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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
SPECTRUM OF GENE MUTATIONS IN THE PATIENTS
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
SPECTRUM OF GENE MUTATIONS IN THE PATIENTS
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: ismailkks1979@gmail.com
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
Department of Genetics
5. Dr Khushi Muhammad ……………………
Assistant Professor
Department of Genetics
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
population KP Pakistan
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).
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 mid- western 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).
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
population KP Pakistan
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
(%)
Prevalence (%) Prevalence (%) of deafness in age groups
3-10 years 11-17 years
Males Females Males Females Males Females
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
Codon Domain Probability
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
(%)
Prevalence (%) Prevalence (%) of deafness in age groups
3-10 years 11-17 years
Males Females Males Females Males Females
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
Codon Domain Probability
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 (%)
Prevalence (%) Prevalence (%) of deafness in age groups
3-10 years 11-17 years
Males Females Males Females Males Females
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
5 1442 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
8 1545 G>A Transition Homoplasmy 0.4 0.26 0.13 0.109 0.050 0.150 0.079
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
13 1598 G>A Transition 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 (%)
Prevalence (%) Prevalence (%) of deafness in age groups
3-10 years 11-17 years
Males Females Males Females Males Females
1 1671 G>A Transition Heteroplasmy 0.8 0.542 0.257 0.227 0.098 0.314 0.158
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
4 1754 G>A Transition Homoplasmy 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
8 1888 G>A Transition Homoplasmy 0.4 0.271 0.128 0.113 0.049 0.157 0.078
9 1899 G>A Transition Heteroplasmy 0.4 0.271 0.128 0.113 0.049 0.157 0.078
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
population KP Pakistan
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 (%)
Prevalence (%) Prevalence (%) of deafness in age groups
3-10 years 11-17 years
Males Females Males Females Males Females
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
Table. Bioedit file of GJB2 gene of deaf patients from Khyber Pakhtunkhwa showing insertion, deletion, missense and nonsense mutations.
100
Table. Bioedit file of GJB2 gene of deaf patients from Khyber Pakhtunkhwa
showing insertion, deletion, missense and nonsense mutations.
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.
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
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
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
ab114 MDWGTLQTILGGVNEHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
**************:*********************************************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
ab114 KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEVKDIE-
******************************************************.****
CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
ab114 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
************************************************************
CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
ab114 FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
***********************************************
Fig. Protein alignment of GJB2 gene of a deaf patient sample ID Ab114, the
mutations are highlighted.
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
ab20 MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
************************************************************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHV-AYRRHEKKRKFIKGEIKSEFKDIE
ab20 RTCATITTSPSPTSGYGPCSSSCPRQRSWPCTWPTGDMRRRGSSSRGRRVNLRTSRRSMP
:. . * . . . : : . **: .. : : ::::. :.:
CRs EIKTQKVRIEGSLWWTYTSSIFFR-VIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTV
ab20 RRSASKAPCGGPTQAASSSGSSSKPPSCTSSMSCTMASPCSGWSATPGLVPTLW----TA
. .:.*. *. : :*. : ::: .: .*:* : . * *.
CRs DCFVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
ab20 LCPGPRRRLSSQCSLQCLEFASCMSLN-----CVICLDIVLGSQKSQFX
* .* .: :: : .:. * * : : *..*. .*
Fig. Protein alignment of GJB2 gene of a deaf patient sample ID Ab20, the
mutations and frame shift mutation position are highlighted.
106
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
ab36 MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
************************************************************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHV-AYRRHEKKRKFIKGEIKSEFKDIE
ab36 RTCATITTSPSPTSGYGPCSSSCPRQRSWPCTWPTGDMRRRGSSSRGRRVNLRTSRRSMP
:. . * . . . : : . **: .. : : ::::. :.:
CRs EIKTQKVRIEGSLWWTYTSSIFFR-VIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTV
ab36 RRSASKAPCGGPTQAASSSGSSSKPPSCTSSMSCTMASPCSGWSATPGLVPTLW----TA
. .:.*. *. : :*. : ::: .: .*:* : . * *.
CRs DCFVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
ab36 LCPGPRRRLSSQCSLQCLEFASCMSLN-----CVICLDIVLGSQKSQFX
* .* .: :: : .:. * * : : *..*. .*
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
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
ab70 MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEGWGDEQADFVCNTLQPGC
****************************************** *****************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHV-AYRRHEKKRKFIKGEIKSEFKDIE
ab70 RTCATITTSPSPTSGYRPCSSSCPRQRSWPCTWPTGDMRRRGSSSRDRRVNLRTSRRSKP
:. . * . . . : : . **: .. : : ::::. :.
