department of genetics hazara university mansehra …

SPECTRUM OF GENE MUTATIONS IN THE PATIENTS

WITH NON SYNDROMIC HEARING LOSS RESIDING IN

KHYBER PAKHTUNKHWA

MUHAMMAD ISMAIL KHAN

DEPARTMENT OF GENETICS HAZARA UNIVERSITY MANSEHRA

2019


WITH NON SYNDROMIC HEARING LOSS RESIDING IN

KHYBER PAKHTUNKHWA

This research study has been conducted and reported as partial fulfillment of

the requirements of PhD degree in Genetics awarded by Hazara University

Masnsehra, Pakistan

SUBMITTED BY MUHAMMAD ISMAIL KHAN

PhD Scholar

RESEARCH SUPERVISOR PROF. HABIB AHMAD PhD (TI)

Vice Chancellor Islamia College University Peshawar

CO-SUPERVISOR DR. MUHAMMAD SHAHID NADEEM

Assistant Professor

DEPARTMENT OF GENETICS

HAZARA UNIVERSITY MANSEHRA 2019

HAZARA UNIVERSITY MANSEHRA

DEPARTMENT OF GENETICS


WITH NON-SYNDROMIC HEARING LOSS RESIDING IN

KHYBER PAKHTUNKHWA

BY

MUHAMMAD ISMAIL KHAN

This research study has been conducted and reported as partial

fulfillment of the requirements of PhD degree in Genetics awarded

by Hazara University Mansehra, Pakistan

The Monday 19, February 2018

HAZARA UNIVERSITY, MANSEHRA

APPROVAL SHEET OF THE MANUSCRIPT

PHD THESIS SUBMITTED BY

Name Muhammad Ismail Khan

Fathers Name Shah Hosh Khan

Date of Birth 01-04-1979

Postal Address Department of Genetics, Hazara University Garden

Campus, Mansehra

Permanent Address Village, Tehsil and P/O Khwaza Khela Mohallah Barkalay District Swat

Telephone: 03459991077 Residence: 0946-745508

Email: [email protected]

PhD Thesis title Spectrum of gene mutations in the Patients with non-syndromic hearing loss

residing in the Khyber Pakhtunkhwa

Language in which the thesis has been written English

APPROVED BY

1. Prof. Dr. Habib Ahmad TI …………………….

(Supervisor)

2. Dr. M. Shahid Nadeem …………………….

(Co-supervisor)

RECOMMENDED BY

1. Prof. Dr. Manzoor Hussain …………………….

Dean Faculty of Sciences

2. Prof. Dr. Habib Ahmad TI …………………….

Department of Genetics/ Supervisor

3. Dr. Hakim Khan …………………….

Associate Professor/Chairman

Department of Genetics

4. Dr. Inamullah …………………….

Associate Professor


5. Dr Khushi Muhammad ……………………

Assistant Professor


Author’s Declaration

I Muhammad Ismail Khan here by state that my Ph.D thesis titled “Spectrum

of gene mutations in the patients with Non-Syndromic Hearing Loss residing

in Khyber Pakhtunkhwa” is my own work and has not been submitted

previously by me for taking any degree from this University (Hazara

University Mansehra) or anywhere else in the country/world.

At any time if my statement is found to be incorrect even after my graduation,

the University has the right to withdraw my degree.

Muhammad Ismail Khan

February 19, 2018

Plagiarism Undertaking

I solemnly declare that research work presented in the thesis titled “Spectrum

of gene mutations in the patients with Non-Syndromic Hearing Loss residing

in Khyber Pakhtunkhwa” is solely my research work with no significant

contribution from any other person. Small contribution/help where taken has

been duly acknowledged and that complete thesis has been written by me.

I understand the zero tolerance policy of the HEC and (Hazara University,

Mansehra) towards plagiarism. Therefore I as an author of the above titled

thesis declare that no portion of my thesis has been plagiarized and any

material used as reference is properly referred/cited.

I undertake that if I found guilty of any formal plagiarism in the above titled

thesis even after award of Ph.D degree, the University reserves the rights to

withdraw/revoke my Ph.D degree and that HEC and the University has the

right to publish my name on the HEC/University Website on which names of

students are placed who submitted plagiarized thesis.

Student/Author Signature: __________________

Name: Muhammad Ismail Khan

Dedications

This thesis work is dedicated to my uncles Bakhtmand Khan,

Afareen Khan and my beloved father Shah Hosh Khan,

the strongest people, I ever seen in my life

i

CONTENTS

List of Tables………………………………………………….…………………….iii

List of Figures………………………………………………………………………v

List of abbreviations………………..…………………………….………………viii

Acknowledgement…………………………………………………………………..x

Abstract……………………………………………………………………………..xii

Chapter 1 .................................................................................................................... 1

INTRODUCTION.................................................................................................... 1

1.1. Background Information ............................................................................... 1

1.2. Genetic Epidemiology of Deafness .............................................................. 4

1.2.1. The Human ear ........................................................................................ 6

1.2.2. Types of hearing loss ............................................................................... 7

1.2.3. Genes involved in hearing loss ............................................................ 10

1.2.4.1. GJB2 Gene ............................................................................................ 15

1.2.4.2. GJB6 Gene ............................................................................................ 18

1.2.4.3. GJB3 Gene ............................................................................................ 19

1.3. Mitochondrial DNA ..................................................................................... 19

1.3.1. The mitochondrial genetics .................................................................. 19

1.3.2. Molecular mechanism of mtDNA mutations causing deafness: .... 21

1.3.3. MTRNR1.................................................................................................. 22

1.3.4. MTRNR2.................................................................................................. 24

1.3.5. MT-TV Gene ........................................................................................... 24

1.3.6. Other mitochondrial genes involved in deafness ............................. 25

MATERIALS AND METHODS.......................................................................... 26

2.1. Sample collection .......................................................................................... 26

2.2. DNA extraction ............................................................................................. 27

2.3. Gel electrophoresis ....................................................................................... 27

2.4. Spectophotometry ........................................................................................ 27

2.5. PCR amplification ......................................................................................... 28

2.6. Gel Electrophoresis of PCR Product .......................................................... 29

ii

2.7. Protocol for gene clean ................................................................................. 31

2.8. DNA Sequencing and sequence analyses ................................................. 31

2.9. Condition for PCR sequencing ................................................................... 32

Chapter 3 .................................................................................................................. 33

RESULTS ................................................................................................................. 33

3.1. Deaf patients in the KP ................................................................................ 33

3.2. Collection of samples ................................................................................... 38

3.3. Analysis of GJB2 ........................................................................................... 39

3.4. Analysis of GJB6 ........................................................................................... 49

3.5. Analysis of Mitochondrial Genes ............................................................... 55

Chapter 4 .................................................................................................................. 63

DISCUSSION ......................................................................................................... 63

Appendix-1 .............................................................................................................. 94

Appendix-2 ............................................................................................................ 112

Appendix-3……………………………………………………………………….127

Appendix-4 ............................................................................................................ 170

Appendix-5 ............................................................................................................ 184

Appendix-6 ............................................................................................................ 190

Appendix-7 ............................................................................................................ 193

Appendix-8 ............................................................................................................ 208

iii

LIST OF TABLES

TABLE DETAILS PAGE

Table. 1. Table.1. Gender wise breakup of people of Khyber

Pakhtunkhwa (Anonymous, 2017).

1

Table.2. Prevalence of gene mutations with non-syndromic hearing

loss in developed countries

12

Table .3. Primers used for the GJB2, GJB6 and mitochondrial genes 28

Table. 4. Reagents and their volumes used for PCR amplfication 29

Table .5. Reagents for Sanger sequencing 31

Table .6. District and gender/age wise census of deaf people in Khyber

Pakhtunkhwa

35

Table .7.1(a). Indels mutations reported in GJB2 gene in deaf patients

Khyber Pakhtunkhwa (KP)

40

Table. 7.1(b) Indels mutations reported in GJB2 gene gender/age wise in

deaf patients, Khyber Pakhtunkhwa

41

Table .7.2(a) Missense/ Nonsense Mutations in GJB2 Gene in deaf Patients

of Khyber Pakhtunkhwa (Known)

43

Table .7.2(b) Prevalence of Missense/ Nonsense mutations in GJB2 gene

(gender/ age wise) in deaf population Khyber Pakhtunkhwa

(Known mutations)

44

Table. 7.3(a) Missense mutations in GJB2 gene in deaf patients Khyber

Pakhtunkhwa (novel mutations)

45

Table .7.3(b) Missense mutations in GJB2 gene in deaf patients Khyber

Pakhtunkhwa (novel mutations)

46

Table .8.1(a) Indels Mutations of GJB6 Patients samples in Khyber

Pakhtunkhwa showing mutation names, their codon,

Domain, Pathogenicity and Prevalence. (Novel Mutations)

50

iv

Table. 8.1(b) Gender and age wise incidence of indels mutations in GJB6

gene

51

Table. 8.2(a) Missense mutations in GJB6 gene (Novel variants) 53

Table. 8.2(b) Missense mutations in GJB6 gene (Novel mutations) 54

Table. 9. Mutations in MTRNR1 gene, its frequency and prevalence in

gender/age wise distribution in deaf patients KP Pakistan

57

Table. 10. Gender/age wise mutation prevalence in MTRNR2 gene in

deaf patients of KP Pakistan

59

Table .11. Mutations in MT-TV Gene of the deaf patients, the mutation

type, pathogenicity, frequency and gender/ age wise

prevalence distribution

61

v

LIST OF FIGURES

FIGURE DETAILS PAGE

Figure. 1 Causes of prelingual hearing loss in developed countries (Shearer

et al., 2017)

4

Figure. 2 Structure of outer and inner ear and its various parts (Lisa, 2014) 7

Figure. 3 Different types of hearing loss (Lisa, 2014) 8

Figure. 4 Different types of hearing loss and their hearing level (Richard et

al., 2008)

9

Figure. 5 Representation of the inner ear hair cell and supporting cells of the

cochlea along with GJB2, GJB6 genes and potassium ion recycling

(Rabionet et al., 2000)

13

Figure. 6 Schematic representation of β connexin molecule coded by GJB2,

GJB6 and GJB3 respectively (Rabionet et al., 2000)

14

Figure.7 Schematic representation of β-connexin Protein (Rabionet et al.,

2000)

15

Figure.8 Schematic representation of GJB and its domains (Rabionet et al.,

2000)

16

Figure.9 Connexin 30 protein (GJB6) and various mutations at amino acid

level (Pandya et al., 2003)

18

Figure.10 Human mitochondrial genome (Varinderpal et al., 2014) 20

Figure. 11 Comparison of 16SrRNA of bacterial and 12SrRNA of humans

(Yu et al., 2013)

23

Figure.12 Figure.12. Recording family history on the informed consent 34

Figure 13 Gel electrophoresis photograph of Isolated DNA from the buccal

swab of eight deaf patients

28

Figure.14 PCR conditions for exon-2 of GJB2 gene 29

Figure.15 (A) Gel picture of Gradient PCR (B) Gel picture of 791 bp PCR

product (GJB2 gene) (C) PCR product of 795 bp mtDNA

31

Figure.16 Agarose gel electrophoreses photograph of 10 PCR products of

795 bp mtDNA fragments along with 1000bp marker

39

vi

Figure.17 Gel photograph of four PCR products of 795 bp mtDNA 40

Figure. 18 Conditions for Sanger sequencing 42

Figure.19 Prevalence of gene mutations in deaf patients gender/age wise

sampled population in Khyber Pakhtunkhwa Pakistan

43

Figure.20 Incidence of mutations in selected genes in deaf population KP 44

Figure.21 Figure.21. District wise male deaf population, KP Pakistan 44

Figure.22 District wise female deaf population, KP Pakistan 45

Figure.23 District wise age/sex class males (3-10 yrs) of deaf population KP

Pakistan.

45

Figure.24 District wise age/sex class males (11-17) years of deaf population KP

Pakistan

46

Figure.25 District wise girls’ age group of 3-10 years in deaf population in KP 46

Figure.26 District wise female age group of 11-17years deaf population KP

Pakistan

47

Figure.27 Total deaf people gender/ age wise in KP Pakistan 47

Figure.28 Prevalence of mutations in GJB2 gene, gender/age wise in deaf

population KP Pakistan

56

Figure.29 Prevalence of indel mutations in GJB2 gene in deaf patients KP,

Pakistan

57

Figure.30 Missense/nonsense mutations in GJB2 gene in deaf patients KP,

Pakistan

57

Figure.31 Missense mutations in GJB2 gene in deaf patients KP, Pakistan

(Novel mutations)

58

Figure.32 Prevalence of mutations in GJB6 gene gender/age wise in deaf

population KP

58

Figure.33 Incidence of indel mutations in GJB6 gene (Novel mutations) 59

Figure.34 Missense mutations in GJB6 gene (Novel mutations) 59

Figure. 35 Prevalence of mutations in MTRNR1 gene gender/age wise in

deaf population of KP Pakistan

62

vii

Figure.36 Percentage wise mutations incidence in MTRNR1 gene in deaf

patients KP, Pakistan

64

Figure.37 Gender/age wise mutations prevalence in MTRNR2 gene in deaf


67

Figure.38 Prevalence of mutations distribution in MTRNR2 gene in deaf

patients KP, Pakistan

70

Figure.39 Gender/age wise mutations prevalence of MT-TVgene in deaf

population KP, Pakistan

72

Figure.40 Mutations prevalence in MT-TV gene in deaf population KP,

Pakistan

75

viii

ABBREVIATIONS

COMPLETE WORDS/ Meaning

Α Alpha

Β Beta

Cx Connecsine/Connexin

Del Deletion

DFNB1 Non-Syndromic Neuro Sensory Deafness

EDTA Ethylene diamine tetra-acetic acid

F Female

Fig Figure

GJB Gap Junction Beta

HGMD Human genes mutation database

HL Hearing loss

HN Humanin

Indels Insertion deletion

Ins Insertion

KP Khyber Pakhtunkhwa

M Male

MilliQ water Double distilled water

µl Microliter

NGS Next generation sequencing

mtDNA Mitochondrial DNA

NMD Non mediated Decay

NCBI National Center of Biotechnology Information

NSHL Non-syndromic hearing loss

ix

PCR Polymerase Chain Reaction

PK Pakistan

PK Protinase K

RNA. Ribonuclic acid

ROS Reactive oxygen species

Taq Thermus aquaticus

TAE Tris acetic acid EDTA

x

ACKNOWLEDGEMENT

First of all, I would like to pay my deepest gratitude to Almighty ALLAH,

Who blessed me with motivation and strength for completing my PhD

research. Peace and blessings of Allah be upon the Holy Prophet Muhammad

(Peace be upon Him), who is a source of guidance and knowledge for

humanity.

It really a matter of great honor and inclination for me to offer profound and

cordial gratitude to my supervisor and mentor Professor Dr. Habib Ahmad

and co-supervisor Dr. Muhammad Shahid Nadeem for their kind and

encouraging behavior throughout my PhD. They were always there for any

advice, guidance, and recommendation. I am very grateful to them for their

privileges, which were awarded to me during the project.

I would like to extend gratitude to my foreign advisor Prof. Dr. Hong Xue

who provided me the opportunity to work at the laboratory in her research

lab at division of life sciences, Hong Kong University of science and

technology, Hong Kong. Appreciation is extended to Ata Ullah Khan, Flora

mat and Peggy Lee for their help and support during my research work at

Hong Kong University of science and technology, Hong Kong.

My appreciation also goes to Prof. Dr. Hakim Khan, chairman Department of

genetics and Prof. Dr. Manzoor Hussain, dean faculty of science Hazara

University, Mansehra for their helpful discussion and guidance. I am also

very thankful to my teachers, Dr Inamullah, Dr Fida Abassi, Dr Muhammad

Jameel, Dr Aziz, Dr Muhammad Islam and friends Dr Ishtiaq Hassan and Dr

amjad Ali for their valuable help during my PhD studies.

Special thanks are extended to my valuable friends, Dr. Muhammad Tariq,

Mr. Rahmanullah, Mr.Shafee Ur Rahman, Mr. Fazal Rahim, Mr. Murad Ali

Rahat, Mr. Rahmat Ali, Mr. Muhammad Hanif, Mr. Faisal Khan, Mr.Bilal, Mr.

Hussain, Dr Muhammad Iqbal (UK) and Dr Mohammad Tariq (Belgium) for

xi

their full time support and encouragement. I express my sincere gratitude to

colleagues, technical and administrative Staff especially, Mr. Jawad,

Mr.Amjad and Muhammad Sabir at the Department of genetics, Hazara

University Mansehra and Peggy Lee at Hong Kong University of science and

technology Hong Kong. I am also very thankful to my cousin Dr Ishtiaq

Ahmad, for his technical help in the thesis.

It is very important to acknowledge the administration of government deaf

special schools as well as the private sector and NGOs based deaf special

schools throughout the Khyber Pakhtunkhwa. I am very thankful to the

faculty and the students of these schools for their cooperation in sampling

and for giving basic information to research team. This research work would

not be possible without their cooperation and help. I offering special thanks to

all of them. Thank you very much for your help and cooperation.

I would like to thank my family for their love and cooperation, especially to

my father Shah Hosh Khan, my mothers, sisters and my brothers, Rahamdal

Khan, Fazal Qayum Khan and Dr Imad-ud-Din. They always encouraged me

and provided full support to pursue my doctoral studies. I am also thankful

to my wife, who is being disturbed by long lasting PhD project, she always

supported me patiently all the way.

I acknowledge management of Hazara University and the ethnogenetic

project of Department of Genetics Hazara University Mansehra for facilitating

me to pursue doctoral studies. I am also very much thankful to the Higher

education commission (HEC) of Pakistan. This research would not have been

possible without the financial support provided by the Higher Education

Commission (HEC), Pakistan, under International Research Support Initiative

Program (IRSIP).

Muhammad Ismail Khan

xii

ABSTRACT

Deafness or hearing loss is one of the most prominent genetic disorders in

human beings. Hearing loss is caused by a number of environmental and

genetic factors. The genetic factors involve about 130 genes which have role in

hearing loss. Among them, the mutations in channel protein connexin genes

GJB2, GJB6 and in mtDNA genes resulting in hearing losses. The GJB2 and

GJB6 genes codes for connexin-26 and connexin-30 proteins, which help the

potassium K+ ions recycling in the inner ear cells and activates/trigger the

neurotransmitters.The neurotransmitters are signaling molecules which here

receive and transfer, the nerve impulses between the central nervous system

and sense organs, recognizing sound accordingly. For unraveling the

mechanism of Non-syndromic Hearing Loss (NSHL), a precise laboratory

protocols was established and employed, for identification two nuclear genes

i.e. exon2 of GJB2, the exon1 of GJB6 gene, and detection of mutations in three

mitochondrial genes viz. MTRNR1, MTRNR2 and MT-TV. For elaborating the

pattern of mutations in NSHL patients, 1500 oral swabs were collected from

the deaf patients belonging to Abbottabad, Bannu, Charsadda, Haripur,

Mansehra, Mardan, Peshawar, Swabi and Swat districts of Khyber

Pakhtunkhwa Province (KP), Pakistan. We observed mutations in 5 genes i.e.

2 nuclear (GJB2 and GJB6) and 3 mitochondrial genes (MTRNR1, MTRNR2

and MT-TV) in 700 (47%) out of the total 1500 deaf patients. Whereas, the rest

of deaf patients (800) might be having mutations in other deafness related

genes. We observed higher incidence of deafness related gene mutations in

males (68%) as compared to the females (32%). The mutations in GJB2 and

GJB6 genes showed prevalence of 1.6 and 0.67%, respectively whereas, in

mitochondrial genes i.e. MTRNR1, MTRNR2 and MT-TV, the mutation rate

was 0.8, 0.73 and 0.53%, respectively. The protocol includes the isolation of

total genomic DNA from the oral swab epithelial cells through modified

phenol-chloroform method of DNA extraction. The DNA was amplified

through thermo scientific polymerase chain reaction (PCR) and gene cleaned

xiii

through manual washing of PCR product with 75% ethanol with step wise

centrifugation. The sequencing was carried out in gene analyzer machine,

through Sanger’s sequencing method. After sequencing of desired genes, all

sequences were verified and confirmed by comparison with reference

sequences at NCBI gene bank. We identified some known and many novel

mutations in sampled deaf patients including indel, missense and nonsense

mutations in targeted genes. The identified mutations in GJB2 gene include

V27C, D46E, N54K, K61R, E110G, A78S, A78P, D66N, W77C, W77L, K15E,

K103N, V153I, 120F, F115V, D46A, V38A, W24*, E119* c.327G>A, c.186C>T,

c.228A>T, c.120A>G and c.240G>A, . The identified mutations in GJB6 gene

were c.41delA, c.42delC, c.43delA, c.31delG, c.ins374-375(16nt), c.ins320-

321(19nt), p.K15Q, p.A88T, p.A92D and p.A149S mutations. The mutations in

MTRNR1 gene were, 1349 T>G, 1420T>G, 1438A>G, 1440 G>A, 1442 G>A,

1492 A>C, 1544 A>T, 1545 G>A, 1546A>T, 1554G>A, 1575 T>G, 1577A>G and

1598 G>A variants in MTRNR1 gene. The mutations identified in MTRNR2

gene included, 1671 G>A, insT>1711, 1735 A>C, 1754 G>A, 1811 A>G, 1814

A>C, delT> 1872, 1888 G>A, 1899 G>A, insT>1960 and insG>1990. Similarly,

the mutations identified in MT-TV gene included, 1604G>T, 1604G>A,

1606G>A, 1609T>G, 1610 A>C, 1625 A>C, 1641G>T, and 1644G>A. Analyses

of the mutations data revealed that these mutations cause frame shift,

missense and nonsense mutational changes in the gene expression and

thereby result in hearing losses. It was further confirmed by protein

alignment, that these mutations also changes the structural configurations of

Cx26 and cx30 proteins, as well as affect the mitochondrial DNA dysfunction,

which impair sound recognition mechanism. Our study provides reliable

protocols for DNA extraction, gene cleaning and sequencing of concerned

responsible genes for hearing loss and thereby screening deaf patients on one

hand, and on the other hand we have established a baseline for gene

mutations in deaf patients of Khyber Pakhtunkhwa. These findings can be

used for genetic counselling, disease diagnostics and gene therapy etc.

1

Chapter 1

INTRODUCTION

1.1. Background Information

Khyber Pakhtunkhwa, a province of diverse ethnic populations, is situated in

north west of Pakistan. This territory was previously recognized by the

North-West Frontier Province (NWFP), since the British rule in Indo-Pak

subcontinent. Khyber Pakhtunkhwa borders the newly established Gilgit

Baltistan province to its north east, Afghanistan to the north-west, Pakistan

administered Kashmir to the east and Punjab to the south. Peshawar is the

capital of this province, locally referred to as ‘Pekhawar’. Khyber

Pakhtunkhwa encompasses a region of 1, 28,961 km² on the map of Pakistan.

According to the 2017 Census of Pakistan, the total population of Khyber

Pakhtukhwa including Federally Administred Tribal Areas (FATA) is

35,525,047. Among them the male, female and transgender represent 50.73,

49.26 and 0.003% individuals, respectively (Table.1). According to the 2017-

Census Report the total count of disabled persons was about 840,000 (0.4%)

persons in Pakistan (Anonymous, 2017). According to the available record the

incidence of deaf people in Khyber Pakhtunkhwa is increasing with the

passage of time, e.g. in the 1998 Census, Report 7.69% of the disable persons

were deaf & mute, which raised to 11.43% as recorded in total disable

population Census, 2017 (Anonymous, 2017).

