HPFH mutation

Delta Beta thalassemia

Alpha Thalassemia Prevalence in India:1% to 18%

Genetic Alterations

Deletions

Single Gene Deletions: Alpha 3.7 Kb Deletion (α3.7 ) ,

Alpha 4.2 Kb Deletion (α4.2 )

Double Gene Deletions: South African (--SA ) ,

South East Asian (--SEA) Point Mutations

Constant Spring Mutation (ααCS )

Hb Pakse Mutation (ααPS )

Triplication : (ααα3.7)

A : Mutant tube 1.8Kb

B : Normal tube 1.8 Kb

1 & 3 : Homozygous Mutant

2 & 4 : Heterozygous

t

1 & 3 : Homozygous Mutant

2 & 4 : Heterozygous

Gap PCR Alpha thalassemia 3.7 del

Alpha Mutations in Beta thalassemia

presenting as TI

0%

20%

40%

60%

80%

Beta Homozygous

Beta Heterozygous

36%

4% 0

60%

Alpha Deletion

Alpha Triplication

Alpha del:α3.7 =90% α4.2 =5% --SA =5%

--SEA not seen in India

Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S. Agarwal, M. Pradhan, DR

Choudhry, VP Choudhyry, R. Saxena. Haematologica 2006;91(9):12790-1280.

Alpha mutations in E beta thalassemia Mutations Total (240) Severe (132) Non severe (108) P value

Deletions 19 (7.9%) 10 (7.5%) 9 (8.3%) NS

Single Gene Deletion (α3.7)

Heterozygous

Homozygous

18 (7.4%)

14 (5.8%)

4 (1.6%)

10 (7.5%)

10 (7.5%)

0 (0%)

8 (7.4%)

4 (3.7%)

4 (3.7%)

NS

NS

0.03

Double Gene Deletions (--SA ) 1 (0.4%) 0 (0%) 1 (0.9%) NS

Point Mutations 9 (3.8%) 0 (0%) 9(8.3%) 0.02

ααCS 6 (2.1%) 0(0%) 6(4.6%) 0.03

ααPS 3 (1.3%) 0(0%) 3(2.8%) 0.04

Triplication 11(4.6%) 8(6.1%) 3(2.8%) 0.002

Poly A (AATAAA-AATA--)^ 2 (0.8%) 0(0%) 2(1.8%) NS

Total 41 (17.1%) 18 (16.7%) 23(21.3%) NS

α4.2 ; --SEA not present

Effect of alpha gene numbers on Phenotype of HbS-β (n=75)

0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

100%

Sb without alpha Del

Sb with alpha Del

Sb with Alpha Trip

Alpha 3.7 del had milder phenotype

Alpha triplication had severe phenotype

Phenotypic effect of α-globin gene numbers on Indian sickle β-thalassemia patients. Pandey SK, Pandey S, Ranjan R, Shah V,

Mishra RM, Sharma M, Saxena R. J Clin Lab Anal. 2014, 28(2):110-3.

α3.7 =89% ; α4.2 =11%

--SA ; --SEA not present

Hb D

Epidemiology

Hemoglobin D

Punjab

disease

SD hemoglobinopathy

Homozygous HbD Punjab: mild hemolytic anemia

Heterozyg D: clinically silent

Double hetero D and S:

Pakistan, Iran, UAE and Mexico: clinical presentation of Hb SD-disease

similar to severe form of sickle cell anemia

AIIMS study on 15 pts of HbD-S showed the severity of disease with

thalassemia intermedia.

S.Pandey , R.Ranjan , T.Seth , V.Shah , R.M.Mishra , S.W.Pandey , R.Saxena Clinical-hematological profile of

Indian hemoglobin sickle cell D Punjab patients RRBS, 6(2), 2012 [41-43]

Effect of Alpha deletions in HbSD

N=10 N=5

S.Pandey , R.Ranjan , T.Seth , V.Shah , R.M.Mishra , S.W.Pandey , R.Saxena Clinical-hematological profile of

Indian hemoglobin sickle cell D Punjab patients RRBS, 6(2), 2012 [41-43]

α3.7 =100% α4.2 ; --SA ; --SEA not present

Xmn-1 polymorphism Xmn-1 polymorphism :C-T variation at position -158 upstream of G gamma gene which is detected by restriction enzyme Xmn-1