CRs EIKTQKVRIEGSLWWTYTSSIFFR-VIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTV
ab70 RRSASKAPCGGPTQAASSSGSSSKPPSCTSSMSCTTASPCSGWSATPGLVPTLW----TA
. .:.*. *. : :*. : ::: . .*:* : . * *.
CRs DCFVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
ab70 LCPGPRRRLSSQCSLQCLEFASCMSLN-----CVICLDIVLGSQKSQFX
* .* .: :: : .:. * * : : *..*. .*
Fig. Protein alignment of GJB2 gene of a deaf patient sample ID Ab70, the indel
mutations and frame shift mutation position are highlighted.
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
ab42 MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
************************************************************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
ab42 KNVCYDHYFPISHIRLWSLQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
*****************:******************************************
CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
ab42 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMNDGFSMQRLVKCNAWPCPNTVDC
************************************* **********************
CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
ab42 FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
***********************************************
Fig. Protein alignments of GJB2 gene of a deaf patient sample ID Ab42, the
missense mutations are highlighted.
108
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
ab43 MDWVTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
*** ********************************************************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
ab43 KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
************************************************************
CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
ab43 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYIFYVMYDGFSMQRLVKCNAWPCPNTVDC
********************************:***************************
CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
ab43 FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
***********************************************
Fig. Protein alignments of GJB2 gene of a deaf patient sample ID Ab43, the
missense mutations are highlighted.
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
ab101 MDWGTLQTILGGVNKHSTSIGKIWITVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
************************:***********************************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
ab101 KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEVKDIE-
******************************************************.****
CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
ab101 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
************************************************************
CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
ab101 FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
***********************************************
Figure. Protein alignment of GJB2 gene of a deaf Patient sample ID Ab101, the
missense and deletion mutations are highlighted.
109
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
ab106 MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
************************************************************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
ab106 XNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
***********************************************************
CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
ab106 IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNGWPCPNTVDC
**************************************************.*********
CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
ab106 FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
***********************************************
Figure. Protein alignment of GJB2 gene of a deaf patient sample ID Ab106, the
nonsense and missense mutations are highlighted.
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
10S MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVAAAKEVWRDEQADFVCNTLQPGC
*************************************.****** ***************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
10S KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
************************************************************
CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
10S IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
************************************************************
CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
10S FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
***********************************************
Figure. Protein alignment of GJB2 gene of a deaf patient sample ID 10S, the
missense mutations are highlighted.
110
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
128AB MDWGTLQTILGGVN-HSTRIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
************** *** *****************************************
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
128AB KNVCYDHYFPISHIRLWSLQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
*****************:******************************************
CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
128AB IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLGKCNAWPCPNTVDC
********************************************** *************
CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
128AB FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
***********************************************
Figure. Protein alignment of GJB2 gene of a deaf patient sample ID 128AB, the
deletion and missense mutations are highlighted.
CLUSTAL 2.0.12 multiple sequence alignment
CRs MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGC
30S MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFGCNTLQPGC
*************************************************** ********
CRs KNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
30S KNLCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKSEFKDIEE
**:*********************************************************
CRs IKTQKVRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
30S IKTQ-VRIEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDC
**** *******************************************************
CRs FVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPVX
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
Figure. Exon1 of GJB6 gene sequence peaks of deaf patient sample ID. G6CH32
117
Figure. Exon1 of GJB6 gene sequence peaks of deaf patient sample ID. G6CH33
.
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
CLUSTAL 2.0.12 multiple sequence alignment
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.
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH6 MDWGTLHTFIGGVXXHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
************* *********************************************
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH6 KNVCYDHFFPASHIRLWALQLIFNSTPELLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
**********.************ *** ********************************
Crs IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC
G6CH6 IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC
************************************************************
Crs FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK
G6CH6 FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK
************************************************************
Crs QNEMNELISDSGQNAITGFPSX
G6CH6 QNEMNELISDSGQNAITGFPSX
Figure. Protein alignments in GJB6 gene deaf patient sample ID.G6CH6 showing
the N14X, K15X, V71A, V84N and A88E mutations.
121
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH7 MDWGTLHTFIGGVXQHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
************* :*********************************************
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH7 KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
************************************************************
Crs IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC
G6CH7 IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC
************************************************************
Crs FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK
G6CH7 FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK
************************************************************
Crs QNEMNELISDSGQNAITGFPSX
G6CH7 QNEMNELISDSGQNAITGFPSX
**********************
K15Q
Figure. Protein alignments in GJB6 gene deaf patient sample ID.G6CH7.The
mutation K15Q is highlighted.