Table.1. Gender wise breakup of people of Khyber Pakhtunkhwa

(Anonymous, 2017)

S. No. Population

Total Percentage

1 Male 18,023,937

50.73

2 Female 17,500,170

49.26

3 Transgender 940 0.002

Total 35,525,047

2

Genetic structure of human population never stays intact for a long duration,

as it is mostly dependent on changeable cultures and geographic

displacements. The effect of these factors results into the translation of

biological pattern through marriage or mating and natural selection

(Abouelhoda et al., 2016; Bassi & Maia, 1985).

The frequencies of genetic disorders play a great role in shaping human

populations. The other important factors which affect human populations

include assortative/consanguineous marriages, birth rate, mortality,

livelihood and geographical conditions (Mikhail, 2014; Berrettini et al., 2008;

Giordano et al., 2002). In human populations, the genetic disorders are

actually not all that common, their nature, prevalence and distribution are

different in different regions of the world (Jagannathan & Bradley, 2016;

Inoue et al., 2002; El-Hazmi, 1999). The genetic disorders are the basic leading

causes of childhood death of the newborns in many countries like Oman,

Kuwait, Qatar, Saudi Arabia and Bahrain (Hamamy & Alwan, 1997).

The consanguineous marriages are mostly practiced in various parts of central

west and south Asia, North and sub-Saharan Africa, and the Middle East,

while less than 0.5% consanguinity is reported in the developed world like

North America, Europe, and Austria (Tabor et al., 2014; Burchard et al., 2003;

Bittles, 1998). Moreover, first cousin unions have been long established

accustomed firmly among Pakistani families (Khan et al., 2016; Hussain &

Bittles, 1998).

In the available literature, various reports show the tendency in a population

to practicise inbreeding. The cultural beliefs play a major role to boost

consanguinity at a large scale (Jagannathan & Bradley, 2016; Tabor et al., 2014;

Burchard et al., 2003). Still, illiteracy, unemployment and rural life are

concerned factors for the high rates of consanguineous marriages (Kir et al.,

2005; Bittles, 2001; Bittles, 1998). From published articles it is clear that the

mean inbreeding coefficient denoted by “F” is an important variable to

3

determine the molecular and genetic diversity in studies of human’s

population (Leutenegger et al., 2002; Hussain & Bittles, 1998). For this purpose

the researchers have calculated the approximate value of the mean inbreeding

coefficient (F) for different world population. For example, the mean

inbreeding coefficient value (F) of Japanese, Turkish and American

populations is less than 0.002F, whereas “F” in Kuwait, is greater than 0.02F.

Unfortunately, in Pakistan the mean inbreeding coefficient value is highest

and is approximately 0.0331F (Javed et al., 2014; Bork et al., 2001; Hussain &

Bittles, 1998).

In human populations, genetic and molecular studies have made important

contributions to disease and health (Knecht et al., 2017; Haan, 2003). Such

studies have shown how physiological and morphological processes are set

up, their role development and their functions are controlled by the genetics

system, and these informations are stored in the genome (Lek et al., 2016;

Guttmacher & Collins, 2002). Any change or mutations in the genes affect

directly the phenotypes of organisms. Although many mutations occur are

harmless and can be considered polymorphism, and other mutations are

pathogenic and cause diseases that can be diagnosed phenotypically by these

mutations (Stenson et al., 2014; Miglani, 2002; Lewin, 2000). Because of their

effects, the characterization and identification of these mutations can give

insights into the treatment of these diseases (Usher & McCarroll, 2015;

Phillips & Hamid, 1999). Due to these reasons, geneticists are tracing genetic

mutations through the family pedigree analysis (Liu et al., 2013; Levitan &

Montagu, 1977). Geneticists later diagnosed and identified different genetic

disorders through working on the positional cloning of the concerned genes.

These contributions produced new techniques for the detection and

identification of genes responsible for Mendelian disorders (Amberger et al.,

2015; Tabor et al., 2014).

Genetic diseases can be divided into four (4) major categories, including

chromosomal disorders, single gene disorders, mitochondrial disorders and

4

polygenic disorders. Within these categories, single gene disorders are occur

extensively and obey clear pedigree style or pattern of inheritance, including

autosomal recessive, autosomal dominant, X-linked recessive, X linked

dominant, Y linked and mtDNA mutations (Amberger et al., 2015;De

keulenaer et al., 2012; Amudha et al., 2005).

1.2. Genetic Epidemiology of Deafness

Hearing loss is a genetic disorder which adversely affects human social life. It

is the most serious and important neurosensory impairment disorder (Downs,

1995; Davis et al., 1986). More than 120 million people throughout the world

are affected by hearing loss. Approximately, 50%–60% of these cases have a

genetic origin, whereas the remaining is caused by environmental factors,

such as the use of ototoxic drugs, injury and trauma. However, with the

passage of time as the public awareness of the problem and technical

advancement in molecular characterization were established, the

environmental factors causing hearing loss have been minimized compared to

the relative proportion resulting from inherited deafness or hearing loss

(Khan et al., 2016; Yu et al., 2013; Cunningham et al., 2005; Kemper & Downs,

2000; Adams et al., 1999). About one in every thousand (1/1000) kids has

some type of prelingual hearing impairment, whereas one in two thousand

(1/2,000) newborns have genetic mutations responsible for hearing loss

(Barbara et al., 2015).

Deafness is classified into syndromic and non-syndromic forms. The

syndromic form is associated with other clinical manifestations, whereas the

non-syndromic form is not related to other clinical diseases. About 80% of

genetic deafness is non-syndromic, whereas the remaining 20% is syndromic

hearing loss (Amina et al., 2016; Cohen et al., 2003). The non-syndromic

genetic hearing loss is divided into four types. The autosomal recessive forms

accounts for 60%–75% of the cases, autosomal dominant inheritance represent

20-30%cases, and approximately 2% are due to mitochondrial and X linked

5

mutations (Shearer et al., 2017). In Figure.1, the causes of prelingual hearing

loss in developed countries were shown.

Figure. 1. Causes of prelingual hearing loss in developed countries (Shearer et al., 2017)

6

1.2.1. The Human ear

The human ear is the sensory organ which is concern with and recognizes the

hearing of sound waves. The human ear is consisting of three (3) parts

including the outer ear, the middle ear and inner ear. These parts of the ear

are morphologically and physiologically different from each other (Bitner-

Glindzicz, 2002).

(a) Outer or external ear: It is consisting of an auricle or pinnae. It is

projected to outside and connected to head, and to the inner side, it is

connected to the tympanic membrane. The function of the outer ear is

to collect the sound waves from outside and transfer them to the inner

tympanic membrane.

(b) Middle ear: The middle ear is consisting of auditory ossicles. The

auditory ossicles generally consist of three (3) small bones present in

chain inside the temporal bone, which is an air filled cavity. These

small bones are called the malleus, incus and stapes, which are

collectively known as the auditory ossicles. The function of auditory

ossicles is to transfer the sound waves from the temporal bone to the

inner ear (Green et al., 2003).

(c) Inner ear: The inner ear consists of special fluid passages and cavities

which are present in the temporal bone in the inner ear. The inner ear

contains two (2) functional units known as the vestibular apparatus

and a snail- like unit called the cochlea (Yan & liu., 2008; Robertson et

al., 1998). The vestibular apparatus further consists of semicircular

canals and vestibules. The sensory organs for balance (postural

equilibrium) are present inside the vestibular apparatus, whereas the

cochlea contains sensory organs needed for hearing (Guan, 2011;

Griffith & Wangemann, 2011).

In Figure.2, the structure of the outer and the inner ear and its various

parts along with chochlea and the mechanism, how they work are shown.

7

Figure. 2. Structure of outer and inner ear and its various parts (Lisa, 2014)

1.2.2. Types of hearing loss

Hearing loss generally consists of three types. These include conductive

hearing impairment, sensorineural hearing impairment and mixed hearing

impairment (Davis et al., 1986)

The defect in the external ear or in the middle ear both structurally and

functionally results in conductive hearing loss.

Similarly, when the defect or impairment is present in the inner ear or in

hearing nerves, it is called sensorineural hearing loss. By contrast, when the

defect is present in middle and inner ear, the phenomena is called mixed

hearing impairment (Richard et al., 2010).

8

Basically, sensorineural hearing loss is usually permanent whereas the

conductive impairment can be repaired or cured by surgery or medical

treatment. (Avraham, 2003). In Figure.3, the different types of hearing loss,

the conductive, sensorineural and mixed hearing loss and concerned parts of

the human ear which are involved in the hearing impairment are shown.

Figure.3. Different types of hearing loss (Lisa, 2014)

Similarly, on the basis of severity, age and causes the hearing loss may be

classified as mild, moderate, severe and profound hearing loss. Usually

hearing loss problems occuraning before the development of speech is called

prelingual, and what its occurrence after the acquisition of speech is post

lingual (Yamasoba et al., 2013; Marazita et al., 1993). Generally prelingual

hearing loss is severe and permanent, whereas post lingual hearing

impairment is usually mild progressive and moderate. Approximately 1/800

people suffer from profound or severe hearing loss at the time of birth or at

early stage of infancy. Basically prelingual hearing loss is less frequent in

occurrence than post lingual hearing impairment (Morton & Nance., 2006).

Normal humans can hear and recognize sounds in the 0-15 dB frequency

range, although the individuals having the slight hearing loss can recognize

9

the sound in 16-25dB. In the case of mild hearing loss, the patient can hear

sounds in the 26-40 dB frequency range. Similarly, moderate hearing loss

patients can hear sounds in the range of 41-55 dB frequency whereas, deaf

patient having a severe or profound hearing impairment can recognizes the

sound in 56-90 dB frequency or above (Lisa, 2014; Guan, 2011; Rouillon et al.,

2006).

The Figure.4 shows the different frequency level of hearing loss, the normal

hearing, mild hearing loss, moderated hearing loss, severe hearing loss and

profound hearing loss.

Figure. 4. Different types of hearing loss and their hearing level (Richard et al.,

2008)

Molecular geneticists working on the structure and function of the human ear

revealed that more than 50-100 genes contribute into the structure and

functions of the human ear. Mutations in these genes result deafness or

hearing loss (Sloan-Heggen et al., 2016; Nance, 2003).

https://www.ncbi.nlm.nih.gov/books/NBK1434/

10

1.2.3. Genes involved in hearing loss

Non-syndromic genes or loci are being discovered very rapidly. Up to now,

125 loci have been discovered, including 71 autosomal recessive, 54 autosomal

dominant, five (5) are X-linked, two (2) are modifiers and one (1) is Y linked

gene or loci. Among all of these loci, some occur in the same gene, whereas

some have gene etiologies (De keulenaer et al., 2012; Estivill et al., 1998; Kelley

et al., 1998). Although nonsyndromic genes for deafness only affect hearing,

their expression is not limited only to the inner ear, but we can say that inner

ear is more sensitive to these reported mutations (Yan & Liu, 2008).

Hereditary hearing loss ranges from mild to profound in occurrence.

Autosomal recessive and sex-linked hearing losses are usually more profound

and severe than dominant mutation hearing loss (Shafique et al., 2014). In

humans, hearing loss onset and progression may occur in infancy and

childhood whereas progressive hearing loss affects large proportion of human

population (De keulenaer et al., 2012).

The first non-syndromic hearing loss gene was reported in 1993. (Marazita et

al., 1993). Due to the use of new advance techniques application in molecular

genetics research, a number of hearing loss genes were mapped.

Approximately more than hundred (100) loci and more than sixty (60) genes

having a role in normal hearing mechanism were mapped. Many of these

genes function inside the cochlea, and mutations in these genes cause cochlear

dysfunction resulting in hearing loss (Avraham, 2003).

Because this abnormal condition causes indistinguishable phenotypes, the use

of genetic screening is necessary to identify the biological basis for these

abnormal conditions.

Genetic screening is mostly applicable to non-syndromic hearing loss, as

these conditions mostly have an indistinguishable phenotype. For the

identification of different alleles of specific genes which are concerned with

11

hearing loss, targeted genetic screening is applicable and focused for these

genes. Beside these conditions, the specific mutational prevalence and the

ethnic origin of individuals are also important factors for the genetic

screening as in the case of mitochondrial DNA, which is characterized by

prominent distant lineages and different haplogroups, each of these

haplogroups possess a specific set of SNPs (Yamasoba et al., 2013; Yu et al.,

2013; Ruiz-Pesini & Wallace, 2006).

As with autosomal recessive hearing loss, the most important causative genes

with respect to its prevalence are GJB2, SLC26A4, MYO15A, OTOF, CDH23,

and TMC1, among others. More than twenty (20) mutations for each of these

genes have been identified and reported. Among them the genes which have

autosomal dominant mutation pattern are common WFS1, MYO7A, and

COCH loci. Many of these genes also have role in causing syndromic hearing

loss (Rabionet et al., 2000).

Recently, a number of non-syndromic loci have been discovered. Up to this

time, more than one hundred and twenty five (125) loci had been reported.

Among them seventy one (71) loci were autosomal recessive, fifty four (54)

were autosomal dominant, five (5) were x-linked, one (1) is Y-linked and two

(2) loci are modifiers. Many of these mutations appear within a common gene,

whereas some of them have an unknown etiology. On the basis of the severity

caused by the mutations in deafness related genes, those showing autosomal

recessive pattern of inheritance usually produced prelingual and severe

hearing loss frequencies, whereas those showing an autosomal dominant

pattern of inheritance are generally less severe and mostly post lingual in

occurrence. It is important to note that X-linked mutations generally affect

male individuals more frequently than females (Khan et al., 2016; Petit, 2006).

In Table.2, the prevalence of different genes mutations in deaf patients with

non-syndromic hearing loss in developed countires were shown.


12

Table.2. Prevalence of gene mutations with non-syndromic hearing loss in

developed countries

S. No. Non-syndromic hearing loss Prevalence of mutations reported

1 GJB2 gene 59%

2 GJB6 gene 5%

3 Cadherin gene 23 mutations

4 Myosine Gene 5%

5 Pendrin gene 5%

6 Sterosilin gene 5%

7 Otoferlin gene 3%

8 mtDNA (MTRNR1) 3%

Research on genetic variation is not only important for the development of

pathogenic treatment approaches, but can also be used for faster better

counselling about these genes in deaf patients. (Petersen & Willems, 2006;

Petit, 2006).

The genetic heterogeneity of deafness related genes is the main problem in the

diagnosis of deaf patient’s cases. Similarly, the prelingual or early age

deafness and the role of certain genetic factors are problems that were less

studied. Because cousin marriages in Pakistan are still common, the

prelingual and early age onset of hearing loss is very important problem.

Consanguineous marriages increase the infant mortality rate as well as

increase the chances of hereditary diseases (Bork et al., 2001; Stenton, 1999).

According to the United Nation organization (WHO) report, approximately

250 million people suffer from hearing loss of different levels. As a whole

this number constitutes 4.2% of the world population (Yan & Liu, 2010;

Nance, 2003; Smith et al., 2005; Morton & Nance, 2006).

13

1.2.4. Channel protein genes and hearing loss

Connexin is a very important class of transmembrane proteins.Connexin

proteins form the hexameric gap junction hemi-channel connexon in such a

way that that the six monomers of connexin proteins combines and form

connexon.

Figure. 5. Representation of the inner ear hair cell and supporting cells of the cochlea

along with GJB2, GJB6 genes and potassium ion recycling (Rabionet et al., 2000)

Connexin proteins are attached and embedded on the surface of neighbouring

cells to form intercellular channels as transmembrane proteins, Connexin acts

to recycle potassium ions in the cochlea hair cells. The gap junction’s channels

allow a passage for passing the molecules, whose size is less than 1000

Daltons between the adjacent cells. The functions of these small channels are

very important because they perform various duties in the living body

including cooperation in the metabolism of ions, the propagation of electrical

cells, localized buffering, growth control and cellular differentiation (Wu et al.,

2004; Kumar & Gilula., 1996).

14

There are 13 connexin genes which encode different types of channel proteins

in mammals. These 13 genes are grouped into two classes of genes on the

basis of their primary structures, alpha genes encode alpha connexin protein,

whereas the beta genes produce beta connexin. Generally four types of gap

junction channel proteins (connexin-26, connexin-30, connexin-31 and

connexin-32) are encoded by GJB2, GJB6, GJB3 and GJB1 respectively (Maeda

et al., 2009). A mutation in any one of these genes causes abnormal channels

proteins that cannot perform their functions very well and produce hearing

loss. Figure.6, presents a diagrammatic representation showing connexin beta

molecules. The positions of dominant mutations in hearing loss are indicated

in the figure.

Figure. 6. Diagram shows the schematic representation of β connexin

molecule coded by GJB2, GJB6 and GJB3 respectively (Rabionet et al., 2000)

All of these four genes follow different pattern of inheritance. For example,

mutations in GJB2 and GJB3 follow an autosomal recessive inherited pattern,

other in the GJB2, GJB3, and GJB6 genes follow an autosomal dominant

inheritance pattern, while mutations in GJB1 gene follow an X-linked

inheritance. All these genes may cause syndromic and non-syndromic

hearing loss, as well. For example, GJB2 gene mutations cause deafness as

well as skin disease keratoderma, the GJB3 gene causes erythrokeratoderma

varibilis along with deafness, and GJB1 gene also causes peripheral



15

neuropathy along with hearing loss (Bitner-Glindzicz, 2002). In addition the

GJB2, GJB3 and GJB6 are also involved in non-syndromic hearing loss (Sloan-

Heggen et al., 2016; Maeda et al., 2009; Kovacs et al., 2007; Lee et al., 2000).

1.2.4.1. GJB2 Gene

GJB2 gene codes for connexin-26, which is a gap junction protein called β--2

polypeptide. The connexin-26 channel protein is expressed inside the inner

ear in the snail- like structure cochlea, which contain the sense organs for

hearing. The connexin-26 is expressed in the discrete region of the cochlea

(Green et al., 2003; Denoyelle et al., 1999). The GJB2 gene, which encodes the

channel protein, is also considered as tumor suppressor gene of class 2,

because connexin-26 is down regulated in tumor tissue. On the other hand,

connexin-26 is upregulated very strongly in the synchronized cells, mostly in

the cell cycle phases especially in G2 and S (Cohn & Kelley, 1999; Denoyelle et

al., 1999).

Connexin-26 and connexin-30 are coded by the GJB2 and GJB6 genes,

respectively. These two genes have 77% identity in amino acids segment and

both of the genes regulate potassium ions recycling in inner hair cells,

diffusion of ions transfer, second messenger and metabolites among the cells.

It is interesting to note that connexin-26 is the only channel protein with a

known and reported structure. The Figure .7, shows schematic representation

of a β-connexin molecule coded by GJB2 gene. IC1, IC2 and IC3 are stand for

intracellular domains of the connexin protein, The EC1 and EC2 stand for

extracellular domains and TM1, TM2, TM3 and TM4 are the transmembrane

domains of the connexin molecule.




16

Figure.7. Schematic representation of β-connexin Protein (Rabionet et al., 2000)

GJB2 is the most important connexin-26 protein gene as it contributes to 50%

of all cases of hearing impairment. Presently, more than 110 mutations have

been reported in the GJB2 gene (Nance, 2003; Gasparini et al., 2000). All of

these mutations cause hearing loss, but the most important and most frequent

types in many world populations are the 35delG, 167delT and 235delC

mutations. The 35delG mutation is the most prominent and frequent in

European populations and account for approximately 70% of all GJB2 gene

mutations (Snoeckx et al., 2005). The carrier frequency of 35delG mutation in

the midwestern United States (USA) is 2.5 % (Green et al., 1999). Similarly

the other frequent mutations in the GJB2 gene are 167delT in Ashaknazi

Jewish (Morell et al., 1998) and 235delC mutation in Southeast Asians

populations. Many studies revealed that the patients who express severe to

profound hearing loss are referred to cochlear implant surgery (Ohtsuka et al.,

2003; Yan et al., 2003; Minarik et al., 2003).

In Figure.8, diagram shows the channel protein conexin (GJB) and its domains

a single unit of connexon and a gap junction. IC1 and IC3 stand for

17

intracellular domains, the EC1 and EC2 stand for extracellular domains, and

TM1, TM2, TM3 and TM4 are stand for transmembrane domains of the

connexin protein.

Fig 8. Schematic representation of GJB and its domains (Rabionet et al., 2000)

It is important to note that the 235delC mutation in the GJB2 gene is not

reported in South Asian population that is India, Pakistan, Bangladesh and Sri

Lanka. The most prevalent mutations in these countries were W24X and

W77X (Scott et al., 1998). Thus, it is important to note that the segregating

pattern in hearing loss is a good indication to recognize and detect those gene

factors which are important in understanding the hearing problems at

molecular and genetic level in Pakistani populations (Shahid et al., 2007).

The GJB2 gene has two (exons), Exon1 and Exon 2. The first exon is

nonfunctional as it forms a truncated protein, whereas the second exon is

functional and encodes the gap junction beta (β) 2 connexin protein. The

second exon of GJB2 gene is 791 bp in size and functional. An intron is present

between these two exons (Gasparini et al., 2000).

18

1.2.4.2. GJB6 Gene

The GJB6 Gene encodes channel protein gene Connexin-30. This protein also

functions along with GJB2 to recycle Potassium ions K+ in the inner ear

cochlear hair cells. The GJB6 gene is situated about 800 kb centromic to GJB2

gene on chromosome 13q12. The GJB6 and GJB2 genes are expressed in the

inner ear cochlear cells (Lautermann et al., 1999), together contributing 8% to

the hearing mechanism as a whole. The GJB6 gene has five (5) exons, with

only the first exon being functional. The first part of functional exon 785 bp

was amplified and screened for deafness related mutations. (Pandya et al.,

2003; Rabionet et al., 2000). The Figure.9, shows the structure of connexin-30

and different mutations at the amino acid level.

Figure. 9. Connexin 30 protein (GJB6) and various mutations at amino acid

level (Pandya et al., 2003)

19

1.2.4.3. GJB3 Gene

GJB3 is another gene that encodes connexin31, a gap junction channel protein.

Mutations reported in GJB3 showed that this gene also contribute in hearing

mechanism, thus to hearing loss both syndromic and non-syndromic.

Syndromically, it causes erythrokeratodermia variabilis as well as hearing loss,

whereas, non-syndromically the GJB3 gene causes an autosomal dominant or

recessive pattern of inheritance in hearing loss. The GJB3 gene contains only

one single exon (Maeda et al., 2009).

Various reports about different world population showed that three amino

acids changes reported at different positions in connexin-31 protein causes

hearing loss in deaf patients. These include a T>G change at position 529

which creates a Y>D amino acid change at codon 177 resulting hearing loss.

Similarly, the C1227T mutation results the 49delk mutation in deaf patients. In

addition a deletion mutation at position 144-146delGAA causes the R32W

amino acid change .To date the exact mechanism behind GJB3 mutations that

cause hearing loss is still unknown (Martin et al., 1999; (Denoyelle et al., 1999).

1.3. Mitochondrial DNA

Mutations in mitochondrial DNA (mtDNA) genes also contribute to hearing

loss both syndromic and nonsyndromic cases. The mtDNA contain 37 genes.

The genes for rRNAs and tRNAs basically contribute to normal hearing,

whereas mutations in these genes cause deafness. The first locus discovered

has a mutation linked to hearing loss been a mitochondrial gene. (Griffith &

Wangemann, 2011; Guan 2011; Fischel-Ghodsian, 1999).