650 bp

450 bp

200 bp

1 2 3 4 5 6 25bp 7 8 9 10 11 12

PCR RFLP: Lane 1,4,5,8,9,10,11 heterozygous; 2,3,6,12 homozygous & lane 7 is normal

Xmn-1 (+/+) Xmn-1 (+/-) Xmn-1 (-/-) P value

HbF 36.2+4.7 32.1+5.2 17.6+4.8 <0.001

JK Science Vol. 8 No. 3, July-September 2006

HPFH and Delta Beta deletions based on origin

HPFH types

HPFH-1 – African

HPFH-2 – Ghanaian

HPFH-3 – Asian Indian

HPFH-4 – Southern Italian

HPFH-5 – Italian

HPFH-6 – Vietnamese

HPFH-7 – Kenyan

SEA-HPFH – Southeast Asian

Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by

enzymatic amplification JE Craig, RA Barnetson, J Prior, JL Raven and SL Thein Blood 1994 83:1673-1682

DELTA BETA Types

Spanish (δβ)o

Sicilian (δβ)o

Chinese (δβ)o

Asian Indian Inv. Deletion Gγ(Aγδβ)0

Turkish (δβ)o

Japanese (δβ)o

Black (δβ)o

HbE beta thalassemia: Prevalence of HbF modifiers

Genotype Total Non severe (108) Severe (132) P value

#c.211 C-T(XmnI) +/+ 11(4.6%) 5(4.6%) 6(4.7%) NS

#c.211 C-T (XmnI)+/- 94(39.2%) 37(34.2%) 57(43%) NS

HPFH-3 (HPFH) 6(2.5%) 6(5.6%) 0(0%) <.001

Asian Indian Inv/Del

Gγ(Aγδβ)0 [delta beta)

2(.83%) 2(1.9%) 0(0%) NS

More than one modulator 7(2.9%) 7(6.5%) 0(0%) <.001

Negative for modulator 121(50.4%) 54(50%) 67(50.7%) NS

β-Globin gene cluster polymorphisms are strongly associated with severity of HbE/β0-thalassemia. R Saxena et al Clin Genet 2007: 72:

497–505

*2 Asian Indian Inv/Del Gγ(Aγδβ)0 had +/+, 3 HPFH-3 had +/-, 2 HPFH3 had +/+ Xmn-I pol.

Note: A study from Thialand in 207 mild and 307 severe patients concluded that T allele of XmnI pol. is more likely

associated with mild disease.

Effect of Xmn-1 on Phenotype of HbS beta(n=75)

Modulating Effect of the −158 Gγ (C→T) Xmn1 Polymorphism in Indian Sickle Cell Patients. S. Pandey, Sweta

Pandey, Rahasya MM and R Saxena. Mediterr J Hematol Infect Dis. 2012; 4(1): e2012001.

Tertiary modifiers a: HFE mutations Iron Overload

C282 Y

H63D

S65C

b: Uridine diphosphoglucuronate glucuronosyltransferase (UGT)

polymorphisms Hyperbilirubinemia

TA7TAA

TA8TAA

211(G-A)

1456 (T-G)

1091(C-T)

686(C-A)

Iron Overload in Thalassemia Intermedia

A major problem

Does not correlate with number of BT

Role of modifier genes speculated

HFE gene: commonest gene implicated:HFE gene protein binds β2-

microglobulin and decreases affinity of TFR receptor

Transferrin receptor gene

Tapasree Goswami, and Nancy C. Andrews J. Biol. Chem. 2006;281:28494-28498

HFE mechanism

Thal Intermedia

Prevalence of HFE mutation H-63D (AIIMS)

0

2

4

6

8

10

12

14

16

18

%

HFE mutation H63D predicts risk of iron over load in thalassemia intermedia irrespective of blood transfusions. Sharma V, Panigrahi

I, Dutta P, Tyagi S, Choudhry VP, Saxena R. IJPM 2007 Jan;50(1):82-5

Thalassemia Intermedia: HFE and Ferritin

0

10

20

30

40

50

60

70

80

90

100

HFE +VE HFE-VE

<500 ng/ml

>500 ng/ml%

Mean BT/ Pt: 3.83 4.52

Mean Age (yrs): 11.3 10.27 HFE mutation H63D predicts risk of iron over load in thalassemia intermedia irrespective of blood transfusions. Sharma V, Panigrahi