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH10 MDWGTLHTFIG-VSQHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
*********** *.:*********************************************
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH10 KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDID-
**********************************************************:
Crs IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC
G6CH10 IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC
************************************************************
Crs FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK
G6CH10 FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK
************************************************************
Crs QNEMNELISDSGQNAITGFPSX
G6CH10 QNEMNELISDSGQNAITGFPSX
**********************
Figure. Protein alignments in GJB6 gene deaf patient sample ID.G6CH10.The
mutations are highlighted.
122
CLUSTAL 2.0.12 multiple sequence alignment
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.
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH4 MDWGTLHTFIGGVNNTPPASGRCGSQSSLFSESSSWWLP--RKCGVTSKTTSSATHCNRH
**************: ..: *: :: . :. . * .: . *
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH4 AKMCAMTTFSRCHTSGCAPS--SSSSPPQRCWWTCMWPTTGTKPLASSGEERRGMISKTR
::* *. .* * . *:*. : *. **:*..: .
Crs IKKQKVRIEGSLWWTYTSSIFFRIIFEAAFMYVFYFLYNGYHLPWVLKCGIDPCPNLVDC
G6CH4 TLKSRRFGRGRCGGRTPAASFSESSLKQPLCMCFTSFTMGTTCPGCN-VGLTPAPTLLTA
*.: .* .:: * . :: .: * : * * *: *.*.*: .
Crs FISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALKESK
G6CH4 LFLGQQRRPCLPFLFLRLFACCLTWQSCATCCKCVLGDQREHRRKKITPIMPRRVSRMKM
:: .:. :.:::: . *: : . * : *. :* : *.:. : .
Crs QNEMNELISDSGQNAITGFPSX
G6CH4 SFQIVVKMQSQVSQAX------
. :: :... .:*
Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH4, the
frame shift and deletion mutations were highlighted.
123
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH6 MDWGTLHTFIGGVNTLHQHREGVDHSHLYFPSHDPRGGCPGSVG--RARGLRLQHTATGM
**************. * : ::: .. .* . ..: : .*
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
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.
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH7 MDWGTLHTFIGGVTTLHQHREGVDHSHLYFPSHDPRGGCPGSVG--RARGLRLQHTATGM
*************.. * : ::: .. .* . ..: : .*
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH7 QKCVLPLFPGVPHP--------AVGPPADLRLHPSAAGGHACGLLQARNHSQVQARREEE
:: * *.* *..** * * * *...: : : *:
Crs IKKQK-VRIEGSLWWTYTSSIFFRIIFEAAF--MYVFYFLYNGYHLPWVLKCGIDPCPNL
G6CH7 FQRHRGHKAEGSDRGVAVVDVHQQHLFPNHLSSLYVCVLLPLQWVPPALGVEMWDPLPQP
::::: : *** . . .:. : :* : :** :* : * : ** *:
Crs VDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALK
G6CH7 CLLYFANREDR----VYHFYDFCVCDLHAARGRVVLPAAESVFEIKESTDAKKSPQSCPK
:::. :: :: : :* * . * .. . *.: *: *: . *
Crs ESKQNEMNELISDSGQNAITGFPSX-
G6CH7 GEAENEADFRWSKCN----HRFPKLX
. :** : *... **.
Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH7, the frame shift and indel mutations were highlighted.
124
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH11 MDWGTLHTFIGGVTTLHQHREGVDHSHLYFPSHDPRGGCPGSVG--RARGLRLQHTATGM
*************.. * : ::: .. .* . ..: : .*
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH11 QKCVLPLFPGVPHP--------AVGPPADLRLHPSAAGGHACGLLQARNHSQVQARIEEL
:: * *.* *..** * * * *...: : : *
Crs IKKQK-VRIEGSLWWTYTSSIFFRIIFEAAF--MYVFYFLYNGYHLPWVLKCGIDPCPNL
G6CH11 FQRHRGHKAEGSDRGVAVVDVHQQHLFPNHLSSLYVCVLLPLQWVPPALGVEMWDPLPQP
::::: : *** . . .:. : :* : :** :* : * : ** *:
Crs VDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALK
G6CH11 CLLYFANREDR----VYHFYDFCVCDLHAARGRVVLPAAESVFEIKESTDAKKSPQSCPK
:::. :: :: : :* * . * .. . *.: *: *: . *
Crs ESKQNEMNELISDSGQNAITGFPSX-
G6CH11 GEAENEADFRWSKCN----HRFPKLX
. :** : *... **.