1.3.1. The mitochondrial genetics

Mitochondria are intracellular organelles present in all mammalian cells. The

function of mitochondria is to produce energy in the form of ATP by

oxidative photophosphorylation. The mitochondrial genome is a circular,


20

double stranded DNA molecule of 16.6 Kb size in humans and contains 37

genes (Elstner et al., 2008). These mitochondrial genes code for 13 essential

proteins of the oxidative phosphorylation machinery, 2 rRNAs (12SrRNA and

16SrRNA) and 22 tRNAs molecules. The rRNAs and tRNAs molecules are

used for the maintenance and expression of mtDNA protein synthesis, protein

import, proteolysis, fatty acid oxidation and citric acid cycle metabolism.

Therefore, the role of these RNAs is very important and essential for normal

mitochondrial function (Varinderpal et al., 2014).

Mitochondrial genes contribute in hearing loss. Mitochondrial DNA

mutations are maternally inherited and have a role in syndromic and non-

syndromic deafness (Griffith & Wangemann, 2011; Fischel-Ghodsian, 1999).

The mitochondrial DNA also does not undergo recombination and as a result,

mutations accumulate sequentially within by maternal lineages (Yu et al.,

2013). Furthermore, being located in the inner membrane, the proximity of

reactive oxygen species (ROS), produced during oxidative phosphorylation.

These free radicals directly enter into mtDNA molecule, causes mutations in it

which results into hearing loss. For this reason and due to having a simple

DNA repair and protection system, it experiences a high mutation rate. The

mtDNA mutations responsible for hearing loss are generally homoplasmic in

nature, with the ribosomal and transfer RNAs genes usually being involoved

(Varinderpal et al 2014; Yu et al., 2013). Figure.10, presents a diagram of the

human mitochondrial genome with the inner circles of diagram showing the

products of MTRNR2, MT-TV and MTRNR1genes that are 16SrRNA, valine-

tRNA and 12SrRNA, respectively. Similarly, the white bars represent the

basic frame reading and the genes for 22 tRNAs.

21

Fig. 10. Human mitochondrial genome (Varinderpal et al., 2014)

1.3.2. Molecular mechanism of mtDNA mutations causing deafness:

Mutations in mtDNA can cause morphological and physiological changes in

the mitochondria. Due to these mutations, the structure of RNAs change,

there by minimizing the steady state level of tRNA amd mRNA and also

altering the tRNA modification. This effect has been confirmed in cybrid cells,

where cybrid cells containing mtDNA mutations exhibited a failure in the

metabolism of tRNA and the synthesis of protein (Guan, 2011). As a result,

ATP synthesis became reduced. The ATP synthesis is also reduced due to

ROS species as these free oxygen radicals absorb the free oxygen, reduces its

availability for kreb, s cycle and electron transport chain. ROS species also

destroy the hair cells and neurons of the cochlea of inner ear (McKenzie et al.,

2004). These actions triggers by mitochondria and at last, the mitochondrial

permeability transition pore opens, activating the programmed cell death

apoptosis, and hence causes hearing loss (Guan, 2011; Jacobs et al., 2005).

22

1.3.3. MTRNR1

The mitochondrial MTRNR1 gene which encodes the 12s rRNA has an

important role. It is considered to be a hot-spot area for pathogenic mutations

of non-syndromic and aminoglycoside induced hearing loss (Elstner et al.,

2008).

In mitochondrial DNA, different mutations are potential susceptibility factors

for hearing loss. The mechanism of aminoglycoside induced hearing loss is

this that in bacteria, the antibiotic aminoglycoside attaches to the 30s

ribosomal subunit of ribosome, making the ribosome unavailable for

translation, and hence, disrupting protein synthesis (Jana & Deb, 2006). In the

case of eukaryotes, the 30s ribosomal subunit counterpart of bacteria is the 12s

rRNA. Mutations in the 12S rRNA gene MTRNR1 alter the secondary

structure of 12S rRNA, making it a high affinity hotspot area for

aminoglycosides action results in mitochondrial dysfunction. Several different

mutations in the 12S rRNA gene have been linked to hearing loss, among

them, the A1555G mutation is the most commonly reported mutation

responsible for the non- syndromic and aminoglycoside induced hearing loss

in various populations around the world (Berrettini et al., 2008).

The A1555G and C1494T mutations in the 12S rRNA gene have been

confirmed to cause non- syndromic as well as aminoglycoside induced

deafness (Guan, 2011; Jacobs et al., 2005). The A1555G site in MTRNR1 gene is

located in the aminoacyl-tRNA acceptor site of the small subunit of ribosome

which is conserved from bacteria to mammals. Therefore, mutations at this

specific site cause the disruption of protein synthesis, resulting mitochondrial

dysfunction (Prezant et al., 1993).

The C1494T and A1555G mutations are the main causes of aminoglycoside

induced deafness and non-syndromic hearing impairments. These mutations

change the conformational structure of 12S rRNA, making it closely resemble

with the bacterial 12S rRNA, which is a target region for antibiotics such as

23

aminoglycosides gentamicin, kanamycin, and streptomycin. The 12SrRNA,

A1555G and C1494T mutations were reported for the first time in 1993 in a

family pedigree, demonstrating that these mutations are involved in

maternally transmitted non- syndromic hearing loss (Yu et al., 2013; Prezant et

al., 1993). As the pathogenicity of these mutations is further confirmed in

various global populations with different ethnic and geographic origins in

sporadic cases and in vitro studies, they have been confirmed as mutations for

HL (Giordano et al., 2002, Inoue et al., 2002; Prezant et al., 1993).

Numerous other studies have tried to detect other mitochondrial mutations

which are pathogenic and related to aminoglycoside ototoxicity. Mutation

A1555G in MTRNR1 gene is situated at the acceptor site of the aminoacyl

tRNA of the small ribosomal subunit and it is very clear that this acceptor site

(A-site) is highly conserved region in mtDNA of different organism from

bacteria to mammals showing high similarity and conservation. (Yu et al.,

2013; Ruiz-Pesini & Wallace, 2006).

In Figure.11, the secondary structure of small ribosomal subunit decoding site

of rRNAs is compaired to the bacteria (16SrRNA) and the human (12SrRNA).

The conserved region of rRNAs of both bacteria and humans are indicated.

Figure. 11. Comparison of 16SrRNA of bacterial and 12SrRNA of humans (Yu

et al., 2013)

24

1.3.4. MTRNR2

MT-RNR2 gene encodes the large 16S mitochondrial ribosomal RNA (Kearsey and

Craig, 1981). MTRNR2 also harbors a short ORF (Open reading frame) that is

translated into the 24-amino acid humanin (HN) peptide that was originally

identified due to its antiapoptotic properties.So when mutations occurred in the

MTRNR2 gene, it affects, the anti apoptotic properties, hence the apoptosis triggers

resulting into programmed cell death and so hearing loss. The cytoprotective effects

of Humanin (HN) appear to be mediated by specific receptors. Among its related

pathways are ribosome biogenesis in eukaryotes and viral mRNA translation (Lee et

al., 2013).

1.3.5. MT-TV Gene

The MT-TV gene is a small locus consisting of only sixty nine (69) nucleotides.

The position of this gene in the mtDNA map is from 1602 to 1670. The

function of MT-TV gene is to encode valine tRNA. When mutations occur in

MT-TV gene, the Valine tRNA can not perform their normal function and

affects translation.

The G1606A mutation in the MT-TV gene was reported in a heteroplasmic

state for the first time in a man aged 48 years who was affected by hearing

impairment, eye sight loss and mild weakness of muscles. (Tiranti et al., 1998).

The same mutation was reported in a 37 year old woman, who was affected

by hearing impairment as well as hypothyroidism, Ataxia and retinitis

pigmentosa (Sacconi et al., 2002).

The mutation G1606A, is situated at the acceptor arm of valine-tRNA.

Furthermore, the phenotypic expression analysis of this mutation shows that

it disrupts the secondary and tertiary structure of the valine-tRNA. Mutations

in MT-TV gene were further analyzed in a single muscle fiber, the high level

of mitochondrial mutant DNA in the cytochrome oxidase negative fibers

proved that the mutation was the cause of the clinical symptoms (Schon et al.,

1997).

https://www.omim.org/entry/561010#9


http://pathcards.genecards.org/card/ribosome_biogenesis_in_eukaryotes

http://pathcards.genecards.org/card/viral_mrna_translation



25

1.3.6. Other mitochondrial genes involved in deafness

A number of other mitochondrial genes are also involved in hearing loss.

Some of these genes and their mutations include: A3243G in the tRNALeu

(UUR) gene, A4295G in the tRNA-Ile gene, G8363A in the tRNALys gene,

T12201C in the tRNAHis gene, C3388A in the ND1 and G8078A in the CO2

genes. They are tRNAs gene mutations which have role in normal hearing

mechanism and so in the hearing loss when mutations occur in these genes

(Varinderpal et al., 2014; Griffith & Wangemann, 2011; McKenzie et al., 2004).

1.4. Objectives

1. Collection of saliva samples from selected deaf individuals and

isolation of total DNA

2. PCR amplification of deafness related genes and purification of

amplified product from agarose gel

3. Nucleotide sequence analysis of amplified genes and comparison of

sequences with normal controls to detect the mutation(s)

4. Determine frequency of mutations causing deafness in Pakistani

populations

5. Implications of the genetic data for studies of hearing loss in Pakistani

populations

6. Results to direct future work on deafness from genetic stand point

26

Chapter 2 MATERIALS AND METHODS

2.1. Sample collection

Oral swabs were collected from 1500 young deaf volunteers enrolled in

special education schools in different districts of Khyber Pakhtunkhwa

Province of Pakistan. The districts from which sampling was done, included

districts Abbotabad, Bannu, Charsadda, Haripur, Mansehra, Mardan,

Peshawar, Swabi and Swat. The oral swabs were collected in sterile cups. The

collection was made after cleaning mouth through brushing their teeth.The 3

ml 5% sugar solution was given to each volunteer and were instructed to keep

the solution in their mouth, rinse it for two to three minutes and spit it into

the sterile cups. The collected samples were stored at -20 °C until processing

for extraction of DNA. A consent form was signed from each volunteer and

the institution’s Heads. In the case of students having 3-5 years age or could

not signed the consent form, the consent form was signed from their parents.

Family history and consent form information were taken from a patient (Figur

12).

Figure.12. Recording family history on the informed consent

27

2.2. DNA extraction

The standard phenol chloroform method was used for DNA extraction (Aidar

& Line, 2007). The step wise procedure of DNA extraction is given at

Appendix 6.

2.3. Gel electrophoresis

The DNA was isolated and analyzed through 1% agarose gel and high quality

RNA free DNA was extracted as shown in Fig.13. The gel preparation

procedure is provided in Appendix-6.

Figure 13. Gel electrophoresis photograph of Isolated DNA from the buccal

swab of eight deaf patients

2.4. Spectophotometry

The quantity or concentration of DNA samples was tested on nanodrop

spectrophotometre. The results of spectrophotometry of these samples

showed that they contain appropriate amount of DNA (See Appendix-7).

Some samples contain a very high amount of DNA, so these samples were

diluted to specific level of concentration. The best DNA concentration for

general PCR is in the range of 20-150 ng/ul, whereas, the best range of DNA

concentration for real time PCR is 4ng/ul.

28

2.5. PCR amplification

The DNA samples were PCR amplified. The PCR condtions set up for getting

quality results and gel pictures of PCR amplification were shown in Figure.

14—17. The selected genes region for amplification included, the most

commonly reported frame shift, missense/nonsense mutations reported in

world population data.The genomic DNA was used as a template to amplify

the 2nd exon of GJB2, the 1st exon of GJB6 genes and regions from

mitochondrial DNA genes, MTRNR1, MTRNR2 and MT-TV. The designed

primers for all of these genes are listed in Table.3 and the reagents used for

PCR amplification are listed in Table 4. The PCR amplification condtions for

GJB2 gene are given in Figure.14

Table.3. Primers used for the GJB2, GJB6 and mitochondrial genes

Gene Primer Primer Sequence

GJB2 Forward 5ʹ ACTGTTCTGTCCTAGCTAGTGATTC 3ꞌ

Reverse 5ʹ CTGTCTAGGTCTTAATCTAACAACTG 3ꞌ

GJB6 Forward 5’ ATGAAGCTTATGGATTGGGGGACGCTG 3’

Reverse 5’ATGCTCGAGGCGCTTGGGAAACCTGTGATTG 3’

MTRNR1/

MTRNR2/

MT-TV

Forward 5ʹCGATCAACCTCACCACCTCTT3'

Reverse 5ʹCTTGGACAACCAGCTATCACCA3'

Figure.14. PCR conditions for exon-2 of GJB2 gene

29

Table.4. Reagents and their volumes used for PCR amplfication

SR. NO. NAME OF REAGENTS ADDED VOLUME OF REGENTS

1 DNTPs 2.0 μl

2 Mgcl2 2.5 μl

3 10x Taq Buffer 2.5 μl

4 Forward Primer 2.0 μl

5 Reverse Primer 2.0 μl

6 Taq DNA Polymerase 0.5 μl

7 Template 1.5 μl

8 ddH20 12 μl

Total Volume 25 μl

2.6. Gel Electrophoresis of PCR Product

The quality of PCR amplified product was checked on 2% agarose gel. The gel

electrophoresis pictures of gradient PCR, PCR product 791bp and 795bp PCR

products of mtDNA along with 1kb ladder, respectively were shown in

Figures 15.

30

Figure.15. (A) Gel picture of Gradient PCR (B) Gel picture of 791 bp PCR product (GJB2 gene) (C) PCR product of 795 bp mtDNA

A

B

C

31

2.7. Protocol for gene clean

Agarose gel bands were rescued and the genes were cleaned at human

genetics lab, Hazara University Mansehra, through protocol of gene elution

kit (Tiangen gene elution kit). The duplicates samples of bands were also gene

cleaned manually method, which is step wise washing of PCR products with

75% ethanol, and centrifugation at the biosciences lab, Hong Kong University

of science and technology, Hong Kong.

2.8. DNA Sequencing and sequence analyses

The purified PCR products were sequenced both by gene analyzer, through

Sanger sequencer at the Division of Life Sciences, Hong Kong University of

Science and Technology (HKUST) and Macrogen (Korea). The amount of

reagents and PCR conditions of Sanger Sequencing are given in Table 5 and

Figure 16, respectively.

Table .5. Reagents used in Sanger sequencing PCR

S.No. Reagents Amount (1X)

1 milliQ Water 8.53 ul

2 PCR Buffer 3 ul

3 Primer ( Forward primer) 0.72ul

4 Big Dye 0.75

5 Template 2ul

6 Total 15ul

32

2.9. Condition for PCR sequencing

The PCR conditions were through out kept standared except, the extension

temperature wich was only one step. The holding temperature was kept 16

oC. The conditions for Sanger sequencing are shown in Figure18.

Figure. 16. PCR conditions for Sanger sequencing

After sequencing PCR, the gene clean was done manually. The samples were

kept for drying the whole night at room temperature and were kept in HiPi

buffer (5 ul Formamide+ 10ul water) for preventing DNA pairing. Before

using gene analyzer, the samples were denaturated at 95oC for 2 minutes. The

sequences thus obtained were compared with the standered reference

sequences at NCBI Gene Banks for analysis.

33

Chapter 3

RESULTS

3.1. Deaf patients in the KP

The results were based on the analyses of 1500 deaf volunteers. As a whole,

the seven hundred out of fifteen hundred (47%) sampled population had

mutations in the selected genes. The remaining 800 deaf patients (53%) might

be having mutations in other deafness related genes. The genderwise

distribution of hearing loss in population (Fig. 17) shows that incidence of

deafness related gene mutations were higher in males (68%) as compared to

the females (32%).

Figure.17. Prevalence of gene mutations in deaf patients gender/age wise

sampled population in Khyber Pakhtunkhwa Pakistan

However, the incidents of selected genes mutations prevalence, individualy in

each gene were given (Fig.18). The prevalence percentages of targeted genes

in deaf sampled population were shown.

0

200

400

600

800 700

474

226 200

274

87

139

Total Males Females 3-10 yrs (M) 11-17 yrs (M) 3-10 yrs (F) 11-17 yrs (F)

34

Figure.18. Incidence of mutations in selected genes in deaf population

Furthermore we did not come across with any of the intersex individuals

suffered from hearng losses. Information about the distribution of deaf

patients in various districts of Khyber Pakhtunkhwa is provided in Table.6.

No targeted genes mutations

53%

GJB2 Gene 13.00%

GJB6 Gene 8%

MTRNR1 Gene 16.00%

MTRNR2 Gene 5.80% MT-TV Gene

3.67%

No targeted genesmutations

GJB2 Gene

GJB6 Gene

MTRNR1 Gene

MTRNR2 Gene

MT-TV Gene

35

Table.6. District and gender/age wise census of deaf people in Khyber

Pakhtunkhwa

District Patients gender Age groups (years)

Male Female

Male Female 3-10 11-17 3-10 11-17

Abbottabad 133 67 85 48 40 27

Bannu 175 25 60 115 08 17

Charsada 100 100 65 35 52 48

Haripur 134 166 90 44 15 51

Mansehra 25 75 11 14 30 45

Mardan 35 65 12 23 08 57

Peshawar 166 34 22 144 05 29

Swabi 72 28 20 52 11 17

Swat 177 23 62 115 17 06

KP 1017 483 427 590 186 297

The results revealed that the highest incidence of male deaf patients (17.4%)

was recored for Swat District and the lowest incidence (2.46%) was reported

for District Mansehra as shown in Fig.19.

Figure.19. District wise male deaf population, KP Pakistan

Abbottabad

BannuCharsada

HaripurMansehra

MardanPeshawar

SwabiSwat

0

50

100

150

200

133

175

100

134

25 35

166

72

177 Abbottabad

Bannu

Charsada

Haripur

Mansehra

Mardan

Peshawar

Swabi

36

Whereas, the highest incidence of female patients, among all the districts was

34.37% in District Haripur and the lowest incidence recorded thereby was

4.76% in Swat District (Fig.20).

Figure.20. District wise female deaf population, KP Pakistan

So far, the distribution of hearing loss in deaf patients with respect to the

gender as well as age groups is concerned, in the age group of 3-10 year

males, the highest frequency of 21.08% was recorded in Haripur District

(Fig.21) whereas, the lowest incidence of 2.58% deaf patients were recorded in

the 3-10 year male group in Mansehra District.

Figure.21. District wise age/sex class males (3-10 yrs) of deaf population

Abbottabad

BannuCharsada

HaripurMansehra

MardanPeshawar

SwabiSwat

0

50

100

150

200

67

25

100

166

75 65 34 28 23

Abbottabad

Bannu

Charsada

Haripur

Mansehra

Mardan

Peshawar

Swabi

Swat

Abbottabad

BannuCharsada

HaripurMansehra

MardanPeshawar

SwabiSwat

0

20

40

60

80

100 85

60 65

90

11 12

22 20

62 Abbottabad

Bannu

Charsada

Haripur

Mansehra

Mardan

Peshawar

Swabi

37

The highest percentage of deaf patient’s was 24.41%, observed in Peshawar

District, in the age group of 11-17 year males. Whereas the lowest percentage

of 2.37% in 11-17 year males was observed in Mansehra District (Fig.22).

Figure.22. District wise age/sex class males (11-17) years of deaf population

With respect to female patients the age group of 3-10 years, the highest

prevalance of 27.96% was in the District Charsada whereas, the lowest

percentage of this group was 2.69%recored in District Peshawar (Fig.23).

Figure.23. District wise girls’ age group of 3-10 years in deaf population in KP

Abbottabad

BannuCharsada

HaripurMansehra

MardanPeshawar

SwabiSwat

0

20

40

60

80

100

120

140

160

48

115

35

44

14

23

144

52

115 Abbottabad

Bannu

Charsada

Haripur

Mansehra

Mardan

Peshawar

Swabi

Swat

Abbottabad

BannuCharsada

HaripurMansehra

MardanPeshawar

SwabiSwat

0

10

20

30

40

50

60

40

8

52

15

30

8 5

11

17

Abbottabad

Bannu

Charsada

Haripur

Mansehra

Mardan

Peshawar

Swabi

Swat

38

The highest incidence of females in the age group of 11-17 years was 19.2%,

which was recorded for District Mardan, whereas the lowest percentage of

female patients of the same age group was 2.02 recorded in District Swat

(Fig.24).

Figure.24. District wise female age group of 11-17years deaf population KP

3.2. Collection of samples

Information about the distribution of deaf patients in various districts of

Khyber Pakhtunkhwa are provided in Table.6. The total number of deaf

patient samples, number of males/ females and their age groups in Khyber

Pakhtunkhwa were given in Fig. 25.

Figure.25 Total deaf people gender/ age wise in KP Pakistan

Abbottabad

BannuCharsada

HaripurMansehra

MardanPeshawar

SwabiSwat

0

20

40

60

27 17

48 51 45

57

29 17

6

Abbottabad

Bannu

Charsada

Haripur

Mansehra

Mardan

Peshawar

Swabi

39

3.3. Analysis of GJB2

Twentyfour (24) mutations in GJB2 gene were identified in deaf patients

including indels, missense and nonsense mutations. Among them, five

mutations were indel mutations, 17 were missense mutations and two (2)

were nonsense mutations. The total deaf individuals having mutations in

GJB2 gene were shown in Fig.26.

Figure.26. Prevalence of mutations in GJB2 gene, gender/age wise in deaf


According to our findings, five (5) indel mutations were found in GJB2 gene;

in which three (3) were deletions and two (2) were insertion mutations, i.e.

c.181delA, c.138-138 del T, c.161-162insA, c.324insG and c.del70 (37nt).

The indel mutations in GJB2 gene were given in Table 7.1(a) along with

mutation site/amino acid changes, codon, domain, pathogenicity and

phenotype frequency. Whereas, in Table. 7.1 (b), the gender and age wise

prevalence of these mutations in percent were given. However, the

prevalence wise indel mutations in GJB2 gene were given in Fig.27.

total mutations

Males

Females3-10 yrs (M)

11-17 yrs (M)3-10 yrs (F)

11-17 yrs (F)

0

50

100

150

200195

133

63 56

77

24

39

total mutations

Males

Females

3-10 yrs (M)

11-17 yrs (M)

3-10 yrs (F)

11-17 yrs (F)

40

Table 7.1(a). Indels mutations reported in GJB2 gene in deaf patients Khyber Pakhtunkhwa

S.No. Mutation Name Mutation site Amino acid change Codon Domain Probability score

(HGMD)

Pathogenecity Incidence (%)

1 D46Efs*36 c.138-138 del T Frame shift 46 EC1 1 Pathogenic 0.8

2 N54Kfs*48 c.161-162insA Frame shift 54 EC1 1 Pathogenic 0.8

3 K61Rfs*21 c.181delA Frame shift 61 EC1 1 Pathogenic 0.6

4 E110G fs*5 c.324insG Frame Shift 110 IC2 1 Pathogenic 0.4

5 V27Cfs*8 c.del70(37nt) Frame shift 27 EC1 1 Pathogenic 0.2

41

Table 7.1(b) Indels mutations reported in GJB2 gene gender/age wise in deaf patients, Khyber Pakhtunkhwa

S. No. Mutation

Name

Mutaion site Frequency

(%)

Prevalence (%) Prevalence (%) of deafness in age groups

3-10 years 11-17 years

Males Females Males Females Males Females

1 D46E fs*36 c.138-138 del T 0.8 0.542 0.257 0.227 0.098 0..314 0.158

2 N54K fs*48 c.161-162insA 0.8 0.542 0.257 0.227 0.098 0..314 0.158

3 K61R fs*21 c.181delA 0.6 0.406 0.193 0.170 0.074 0.235 0.118

4 E110G fs*5 c.324insG 0.4 0.271 0.128 0.113 0.049 0.157 0.078

5 V27C fs*8 c.del70(37nt) 0.2 0.135 0.064 0.056 0.024 0.078 0.039

42

Figure.27. Prevalence of indel mutations in GJB2 gene in deaf patients KP, Pakistan

The missense and nonsense mutations and their prevalence in sampled population

were listed in Tables, 7.2(a), 7.2(b), 7.3(a) and 7.3(b) respectively. Among them six

(6) mutations were already known mutations in the world deaf population data

whereas, eleven (11) mutations were recorded as novel variants. However, the

identified two (2) nonsense mutations were already known in world population.