I, Dutta P, Tyagi S, Choudhry VP, Saxena R. IJPM 2007 Jan;50(1):82-5

Genotype No. of patients with >1000ng/ml/Total no. of patients

H63D/H63D 16/18(88.8%)

wt/wt 65/189(34.4%)

Incidence of higher ferritin levels due to H63D

mutation

Thalassemia Intermedia: Jaundice

homozygous (n=76) Heterozygous (n=17)

Pallor 69 (90.8%) 17 (100%)

Fatigue 35 (46%) 12 (70.5%)

Jaundice 32 (42.1%) 12 (70.5%)

Failure to gain wt. 16 (21.1%) 05(29.4%)

Spleen NP < 6cm 6 cm

05 (6.6%) 52 (68.4%) 19 (25%)

02 (11.8%) 07 (41.2%) 08 (47%)

Liver NP < 6 cm 6 cm

05 (6.6%) 62 (81.5%) 09 (11.8%)

01 (5.9%) 15 (88.2%) 01 (5.9%)

Molecular Characterization Of Thalassemia Intermedia In Indians. I. Panigrahi, S Agarwal, M. Pradhan, DR Choudhry, VP

Choudhry, R Saxena. Haematologica 2006 91(9):1279-1280.

UGT (Uridine Diphosphate Glucuronosyl

Transferase-1) Polymorphism

Gene for UDP-glucuronosyltransferase-1 (UGT1*1):

enzyme responsible for hepatic glucuronidation of

bilirubin

The increased level of bilirubin has been related to a

polymorphism of the promoter of the UGT1*1 gene

Frequency of UGT polymorphisms in Patients

with E beta & controls

Genotype Patients Controls P value

A(TA)6TAA/A(TA)6TAA 42(17.5%) 61(61%) .001

A(TA)6TAA/A(TA)7TAA 55(22.9%) 20(20%) NS

A(TA)7TAA/A(TA)7TAA 23(9.5%) 1(1%) .001

A(TA)6TAA/A(TA)8TAA 4(1.6%) 0(0%) NS

A(TA)7TAA/211(G-A) 44(18.3%) 0(0%) .001

A(TA)6TAA/211(G-A) 43(17.9%) 13(13%) NS

211(G-A)/211(G-A) 15(6.3%) 0(0%) .001

Genotype T. Bilirubin Ind. Bilirubin

(TA)6/(TA)6 3.6±1.5 2.5±1.5

(TA)6/(TA)7 3.7±1.1 2.5±1.0

(TA)6/(TA)8 4.5±1.8 3.4±1.7

211(G-A)/(TA)6 3.6±1.0 2.6±1.0

(TA)7/(TA)7* 6.1±1.6 5.3±1.7

211(G-A)/211(G-A)* 6±1.1 5.1±1.0

211(G-A)/(TA)7* 8.0±2.4 7.5±2.3

Effect of UGT1A1 pol. on serum bilirubin levels

in HbE/β thalasssemia patients

*P value = <.05

Hb E/beta-thalassaemia: a common & clinically diverse disorder. Nancy F. Olivieri et al Indian J Med

Res 134, October 2011, pp 522-531

Summary Coexistence of hemoglobinopathy with beta thalassemia gene

makes the phenotype :

severer than the homozygous hemoglobinopathy

milder than thalassemia major

Thalassemia intermedia itself shows marked phenotypic variability:

Severity is increased by:

5 common beta thal mutations: IVS1-5,IVS1-1, Cd8/9, Cd41/42,619bp

del

Alpha triplications: ααα3.7

Beta LCR Polymorphisms: 5’ Silencer –

(AT)8(T)7

(AT)7(T)7

HS4- A Allele

Summary(contd.)

Mild phenotype by coexistence of :

Alpha deletions: -α 3.7, -α 4.2, --SA

Xmn-1 polymorphism

Beta Mutations: Cap+1 A/C; -88 C/T

HPFH: HPFH-3

Beta LCR Polymorphism: HS1- (CA)11(TA)7

HS2-(AT)9N12(AT)10

Summary(contd.)

Iron overload is high in HFE gene mutation (H63D)

Chance of occurance of jaundice is

higher in presence following polymorphisms;

A(TA)7TAA/A(TA)7TAA

A(TA)7TAA/211(G-A)

211(G-A)/211(G-A)

It is lower in following polymorphism

A(TA)6TAA/A(TA)6TAA

Acknowledgements

• Department of Science and Technology

• Indian Council of Medical Research