Figure. Protein alignments of GJB6 gene of a deaf Patient sample ID G6CH11, the frame shift and indel mutations were highlighted.
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH13 MDWGTLHTFIGGVTTLHQHREGVDHSHLYFPSHDPRGGCPGSVG--RARGLRLQHTATGM
*************.. * : ::: .. .* . ..: : .*
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH13 QKCVLPLFPGVPHP--------AVGPPADLRLHPSAAGGHACGLLQARNHSQVHAMREEE
:: * *.* *..** * * * *...: . *:
Crs IKKQK-VRIEGSLWWTYTSSIFFRIIFEAAF--MYVFYFLYNGYHLPWVLKCGIDPCPNL
G6CH13 FQRHRGHKAEGSDRGVAVVDVHQQHLFPNHLSSLYVCVLLPLQWVPPALGVEMWDPLPQP
::::: : *** . . .:. : :* : :** :* : * : ** *:
Crs VDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALK
G6CH13 CLLYFANREDR----VYHFYDFCVCDLHAARGRVVLPAAESVFEIKESTDAKKSPQSCPK
:::. :: :: : :* * . * .. . *.: *: *: . *
Crs ESKQNEMNELISDSGQNAITGFPSX-
G6CH13 GEAENEADFRWSKCN----HRFPKLX
. :** : *... **.
Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH13, the
frameshift and indel mutations are highlighted.
125
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH14 MDWGTLHTFIGGVTTLHQHREGVDHSHLYFPSHDPRGGCPGSVG--RARGLRLQHTATGM
*************.. * : ::: .. .* . ..: : .*
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH14 QKCVLPLFPGVPHP--------AVGPPADLRLHPSAAGGHACGLLQARNHSQVQARREEE
:: * *.* *..** * * * *...: : : *:
Crs IKKQK-VRIEGSLWWTYTSSIFFRIIFEAAF--MYVFYFLYNGYHLPWVLKCGIDPCPNL
G6CH14 FQRHRGHKAEGSDRGVAVVDVHQQHLFPNHLSSLYVCVLLPLQWVPPALGVEMWDPLPQP
::::: : *** . . .:. : :* : :** :* : * : ** *:
Crs VDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALK
G6CH14 CLLYFANREDR----VYHFYDFCVCDLHAARGRVVLPAAESVFEIKESTDAKKSPQSCPK
:::. :: :: : :* * . * .. . *.: *: *: . *
Crs ESKQNEMNELISDSGQNAITGFPSX-
G6CH14 GEAENEADFRWSKCN----HRFPKLX
. :** : *... **.
Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH14, the
frameshift and indel mutations are highlighted.
CLUSTAL 2.0.12 multiple sequence alignment
Crs MDWGTLHTFIGGVNKHSTSIGKVWITVIFIFRVMILVVAAQEVWGDEQEDFVCNTLQPGC
G6CH15 MDWGTLHTFIGGVTTLHQHREGVDHSHLYFPSHDPRGGCPGSVG--RARGLRLQHTATGM
*************.. * : ::: .. .* . ..: : .*
Crs KNVCYDHFFPVSHIRLWALQLIFVSTPALLVAMHVAYYRHETTRKFRRGEKRNDFKDIED
G6CH15 QKCVLPLFPGVPHP--------AVGPPADLRLHPSAAGGHACGLLQARNHSQVQARREEE
:: * *.* *..** * * * *...: : : *:
Crs IKKQK-VRIEGSLWWTYTSSIFFRIIFEAAF--MYVFYFLYNGYHLPWVLKCGIDPCPNL
G6CH15 FQRHRGHKAEGSDRGVAVVDVHQQHLFPNHLSSLYVCVLLPLQWVPPALGVEMWDPLPQP
::::: : *** . . .:. : :* : :** :* : * : ** *:
Crs VDCFISRPTEKTVFTIFMISASVICMLLNVAELCYLLLKVCFRRSKRAQTQKNHPNHALK
G6CH15 CLLYFANREDR----VYHFYDFCVCDLHAARGRVVLPAAESVFEIKESTDAKKSPQSCPK
:::. :: :: : :* * . * .. . *.: *: *: . *
Crs ESKQNEMNELISDSGQNAITGFPSX-
G6CH15 GEAENEADFRWSKCN----HRFPKLX
. :** : *... **.