The analyses figures and bioedit data tables of GJB2 gene were listed at Appendix-

1. The known missense/nonsense variants in our finding are given in Fig.28,

whereas, in Fig.29, the novel missense mutations observed in the GJB2 gene of deaf

patients.

Prevalence, 2.80%

D46E del, 0.80%

N54K ins, 0.80%

K61R del, 0.60%

E110G ins, 0.40%

V27C del, 0.20%

Prevalence

D46E del

N54K ins

K61R del

E110G ins

V27C del

43

Table 7.2 (a). Missense/ Nonsense Mutations in GJB2 Gene in deaf Patients of Khyber Pakhtunkhwa (Known)

S.No. Mutation

Name

Mutation

site

3-letter

Codon

Amino acid

change

Codon Domain Probability

Score

Pathogenecity Incidence

%

1 A78S 232G>T GCU→UCU Ala→Ser 78 TM2 0.999 Pathogenic 1

2 D66N 196G>A GAU→AAU Asp→Asn 66 EC1 0.999 Pathogenic 0.8

3 W24* 71G>A UGG→UGA Trp→Stop 24 TM1 1 Pathogenic 0.4

4 c.327G>A 327G>A - None None IC2 1 Pathogenic 0.6

5 A78P 232G>C GCG→CCC Ala→Pro 78 TM2 0.999 Pathogenic 0.4

6 E119* 355G>T GAA→UAA Glu→Stop 119 IC2 1 Pathogenic 0.4

7 W77L 230G>T UGG→UGU Try→Leu 77 TM2 0.999 Pathogenic 0.4

8 W77C 231G>T UGG→UGU Try→Cys 77 TM2 0.999 Pathogenic 0.4

44

Table 7.2 (b). Prevalence of Missense/ Nonsense mutations in GJB2 gene (gender/ age wise) in deaf population Khyber

Pakhtunkhwa (Known mutations)

S. No. Mutation

Name

Mutaion

site

Frequency

(%)




1 A78S 232G>T 1 0.678 0.322 0.284 0.124 0.393 0.198

2 D66N 196G>A 0.8 0.542 0.257 0.227 0.098 0.314 0.158

3 W24* 71G>A 0.4 0.271 0.128 0.113 0.049 0.157 0.073

4 c.327G>A 327G>A 0.6 0.406 0.193 0.170 0.074 0.235 0.118

5 A78P 232G>C 0.4 0.271 0.128 0.113 0.049 0.157 0.073

6. E119* 355G>T 0.4 0.271 0.128 0.113 0.049 0.157 0.073

7 W77L 230G>T 0.4 0.271 0.128 0.113 0.049 0.157 0.073

8 W77C 231G>T 0.4 0.271 0.128 0.113 0.049 0.157 0.073

45

Table 7.3 (a). Missense mutations in GJB2 gene in deaf patients Khyber Pakhtunkhwa (novel mutations)

S.No. Mutation

Name

Mutation

Site

3-letters

Codon

Amino acid

change

Codon Domain Probability Pathogenecity Frequency

(%)

1 K15E 43A>G AAA→GAA Lys →Glu 15 IC1 0.995 Pathogenic 1.2

2 K103N c.309G>T AAA→AAU Lys→Asn 103 IC2 0.999 Pathogenic 0.8

3 c.186C>T c.186C>T - - - EC1 1 Pathogenic 0.4

4 V153I 457G>A GUU→AUU Val→Ile 153 TM3 0.815 Pathogenic 0.6

5 I20F 58A>T AUU→UUU Ile→Phe 20 TM1 0.999 Pathogenic 0.4

6 F115V 343T>G UUU→GUU Phe→Val 115 IC2 0.999 Polymorphim 0.4

7 D46A 137A>C GAU→GCU Asp→Ala 46 EC1 0.999 Pathogenic 0.4

8 V38A 113T>C GUU→GCU Val→Ala 38 TM1 0.999 Pathogenic 0.4

9 c.120A>G 120A>G - None None TM1 0.999 Pathogenic 0.4

10 c.228A>T 228A>T - None None TM4 0.999 Pathogenic 0.4

11 c.240G>A 240G>A - None None TM4 1 Pathogenic 0.4

46

Table 7.3(b). Missense mutations in GJB2 gene in deaf patients Khyber Pakhtunkhwa (novel mutations)

S. No. Mutation Name

Frequency

(%)

Prevalence (%) Prevalence(%) of deafness in age groups

Males Females 3-10 years 11-17 years

Males Females Males Females

1 K15E 1.2 0.813 0.386 0.341 0.148 0.471 0.237

2 K103N 0.8 0.542 0.257 0.227 0.098 0.314 0.158

3 c.186>T 0.4 0.271 0.128 0.113 0.049 0.157 0.078

4 V153I 0.6 0.406 0.193 0.170 0.074 0.235 0.111

5 I20F 0.4 0.271 0.128 0.113 0.049 0.157 0.078

6 F115V 0.4 0.271 0.128 0.113 0.049 0.157 0.078

7 D46A 0.4 0.271 0.128 0.113 0.049 0.157 0.078

8 V38A 0.4 0.271 0.128 0.113 0.049 0.157 0.078

9 c. 120A>G 0.4 0.271 0.128 0.113 0.049 0.157 0.078

10 c. 228A>T 0.4 0.271 0.128 0.113 0.049 0.157 0.078

11 c.240G>A 0.4 0.271 0.128 0.113 0.049 0.157 0.078

47

Figure.28. Missense/nonsense mutations in GJB2 gene in deaf patients KP,

Pakistan

Prevalence, 4%

A78S, 1.00%

D66N, 0.80%

W24*, 0.40% c.327G>A,

0.60%

A78P, 0.40% E119*, 0.40% W77L, 0.40%

W77C, 0.40%

Prevalence A78S D66N W24* c.327G>A A78P E119* W77L W77C

48

Figure.29. Missense mutations in GJB2 gene in deaf patients KP, Pakistan

(Novel mutations)

Prevalence, 5.80%

K15E, 1.20%

K103N, 0.80%

c.186 C>T, 0.40%

V153I, 0.60%

I20F, 0.40% F115V, 0.40%

D46A, 0.40%

V38A, 0.40% c.120 A>G, 0.40%

c.228 A>T, 0.40%

c.240 G>A, 0.40%

Prevalence K15E K103N c.186 C>T V153I I20F

F115V D46A V38A c.120 A>G c.228 A>T c.240 G>A

49

3.4. Analysis of GJB6

We identified ten (10) mutations in GJB6 gene in deaf sampled population. Among

them six variants were indel mutations and four were missense mutations. The

figures and bioedit data tables of GJB6 gene analysis were given at Appendix-2.

The total number of deaf patients, gender and age class wise having mutations in

GJB6 gene in sampled population were given in Fig.30.

Figure.30. Prevalence of mutations of GJB6 gene in gender/age wise in deaf

population KP

The Tables. 8.1(a), shows the indel mutations in GJB6, the mutation site/amino

acid changes, codon, domain, pathogenicity and incidence frequency of these

mutations in GJB6 gene were mentioned. Whereas, in Table. 8.1 (b) (Fig.31), the

gender and age wise incidence of these mutations were given.

MalesFemales

3-10 yrs(M)

11-17 yrs(M)

3-10 yrs(F)

11-17 yrs(F)

0

20

40

60

80

100

123

83

40 35

48

16 24

Males

Females

3-10 yrs (M)

11-17 yrs (M)

3-10 yrs (F)

11-17 yrs (F)

50

Table 8.1(a) Indel mutations recorded in GJB6 gene (Novel Mutations)

S. No. Mutation

Name

Mutaion

site

3-letter

Codon

Amino

acid

change


Score

(HGMD)

Pathogenicity Incidence

(%)

1 N14K fs*21 c.42delC None Frame Shift 14 IC1 1 Pathogenic 2

2 N14T fs*21 c.41delA None Frame Shift 14 IC1 1 Pathogenic 1.8

3 G12V fs*23 c.31delG None Frame Shift 12 IC1 1 Pathogenic 0.6

4 K15N fs*20 c.43delA None Frame Shift 15 IC1 1 Pathogenic 0.4

5 K125N fs*28 c.ins374_375

(16nt)

None Frame Shift 125 IC2 1 Pathogenic 0.2

6 R108N fs*13 c.ins320_321

(19nt)

None Frame Shift 108 IC2 1 Pathogenic 0.2

51

Table 8.1(b). Gender and age wise incidence of indels mutations in GJB6 gene

S. No. Mutation

Name

Mutaion

site

Frequency

(%)




1 N14K fs*21 42delC 2 1.356 0.644 0.569 0.248 0.786 0.396

2 N14T fs*21 41delA 1.8 1.220 0.579 0.512 0.222 0.707 0.356

3 G12V fs*23 31delG 0.6 0.406 0.193 0.170 0.074 0.235 0.118

4 K15N fs*20 43delA 0.4 0.271 0.128 0.113 0.049 0.157 0.078

5 K125N fs*28 c.ins374_37

5 (16nt)

0.2 0.135 0.064 0.056 0.024 0.078 0.039

6 R108N fs*13 C.ins320_3

21 (19nt)

0.2 0.135 0.064 0.056 0.024 0.078 0.039

52

Figure.31. Incidence of indel mutations in GJB6 gene (Novel mutations)

The missense mutations in GJB6 gene of sampled population, were listed in Table

8.2(a), the missense mutations, their mutation sites, aminoacids changes, codon,

domain, pathogenicity and pevalence were mentioned in the table. Furthermore,

the gender/age wise prevalence of missense mutations were given in Table. 8.2

(b).

Prevalence 5%

N14K 2%

N14T 1.80%

G12V 0.60%

K15N 0.40% K125N

0.20% R108N 0.20%

Prevalence

N14K

N14T

G12V

K15N

K125N

R108N

53

Table.8.2 (a): Missense mutations in GJB6 gene (Novel mutations)

S. No. Mutation Name Mutation site 3-letter Codon Amino acid

change


Score(HGMD)

Pathogenicity Frequency

(%)

1 K15Q 43A>C AAA→CAA Lys→Gln 15 IC1 0.986 Pathogenic 1.8

2 A88T 262G>A GCU→ACU Ala→Thr 88 TM2 0.999 Pathogenic 0.4

3 A92D 275C>A GCU→GAU Ala→Asp 92 TM2 0.999 Pathogenic 0.4

4 A149S 445G>T GCU→UCU Ala→Ser 149 TM3 0.999 Polymorphism 0.4

54

Table 8.2(b). Missense mutations in GJB6 gene in gender/ age class wise in deaf patients (Novel mutations)

S. No. Mutation

name

Mutation

site

Frequency

(%)

Prevalence (%) Prevalence(%) of deafness in age groups

Male

Female 3-10 years 11-17 years

Male Female Male Female

1 K15Q 43A>C 1.8 1.220 0.579 0.512 0.222 0.707 0.356

2 A88T 262G>A 0.4 0.271 0.128 0.113 0.049 0.157 0.078

3 A92D 275C>A 0.4 0.271 0.128 0.113 0.049 0.157 0.078

4 A149S 445G>T 0.4 0.271 0.128 0.113 0.049 0.157 0.078

55

The missense mutations in GJB6 gene of deaf sampled population were illustrated

in Fig.32, along with the prevalence and epidemic of individual mutations.

Figure.32. Missense mutations in GJB6 gene (Novel mutations)

3.5. Analysis of Mitochondrial Genes

Three (3) mitochondrial genes were analyzed, i.e. MTRNR1, MTRNR2 and MT-

TV. These genes code 12S rRNA, 16S rRNA and Valine tRNA respectively. These

genes are located adjacent to each other in the mitochondrial genome. We

identified Thirteen (13) mutations in MTRNR1 gene, eleven (11) mutations in

MTRNR2 gene and eight (8) mutations in MT-TV gene respectively. In these

mutations some were also already known and reported in the world population

data whereas, many were novel mutations. In the Tables, 9, 10 and Table.11, the

prevalence, pathogenicity and gender/age wise incidences of these mutations are

listed. However, the analysis of figures and bioedit data tables of MTRNR1,

MTRNR2 and MT-TV genes were given at appendix-3, 4 and appendix-5

respectively.

Prevalence, 3.00%

K15Q, 1.80%

A88T, 0.40% A92D, 0.40% A149S, 0.40%

Prevalence

K15Q

A88T

A92D

A149S

56

The frequency prevalence of mutations in MTRNR1 gene in gender as well as in

age classes were given in Table.9; Fig.33 &Fig.34. Similarly, in case of MTRNR2

gene, the mutations frequency and its prevalence in gender and age groups were

given in (Table.10; Fig.35 & Fig.36). The mutations prevalence of MT-TV gene in

gender/ age wise as well as individual mutations incidence were given in

(Table.11; Fig.37, Fig.38).

57

Table 9. Mutations in MTRNR1 gene, its frequency and prevalence in gender/age wise distribution in deaf patients KP

Pakistan

S. No.

Mutation Name

Mutaion type

Nature Frequency (%)




1 1349 T>G Transversion Homoplasmy 0.8 0.53 0.25 0.222 0.096 0.307 0. 153

2 1420 T>G Transversion Heteroplasmy 4.6 3.08 1.52 1.293 0.585 1.786 0.934

3 1438 A>G Transition Homoplasmy 6 4.06 1.932 1.704 0.774 2.355 1.188

4 1440 G>A Transition Homoplasmy 0.4 0.26 0.13 0.109 0.050 0.150 0.079


6 1492 A>C Transversion Heteroplasmy 0.4 0.26 0.13 0.109 0.050 0.150 0.079

7 1544 A>T Transition Heteroplasmy 0.8 0.53 0.25 0.222 0.096 0.307 0.153


9 1546 A>T Transition Heteroplasmy 0.6 0.40 0.19 0.167 0.073 0.232 0.116

10 1554 G>A Transition Heteroplasmy 0.6 0.40 0.19 0.167 0.073 0.232 0.116

11 1575 T>G Transversion Homoplasmy 0.4 0.26 0.13 0.109 0.050 0.150 0.079

12 1577 T>G Transversion Homoplasmy 0.6 0.40 0.19 0.167 0.073 0.232 0.116


58

Figure.33. Prevalence of mutations in MTRNR1 gene gender/age wise in deaf

population of KP Pakistan

Figure.34. Percentage wise mutations incidence in MTRNR1 gene in deaf patients

KP, Pakistan

Males

Females

3-10 yrs (M)

11-17 yrs(M)

3-10yrs(F)

11-17yrs(F)

0

50

100

150

200

240

162

78 68

94

30

48 Males

Females

3-10 yrs (M)

11-17 yrs (M)

3-10yrs(F)

11-17yrs(F)

A1438G, 6.00%

T1420G, 4.60%

A1544T, 0.80%

T1349G, 0.80%

G1598A, 0.60%

G1554A, 0.60%

A1546T, 0.60%

A1577G, 0.40%

T1575G, 0.40%

A1492C, 0.40%

G1440A, 0.40%

G1442A, 0.40%

A1438G

T1420G

A1544T

T1349G

G1598A

G1554A

A1546T

A1577G

T1575G

A1492C

G1440A

G1442A

59

Table 10. Gender/age wise mutation prevalence in MTRNR2 gene in deaf patients of KP Pakistan

S. No.

Mutation Name

Mutaion type Nature Frequency (%)





2 Ins T 1711 Pathogenic Homoplasmy 0.2 0.135 0.064 0.056 0.024 0.078 0.039

3 1735 A>C Transversion Heteroplasmy 0.6 0.406 0.193 0.170 0.074 0.235 0.118


5 1811 A>G Transition Homoplasmy 1.4 0.949 0.450 0.398 0.173 0.550 0.276

6 1814 A>C Transversion Homoplasmy 0.8 0.542 0.257 0.227 0.098 0.314 0.158

7 del T 1872 Pathogenic Homoplasmy 0.2 0.135 0.064 0.056 0.024 0.078 0.039



10 ins T>1960 Pathogenic Homoplasmy 0.2 0.135 0.064 0.056 0.024 0.078 0.039

11 insG>1990 Pathogenic Homoplasmy 0.2 0.135 0.064 0.056 0.024 0.078 0.039

60

Figure.35. Gender/age wise mutations prevalence in MTRNR2 gene in deaf


Figure.36. Prevalence of mutations distribution in MTRNR2 gene in deaf patients

KP, Pakistan

Males

Females

3-10 yrs(M)

11-17 yrs(M)

3-10 yrs(F)

11-17 yrs(F)

0

20

40

60

87

59

28 25

34

11

17 Males

Females

3-10 yrs (M)

11-17 yrs (M)

3-10 yrs (F)

11-17 yrs (F)

A1811G, 1.40%

G1671A, 0.80%

A1814C, 0.80%

A1735C, 0.60%

G1754A, 0.60%

G1888A, 0.40%

G1899A, 0.40%

1990 insG, 0.20% 1711 insT, 0.20%

1960 insT, 0.20%

1872 delT, 0.20% A1811G

G1671A

A1814C

A1735C

G1754A

G1888A

G1899A

1990 insG

1711 insT

1960 insT

1872 delT

61

Table .11. Mutations in MT-TV Gene of the deaf patients, the mutation type, pathogenicity, frequency and gender/ age

wise prevalence distribution

S. No.

Mutation Name

Mutaion type

Nature Pathogenicity Frequency (%)




1 G1604A Transition Homoplasmy Possibly benign 0.33 0.223 0.106 0.093 0.040 0.129 0.065

2 G1604 T Transversion Heteroplasmy Possibly benign 0.33 0.223 0.106 0.093 0.040 0.129 0.065

3 G1606A Transition Heteroplasmy Confirmed

Pathogenic

0.66 0.447 0.212 0.187 0.081 0.259 0.130

4 T1609G Transversion Heteroplasmy Possibly benign 0.66 0.447 0.212 0.187 0.081 0.259 0.130

5 A1610C Transversion Homoplasmy Possibly benign 0.66 0.447 0.212 0.187 0.081 0.259 0.130

6 A1625C Transversion Homoplasmy Likely benign 0.33 0.223 0.106 0.093 0.040 0.129 0.065

7 G1641T Transversion Homoplasmy Possibly

Pathogenic

0.33 0.223 0.106 0.093 0.040 0.129 0.065

8 G1644A Transition Heteroplasmy Confirmed

Pathogenic

0.33 0.223 0.106 0.093 0.040 0.129 0.065

62

Figure.37. Gender/age wise mutations prevalence of MT-TVgene in deaf

population KP, Pakistan

Figure.38. Mutations prevalence in MT-TV gene in deaf population KP, Pakistan

Males

Females

3-10 yrs(M)

11-17 yrs(M)

3-10 yrs(F)

11-17 yrs(F)

0

5

10

15

20

25

30

35

40

55

37

18 16

21

7

11 Males

Females

3-10 yrs (M)

11-17 yrs (M)

3-10 yrs (F)

11-17 yrs (F)

Prevalence, 3.63%

G1606A, 0.66%

T1609G, 0.66%

A1610C, 0.66%

G1604A, 0.33%

G1604T, 0.33%

A1625C, 0.33% G1641T, 0.33%

G1644A, 0.33% Prevalence

G1606A

T1609G

A1610C

G1604A

G1604T

A1625C

G1641T

G1644A

63

Chapter 4

DISCUSSION

Hearing loss or deafness is a major physical disability which harms the quality of

life and handicaps the subjects socially. According to an estimate, one out of

thousand in people are affected with deafness (Khan et al., 2016; Yu et al., 2013;

Cunningham et al., 2005; Kemper & Downs, 2000; Adams et al., 1999). Many

genetic and environmental factors are thought to contribute to deafness. For

instance, a number of genes are known to be involved in the hearing mechanism,

almost 130 genes are identified for having role in normal hearing. Mutations in

these genes causing hearing losses up to various extents depending upon the

involvement of genes and their associated environment. The most frequent

causative genes in order of frequency are GJB2, SLC26A4, MYO15A, OTOF, CDH23

and TMC1. The GJB2 gene is the most important gene, which code for channel

protein connexin-26 and is susceptable to frame shift mutations, resulting in

impaired hearing mechanism. The incidences of different gene mutations in

hearing loss in developed countries were given at Table.2 (Rabionet et al., 2000;

Morell et al., 1998; Denoyelle et al., 1997; Hilgert et al., 2009).

The results presented over here are based on the screening of sum 1500 deaf

patients recored from Abbotabad, Bannu, Charsadda, Haripur, Mansehra,

Mardan, Peshawar, Swabi and Swat districts of Khyber Pakhtunkhwa Pakistan.

The screening was done for five genes involved in hearing loss; among two of

these genes (GJB2, GJB6) were nuclear and the three, i.e. MTRNR1, MTRNR2 and

MT-TV were mitochondrial in origin. Our results on the incidence of mutations in

targeted individuals of the associated communities of the deaf patients and

information based on their culture, religious beliefs, customs, marriage practices,

literacy, economical situations and conservative status of the marriages, especially

first cousin marriages were also taken into consideration. We noted that the

research area is consisting of male dominanated society. Among deaf patients, the

64

number of males, female patients and their district wise age group provided clues

about the increase of awareness and literacy rate and vice versa. We presume that

the enrollement of deaf girls in the special schools are proportionately reflecting

the socioeconomic background of the people residing in that particular district.

The less number of female patients enrolled in a special schools means that the

people of that specific area are culturally conservative, less educated and low

earning. Similarly, the age groups of deaf patients in different districts give a clue

that where the young and small age group deaf patients are reported extensively,

means the literacy rate of the people of the area is low, having no idea about the

demerits of cousin marriage practices and hence the, small age group (3-10 years)

deaf patients children are greater in number in the special schools, as a result of

inbreeding depression. On the other hand, when the deaf patient students of older

age groups (11-17 years) are reported extensively in an area as compare to the

small age group children, signifies that the small age group individuals are

minimum or physically normal, gave a clue that the people of the area are more

educated, civilized and having the awareness of the demerits of cousin marriages

and appreciate cross marriages.

Five gene mutations which were confirmed for involvement in the hearing

impairment were extracted and amplified through optimized DNA protocol,

extracted from the epithelial cells of oral swab through a simple and economically

cheap procedure (Aidar & Line, 2007). Usually the DNA is extracted from the

blood cells but the DNA extraction from the blood cells faces some technical

problems. Blood samples collection is difficult as the patients are usually highly

conserved, having less awareness and avoiding giving blood. Also few blood

components inhibit PCR when the DNA is not extracted properly (Guescini et al.,

2008). On the other hand the advantage of collection of DNA from oral swab has a

merit as the saliva contain high amount of mucin and is rich in epithelial cells

which are extracted properly without contaimination (Schenkles et al., 1995).

65

Out of the 1500 DNA samples of deaf patients analyzed for five genes, we

observed that the total number of mutations recorded in 700 deaf patients

contributing 47% share to the deaf population. Among the analyzed samples, 1017

were males and 483 were females. All the collected samples of deaf patients belong

to non-syndromic hearing loss, as these deaf patients are studying only in special

schools for deaf students. The total number of deaf patients, gender as well as the

age classes and the mutations found in them were given in Figure.17.

The age and sex wise groups of these patients were 3-10 and 11-17 years, males

and females. Among these, the total number of males 3-10 years age group were

427 and 11-17 years males were 590 whereas, the females of 3-10 years age group

were 186 and 11-17 years age females were 297 in numbers. Among the male

patients, the distict Swat possess, the highest value (17.40%), whereas, the lowest

number of male patients were in district Mansehra, 2.46% (Figure.19). Similarly,

the highest numbers of female deaf patients were reported in District Haripur,

34.37% and the lowest number of females patients were reported in Swat district,

4.76% (Figure. 20). The overall informations of gender and age classes of deaf

patients in Khyber Pakhtunkhwa were given in Table.6.

Overall, twenty four 24 mutations were found in GJB2 gene in deaf patients with

over all prevalence of 1.6%. Among them five mutations were indel (three are

deletions and two are insertions)(Table: 7.1(a), 7.1(b) Figure, 27), 17 were missense

and two(2) were nonsense mutations (Table.7.2 (a), 7.2(b) Figure.28).The

contribution of GJB2 gene mutations in 47% deaf population was 13% (Figure.18).

We found the indel mutations c.138delT, c.170del (37nt), c.161-162insA and

c.324insG in deaf patients were also already reported (Known) in the world

populations (Hayashi et al., 2011; Choi et al.,2009; Dai et al.,2009; Mani et al.,2009;

Yilmaz& Christofori,2009; Putcha et al., 2007; snoeckx et al.,2005; Richard et al.,

2004). However, the c.181delA mutation is a novel mutation in our studied

66

samples (Figure.30 & Figure.31). The missense/nonsense identified mutations in

studied samples are p.A78S, p.A78P, p.E119*, p.W77L, p.W77C, p.D66N, and

p.W24*. These mutations are also already known in the world deaf population

(Putcha et al., 2007; li et al., 2005; Wu et al., 2004; Hwa et al., 2003; Maestrini et al.,

1999; Kelly et al., 1998).

However, the missense mutations p.K103N, p.V153I, p.I20F, p.F115V, p.D46A,

p.V38A, c.120A>G, c.186C>T, c.120A>G, c.228A>T and c.240G>A are novel

mutations reported in sampled population (See Tables 7.3(a), 7.3(b) Figure.28). All

of these mutations are pathogenic mutations as there mutation taster probability

score is about ‘1’ at mutation taster score database and were listed at human gene

mutations database (HGMD). All these mutations change the amino acid sequence,

causing frame shift mutations or splice site changes and may modify the protein

structures.The physiochemical properties of these mutations at aminoacid level,

results in abnormal protein molecules and also leading to NMD (non- mediated

protein decay. These mutations along with theire calculated physiochemical

properties are listed at HGMD.

Among the 700 deaf patients, the 195 individuals have mutations in GJB2 gene.

Among them 133 were males and 63 were females. Furthermore, in the age group

of 3-10 years males, the 56 individuals have mutations whereas, in age group 11-17

years, the 77 patients have mutations in GJB2 gene. However, in case of females’

age group 3-10 years, the 24 patients have mutations, whereas, in the age group of

11-17 years females, 39 deaf patients have mutations in GJB2 gene (Figure.28). The

known and novel mutations (indel, missense/nonsense) found in GJB2 gene are

given in Figures, 29-31. We did not found the confirmed mutations in GJB2 gene in

the world deaf populations that are 35delG, 167delT, 235delC in our studied

samples. But we have found some other mutations, many of them are (Known) in

the world populations and some are novel mutations (Tables 7.1(a), 7.1(b), 7.2(a),

7.2(b), 7.3(a), 7.3(b) and (Figures, 26-29).

67

The GJB6 gene comprises of five exons. The exon1 is functional coding exon and

codes for connexin-30 channel protein whereas; the other four exons are non-

functional. We have amplified the 1st coding exon as it has only the normal protein

product connexin-30. Ten mutations were found in the exon1 of GJB6 gene with

overall prevalene of 0.66% in deaf patients. However, The contribution of GJB6

gene mutations in 47% deaf population was 8.20 %( Figure.18) in selected deaf

patients. Among these mutations six (6) were indels mutations i.e. c.41delA,

c.42delC, c.43delA, c.31delG, c.ins 374-375(16nt) and c.ins 320-321(19nt). These

mutations cause frame shift mutataions (Table 8.1 (a) (Figure.31). The indel

mutations, c.41delA, c.42delC, c.43delA, c.ins 374-375(16nt) and c.ins 320-321(19nt)

are novel mutations whereas, the c.31delG is also known in the world deaf

population (Lamartine et al., 2000). Similarly, four (4) mutations i.e. p. K15Q,

p.A88T, p.A92D and p.A149S are missense mutations and are novel (Tables. 8.1(a),

8.1(b), 8.2(a), and 8.2(b) (Figure,32).

The GJB6 gene mutations were reported in 123 deaf patients. Among them 83 were

males and 40 were females. So far as the age groups concerned; the 35 male

individuals having 3-10 years age have mutations, whereas, the 48 male patients

having 11-17 years age, have mutations in GJB6 gene. However, in the case of

female patients of 3-10 years, the 16 patients and the female patients of 11-17 years,

the 24 patients have mutations in GJB6 gene as given in Fig.32. The indel and

missense mutations found in GJB6 gene mutations (novel as well as known) are

shown in Figure, 31 & Figure, 32.

We have found 13 mutations in MT-RNR1 gene of deaf patients with over all

prevalence of 0.8% in deaf population. However, the contribution of MTRNR1

gene mutations in 47% deaf population is 16%(Table.9, Figure.18).The mutations

found in MTRNR1 gene were include, 1349 T>G, 1420T>G, 1438A>G, 1440 G>A,

1442 G>A, 1492 A>C, 1544 A>T, 1545 G>A, 1546 A>T, 1554 G>A, 1575 T>G,

1577A>G and 1598 G>A. Among these mutations, the 1438A>G and 1420T>G are

68

more prevalent comprising of 6% and 4.6% respectively in our research data. The

1349T>G, 1438A>G, 1440G>A , 1442G>A and 1598G>A are also known in world

deaf population (Lu et al., 2010; Rydzanicz et al.,2009; Konings et al., 2008; Chen et

al.,2008; Wang et al.,2006; Wang et al., 2006; Starikovskaya et al.,2005; Li et al., 1998).

However, the mutations 1420T>G, 1492 A>C, 1544 A>T, 1545 G>A, 1546 A>T,

1554G>A, 1575 T>G and 1577A>G are novel mutations in our data (Table.9,

Figures.33 & Figure.34).

In MTRNR1 gene, the total 240 deaf individuals have mutations. Among them, the

163 were males and 77 were females. However, in the age group of 3-10 years

males were 68 and the males’ patients of 11-17 years were 94. Similarly, the

females of age group 3-10 years were 30 and 11-17 years age were 48 (Figure.33 &

Figure.34).

It is very important to note that the C1494T and A1555G mutations are not

reported in our samples at all, which are confirmed mutations in MTRNR1 gene in

world deaf population data. These mutations are responsible for syndromic and

non-syndromic hearing loss. They cause aminoglycoside induced and non-

syndromic hearing loss through bringing the change in conformational structure

of 12S rRNA, which become closely resemble with bacterial 12S rRNA after

mutations. The bacterial 12S rRNA is a target region to antibiotics such as

aminoglycosides gentamicin, kanamycin, and streptomycin and hence the after the

antibiotic treatment these drugs targets the human 12S rRNA instead of bacterial

r RNA, damages it and as a result the concerned cells can not perform their normal

function and goes to programmed cell death known as apoptosis.

In our study, we reported the G1554A, which is very close to A1555G mutation,

we may say that this possible new mutation may cause aminoglycoside induced

hearing loss on molecular basis as this study area is not previously screened for

69

any mitochondrial genes related to deafness. The different tribes living together in

Khyber Pakhtunkhwa also prove the concepts of private mutations (Ruiz-Pesini &

Wallace 2006; Nance, 2003).

In MT-RNR2 gene, 11 mutations were found with over all prevalence of 0.73% in

deaf patients. Whereas, the contribution of MTRNR2 gene mutations in 47% deaf

patients samples was 5.80%.

The mutations found in MTRNR2 gene were, 1671 G>A, 1735 A>C, 1754 G>A,

1811 A>G, 1814 A>C, 1888 G>A, 1899 G>A, 1711>insT, 1872>delT, 1960>insT and

1990insG. Nine mutations of these were novel mutations whereas, two were

known in the world deaf population data (Table.10, Figures.35 & Figure.36). In

addition to point mutations we also detected some indels mutations as well in the

MT-RNR2 gene, as listed. These indels mutations are rarely reported in

mitochondrial DNA of world data and are novel in studied samples.

The variants 1811A>G and 1888G>A were also known in the world deaf

population data (Janssen et al., 2006; Zhao et al., 2004; Lehtonen et al., 2003; Maasz

et al., 2003; Herrnstadt et al., 2002). However, the mutations, 1671 G>A, 1814 A>C,

1735 A>C, 1754 G>A, 1899 G>A, 1711>insT, 1872>delT, 1960>insT and 1990insG

are novel mutations in our studied samples.

The MTRNR2 gene mutations were reported in 87 deaf individuals where 59 were

males and 28 were females. In the case of males, the age groups 3-10 years were 25

and the males of 11-17 years age were 34. However, the females having age group

3-10 years were 11 and the females aged 11-17 years were 17 in number (Figure,35

& Figure,36).

In MT-TV gene, we found eight (8) mutations with over all prevalence of 0.53%in

deaf population. However, the contribution of MT-TV gene mutations in 47% deaf

patients was 3.67 %( Table.11). This gene is less involved in hearing loss as

70

compared to other mitochondrial genes in our research patients’ samples. The

mutations found in MT-TV gene were, 1604G>T, 1604G>A, 1606G>A, 1609 T>G,

1610 A>C, 1625 A>C, 1641 G>T, and 1644G>A. The mutations 1606G>A and

1644G>A were also known in world deaf population data associated with other

diseases as well (Fraidakis et al., 2014; Bannwarth et al., 2013; Nishigaki et al., 2010;

Tanji et al., 2008; Menotti et al., 2004; Sacconi et al.,2002; Tiranti et al., 1998).

However, the mutations, 1604G>T, 1604G>A, 1609 T>G, 1610 A>C, 1625 A>C and

1641 G>T were novel mutations in MT-V gene in our studied deaf patients

samples (Figure.37 & Figure.38). In 55 deaf individuals, mutations were found in

MT-TV gene, where 37 were males and 18 were females. Among the males, the 3-

10 years age group were 16 and in males aged 11- 17 years were 22 deaf

individuals. Similarly, in females’ age 3-10 years were 7 and in females aged 11-17

years, were 11 deaf patients (Figure.37 & Figure.38). Some samples have only one

mutation whereas; many samples have more than one mutation per sample.

Unlike the unpredictable structures of rRNA, the scoring system for tRNA is

available. According to the scoring system of valine tRNA, the variants reported in

deaf patients samples are classified as, confirmed pathogenic, possibly pathogenic,

possibly benign and likely benign mutations (Table.11). The molecular mechanism

and functional studies of these new reported variants in MT-V gene will further

describe their role in hearing mechanism and so in hearing loss.

Some identified mutations in the concerned deafness related genes in our research

ata were different from the known world deaf population. This is because, as this

area was not previously screened for any deafness related genes. Moreover, the

different tribes living together in Khyber Pakhtunkhwa further proves the concept

of private mutations in this area as private mutations discussed in other world

reported data (Jacobs et al., 2005; Nance, 2003). As a whole, the contribution of

targeted genes mutations in our project shows that as whole 47% deaf populations

71

have mutations in these genes, whereas, the remaining deaf samples may have

other deafness related genes mutations (Figure.18).

From the above study, it is concluded that the indels mutations were found in the

population during the screening of deaf patients data, which leads to frame shift

mutations, causing splice site changes, followed by non-mediated protein decay

(NMD) and hence cause a drastic change in protein structure. There were some

substitutions of nucleotides which in turn replaced some amino acids or terminate

the nucleotide chain, results into conformational changes in protein structure and

causing hearing loss.

It also concluded that there are more than 130 genes involved in hearing

mechanism. The screenings of other genes are also important for the best

elucidating hearing loss in Khyber Pakhtunkhwa Pakistan. The better genetic

counselling in the cousin marriages as well as proper diagnosis may further

improve the treatment through protein therapy and the application of gene

therapy.

72

Recommendations

Our study was based on scereening of 1500 DNA samples from the deaf

populations of KP wherein 1017 and 483 samples were collected from male

and female, respectively. The higher incidence of deafness in male gender is

due to the male dominant society and conservative culture of the area, the

female there were not allowed to go to schools, and also the collection of

samples from the girls is generally not allowed. It is imperative to launch a

proper awareness compaign for taking care and councelling about the

inherited diseases, cousin marriages and the demerits of conservative

culture.

We concluded that 700 of the screened deaf patients almost 47% had

mutations in GJB2, GJB6, MTRNR1, MTRNR2 and MT-TV genes. Whereas,

the remaining 53%patients, might be having other genes mutations, which

need proper screening. Hence the diagnosis and screening of the deaf

people of KP through other deafness related genes is also recommended.

We reported many variant SNPs and mutations in the deaf population of

the area, so it is recommended that the screening of normal population as

well as deaf population as a whole is necessary for the identification of

pathogenic mutations and their physiological role in causing deafness in

any stage of life.

Personalized whole genome and trascriptomic analyses of the population

are imperative for creating molecular database for deafness related

information at all levels.

Further use of the recent technologies, analytical softwares, and designing

system for personalized medicine needs to be introduced.

Though the information we got from 3 mitochondrial genes are more

imprrtant but needs further elaboration through the whole genome

sequencing of mitochondria.

73

Genetic councelling needs to be introduced for minimizing hearing losses in

the population.

The ethical issues related to gene cloning and gene therapy needs profer

strategy for curing the hearing losses and other genetic disorders.

74

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93

Appendix-1 Tables and Figures of GJB2 gene analysis of deaf patients KP Pakistan

94

Appendix - 1

Table. Bioedit file of GJB2 gene of deaf patients of Hazara Division showing

insertion, deletion, missense and nonsense mutations.

95

Table. Bioedit file of GJB2 gene mutations from Khyber Pakhtunkhwa showing deletion mutations in the deaf patients sample ID. Ab83 and sample ID. Ab60 as

well as many missense/Nonsense mutations.

96

Table. Bioedit file of GJB2 gene of deaf patients from Khyber Pakhtunkhwa showing some insertion mutations as well as Missense/Nonsense mutations.

97

Table. Bioedit file of GJB2 gene of deaf patients from Khyber Pakhtunkhwa

showing insertion, deletion, missense and nonsense mutations.

98

Table. Bioedit file of GJB2 gene of deaf patients from Khyber Pakhtunkhwa showing insertion, deletion, missense and nonsense mutations.

99


100

Table. Bioedit file of GJB2 gene of deaf patients from Khyber Pakhtunkhwa

showing insertion, deletion, missense and nonsense mutations.


101

Fig. Different Peaks showing the normal as well as mutated Peaks of Exon11 of GJB2

Gene in Deaf Patient Sample ID.G2AB2

102

Mutation T343G (Homozygous) Mutation G355T (Homozygous)

Fig. Alignment of GJB2 Gene in Patient sample ID Ab114 and its mutation Peaks

shows the mutations T343G and G355T. Both are homozygous mutations. The black line among the peaks represents the position of mutations.

Fig. Protein Alignment of GJB2 Gene of a Deaf Patient sample ID Ab114.

Mutations K15E, F115V and 120delE can be seen.

103

Fig. DNA sample Alignment of GJB2 Gene Deaf Patient Sample ID. Ab78. The

mutation was represented by green colour where guanine is replaced by adenine. The black line shows the position of the mutation.

Fig. Protein alignment of GJB2 gene of a deaf patient sample ID Ab78 showing the mutation V153I highlighted.

104

CLUSTAL 2.0.12 multiple sequence alignment

CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC

ab50 MDCGTLQTILGGVNKHSTSFGKIWLTVLFIFRIMILAAGAKEVWGDEQADFVCNTLQPGC

** ****************:****************...*********************

CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKG---EIKSEFKD

ab50 KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGGDKEIGHRGDQ

************************************************* ** . .:

CRs IEEIKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGF-SMQRLVKCNAWPCPN

ab50 NPEGPHRRLPVVDLHKQHLLPGHLRSRLHVRLLCHVRRLLHAAAGEVQRLALS------Q

* ::: : . .. : :. : :* ::: . .:***. . :

CRs TVDCFVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

ab50 HCGLLCVPAHEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

. : . *****************************************

Fig. Protein Alignment of GJB2 gene of a deaf patient sample ID Ab50, the

mutations and frame shift mutation positions are highlighted.



ab52 MDWSSRCRSWG-VTNTPPALERSSSPSSSFFALSSLWLQRR--CGEMSRPTLSATPCSQA

***.: * *.: ..:: : . :* : * : : *: . :. * . .

CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE

ab52 AERVLRSLLPHLPHPAMGPAADLRVHASAPSGHARGLPETEEEEVHQGGDKEIGHRGDQN

: :* . : .: . . . *::. . *: : .:. ::

CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC

ab52 PEGPHRRLP---VVDLHKQHLLPGHLRSRLHVRLLCHVRRLLHAAAGEVQRLACPNTVDC

: : *: .. :: :.: : : : : : .*******

CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

ab52 FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

***********************************************

Fig. Protein alignment of GJB2 gene of a deaf patient sample ID Ab52, the

mutations and frame shift mutation positions are highlighted.

105



ab114 MDWGTLQTILGGVNEHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC

**************:*********************************************


ab114 KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEVKDIE-

******************************************************.****


ab114 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC

************************************************************



***********************************************


mutations are highlighted.



ab20 MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC

************************************************************

CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHV-AYRRHEKKRKFIKGEIKSEFKDIE

ab20 RTCATITTSPSPTSGYGPCSSSCPRQRSWPCTWPTGDMRRRGSSSRGRRVNLRTSRRSMP

:. . * . . . : : . **: .. : : ::::. :.:

CRs EIKTQKVRIEGSLWWTYTSSIFFR-VIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTV

ab20 RRSASKAPCGGPTQAASSSGSSSKPPSCTSSMSCTMASPCSGWSATPGLVPTLW----TA

. .:.*. *. : :*. : ::: .: .*:* : . * *.

CRs DCFVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

ab20 LCPGPRRRLSSQCSLQCLEFASCMSLN-----CVICLDIVLGSQKSQFX

* .* .: :: : .:. * * : : *..*. .*


mutations and frame shift mutation position are highlighted.

106




************************************************************


ab36 RTCATITTSPSPTSGYGPCSSSCPRQRSWPCTWPTGDMRRRGSSSRGRRVNLRTSRRSMP

:. . * . . . : : . **: .. : : ::::. :.:


ab36 RRSASKAPCGGPTQAASSSGSSSKPPSCTSSMSCTMASPCSGWSATPGLVPTLW----TA

. .:.*. *. : :*. : ::: .: .*:* : . * *.



* .* .: :: : .:. * * : : *..*. .*

Fig. Protein alignment of GJB2 gene of a deaf patient sample ID Ab36, the frame

shift mutation position is highlighted.

CLUSTAL 2.0.12 multiple sequence

CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQ---ADFVCNTLQ

ab39 MDWGTLQTIXGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVGEMSRPTLSATPCSQAA

********* ********************************* .: : *.

CRs PGCKNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKD

ab39 RTCATITTSPSPTSGYGPCSSSGPRQRSWPCTWPTGDMRGRGSSSRGRRVN--LRTSRRS

* .: . . .: : * *. : .: * ..: : :: :::. :.

CRs IEEIKTQKVRIEGSLWWTYTSSIFFR-VIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPN

ab39 KPRRSASKAPCGGPTQAASSSGSSSKPPSCTSSMSCTTASPCSGWSAT----PGLVPTLR

. .:.*. *. : :*. : ::: . .*:* . * .

CRs TVDCFVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

ab39 TALCPGPRRRLSSQCSLQCLEFA-SCMSLN----CVICLDIVLGSQKSQFX

*. * .* .: :: : .: *: ** * : : *..*. .*

Fig. Protein alignment of GJB2 gene of a deaf patient sample ID Ab39, the frame

shift mutation position is highlighted.

107



ab70 MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEGWGDEQADFVCNTLQPGC

****************************************** *****************


ab70 RTCATITTSPSPTSGYRPCSSSCPRQRSWPCTWPTGDMRRRGSSSRDRRVNLRTSRRSKP

:. . * . . . : : . **: .. : : ::::. :.


ab70 RRSASKAPCGGPTQAASSSGSSSKPPSCTSSMSCTTASPCSGWSATPGLVPTLW----TA

. .:.*. *. : :*. : ::: . .*:* : . * *.



* .* .: :: : .:. * * : : *..*. .*

Fig. Protein alignment of GJB2 gene of a deaf patient sample ID Ab70, the indel

mutations and frame shift mutation position are highlighted.




************************************************************


ab42 KNVCYDHYFPISHIRLWSLQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE

*****************:******************************************


ab42 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMNDGFSMQRLVKCNAWPCPNTVDC

************************************* **********************



***********************************************

Fig. Protein alignments of GJB2 gene of a deaf patient sample ID Ab42, the

missense mutations are highlighted.

108



ab43 MDWVTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC

*** ********************************************************


ab43 KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE

************************************************************


ab43 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYIFYVMYDGFSMQRLVKCNAWPCPNTVDC

********************************:***************************



***********************************************

Fig. Protein alignments of GJB2 gene of a deaf patient sample ID Ab43, the




ab101 MDWGTLQTILGGVNKHSTSIGKIWITVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC

************************:***********************************


ab101 KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEVKDIE-

******************************************************.****


ab101 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC

************************************************************



***********************************************

Figure. Protein alignment of GJB2 gene of a deaf Patient sample ID Ab101, the

missense and deletion mutations are highlighted.

109




************************************************************


ab106 XNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE

***********************************************************


ab106 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNGWPCPNTVDC

**************************************************.*********



***********************************************

Figure. Protein alignment of GJB2 gene of a deaf patient sample ID Ab106, the

nonsense and missense mutations are highlighted.



10S MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVAAAKEVWRDEQADFVCNTLQPGC

*************************************.****** ***************


10S KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE

************************************************************


10S IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC

************************************************************


10S FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

***********************************************

Figure. Protein alignment of GJB2 gene of a deaf patient sample ID 10S, the


110



128AB MDWGTLQTILGGVN-HSTRIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC

************** *** *****************************************


128AB KNVCYDHYFPISHIRLWSLQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE

*****************:******************************************


128AB IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLGKCNAWPCPNTVDC

********************************************** *************


128AB FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

***********************************************

Figure. Protein alignment of GJB2 gene of a deaf patient sample ID 128AB, the

deletion and missense mutations are highlighted.



30S MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFGCNTLQPGC

*************************************************** ********


30S KNLCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE

**:*********************************************************


30S IKTQ-VRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC

**** *******************************************************


30S FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX

***********************************************

Fig. Protein alignment of GJB2 gene of a deaf patient sample ID 30S, the missense

and deletion mutations are highlighted.

111

Appendix-2 Figures and tables of GJB6 gene analysis of deaf patients KP Pakistan

112

Appendix.2

Table. Bioedit File of GJB6 gene mutations in deaf patients of Khyber Pakhtunkhwa shows many indel and missense/Nonsense mutations. The deletion and insertion mutations can be seen prominently in the Table

.

113

Table. Bioedit File of GJB6 gene mutations in deaf patients of Khyber Pakhtunkhwa shows many indel and missense/Nonsense Mutations. The two

prominent insertion mutations can be seen in the samples ID. G6CH18 and G6CH35.