Figure. Protein alignments of GJB6 gene of a deaf patient sample ID G6CH15, the frameshift and indel mutations are highlighted.
126
CLUSTAL 2.0.12 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA
384HR TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA
************************************************************
C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG
384HR ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG
**************** ***************** *************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA
384HR GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA
************************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA
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
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA
285ST TGAAGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA
************************************************************
C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG
285ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAAAGTGCTTAGTTGAACAGG
**************** *******************************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA
285ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA
************************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA
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
CLUSTAL 2.1 multiple sequence alignment
C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG
295ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG
**************** *******************************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA
295ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA
************************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA
295ST ACTAAAACCCCCACGCATTTTTTTAGAGGAAACAAGTCGTAACATGGTAAGTGTACTGGA
*********** ******** * ******* *****************************
C AAGTGCACTTGGACGAA
295ST 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
CLUSTAL 2.1 multiple sequence alignment
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
the nucleotide peaks
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
HR333 GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 157
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
HR333 CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 217
*************** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
HR333 CCCTGAAGCGCGTACACACCGCCCGTCACCCTCTCAAGTATACTTCAAAGGACATTTAA 277
***********************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
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
the nucleotide peaks
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
354HR GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 149
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
354HR CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 209
*************** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
354HR CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 269
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
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
CLUSTAL 2.1 multiple sequence alignment
C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA
340HR TGAGGGGGCCAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA
***** *** **************************************************
C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG
340HR ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG
********************************** *************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA
340HR GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA
************************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA
340HR ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA
************************************************************
C AAGTGCACTTGGACGAA
340HR AAGTGCACTTGGACGAA
*****************
146
Figure: Mutation at position A1438G ( Homoplasmy) in sample ID.340HR
Figure. Nucleotide alignments of deaf patient sample ID. 340HR of MTRNR1 gene. The mutation Positions T1420G is highlighted and denoted by black bar in
the nucleotide peaks
147
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
364HR GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 151
**** *******************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
364HR CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAAAATAGAGTGCTTAGTTGAACAGGG 210
********************************* **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299
364HR CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTT 270
***********************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 358
364HR ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 330
***********************************************************
C AAAGTGCACTTGGACGAA
364HR AAAGTGCACTTGGTCGAA
******************
Figure: Novel mutation at position T1349G( Homoplasmy) in sample ID.364HR
Figure. Nucleotide alignments of deaf patient sample ID. 364HR of MTRNR1 gene. The mutation Positions T1349G is highlighted and denoted by black bar in
the nucleotide peaks
148
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
382HR GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAAA 159
**** *******************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
382HR CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 219
*************** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
382HR CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 279
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
382HR CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 339
************************************************************
C AGTGCACTTGGACGAA
382HR 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
CLUSTAL 2.1 multiple sequence alignment
C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 178
383HR TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACCATAGCCCTTATGA 154
***********************************************************
C AACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAG 238
383HR AACTTAAAGGTCGAAGGGGGATTTAGCAGTAAACTGAGAATAGAGTGCTTAGTTGAACAG 214
***************** ***************** ************************
C GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 298
383HR GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 274
************************************************************
C AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 358
383HR AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 334
************************************************************
C AAAGTGCACTTGGACGAA
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
CLUSTAL 2.1 multiple sequence alignment
C GAAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCAT 120
386HR GAAGGCTACAAAGTAAGCGCAAGTACCCACGTAAAGACGTTAGGTCAAGGTGTAGCCCAT 96
************************************************************
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
386HR GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 156
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
386HR CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 216
*************** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
386HR CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 276
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
386HR CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 336
************************************************************
C AGTGCACTTGGACGAA
386HR AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C ATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 178
387HR ATGAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 157
****** *****************************************************
C AACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAG 238
387HR AACTTAAGGGTCGAAGGGGGATTTAGCAATAAACTGAAAGTAGAGTGCTTAGTTGAACAG 217
***************** ***************** *************************
C GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 298
387HR GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 277
************************************************************
C AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 358
387HR AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 337
************************************************************
C AAAGTGCACTTGGACGAA
387HR AAAGTGCACTTGGACGAA
******************
152
CLUSTAL 2.1 multiple sequence alignment
C ATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 178
390HR ATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 154