114

Figure. Sequence of exon1 Peaks of GJB6 gene of deaf patient sample ID G6CH28

115

Figure. Exon1 of GJB6 gene sequence peaks of deaf patient sample ID. G6CH31

116


117


.

118

Fig. Nucleotide alignment of exon1 of GJB6 gene of a deaf patient sample ID G6CH3, the indel mutations are highlighted with green colour.

.

Fig. DNA sample alignments and mutations G445T and A473T in exon1 of GJB6 gene in deaf Patient sample ID. G6CH28 are shown by highlighting with green colour whereas the mutation positions in the peaks are indicated by black bars.

119

Fig. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH23, the

nonsense and missense mutations are highlighted.

Figure. Nucleotide alignment of GJB6 gene of a deaf patient sample ID G6CH28, the nonsense and missense mutations are highlighted.

120


Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC

G6CH4 MDWGTLHTFIGGVNXHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQDDFVCNTLQPAC

************** *********************************:*********.*

Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED

G6CH4 KNVCYDHFFPVSHIRLCPLQLIFVSTPTLLVDMHVAYYRHETTRKFRRGEKRNDFKDIED

**************** .*********:*** ****************************

Crs IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC

G6CH4 IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC

************************************************************

Crs FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK

G6CH4 FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK

************************************************************

Crs QNEMNELISDSGQNAITGFPSX

G6CH4 QNEMNELISDSGQNAITGFPSX

**********************

Figure. The protein alignments in GJB6 gene deaf patient sample ID.G6CH4

showing the K15X, E49D, A78P, A88T and A92D mutations.



G6CH6 MDWGTLHTFIGGVXXHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC

************* *********************************************


G6CH6 KNVCYDHFFPASHIRLWALQLIFNSTPELLVAMHVAYYRHETTRKFRRGEKRNDFKDIED

**********.************ *** ********************************



************************************************************



************************************************************



Figure. Protein alignments in GJB6 gene deaf patient sample ID.G6CH6 showing

the N14X, K15X, V71A, V84N and A88E mutations.

121



G6CH7 MDWGTLHTFIGGVXQHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC

************* :*********************************************


G6CH7 KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED

************************************************************



************************************************************



************************************************************



**********************

K15Q

Figure. Protein alignments in GJB6 gene deaf patient sample ID.G6CH7.The

mutation K15Q is highlighted.



G6CH10 MDWGTLHTFIG-VSQHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC

*********** *.:*********************************************


G6CH10 KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDID-

**********************************************************:



************************************************************



************************************************************



**********************

Figure. Protein alignments in GJB6 gene deaf patient sample ID.G6CH10.The

mutations are highlighted.

122


Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFR--VMILVVAAQEVWGDEQEDFVCNTLQP

G6CH3 MDWGTLHTFIGGCHNTPPASGRCGSQSSLFSESSSWWLPRKCGVTSKRTSSATHCNRDAK

************ :: ..: *: :: . * . . .. **

Crs GCKNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDI

G6CH3 MCAMTTFSR-CPTSGCGPSSSSSPPQRCWWPCMWPTTGTKPLASSSEERRGMISKTYMTL

* . :.: *.* : . : .. :: : *** : : :

Crs EDIKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLV

G6CH3 KSRRFGRGRCGG----RTPAASFSESSLKQPLCMCFTSFTMGTTCPGCN-VGLTPAPTLL

:. : : * * .:: * . :: .: * : * * *: *.*.*:

Crs DCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKE

G6CH3 TALFLGQQRRPCLPFLFLRLFACCLTWQSCATCCKCVLGDQREHRRKKITPIMPRRVSRM

.:: .:. :.:::: . *: : . * : *. :* : *.:. :

Crs SKQNEMNELISDSGQNAITGFPSX

G6CH3 KMSFQIVVKMQSQVSQAX------

. . :: :... .:*

Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH3, the

frame shift and indel mutations were highlighted.



G6CH4 MDWGTLHTFIGGVNNTPPASGRCGSQSSLFSESSSWWLP--RKCGVTSKTTSSATHCNRH

**************: ..: *: :: . :. . * .: . *


G6CH4 AKMCAMTTFSRCHTSGCAPS--SSSSPPQRCWWTCMWPTTGTKPLASSGEERRGMISKTR

::* *. .* * . *:*. : *. **:*..: .


G6CH4 TLKSRRFGRGRCGGRTPAASFSESSLKQPLCMCFTSFTMGTTCPGCN-VGLTPAPTLLTA

*.: .* .:: * . :: .: * : * * *: *.*.*: .


G6CH4 LFLGQQRRPCLPFLFLRLFACCLTWQSCATCCKCVLGDQREHRRKKITPIMPRRVSRMKM

:: .:. :.:::: . *: : . * : *. :* : *.:. : .


G6CH4 SFQIVVKMQSQVSQAX------

. :: :... .:*


frame shift and deletion mutations were highlighted.

123



G6CH6 MDWGTLHTFIGGVNTLHQHREGVDHSHLYFPSHDPRGGCPGSVG--RARGLRLQHTATGM

**************. * : ::: .. .* . ..: : .*


G6CH6 QKCVLPLFPGVPHP--------AVGPPADLQLHPRAAGGHACGLLQARNHSQVQARREEE

:: * *.* *..** * * * *...: : : *:

Crs IKKQK-VRIEGSLWWTYTSSIFFRIIFEAAF--MYVFYFLYNGYHLPWVLKCGIDPCPNL

G6CH6 FQRHRGHKAEGSDRGVAVVDVHQQHLFPNHLSSLYVCVLLPLQWVPPALGVEMWDPLPQP

::::: : *** . . .:. : :* : :** :* : * : ** *:

Crs VDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALK

G6CH6 CLLYFANREDR----VYHFYDFCVCDLHAARGRVVLPAAESVFEIKESTDAKKSPQSCPK

:::. :: :: : :* * . * .. . *.: *: *: . *

Crs ESKQNEMNELISDSGQNAITGFPSX-

G6CH6 GEAENEADFRWSKCN----HRFPKLX

. :** : *... **.

Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH6, the frame shift and indel mutations were highlighted.



G6CH7 MDWGTLHTFIGGVTTLHQHREGVDHSHLYFPSHDPRGGCPGSVG--RARGLRLQHTATGM

*************.. * : ::: .. .* . ..: : .*


G6CH7 QKCVLPLFPGVPHP--------AVGPPADLRLHPSAAGGHACGLLQARNHSQVQARREEE

:: * *.* *..** * * * *...: : : *:



::::: : *** . . .:. : :* : :** :* : * : ** *:



:::. :: :: : :* * . * .. . *.: *: *: . *



. :** : *... **.

Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH7, the frame shift and indel mutations were highlighted.

124




*************.. * : ::: .. .* . ..: : .*


G6CH11 QKCVLPLFPGVPHP--------AVGPPADLRLHPSAAGGHACGLLQARNHSQVQARIEEL

:: * *.* *..** * * * *...: : : *



::::: : *** . . .:. : :* : :** :* : * : ** *:



:::. :: :: : :* * . * .. . *.: *: *: . *



. :** : *... **.

Figure. Protein alignments of GJB6 gene of a deaf Patient sample ID G6CH11, the frame shift and indel mutations were highlighted.




*************.. * : ::: .. .* . ..: : .*


G6CH13 QKCVLPLFPGVPHP--------AVGPPADLRLHPSAAGGHACGLLQARNHSQVHAMREEE

:: * *.* *..** * * * *...: . *:



::::: : *** . . .:. : :* : :** :* : * : ** *:



:::. :: :: : :* * . * .. . *.: *: *: . *



. :** : *... **.


frameshift and indel mutations are highlighted.

125




*************.. * : ::: .. .* . ..: : .*



:: * *.* *..** * * * *...: : : *:



::::: : *** . . .:. : :* : :** :* : * : ** *:



:::. :: :: : :* * . * .. . *.: *: *: . *



. :** : *... **.


frameshift and indel mutations are highlighted.




*************.. * : ::: .. .* . ..: : .*



:: * *.* *..** * * * *...: : : *:



::::: : *** . . .:. : :* : :** :* : * : ** *:



:::. :: :: : :* * . * .. . *.: *: *: . *



. :** : *... **.

Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH15, the frameshift and indel mutations are highlighted.

126


Crs MDWGTLHTFI-GGVNKHSTSIGKV--WITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQ

G6CH18 MDWGTLRTFIRGGQQTLNPHIGRCGSQSSLFSHLSSWWLPRKCGVTSKRTSSATHCNRDA

******:*** ** :. .. **: ::: : * . . .. **

Crs PGCKNVCYDHFFPVS--HIRLWALQLIFVSTPALLVAMHVAYYR-HETTRKFRRGEKRND

G6CH18 KMCAMTTFSR-CPTSGCGPSSSSSPPQRCWWPCMWPTTGTKPLASSETTRKFRLGYKRNY

* . :.: *.* : *.: : . ******* * ***

Crs FKDIEDIKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPC

G6CH18 FKDIEDIKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPC

************************************************************

Crs PNLVDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNH

G6CH18 PNLVDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNH

************************************************************

Crs ALKESKQNEMNELISDSGQNAITGFPSX

G6CH18 ALKESKQNEMNELISDSGQNAITGFPSX

****************************

Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH18, the frameshift and indel mutations are highlighted.

127

Appendix-3 Tables and Figures of MTRNR1 gene analysis of deaf patients KP Pakistan

128

Appendix - 3

Table. Bioedit file of MTRNR1 gene of deaf patients showing different mutations .

129

Figure. Different Peaks of MTRNR1gene in deaf patient Sample ID. DAM29

130

CLUSTAL 2.1 multiple sequence alignment

C GAAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCAT

341HR GAAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCAT

************************************************************

C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA

341HR GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA

************************************************************

C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG

341HR CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG

*************** ***************** **************************

C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA

341HR CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA

************************************************************

C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA

341HR CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA

************************************************************

C AGTGCACTTGGACGAAC

341HR AGTGCACTTGGACGAAC

*****************

Mutation position at T1420G

Figure. DNA Alignments and Peaks of MTRNR1 Gene in Deaf Patient sample

ID.341HR, showing the mutation position of T1420G and A1438G.The mutations are highlighted in the alignment.The mutation position of T1420G is indicated by

black bar in the nucleotide Peaks.

131

CLUSTAL multiple sequence alignment by MUSCLE (3.8)

C AAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCATG

DAM13 AAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCATG

************************************************************

C AGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAAC

DAM13 AGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAAC

************************************************************

C TTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGGC

DAM13 TTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGGC

******************************** ***************************

C CCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAAC

DAM13 CCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAAC

************************************************************

C TAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAAA

DAM13 TAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAAA

************************************************************

C GTGCACTTGGACGAA

DAM13 GTGCACTTGGACGAA

***************

Figure: Normal Position of T1420T in sample ID.DAM13 A1438G (Homoplasmy)

Figure. DNA alignments and peaks of MTRNR1 gene in deaf patients sample ID. DAM13, the normal position of T1420T and mutation Positions of A1438G in

MTRNR1 gene are shown. The mutations are highlighted in the alignment and represented by black bar in the nucleotide Peaks.

132

CLUSTAL multiple sequence alignment by MUSCLE (3.8)

rCRS AAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCA

MTMR11 AAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCA

**********************************************************

rCRS TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA

MTMR11 TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA

************************************************************

rCRS ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG

MTMR11 ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG

********************************** *************************

rCRS GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA

MTMR11 GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA

************************************************************

rCRS ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA

MTMR11 ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA

************************************************************

rCRS AAGTGCACTTGGACGAA

MTMR11 AAGTGCACTTGGACAAA

************** **

Figure. Alignments of deaf patient sample ID.MTMR11. The mutation A1438G(homoplasmy) and G1598A (homoplasmy) in MTRNR1 gene were

shown. The mutation G1598A is illustrated by black Bar

133


C CATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATG

337HR CATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATG

************************************************************

C AAACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACA

337HR AAACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACA

****************** ***************** ***********************

C GGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATT

337HR GGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATT

************************************************************

C TAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTG

337HR TAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTG

************************************************************

C GAAAGTGCACTTGGACGAAC

337HR GAAAGTGCACTTGGACGAAC

********************

Figure. Alignments of deaf patient sample ID.337HR. The mutations T1420G and

A1438G of MTRNR1 gene were shown. The mutation T1420G (heteroplasmy) is

illustrated in peaks by black Bar

134


C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA

340HR TGAGGGGGCCAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA

***** *** **************************************************

C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG

340HR ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG

********************************** *************************

C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA

340HR GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA

************************************************************

C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA

340HR ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA

************************************************************

C AAGTGCACTTGGACGAA

340HR AAGTGCACTTGGACGAA

*****************

Figure. Nucleotide alignments of deaf patient sample ID 340HR in MTRNR1 gene. Different mutation positions were highlighted. The Position A1438G

(homoplasmy) is illustrated by black Bar

135


C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA

364HR GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA

**** *******************************************************

C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG

364HR CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAAAATAGAGTGCTTAGTTGAACAGGG

********************************* **************************

C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA

364HR CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTT

***********************************************************

C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG

364HR ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG

***********************************************************

C AAAGTGCACTTGGACGAA

364HR AAAGTGCACTTGGTCGAA

******************

Figure. Nucleotide alignments of deaf patient sample ID 364HR. Different mutations position in MTRNR1 gene were highlighted. The position of T1349G

(Homoplasmy) is illustrated by black Bar in the peaks

136



384HR TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA

************************************************************


384HR ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG

**************** ***************** *************************



************************************************************


384HR ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGGACTGGA

***************************************************** ******

C AAGTGCACTTGGACGAA

384HR AAGTGCACTTGGACAAA

************** **

Mutation T1577G (homoplasmy) in sample ID.384HR

137

Mutation G1598A (homoplasmy) in sample ID.384HR

Figure. Nucleotide alignments of deaf patient sample ID 384HR of MTRNR1

gene. Different mutations positions were highlighted. The position of T1577G (Homoplasmy) and G1598A (Homoplasmy) are illustrated by black bars in the

peaks

138


C CATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATG

263ST CATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATG

************************************************************

C AAACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACA

263ST AAACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAAAGTAGAGTGCTTAGTTGAACA

****************** ***************** * *********************

C GGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATT

263ST GGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATT

************************************************************

C TAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTG

263ST TAACTAAAACCCCTACGCATTTTTATAAAGGAGACAAGTCGTAACATGGTAAGTGTACTG

************************************************************

C GAAAGTGCACTTGGACGAA

263ST GAAAGTGCACTTGGACAAA

*******************

Mutation G1440A (heteroplasmy)

Figure. Nucleotide alignments of deaf patient sample ID 263STin MTRNR1 gene.

The mutation positions T1420G, A1438G and T1577G are highlighted. The position of T1577G is illustrated by black bars in the peaks

139



285ST TGAAGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA

************************************************************


285ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAAAGTGCTTAGTTGAACAGG

**************** *******************************************


285ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA

************************************************************


285ST ACTAAAACCCCTACGCATTTTTTTAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA

******************** * *************************************

C AAGTGCACTTGGACGAA

285ST AAGTGCACTTGGACGAA

*****************

Mutations A1544T, A1546T (heteroplasmy)

Figure. Nucleotide alignments of deaf patient sample ID 263STof MTRNR1 gene. The mutation Positions T1420G, A1438G and A1544T and A1546T are

highlighted. The position of A1544T and A1546T is illustrated by black bars in the peaks

140


C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG

295ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG

**************** *******************************************


295ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA

************************************************************


295ST ACTAAAACCCCCACGCATTTTTTTAGAGGAAACAAGTCGTAACATGGTAAGTGTACTGGA

*********** ******** * ******* *****************************

C AAGTGCACTTGGACGAA


*****************

Figure. Nucleotide alignments of deaf patient sample ID 263ST of MTRNR1 gene. The mutation Positions T1420G, A1438G and A1544T and A1546T are

highlighted. The position of A1544T and A1546T is illustrated by black bars in the peaks.

T1420G (Homoplasmy) in sample ID.295ST

Figure. Nucleotide alignments of deaf patient sample ID 295ST of MTRNR1 gene. The mutation Positions T1420G is highlighted and denoted by black bar in

the nucleotide peaks

141

T1535C (Heteroplasmy) in sample ID.295ST

Figure. Nucleotide alignments of deaf patient sample ID 295ST of MTRNR1 gene. The mutation Positions T1535C is highlighted and denoted by black bar in the nucleotide peaks

A1544T and A1546T (Heteroplasmy) in sample ID.295ST

Figure. Nucleotide alignments of deaf patient sample ID 295ST of MTRNR1 gene. The mutation Positions A1544T and A1546T are highlighted and denoted

by black bar in the nucleotide peaks

142

G1554A (heteroplasmy)in sample ID.295ST

Figure. Nucleotide alignments of deaf patient sample ID 295ST of MTRNR1 gene. The mutation Positions G1554A is highlighted and denoted by black bar in the

nucleotide peaks


C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180

415CH GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 150

************************************************************

C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240

415CH CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 210

*************** ***************** **************************

C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300

415CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 270

************************************************************

C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360

415CH CTAAAACCCCTACGCATTTTTATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 330

************************************************************

C AGTGCACTTGGACGAA

415CH AGTGCACTTGGACCAA

****************

143

Figure: Novel mutation at position T1420G( Homoplasmy) in sample ID.415CH

Figure. Nucleotide alignments of deaf patient sample ID. 415CH of MTRNR1 gene. The mutation Positions T1420G is highlighted and denoted by black bar in



HR333 GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 157

************************************************************


HR333 CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 217

*************** ***************** **************************


HR333 CCCTGAAGCGCGTACACACCGCCCGTCACCCTCTCAAGTATACTTCAAAGGACATTTAA 277

***********************************************************


HR333 CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 337

************************************************************

C AGTGCACTTGGACGAA

HR333 AGTGCACTTGGACGAA

****************

144

Figure: Novel mutation at position T1420G ( Heteroplasmy) in Sample ID.333HR

Figure. Nucleotide alignments of deaf patient sample ID. 333HR of MTRNR1 gene. The mutation Positions T1420G is highlighted and denoted by black bar in



354HR GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 149

************************************************************


354HR CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 209

*************** ***************** **************************


354HR CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 269

************************************************************


354HR CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 329

************************************************************

C AGTGCACTTGGACGAA

354HR AGTGCACTTGGACGAA

****************

145

Figure: Novel mutation at position T1420G ( Heteroplasmy)in sample ID.354HR

Figure. Nucleotide alignments of deaf patient sample ID. 354HR of MTRNR1

gene. The mutation Positions T1420G is highlighted and denoted by black bar in the nucleotide peaks



340HR TGAGGGGGCCAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA

***** *** **************************************************


340HR ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG

********************************** *************************



************************************************************


340HR ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA

************************************************************

C AAGTGCACTTGGACGAA

340HR AAGTGCACTTGGACGAA

*****************

146

Figure: Mutation at position A1438G ( Homoplasmy) in sample ID.340HR



147



364HR GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 151

**** *******************************************************


364HR CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAAAATAGAGTGCTTAGTTGAACAGGG 210

********************************* **************************

C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299

364HR CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTT 270

***********************************************************

C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 358

364HR ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 330

***********************************************************


364HR AAAGTGCACTTGGTCGAA

******************

Figure: Novel mutation at position T1349G( Homoplasmy) in sample ID.364HR



148


C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA 179

384HR TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA 157

************************************************************

C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG 239

384HR ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 217

**************** ***************** *************************

C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299

384HR GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 277

************************************************************

C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 359

384HR ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGGACTGGA 337

***************************************************** ******

C AAGTGCACTTGGACGAA

384HR AAGTGCACTTGGACAAA

************** **

Figure: Novel mutation at position T1577G(Homoplasmy) in sample ID.384HR

Figure. Nucleotide alignments of deaf patient sample ID. 384HR of MTRNR1 gene. The mutation Positions T1577G is highlighted and denoted by bar in the

nucleotide peaks

149



382HR GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAAA 159

**** *******************************************************



*************** ***************** **************************



************************************************************



************************************************************

C AGTGCACTTGGACGAA


****************

Figure: Novel mutation at position T1349G(Homoplasmy) in sample ID.382HR

Figure. Nucleotide alignments of deaf patient sample ID. 382HR of MTRNR1 gene. The mutation Positions T1349G is highlighted and denoted by bar in the

nucleotide peaks

150


C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 178

383HR TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACCATAGCCCTTATGA 154

***********************************************************

C AACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAG 238

383HR AACTTAAAGGTCGAAGGGGGATTTAGCAGTAAACTGAGAATAGAGTGCTTAGTTGAACAG 214

***************** ***************** ************************

C GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 298

383HR GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 274

************************************************************

C AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 358

383HR AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 334

************************************************************


383HR AAAGTGCACTTGGACGAA

******************

Figure: Novel mutation at position G1598A in sample ID.384HR

Figure. Nucleotide alignments of deaf patient sample ID. 384HR of MTRNR1 gene. The mutation Positions G1598A is highlighted and denoted by bar in the

nucleotide peaks

151


C GAAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCAT 120

386HR GAAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCAT 96

************************************************************


386HR GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 156

************************************************************



*************** ***************** **************************



************************************************************



************************************************************

C AGTGCACTTGGACGAA


****************


C ATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 178

387HR ATGAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 157

****** *****************************************************


387HR AACTTAAGGGTCGAAGGGGGATTTAGCAATAAACTGAAAGTAGAGTGCTTAGTTGAACAG 217

***************** ***************** *************************



************************************************************



************************************************************



******************

152



390HR ATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 154

************************************************************


390HR AACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAG 214

***************** ***************** ************************



************************************************************



************************************************************



******************



121sb GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 153

************************************************************


121sb CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAAAGTGCTTAATTGAACAGGG 213

*************** ***************** ******** ******** ********


121sb CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 273

************************************************************


121sb CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 333

************************************************************

C AGTGCACTTGGACGAA

121sb AGTGCACTTGGACGAA

****************

153



123sb GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 150

************************************************************


123sb CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 210

*************** ***************** ***************************


123sb CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTTA 270

************************************************************


123sb CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGGAAGTGTACTGGAA 330

************************************************************

C AGTGCACTTGGACGAA

123sb AGTGCACTTGGACGAA

****************



249ST GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAAA 153

***********************************************************


249ST CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAATTGAACAGGG 213

*************** ***************** **************************


249ST CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 273

************************************************************


249ST CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 333

************************************************************

C AGTGCACTTGGACGAA

249ST AGTGCACTTGGACGAA

****************

154



262 GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAAA 152

************************************************************


262 CTTAAGGGGCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 211

******** ****** ***************** **************************


262 CCCTGAAGCGCGTACACACCGCCCGTCCCCCTCCTCAAGTATACTTCAAAGGACATTTAA 271

*************************** ********************************


262 CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGGAAGTGTACTGGAA 331

********************************************** *************

C AGTGCACTTGGACGAA

262 AGTGCACTTGGACGAA

****************


C CATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATG 177

263ST CATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATG 150

************************************************************

C AAACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACA 237

263ST AAACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAAAGTAGAGTGCTTAGTTGAACA 210

****************** ***************** * *********************

C GGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATT 297

263ST GGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATT 270

************************************************************

C TAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTG 357

263ST TAACTAAAACCCCTACGCATTTTTATAAAGGAGACAAGTCGTAACATGGTAAGTGTACTG 330

************************************************************

C GAAAGTGCACTTGGACGAA

263ST GAAAGTGCACTTGGACAAA

*******************

155

Figure: Novel mutation at position G1440A(Heteroplasmy) at sample ID.263ST



264ST GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 153

************************************************************

C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240

264ST CTTAAGGGGCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 213

******** ****** ********************************************


264ST CCCTGAAGCGCGTACACACCGCCCCCGTCCCCCCCCAAGTATACTTCAAAGGACATTTAA 273

************************************************************



****************************** *****************************

C AGTGCACTTGGACGAA


****************



266ST ATGAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 152

************************************************************


266ST AACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAG 212

***************** ***************** ************************


266ST GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 272

************************************************************

156


266ST AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 332

************************************************************


266ST AAAGTGCACTTGGACGAA

******************



268ST GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 154

************************************************************

C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG 239

268ST CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 213

********************************* *************************


268ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 273

************************************************************


268ST ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 333

************************************************************

C AAGTGCACTTGGACGAA


*****************



282ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAAATGAGAGTAGATGCTTGAGTTGAACAGG 212

**************** ***************** *************************


282ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTAATACTTCAAAGGACATTA 272

*************************************************************


282ST ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 332

************************************************************

C AAGTGCACTTGGACGAA


*****************

157



283ST GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 152

**** *******************************************************


283ST CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 212

*************** ********************************************


283ST CCCTGAAGCGCGTACACACCGCCCGTCACCCCCCCCAAGTATACTTCAAAGGACATTTAA 272

******************************* ** *************************


283ST CTAAAACCCCTACGCATTTTTTTAGAGGAAACAAGTCGTAACATGGTAAGTGTACTGGAA 332

******************* * ******* ******************************

C AGTGCACTTGGACGAA


****************



285ST TGAAGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA 154

************************************************************


285ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAAAGTGCTTAGTTGAACAGG 214

**************** *******************************************



************************************************************


285ST ACTAAAACCCCTACGCATTTTTTTAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 334

******************** * *************************************

C AAGTGCACTTGGACGAA


*****************

158

Novel mutations at position A1544T and A1546T(Heteroplasmy)in sample ID.285ST



286ST CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 213

************************************************************



************************************************************


286ST CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGGACTGGAA 333

**************************************************** *******

C AGTGCACTTGGACGAA

286ST AGTGGACTTGGACAAA

*** ******* **

Figure: Novel mutation at position G1598A(Homoplasmy)in sample ID.286ST

159



289ST CCCTGAAGCGCGTAAACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 264

************** *********************************************


289ST CTAAAACCCCTACGCATTTATATAAAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 324

************************ ***********************************

C AGTGCACTTGGACGAA


****************


C CCATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTAT 176

293ST CCATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTAT 149

************************************************************

C GAAACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAAC 236

293ST GAAACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAAC 209

******************* ****************************************

C AGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACAT 296

293ST AGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACAT 269

************************************************************

C TTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACT 356

293ST TTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACT 329

************************************************************

C GGAAAGTGCACTTGGACGAA

293ST GGAAAGTGCACTTGGACGAA

********************


C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 239

295ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 215

**************** *******************************************



************************************************************

160


295ST ACTAAAACCCCCACGCATTTTTTTAGAGGAAACAAGTCGTAACATGGTAAGTGTACTGGA 335

*********** ******** * ******* *****************************

C AAGTGCACTTGGACGAA


*****************

Figure: Novel mutation at position T1420G(Homoplasmy)in sample ID.295ST

Figure: Novel mutation at position T1535C(Heteroplasmy)in sample ID.295ST

161




*************** ********************************************



************************************************************



************************************************************

C AGTGCACTTGGACGAA


****************


C CCATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTAT 176

306ST CCATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTAT 159

************************************************************

C GAAACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAAC 236

306ST GAAACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAAC 219

******************* ****************************************

C AGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACAT 296

306ST AGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACAT 279

************************************************************

C TTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACT 356

306ST TTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACT 339

************************************************************

C GGAAAGTGCACTTGGACGAA

306ST GGAAAGTGCACTTGGACGAA

********************



311ST GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCCGAAAACTACGATAGCCCTTATGAAA 157

************************************************************



*************** ***************** **************************



************************************************************

162

C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGG-ACTGGAA 360

311ST CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGGGGTACTGGAA 337

************************************************** ** *******

C AGTGCACTTGGACGAA


****************



325ST TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAA 154

************************************************************

C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 239

325ST ACTTAAGGGTCTAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 214

*********** **** *******************************************


325ST GCCCTGAAGCGCGTACACACCGCCCGTCCCCCTCCTCAAGTATACTTCAAAGGACATTTA 274

**************************** *******************************


325ST ACTAAAACCCCTACCCATTTATATAGAGGAAACAAGTCGTAACATGGTAAGTGTACTGGA 334

************** *************** *****************************

Figure: Novel mutation at position G1538C(Heteroplasmy)in sample ID.325ST

163



PS181 GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 153

************************************************************


PS181 CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 213

************************************************************


PS181 CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 273

************************************************************


PS181 CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 333

************************************************************

C AGTGCACTTGGACGAA

PS181 AGTGCACTTGGACGAA

****************


c CCGATCAACCTCACCACCTCTTGCTCAGCCTATATACCGCCATCTTCAGCAAACCCTGAT 60

208 CCGATCAACCTCACCACCTCTTGCTCAGCCTATATACCGCCATCTTCAGCAAACCCTGAT 60

************************************************************

c GAAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCAT 120

208 GAAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCAT 120

************************************************************

c GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180

208 GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180

************************************************************

c CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240

208 CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240

************************************************************

c CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300

208 CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300

************************************************************

c CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360

208 CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360

************************************************************

c AGTGCACTTGGACGAA

208 AGTGCACTTGGACGAA

****************

164



PS185 CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 211

*************** ***************** **************************


PS185 CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 271

************************************************************


PS185 CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 331

************************************************************

C AGTGCACTTGGACGAA

PS185 AGTGCACTTGGACGAA

****************




*************** ****************** **************************



************************************************************


396CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 335

************************************************************

C AGTGCACTTGGACGAA

396CH AGTGCACTTGGACGAA

****************


C TTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240

407CH TTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGGAGAGTGCTTAGTTGAACAGGG 215

***********************************************************



************************************************************



************************************************************

C AGTGCACTTGGACGAA


****************

165



403CH GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 149

**** *******************************************************


403CH CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 209

********************************* **************************



*************************************************************



************************************************************

C AGTGCACTTGGACGAA


****************




************************************************************



************************************************************



************************************************************

C AGTGCACTTGGACGAA


****************



412CH GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 273

************************************************************


412CH AAATAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 333

** *********************************************************


412CH AAAGTGCACTTGGACGAA

******************

166

Novel mutation at position C1525A ( Homoplasmy) in sample ID.412CH


C CAGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACA 295

421CH CAGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACA 267

************************************************************

C TTTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTAC 355

421CH TTTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTAC 327

************************************************************

C TGGAAAGTGCACTTGGACGAA

421CH TGGAAAGTGCACTTGGACGAA

*********************



422CH GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 277

************************************************************


422CH ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 337

************************************************************

C AAGTGCACTTGGACGAA

422CH AAGTGCACTTGGACGAA

*****************

167


c AGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACAT 296

425CH AGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCCAAGGACAT 271

************************************************************

c TTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACT 356

425CH TTAACTTAAACCCCTACCCCTTTTTATAGAGGAGACAAGTCGTAACATGGGAAGTGTACT 331

************************************************** *********

c GGAAAGTGCACTTGGACGAA

425CH GGAAAGTGCACTTGGACGAA

********************



428CH GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 157

************************************************************

c CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240

428CH CTTAAGGGTCGAAGGGGGATTTAGCAGAAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 217

*************** ***************** **************************


428CH CCCTGAAGGGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 277

************************************************************



************************************************************

c AGTGCACTTGGACGAA


****************




************************************************************



************************************************************



************************************************************

c AGTGCACTTGGACGAA


****************

168



434CH GAGGTGGCAAGAAATGGGCAACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAAA 149

************************************************************



************************************************************



************************************************************


434CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGGA 329

********************************************************** *


c CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240


*************** ***************** **************************



************************************************************



************************************************************

c AGTGCACTTGGACGAA


****************




*************** ***************** **************************



************************************************************



************************************************************

C AGTGCACTTGGACGAA


****************

169

Appendix-4 Tables and Figures of MTRNR2 gene analysis of deaf patients KP Pakistan

170

Appendix - 4

Table. Bioedit file mutations data of deaf patients samples in MTRNR2 gene of Khyber Pakhtunkhwa. The mutation A1811G is illustrated.

Table. Bioedit file mutations data of deaf patients samples in MTRNR2 gene of Khyber Pakhtunkhwa. Different mutations can be seen in the table

171

Table. Bioedit file mutations data of deaf patients samples in MTRNR2 gene of

Khyber Pakhtunkhwa.

172

Figure. Sequence of MTRNR2 of a deaf patient sample ID.MTAB44

.

173

C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACTA

MTAB44 GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACTA

*************************************

C CCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAACC

MTAB44 CCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAACC

************************************************************

C TGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATATA

MTAB44 TGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAGCCAAGCATAATATA

********************************************* **************

C GCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAGGA

MTAB44 GCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAGGA

************************************************************

C GAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCACA

MTAB44 GAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCACA

************************************************************

C CCCGTCTATGTAGCAAAATAGTGGGAAGATTTATAGGTAGAGGCGACAAACCTACCGAGC

MTAB44 CCCGTCTATGTAGCAAAATAGTGGGAAGATTTATAGGTAGAGGCGACAAACCTACCGAGC

************************************************************

Mutation: A1811G (Homoplasmy)

Figure. Nucleotide alignments of MTRNR2 gene of deaf patient sample ID MTAB44.The mutation Positions A1811G is highlighted and the mutation

Position is illustrated by black bar in the peaks

174

C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACT

249ST GAACTAAACCTAGCCCCAAACCCACTCCACCTTACT

** *********************************

C ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC

249ST ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC

************************************************************

C CTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAA

249ST CTGGCGCAATAGATATAGTACCGCTAGGGAAAGATGAAAAA

*****************************************

Mutation: G1671A (Heteroplasmy)

Figure. Nucleotide alignments of MTRNR2 gene of deaf patient sample ID 249ST

and the mutation Positions G1671A is highlighted and the Position of mutation is

illustrated by black bar in the nucleotide Peaks.

175


C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAC

340HR GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAC

***********************************

C TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA

340HR TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA

************************************************************

C CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA

340HR CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAGCCAAGCATAATA

*********************************************** ************

C TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG

340HR TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG

************************************************************

C GAGAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCA

340HR GAGAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCA

************************************************************

C CACCCGTCTATGTAGCAAAATAGTGGGAAGATTTATAGGTAGAGGCGACAAACCTACCGA

340HR CACCCGTCTATGTAGCAAAATAGTGGGAAGATTTATAGGTAGAGGCGACAAACCTACCGA

************************************************************

Figure: Mutation at A1811G (Homoplasmy)

Figure. Nucleotide allignments of MTRNR2 gene of deaf patient sample ID.340HR.The mutation Positions A1811G is highlighted and the mutation

Position is illustrated by black bar in the peaks

176



362HR GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAC

***********************************


362HR TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA

************************************************************


362HR CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA

************************************************************


362HR TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG

************************************************************


362HR GAGAACCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCA

**** *******************************************************

C CACCCGTCTATGTAGCAAAATAGTGGGAAGATTTATAGGTAGAGGCGACAAACCTACCGA

362HR CACCCGTCTATGTAGCAAAATAGTGGGAAGATTTATAGGTAGAGGCGACAAACCTACCGA

************************************************************

Mutation at G1888A (Homoplasmy)

Figure. Nucleotide alignments of MTRNR2 gene of deaf patient sample 362HR.The mutation Positions G1888A is highlighted and the mutation Position

is illustrated by black bar in the peaks

177



428CH GAGGTAAACCTAGCCCCAAACCCACTCCACCTTACT

*** ********************************


428CH ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC

************************************************************

Mutation: C1672G (Homoplasmy)

Figure. Nucleotide alignments of MTRNR2 gene of deaf patient sample ID.428CH.The mutation C1672G is highlighted and the mutation Position is

illustrated by black bar in the peaks

178



422CH GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAA

***********************************


422CH TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA

************************************************************


422CH CCTGGCGCAATAAATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA

************ ***********************************************


422CH TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAA-AACTTTGCAAG

************************************************ ***********

) Mutation: del T1872

Figure. Nucleotide alignments of MTRNR2 gene of deaf patient sample ID.422CH.The mutation positions G1776A and del T1872 is highlighted and the

mutation Position of del T1872 is illustrated by black bar in the peaks

179


rCRS GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACTA

DSB9 GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACTA

*************************************

rCRS CCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAACC

DSB9 CCAGACAACCTTAGCCAAACCATTTACCCAAACAAAGTATAGGCGATAGAAATTGAAACC

******************************** ***************************

rCRS TGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATATA

DSB9 TGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATATA

************************************************************

rCRS GCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAGGA

DSB9 GCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAGGA

************************************************************

rCRS GAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCACA

DSB9 GAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCACA

************************************************************

Mutation: T1738C (Homoplasmy)

Figure. Nucleotide alignments of MTRNR2 gene of deaf patient sample ID.DSB9.The mutation positions T1738C is highlighted and is denoted by black

bar in the peaks

180


rCRS GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACT

DSB11 GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACT

************************************

rCRS ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC

DSB11 ACCAGACAACCTTAACCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC

************** *********************************************

rCRS CTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATAT

DSB11 CTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATAT

************************************************************

rCRS AGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG

DSB11 AGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG

***********************************************************

Mutation: G1719A (homoplasmy)

Figure. Nucleotide alignments of MTRNR2 gene of deaf patient sample ID.DSB11.The mutation Positions G1719A is highlighted and the mutation

Position is denoted by black bar in the peaks

.

181



249ST GAACTAAACCTAGCCCCAAACCCACTCCACCTTACT

** *********************************


249ST ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC

************************************************************

C CTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAA

249ST CTGGCGCAATAGATATAGTACCGCTAGGGAAAGATGAAAAA

*****************************************



289ST GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACT

************************************


289ST ACCAAACAACCTTAGCCAAACCATTTACCCCAATAAAGTATAGGCGATAGAAATTGAAAC

**** ************************* *****************************

C CTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATAT

289ST CTGGCGCAATAAATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCCAGCATAATAT

*********** ************************************* **********

C AGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAGG

289ST AGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAGG

************************************************************



295ST GAACTAAACCTAGCCCCAAACCCACTCCACCTTAC

** ********************************

C TACCAGACAACCTTAGCCAAACCATTTACCCAGATAAAGTATAGGCGATAGAAATTGAAA

295ST TACCAAACAACCTTAACCAAACCATTTACCCAAATAAAGTATAGGCGAAAGAAATTGAAA

***** ********* **************** *************** ***********


295ST CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA

************************************************************


295ST TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAAAAATAACTTTGCAAG

******************************************** ****************

182



285ST GAACTAAACCTAGCCCCAAACCCACTCCACCTTAC

** ********************************


285ST TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAAAAATTGAAA

***** ******************************************** *********


285ST CCTGGCGCAATAGATATTGTACCGCAAGGGAAAGATGAAAAATTTTAACCAAGCATAATA

************ **** ***** ******************** ***** *********


285ST TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG

************************************************************


285ST GAGAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCA

************************************************************

Figure. Mutation at position G1671A (heteroplasmy) in sample I.D.249ST

G1719A (homoplasmy) in sample 295ST Mutation A1808T (heteroplasmy) in 285ST

Figure. Nucleotide alignments of MTRNR2 gene of deaf patients sample IDs 249ST, 285ST, 295ST and 289ST.The mutation Positions of these samples were

highlighted and the mutation Position of some samples is denoted by black bar in the peaks.

Figure. Mutations A1735C

(heteroplasmy) in 289ST

183

Appendix-5 Tables and Figures of MT-TV gene analysis of deaf patients KP Pakistan

184

Appendix 5

Table. Bioedit file data of MT-V gene of deaf patients of KP showing different mutations.

185

.

Figure. Complete sequence of MT-TV Gene of Deaf Patient Sample ID.305ST

186


C CCAGAGTGTAGCTTAACACAAAGCACCCAACTTACACTTAGGAG

305ST CCAGAGTGGAGCTTAACACAAAGCACCCAACTTACACTTAGGAG

********************************************

C ATTTCAACTTAACTTGACCGCTCT

305ST ATTTCAACTTAACTTGACCGCTCT

************************

Mutation: T1609G (heteroplasmy)

Figure. Nucleotide alignments of MT-TV gene of deaf patients sample ID. 305ST.The mutation Positions T1609G is highlighted and the mutation Position is

denoted by black bar in the peaks.


C CCAGAGTGTAGCTTAACACAAAGCACCCAACTTACACTTAGGA

295ST CCAGAGTGTAGCTTAACACAAAGCACCCAACTTACACTTAGGA

*******************************************

C GATTTCAACTTAACTTGACCGCTCT

295ST AATTTCAACTTAACTTGACCGCTCT

************************

Mutation: G1644A (heteroplasmy)

Figure. Nucleotide alignments of MT-TV gene of deaf patients sample ID. 295ST.The mutation Positions T1644A is highlighted and the mutation Position is

denoted by black bar in the peaks.

187


C CCAGAGTGTAGCTTAACACAAAGCACCCAACTTACACTTAGGA

396CH CCATAGTGTAGCTTAACACAAAGCACCCAACTTACACTTAGGA

*** ***************************************

C GATTTCAACTTAACTTGACCGCTCT

396CH GATTTCAACTTAACTTGACCGCTCT

*************************

Mutation: G1604T (heteroplasmy)

Figure. Nucleotide alignments of MT-TV gene of deaf patients sample ID. 396CH.The mutation Positions G1604T is highlighted and the mutation Position

is denoted by black bar in the peaks.

188


C CCAGAGTGTAGCTTAACACAAAGCACCCAACTTACACTT

MTAB48 CCAGAGTGTAGCTTAACACAAAGCACCCAACTTACACTT

***************************************

C AGGAGATTTCAACTTAACTTGACCGCTCT

MTAB48 ATGAGATTTCAACTTAACTTGACCGCTCT

* ***************************

Mutation: G1641T (Homoplasmy)

Figure. Nucleotide alignments of MT-TV gene of deaf patients sample ID. MTAB48.The mutation Positions G1641T is highlighted and the mutation

Position is denoted by black bar in the peaks.

189

Appendix-6 DNA extraction protocol and preparation of agarose gel and gel electrophoresis

190

Appendix-6

DNA extraction Protocol

1. The 1 ml saliva containing epithelial cells was transferred to a 2ml

Eppendorff tube and 100ul lysis solution was then added to each tube. In

addition, 1 ul protinase K and 0.6 ul betamarcaptoethanol (BME) were

added to the lysis/saliva solution

2. All samples were incubated at 56 ͦ C for 90 minutes

3. After incubation, 600 ul of phenol chloroform was added to the samples and

mixed thoroughly

4. The samples were kept at room temperature for 5 minutes and then

centrifuged at 10,000 rpm for 15 minutes

5. The aqueous supernatant was transferred to new Eppendorf tube and the

phenol remaining mixture was discarded

6. Equal volume of isopropanol was added to the samples and the samples

were chilled at -20 ͦ C for 20 minutes

7. The chilled samples were centrifuged at 10,000 rpm for 10 minutes

8. The supernatant was discarded and the DNA pellet was washed with 70%

ethanol, then centrifuged at 8000 rpm for 5 minutes

9. The ethanol was removed and the pellet was air dried

10. 30 ul deionized distilled water was added to the DNA pellet of each

sample, and all samples were incubated at 56 ͦ C for 10 minutes to rehydrate

the nucleic acid.

191

Preparation of Agarose gel and gel electrophoresis

To prepare 50ml gel solution, 1ml 50X TAE and 49ml distilled water were added to

a conical glass. The 0.5 g agarose will be added to the 50ml gel solution and this is

the standard for the preparation of agarose gel. The whole solution apparatus were

heated in the oven for 2 minutes and then kept to cool down until your hand can

bear it. After cool down, the 6ul ethidium bromide was added to the 50ml

solution. The solution was then poured into gel tray and a comb having wells was

adjusted in the gel and left it until become solid. After this the gel tray along with

gel were kept in buffer tank and the comb was removed. The samples were loaded

with samples along with blue dye at 100 volt for 30 minutes. The gel was then

checked on ultra violet and photograph was taken.