************************************************************
C AACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAG 238
390HR AACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAG 214
***************** ***************** ************************
C GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 298
390HR GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 274
************************************************************
C AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 358
390HR AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 334
************************************************************
C AAAGTGCACTTGGACGAA
390HR AAAGTGCACTTGGACGAA
******************
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
121sb GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 153
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
121sb CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAAAGTGCTTAATTGAACAGGG 213
*************** ***************** ******** ******** ********
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
121sb CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 273
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
121sb CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 333
************************************************************
C AGTGCACTTGGACGAA
121sb AGTGCACTTGGACGAA
****************
153
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
123sb GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 150
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
123sb CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 210
*************** ***************** ***************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
123sb CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTTA 270
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
123sb CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGGAAGTGTACTGGAA 330
************************************************************
C AGTGCACTTGGACGAA
123sb AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
249ST GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAAA 153
***********************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
249ST CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAATTGAACAGGG 213
*************** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
249ST CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 273
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
249ST CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 333
************************************************************
C AGTGCACTTGGACGAA
249ST AGTGCACTTGGACGAA
****************
154
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
262 GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAAA 152
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
262 CTTAAGGGGCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 211
******** ****** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
262 CCCTGAAGCGCGTACACACCGCCCGTCCCCCTCCTCAAGTATACTTCAAAGGACATTTAA 271
*************************** ********************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
262 CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGGAAGTGTACTGGAA 331
********************************************** *************
C AGTGCACTTGGACGAA
262 AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
264ST GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 153
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
264ST CTTAAGGGGCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 213
******** ****** ********************************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
264ST CCCTGAAGCGCGTACACACCGCCCCCGTCCCCCCCCAAGTATACTTCAAAGGACATTTAA 273
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
264ST CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 333
****************************** *****************************
C AGTGCACTTGGACGAA
264ST AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C ATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 178
266ST ATGAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGA 152
************************************************************
C AACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAG 238
266ST AACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAG 212
***************** ***************** ************************
C GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 298
266ST GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 272
************************************************************
156
C AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 358
266ST AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 332
************************************************************
C AAAGTGCACTTGGACGAA
266ST AAAGTGCACTTGGACGAA
******************
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
268ST GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 154
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG 239
268ST CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 213
********************************* *************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299
268ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 273
************************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 359
268ST ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 333
************************************************************
C AAGTGCACTTGGACGAA
268ST AAGTGCACTTGGACGAA
*****************
CLUSTAL 2.1 multiple sequence alignment
C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG 239
282ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAAATGAGAGTAGATGCTTGAGTTGAACAGG 212
**************** ***************** *************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299
282ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTAATACTTCAAAGGACATTA 272
*************************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 359
282ST ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 332
************************************************************
C AAGTGCACTTGGACGAA
282ST AAGTGCACTTGGACGAA
*****************
157
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
283ST GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 152
**** *******************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
283ST CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 212
*************** ********************************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
283ST CCCTGAAGCGCGTACACACCGCCCGTCACCCCCCCCAAGTATACTTCAAAGGACATTTAA 272
******************************* ** *************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
283ST CTAAAACCCCTACGCATTTTTTTAGAGGAAACAAGTCGTAACATGGTAAGTGTACTGGAA 332
******************* * ******* ******************************
C AGTGCACTTGGACGAA
283ST AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA 179
285ST TGAAGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA 154
************************************************************
C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGG 239
285ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAAAGTGCTTAGTTGAACAGG 214
**************** *******************************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299
285ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 274
************************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 359
285ST ACTAAAACCCCTACGCATTTTTTTAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 334
******************** * *************************************
C AAGTGCACTTGGACGAA
285ST AAGTGCACTTGGACGAA
*****************
158
Novel mutations at position A1544T and A1546T(Heteroplasmy)in sample ID.285ST
CLUSTAL 2.1 multiple sequence alignment
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
286ST CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 213
************************************************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
286ST CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 273
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
286ST CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGGACTGGAA 333
**************************************************** *******
C AGTGCACTTGGACGAA
286ST AGTGGACTTGGACAAA
*** ******* **
Figure: Novel mutation at position G1598A(Homoplasmy)in sample ID.286ST
159
CLUSTAL 2.1 multiple sequence alignment
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
289ST CCCTGAAGCGCGTAAACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 264
************** *********************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
289ST CTAAAACCCCTACGCATTTATATAAAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 324
************************ ***********************************
C AGTGCACTTGGACGAA
289ST AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
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
********************
CLUSTAL 2.1 multiple sequence alignment
C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 239
295ST ACTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 215
**************** *******************************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299
295ST GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 275
************************************************************
160
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 359
295ST ACTAAAACCCCCACGCATTTTTTTAGAGGAAACAAGTCGTAACATGGTAAGTGTACTGGA 335
*********** ******** * ******* *****************************
C AAGTGCACTTGGACGAA
295ST AAGTGCACTTGGACGAA
*****************
Figure: Novel mutation at position T1420G(Homoplasmy)in sample ID.295ST
Figure: Novel mutation at position T1535C(Heteroplasmy)in sample ID.295ST
161
CLUSTAL 2.1 multiple sequence alignment
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
305ST CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 221
*************** ********************************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
305ST CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 281
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
305ST CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 341
************************************************************
C AGTGCACTTGGACGAA
305ST AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
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
********************
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
311ST GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCCGAAAACTACGATAGCCCTTATGAAA 157
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
311ST CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 217
*************** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
311ST CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 277
************************************************************
162
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGG-ACTGGAA 360
311ST CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGGGGTACTGGAA 337
************************************************** ** *******
C AGTGCACTTGGACGAA
311ST AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAA 179
325ST TGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAA 154
************************************************************
C ACTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 239
325ST ACTTAAGGGTCTAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGG 214
*********** **** *******************************************
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299
325ST GCCCTGAAGCGCGTACACACCGCCCGTCCCCCTCCTCAAGTATACTTCAAAGGACATTTA 274
**************************** *******************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 359
325ST ACTAAAACCCCTACCCATTTATATAGAGGAAACAAGTCGTAACATGGTAAGTGTACTGGA 334
************** *************** *****************************
Figure: Novel mutation at position G1538C(Heteroplasmy)in sample ID.325ST
163
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
PS181 GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 153
************************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
PS181 CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 213
************************************************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
PS181 CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 273
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
PS181 CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 333
************************************************************
C AGTGCACTTGGACGAA
PS181 AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
PS185 CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 211
*************** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
PS185 CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 271
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
PS185 CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 331
************************************************************
C AGTGCACTTGGACGAA
PS185 AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
396CH CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 215
*************** ****************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
396CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 275
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
396CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 335
************************************************************
C AGTGCACTTGGACGAA
396CH AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C TTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
407CH TTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGGAGAGTGCTTAGTTGAACAGGG 215
***********************************************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
407CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 275
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
407CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 335
************************************************************
C AGTGCACTTGGACGAA
407CH AGTGCACTTGGACGAA
****************
165
CLUSTAL 2.1 multiple sequence alignment
C GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
403CH GAGGGGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 149
**** *******************************************************
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
403CH CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 209
********************************* **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
403CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 269
*************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
403CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 329
************************************************************
C AGTGCACTTGGACGAA
403CH AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
408CH CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 210
************************************************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
408CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 270
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
408CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 330
************************************************************
C AGTGCACTTGGACGAA
408CH AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 298
412CH GGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTT 273
************************************************************
C AACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 358
412CH AAATAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGG 333
** *********************************************************
C AAAGTGCACTTGGACGAA
412CH AAAGTGCACTTGGACGAA
******************
166
Novel mutation at position C1525A ( Homoplasmy) in sample ID.412CH
CLUSTAL 2.1 multiple sequence alignment
C CAGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACA 295
421CH CAGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACA 267
************************************************************
C TTTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTAC 355
421CH TTTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTAC 327
************************************************************
C TGGAAAGTGCACTTGGACGAA
421CH TGGAAAGTGCACTTGGACGAA
*********************
CLUSTAL 2.1 multiple sequence alignment
C GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 299
422CH GCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTA 277
************************************************************
C ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 359
422CH ACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGA 337
************************************************************
C AAGTGCACTTGGACGAA
422CH AAGTGCACTTGGACGAA
*****************
167
CLUSTAL 2.1 multiple sequence alignment
c AGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACAT 296