192

Appendix-7 Clinical data of deaf patients samples KP Pakistan

193

Appendix-7 Table: Clinical data of District Abbottabad KP Pakistan

S.No Sample ID DNA Concentration Unit

1 89 22.6 ng/µl

2 76 24.3 ng/µl

3 102 1.9 ng/µl

4 64 14.8 ng/µl

5 16 3 ng/µl

6 125 12.5 ng/µl

7 73 0.1 ng/µl

8 15 43.8 ng/µl

9 135 15 ng/µl

10 18 5.7 ng/µl

11 136 43.4 ng/µl

12 38 5.3 ng/µl

13 74 159.6 ng/µl

14 137 1.8 ng/µl

15 87 10.4 ng/µl

16 65 7.8 ng/µl

17 106 24.6 ng/µl

18 36 112 ng/µl

19 53 0.6 ng/µl

20 47 9.9 ng/µl

21 99 1.7 ng/µl

22 110 177.7 ng/µl

23 129 0.4 ng/µl

24 33 6.2 ng/µl

25 42 51.8 ng/µl

26 112 1.6 ng/µl

27 30 0.4 ng/µl

28 105 0.5 ng/µl

29 60 34.9 ng/µl

30 103 3.8 ng/µl

31 67 5.6 ng/µl

32 45 3.1 ng/µl

33 115 26.4 ng/µl

34 125 2.2 ng/µl

35 98 14.6 ng/µl

36 17 1.1 ng/µl

37 59 1.8 ng/µl

38 9 0.2 ng/µl

39 141 6.7 ng/µl

194

40 100 23.9 ng/µl

41 6 1.5 ng/µl

42 78 571.5 ng/µl

43 66 37.7 ng/µl

44 19 6.1 ng/µl

45 138 5.1 ng/µl

46 79 190.4 ng/µl

47 27 0.8 ng/µl

48 37 13 ng/µl

49 44 34.4 ng/µl

50 104 107.3 ng/µl

51 86 5.6 ng/µl

52 40 182 ng/µl

53 46 5.7 ng/µl

54 2 6.4 ng/µl

55 56 3.7 ng/µl

56 131 15.7 ng/µl

57 120 12.7 ng/µl

58 118 11.4 ng/µl

59 22 4.2 ng/µl

60 111 5.5 ng/µl

61 44 4.2 ng/µl

62 52 94.4 ng/µl

63 55 17.3 ng/µl

64 75 19 ng/µl

65 119 2.3 ng/µl

66 54 49.1 ng/µl

67 58 19.9 ng/µl

68 122 12.9 ng/µl

69 92 92.3 ng/µl

70 124 2.9 ng/µl

71 77 7.8 ng/µl

72 80 131.7 ng/µl

73 121 160.3 ng/µl

74 107 3.5 ng/µl

75 128 23.4 ng/µl

76 97 11.5 ng/µl

77 93 4.7 ng/µl

78 95 3.8 ng/µl

79 48 2 ng/µl

80 133 3.8 ng/µl

81 140 3.6 ng/µl

195

82 5 19 ng/µl

83 117 147.7 ng/µl

84 13 63.2 ng/µl

85 70 14.1 ng/µl

86 10 4.2 ng/µl

87 126 188.7 ng/µl

88 134 148.8 ng/µl

89 69 40.7 ng/µl

90 11 11.8 ng/µl

91 31 13.3 ng/µl

92 50 183.3 ng/µl

93 18 13 ng/µl

94 109 429 ng/µl

95 108 7.8 ng/µl

96 14 1.6 ng/µl

97 28 3.1 ng/µl

98 43 26.2 ng/µl

99 130 4.6 ng/µl

100 123 2.3 ng/µl

101 8 95.7 ng/µl

102 20 626.4 ng/µl

103 19 20.9 ng/µl

104 7 12.9 ng/µl

105 114 66.1 ng/µl

106 57 3.2 ng/µl

107 82 5.3 ng/µl

108 127 2.6 ng/µl

109 41 409.7 ng/µl

110 68 54.7 ng/µl

111 1 4.1 ng/µl

112 101 1704.8 ng/µl

113 83 181.8 ng/µl

114 84 19.7 ng/µl

115 39 199.3 ng/µl

116 24 2.8 ng/µl

117 90 3.9 ng/µl

118 91 163.6 ng/µl

119 21 7.3 ng/µl

120 139 19.6 ng/µl

121 96 5.8 ng/µl

122 3 2.8 ng/µl

123 74 54.6 ng/µl

196

Table: Clinical data of deaf patients Bannu district KP Pakistan


1 3 77.7 ng/µl

2 1 103.2 ng/µl

3 2 112 ng/µl

4 4 180.6 ng/µl

5 6 35.7 ng/µl

6 7 48.3 ng/µl

7 7 21.9 ng/µl

8 8 28.7 ng/µl

9 9 26.7 ng/µl

10 11 58.4 ng/µl

11 12 56.2 ng/µl

12 13 56.8 ng/µl

13 14 48.7 ng/µl

14 15 32.9 ng/µl

15 16 35.7 ng/µl

16 17 31.5 ng/µl

17 18 58.1 ng/µl

18 19 14.1 ng/µl

19 20 36.7 ng/µl

20 21 92.5 ng/µl

21 22 21.8 ng/µl

22 23 28.4 ng/µl

23 24 74.4 ng/µl

24 25 26.5 ng/µl

25 26 56.5 ng/µl

26 27 26.3 ng/µl

27 28 67.6 ng/µl

28 29 36.8 ng/µl

29 29 36.1 ng/µl

30 30 70.3 ng/µl

31 31 150 ng/µl

32 32 22.5 ng/µl

33 33 31 ng/µl

34 34 29.8 ng/µl

35 35 21.8 ng/µl

36 36 30 ng/µl

37 37 63.2 ng/µl

38 38 33.3 ng/µl

39 39 38.1 ng/µl

40 40 17.7 ng/µl

197

Table: Clinical data deaf patients Charsada district KP Pakistan


1 3 246.7 ng/µl

2 4 259.8 ng/µl

3 6 258.9 ng/µl

4 7 194.7 ng/µl

5 8 202 ng/µl

6 9 225.4 ng/µl

7 10 -93.7 ng/µl

8 11 690.7 ng/µl

9 12 93.6 ng/µl

10 13 261.2 ng/µl

11 14 3247.5 ng/µl

12 15 252.9 ng/µl

13 16 862.4 ng/µl

14 17 -9.1 ng/µl

15 18 615.9 ng/µl

16 19 1349.2 ng/µl

17 23 1375.9 ng/µl

18 28 284.2 ng/µl

19 29 350.9 ng/µl

20 30 -2.3 ng/µl

21 34 819.7 ng/µl

22 40 526.3 ng/µl

23 41 1021.2 ng/µl

24 42 350 ng/µl

25 43 558.4 ng/µl

26 47 246.2 ng/µl

27 49 671.5 ng/µl

28 50 858.2 ng/µl

29 51 89 ng/µl

30 52 355.2 ng/µl

31 53 412.6 ng/µl

32 54 1326.1 ng/µl

33 55 303.3 ng/µl

34 57 102.1 ng/µl

35 58 87.4 ng/µl

36 59 264.7 ng/µl

37 56 778.9 ng/µl

38 60 622.3 ng/µl

39 61 453.5 ng/µl

198

40 62 877 ng/µl

41 64 818.7 ng/µl

42 65 326.6 ng/µl

43 66 281.7 ng/µl

44 67 609.5 ng/µl

45 68 205.9 ng/µl

46 69 397.5 ng/µl

47 70 552.2 ng/µl

48 73 -2.2 ng/µl

49 75 632.4 ng/µl

50 76 73.5 ng/µl

51 77 282.8 ng/µl

199

Table: Clinical data of deaf patients Haripur district KP Pakistan


1 1 236.6 ng/µl

2 2 1299.9 ng/µl

3 3 740.6 ng/µl

4 4 1027.1 ng/µl

5 5 228.3 ng/µl

6 6 822.2 ng/µl

7 7 2832.1 ng/µl

8 8 517 ng/µl

9 9 1494.3 ng/µl

10 10 840.2 ng/µl

11 11 108.3 ng/µl

12 15 1650.3 ng/µl

13 19 1859.4 ng/µl

14 20 6064 ng/µl

15 22 957.9 ng/µl

16 25 283.4 ng/µl

17 27 227.4 ng/µl

18 28 151.4 ng/µl

19 29 778.5 ng/µl

20 30 976.7 ng/µl

21 36 728 ng/µl

22 38 727.1 ng/µl

23 39 627.3 ng/µl

24 40 1022 ng/µl

25 41 439.1 ng/µl

26 42 761.4 ng/µl

27 43 1241.8 ng/µl

28 44 2051.3 ng/µl

29 45 1433.4 ng/µl

30 46 546.6 ng/µl

31 47 920.7 ng/µl

32 48 152.8 ng/µl

33 49 167.6 ng/µl

34 50 527 ng/µl

35 52 270.7 ng/µl

36 54 249.5 ng/µl

37 53 183.6 ng/µl

38 55 180.6 ng/µl

39 56 78.9 ng/µl

200

40 31 362.6 ng/µl

41 32 449.7 ng/µl

42 33 268.3 ng/µl

43 34 917.8 ng/µl

44 35 1087.8 ng/µl

201

Table: Clinical data of deaf patients Mardan district KP Pakistan


1 1 299.2 ng/µl

2 2 74.7 ng/µl

3 3 79 ng/µl

4 4 53.1 ng/µl

5 5 458.7 ng/µl

6 6 62.4 ng/µl

7 9 92.5 ng/µl

8 10 184.4 ng/µl

9 11 516 ng/µl

10 12 80.8 ng/µl

11 16 96.2 ng/µl

12 20 55.1 ng/µl

13 21 207.8 ng/µl

14 22 161 ng/µl

15 23 59.8 ng/µl

16 27 53.8 ng/µl

17 30 187.4 ng/µl

18 34 319.4 ng/µl

19 35 107.6 ng/µl

20 36 54.1 ng/µl

21 38 23.5 ng/µl

22 39 229.6 ng/µl

23 42 217.8 ng/µl

24 43 294.3 ng/µl

25 44 210.7 ng/µl

26 46 746.5 ng/µl

27 47 166 ng/µl

28 48 485.5 ng/µl

29 49 184.3 ng/µl

30 50 12 ng/µl

31 53 55.2 ng/µl

32 54 412.1 ng/µl

33 55 409.3 ng/µl

34 57 216.1 ng/µl

35 58 695.2 ng/µl

36 59 833.4 ng/µl

37 60 213.1 ng/µl

38 61 117.9 ng/µl

39 64 192.4 ng/µl

40 62 413.1 ng/µl

202

Table: Clinical data of deaf patients Peshawar district KP Pakistan


1 1 589.3 ng/µl

2 2 264.3 ng/µl

3 3 267.6 ng/µl

4 4 309.9 ng/µl

5 5 308.6 ng/µl

6 6 384.5 ng/µl

7 7 147.9 ng/µl

8 8 220.4 ng/µl

9 9 231.6 ng/µl

10 10 454.5 ng/µl

11 11 356.1 ng/µl

12 13 -29.7 ng/µl

13 14 849.1 ng/µl

14 16 122.3 ng/µl

15 17 763.5 ng/µl

16 18 1146.7 ng/µl

17 19 414.4 ng/µl

18 20 539.4 ng/µl

19 21 287.8 ng/µl

20 22 390.3 ng/µl

21 22 377.2 ng/µl

22 23 379.8 ng/µl

23 25 58.2 ng/µl

24 26 229.6 ng/µl

25 28 412.2 ng/µl

26 29 447 ng/µl

27 30 461.2 ng/µl

28 31 385.1 ng/µl

29 32 989 ng/µl

30 32 937.5 ng/µl

31 33 1464.1 ng/µl

32 34 796.1 ng/µl

33 35 107.1 ng/µl

34 36 219.8 ng/µl

35 37 1134.1 ng/µl

36 38 154.8 ng/µl

37 39 739.9 ng/µl

38 40 208.1 ng/µl

39 41 175.8 ng/µl

203

40 42 128.5 ng/µl

41 44 267.7 ng/µl

42 45 179.8 ng/µl

43 46 28.1 ng/µl

44 47 428 ng/µl

45 48 72.9 ng/µl

46 49 135.8 ng/µl

47 50 125.5 ng/µl

48 51 464.4 ng/µl

49 52 171.2 ng/µl

50 53 161.5 ng/µl

51 54 62.8 ng/µl

52 55 134.3 ng/µl

53 56 211 ng/µl

54 57 49.2 ng/µl

55 58 154.6 ng/µl

56 59 297.5 ng/µl

57 60 88.2 ng/µl

58 61 87.9 ng/µl

59 62 34.9 ng/µl

60 63 62.8 ng/µl

61 64 812 ng/µl

62 65 68.3 ng/µl

63 66 62.3 ng/µl

64 67 57.5 ng/µl

65 68 154.3 ng/µl

66 69 427.2 ng/µl

67 70 355.4 ng/µl

68 71 38.3 ng/µl

69 72 38.7 ng/µl

70 73 68.2 ng/µl

71 74 78.4 ng/µl

72 75 218.5 ng/µl

73 78 430 ng/µl

74 79 107.8 ng/µl

75 80 68.5 ng/µl

76 81 97.6 ng/µl

77 83 95.3 ng/µl

204

Table: Clinical data of deaf patients Swat district KP Pakistan

S.NO Sample ID DNA Concentration Unit

1 1 198.9 ng/µl

2 2 147.4 ng/µl

3 3 187.7 ng/µl

4 4 82.3 ng/µl

5 5 120.9 ng/µl

6 6 250.4 ng/µl

7 7 232.3 ng/µl

8 8 330.5 ng/µl

9 9 190.7 ng/µl

10 10 123.5 ng/µl

11 11 454.5 ng/µl

12 12 330.6 ng/µl

13 13 96 ng/µl

14 14 125.3 ng/µl

15 15 473.6 ng/µl

16 16 233.6 ng/µl

17 17 337.2 ng/µl

18 18 108.8 ng/µl

19 19 345.6 ng/µl

20 20 385.8 ng/µl

21 21 253.7 ng/µl

22 22 338.7 ng/µl

23 23 101.4 ng/µl

24 24 474.6 ng/µl

25 25 500.6 ng/µl

26 26 196.9 ng/µl

27 27 99.3 ng/µl

28 28 199.9 ng/µl

29 29 262.5 ng/µl

30 30 -14.7 ng/µl

31 31 132.4 ng/µl

32 32 194 ng/µl

33 33 499.6 ng/µl

34 34 184.4 ng/µl

35 35 249.2 ng/µl

36 36 534.3 ng/µl

37 37 179 ng/µl

38 38 180.7 ng/µl

39 39 223.9 ng/µl

205

40 40 248.8 ng/µl

41 41 364.6 ng/µl

42 42 160.6 ng/µl

43 43 107.4 ng/µl

44 44 131.3 ng/µl

45 45 132.2 ng/µl

46 46 137.7 ng/µl

47 47 205.1 ng/µl

48 48 426.4 ng/µl

49 49 621.6 ng/µl

50 50 179.5 ng/µl

51 51 261.9 ng/µl

52 52 483.1 ng/µl

53 53 1057.1 ng/µl

54 54 359.7 ng/µl

55 55 298.1 ng/µl

56 56 995.1 ng/µl

57 57 836.6 ng/µl

58 58 39.2 ng/µl

59 59 562 ng/µl

60 60 149.7 ng/µl

61 61 331.2 ng/µl

62 62 666.9 ng/µl

63 64 286 ng/µl

64 65 280.4 ng/µl

65 66 622.4 ng/µl

66 67 827 ng/µl

67 68 -2.6 ng/µl

68 69 1090.4 ng/µl

69 70 382.1 ng/µl

70 71 422.4 ng/µl

71 72 94.5 ng/µl

72 73 302.6 ng/µl

73 74 371.7 ng/µl

74 75 111.6 ng/µl

75 76 387.4 ng/µl

76 77 252.1 ng/µl

77 78 241.9 ng/µl

78 79 172.8 ng/µl

79 80 41.3 ng/µl

80 81 499.7 ng/µl

81 82 658.7 ng/µl

206

82 83 482.4 ng/µl

83 84 177.6 ng/µl

84 85 64.1 ng/µl

85 86 386.8 ng/µl

86 87 261.6 ng/µl

87 88 727.9 ng/µl

88 89 439.1 ng/µl

89 90 707.5 ng/µl

90 92 874.7 ng/µl

91 93 594.3 ng/µl

92 94 284.6 ng/µl

93 95 352.6 ng/µl

94 96 237.3 ng/µl

95 97 365 ng/µl

96 98 450.1 ng/µl

207

Appendix-8 Demographic data of deaf patients KP Pakistan

208

Appendix 8

Table: Demographic data of deaf patients of Abbotabad district KP Pakistan

S. No Name Sex

1 Raja Matiullah M

2 Mubashir M

3 Harbish F

4 Hafza F

5 Azan M

6 Mohsin M

7 Maham F

8 Mashair F

9 Neeruf F

10 Anzar M

11 Zeenat F

12 Alishba F

13 Shah Zaib M

14 Afnan M

15 Abdullah M

16 Nayab F

17 Kinza F

18 Zakya F

19 Ajmal M

20 Iman F

21 Waleed M

22 Awais M

23 Bibi Hawa F

24 Uma Faisal F

25 Hafza F

26 Fatiha F

27 Yasmeen F

28 Samaviya F

29 Umi Hani F

30 Haider M

31 Gul Sher M

32 Maaz M

33 Hamna F

34 Naeem M

35 Aliba Khalid F

36 Zumesha F

37 Maryam F

209

38 Awais M

39 Raheel M

40 Shehroz M

41 Hasnain M

42 Hassan M

43 Shehryar M

44 Afaq M

45 Bilal M

46 Sher Ali M

47 Nadar Ali M

48 Saif Ullah M

49 Anas M

50 Talha M

51 Mubashir M

52 Ihtesham M

53 Abdul Mohiz M

54 Muneer M

55 Fatima F

56 Maryam F

57 Huzaima M

58 Hafza M

59 Sidra F

60 Khudija F

61 Misbah F

62 Pakiza F

63 Zainab F

64 Sumera F

65 Humama F

66 Sara F

67 Shamil M

68 Khizar Hayat M

69 Saad M

70 Abdul Basit M

71 Qasim M

72 Talal M

73 Mohsin M

74 Abbas M

75 Saif ullah M

76 Naveed M

77 Kamran M

78 Sami Ullah M

79 Naveed M

210

80 Isha F

81 Rabia F

82 Laraib F

83 Talha M

84 Abbas M

85 Alif Bashir M

86 Mehmood Zubair M

87 Talha Wajid Khan M

88 Ihsan Imtiaz M

89 Khadir Tahir M

90 Tayyaba Wazir F

91 Amna Niaz Gul F

92 Aqsa Younas F

93 Abdullah M

94 Sohail M

95 Nimra F

96 Ashir M

97 Hassan M

98 Yamaan M

99 Usman M

100 Zeeshan M

101 Warda F

102 Jannat F

103 Uzair M

104 Huzaifa M

105 Mohsin M

106 Ihtesham M

107 Khwaja Hayat M

108 Rehan M

109 Shakir M

110 Kamran M

111 Naeem M

112 Hamza M

113 Nazish F

114 Abdullah M

115 Shah Zeb M

116 Umar M

117 Kashif M

118 Ahmad ali M

119 Naveed M

120 Zeeshan M

121 Mudassir M

211

122 Faisal M

123 Husna M

124 Shanza F

125 Nida F

126 Zohaib M

127 Afzal M

128 Kamran M

129 Bilawal M

130 Umair M

131 Awais M

132 Sharjeel M

133 Hunain M

134 Momina F

135 Irmish F

136 Tayyab M

137 Hamid ali M

138 Ramiz M

139 Abdullah M

140 Hashir M

141 Jibran M

212

Table. Demographic data of deaf patients Bannu district KP Pakistan

S.No Name Father name Age Gender

1 Bilal Amdad ali 18 Male

2 Muhibullah Mirwali ayaz 10 Male

3 Shafiullah Abdullah 12 Male

4 Sohail Abdul Rashid 16 Male

5 Fahad Sanaullah 10 Male

6 Muddasir Mirwali khan 10 Male

7 Fayaz ali Hazrat usman 8 Male

8 Rohib khan Sabghul ullah 5 Male

9 Sufyan Nasir-u-Din 7 Male

10 Gulam Nabi Syed Nabi 10 Male

11 Zarkaish Dila Baz 7 Female

12 Sheraz khan Rahim Dad khan 35 Male

13 Ibrahim Syed Umer 22 Male

14 Anwar Sher dawar 33 Male

15 Muhammad younas Ali khan 28 Male

16 Mir jahan shah Azim shah 26 Male

17 Niaz ali Rabani khan 30 Male

18 Miraj ali shah Asghar ali shah 12 Male

19 Barkat ullah 19 Male

20 Asmat ullah Shah Nawaz 30 Male

21 Sana ullah shah Abdullah shah 18 Male

22 Hukumzad khan 30 Male

23 Sajid Muhammad Daraz khan 33 Male

24 Mahtab ullah Mumtaz khan 25 Male

25 Sajid Rahmat ullah 20 Male

26 Usman ullah Syed mali khan 19 Male

27 Waqar ahmad Sadiq 20 Male

28 Shoaib-ur-Rehman Abdul rehman 35 Male

29 Amroon Sher daraz 29 Male

30 Taswar ali shah Miraz ali shah 34 Male

31 Khalid khan Nawaz khan 27 Male

32 Manzoor shah Dawalaver shah 13 Male

33 Akbar shah Dawalaver shah 11 Male

34 Ubra Dawalaver shah 15 Female

35 Shafia Khan wali shah 14 Female

36 Nisar ali shah Khan wali shah 18 Male

37 Wasi ullah Shah Khan wali shah 16 Male

213

38 Karim ullah Satar khan 20 Male

39 Sameena Hazrat khan 22 Female

40 Nadar Hazrat khan 18 Female

41 Abdullah Ajaz 36 Male

214

Table: Demographic data of deaf patients Charsada district KP Pakistan (1)

Name Class Sex

Madiha ply Group F

Abdullah ply Group M

Zaeed Khan ply Group M

Amin ply group M

Umar ply group M

Fawad Ali ply group M

Zohaib ply group M

Maria ply group F

Zeer Laghal ply group F

Infal ply group F

Muhammd ali ply group M

Amin ullah ply group M

Muhammd ply group M

Haroon Nursaryi B M

Sannan ali Nercri B M

Amin ullah Nercri B M

Salman Nercri B M

Talha Nercri B M

Asia Nercri B F

Asima Nercri B F

Mahwish Nercri B F

Mudasir 1st M

Hasan ali 1st M

Asad ullah 1st M

khusna gul 1st F

Sumia 1st F

Iqra 1st F

Danial 1st M

Talha 1st M

Faheem 1st M

Mathi ullah 1st M

Umar 1st M

Marhaba 4th F

Waqas 4th M

Maliha 4th F

215

Neemra 4th F

Qasam 4th F

Khanzib 4th M

Ismail 4th M

Shan 4th M

Hamad 4th M

Ahmad Ali 4th M

Jawad 6th M

Akif 6th M

Adnan ul Haq 6th M

Manzor 6th M

Akmal shah 6th M

Thajlla 6th F

Radda 6th F

Kalsum 6th F

Saima 6th F

Sania 6th F

Zainab 7th F

Nileem 7th F

Thanweer 7th M

Numan 7th M

Mariam 7th F

Sadaf 7th F

Shamila 7th F

216

Table: Demographic data of deaf patients Charsada district KP Pakistan (2)

S.no Name Father name sex Age

1 Samin-ur-rahman Adbur Rahman M 16

2 Ijaz Ayaz M 16

3 Faizan Nazir ahmad M 16

4 Faisal jamal Qasam shah M 16

5 Sefatullah Noor zada khan M 16

6 Arindar singh Darshan singh M 16

7 Younas khan Haris ullah M 16

8 Adnan khan Karam M 16

9 Kamil Hajji munawar M 16

10 Junaid ullah Moman khan M 16

11 Muzamil Tilwat khan M 16

12 Dilawar Tilawat khan M 16

13 Sohail ahmad Iqbal Muhammad M 16

14 Shahzad Aurangzeb M 16

15 Faizullah Ihsaanullah M 16

16 Muhammad Shafi Said alam M 16

17 Zahoor ahmad Nezar ahmad M 13

18 Asif M 14

19 Zeeshan M 13

20 Zakir Afridi M 13

21 Ikhtesham ul haq Nezamulhaq M 14

22 Usman Mehtab M 14

23 Abbas khan M 14

24 Adil Seyal soz M 13

25 Tariq Zargul M 13

26 Muhammad raheel Sohail M 13

27 Saqib Wisal khan M 13

28 Zamar M 13

29 Yasir Mullah M 13

30 Asif Nana M 14

31 Ihzaf Toor gul M 13

32 Noman Roukhul amin M 13

33 Yaseen Hidyat ullah M 14

34 Subhan Hikmat M 14

35 Umar Nisar M 14

36 Uzair Aishr M 14

37 Sohaib Zahid M 14

38 Zakir ullah Saleem M 13

39 Ahmad ullah Peer khan M 15

217

40 Hamad M 14

41 Inayat Aftab M 14

42 Kismat khan Iqbal M 13

43 Doa khan Shoukat F 13

44 Madina Ghani-ur-rahman F 14

45 Faryal Raj Muhammad F 14

47 Sameena F 14

48 Maria F 14

49 Saqib Zahir M 15

50 m.junaid shahid Shahid naizi M 14

51 Haroon Arif M 14

52 Sumaria Aurangzeb F 15

53 Noor-ul-ullah Sanar gul M 15

54 Safdar Aswan ali M 15

55 Haris khan m.mirdal khan M 15

56 Awais Javed khan M 15

57 Rizwana Nasurallah F 15

58 Amjad Sail baz M 15

59 Rashid Sami ullah M 14

60 Maaz Saad main M 14

61 Abdul samad Shoukat M 14

62 Bilal Sanab gul M 14

63 Haroon Azeem M 14

64 Muhammad awais karni Ferdos M 14

65 Akhtar jan Mehboob khan M 14

66 Irfan ullah Roman khan M 14

67 Hafeez NAIKMAN SHAH M 15

68 Sohail M 15

69 Umar ahmad Monin khan M 15

70 Sami Sharab muhammad M 14

71 Sumara Sanab F 14

72 Sara Ayran F 14

73 Sudais Irfan ullah M 14

75 Wahid gul Mehboob ullah M 13

76 Fatma Jan muhammad F 13

78 Laiba Shah jehan F 12

79 Faheem akbar Said akbar M 14

80 m. sadiq Muheb shah M 13

81 Hilman Awal khan M 12

82 Sohail Nakeebullah M 12

83 Aysha Saju F 12

department of genetics hazara university mansehra …

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