425CH AGGGCCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCCAAGGACAT 271
************************************************************
c TTAACTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACT 356
425CH TTAACTTAAACCCCTACCCCTTTTTATAGAGGAGACAAGTCGTAACATGGGAAGTGTACT 331
************************************************** *********
c GGAAAGTGCACTTGGACGAA
425CH GGAAAGTGCACTTGGACGAA
********************
CLUSTAL 2.1 multiple sequence alignment
c GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
428CH GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 157
************************************************************
c CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
428CH CTTAAGGGTCGAAGGGGGATTTAGCAGAAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 217
*************** ***************** **************************
c CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
428CH CCCTGAAGGGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 277
************************************************************
c CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
428CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 337
************************************************************
c AGTGCACTTGGACGAA
428CH AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
c CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
430CH CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 215
************************************************************
c CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
430CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 275
************************************************************
c CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
430CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 335
************************************************************
c AGTGCACTTGGACGAA
430CH AGTGCACTTGGACGAA
****************
168
CLUSTAL 2.1 multiple sequence alignment
c GAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAGCCCTTATGAAA 180
434CH GAGGTGGCAAGAAATGGGCAACATTTTCTACCCCAAAAAACTACGATAGCCCTTATGAAA 149
************************************************************
c CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 240
434CH CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 209
************************************************************
c CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
434CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 269
************************************************************
c CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
434CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGGA 329
********************************************************** *
CLUSTAL 2.1 multiple sequence alignment
c CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
441CH CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 213
*************** ***************** **************************
c CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
441CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 273
************************************************************
c CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
441CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 333
************************************************************
c AGTGCACTTGGACGAA
441CH AGTGCACTTGGACGAA
****************
CLUSTAL 2.1 multiple sequence alignment
C CTTAAGGGTCGAAGGTGGATTTAGCAGTAAACTAAGAGTAGAGTGCTTAGTTGAACAGGG 240
415CH CTTAAGGGTCGAAGGGGGATTTAGCAGTAAACTGAGAGTAGAGTGCTTAGTTGAACAGGG 210
*************** ***************** **************************
C CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 300
415CH CCCTGAAGCGCGTACACACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGGACATTTAA 270
************************************************************
C CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 360
415CH CTAAAACCCCTACGCATTTATATAGAGGAGACAAGTCGTAACATGGTAAGTGTACTGGAA 330
************************************************************
C AGTGCACTTGGACGAA
415CH 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
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAC
362HR GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAC
***********************************
C TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA
362HR TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA
************************************************************
C CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA
362HR CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA
************************************************************
C TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG
362HR TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG
************************************************************
C GAGAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCA
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
CLUSTAL 2.1 multiple sequence alignment
C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACT
428CH GAGGTAAACCTAGCCCCAAACCCACTCCACCTTACT
*** ********************************
C ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC
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
CLUSTAL 2.1 multiple sequence alignment
C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAC
422CH GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAA
***********************************
C TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA
422CH TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA
************************************************************
C CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA
422CH CCTGGCGCAATAAATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA
************ ***********************************************
C TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG
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
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACT
249ST GAACTAAACCTAGCCCCAAACCCACTCCACCTTACT
** *********************************
C ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC
249ST ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC
************************************************************
C CTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAA
249ST CTGGCGCAATAGATATAGTACCGCTAGGGAAAGATGAAAAA
*****************************************
CLUSTAL 2.1 multiple sequence alignment
C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACT
289ST GAGCTAAACCTAGCCCCAAACCCACTCCACCTTACT
************************************
C ACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAAC
289ST ACCAAACAACCTTAGCCAAACCATTTACCCCAATAAAGTATAGGCGATAGAAATTGAAAC
**** ************************* *****************************
C CTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATAT
289ST CTGGCGCAATAAATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCCAGCATAATAT
*********** ************************************* **********
C AGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAGG
289ST AGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAGG
************************************************************
CLUSTAL 2.1 multiple sequence alignment
C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAC
295ST GAACTAAACCTAGCCCCAAACCCACTCCACCTTAC
** ********************************
C TACCAGACAACCTTAGCCAAACCATTTACCCAGATAAAGTATAGGCGATAGAAATTGAAA
295ST TACCAAACAACCTTAACCAAACCATTTACCCAAATAAAGTATAGGCGAAAGAAATTGAAA
***** ********* **************** *************** ***********
C CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA
295ST CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA
************************************************************
C TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG
295ST TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAAAAATAACTTTGCAAG
******************************************** ****************
182
CLUSTAL 2.1 multiple sequence alignment
C GAGCTAAACCTAGCCCCAAACCCACTCCACCTTAC
285ST GAACTAAACCTAGCCCCAAACCCACTCCACCTTAC
** ********************************
C TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAGAAATTGAAA
285ST TACCAGACAACCTTAGCCAAACCATTTACCCAAATAAAGTATAGGCGATAAAAATTGAAA
***** ******************************************** *********
C CCTGGCGCAATAGATATAGTACCGCAAGGGAAAGATGAAAAATTATAACCAAGCATAATA
285ST CCTGGCGCAATAGATATTGTACCGCAAGGGAAAGATGAAAAATTTTAACCAAGCATAATA
************ **** ***** ******************** ***** *********
C TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG
285ST TAGCAAGGACTAACCCCTATACCTTCTGCATAATGAATTAACTAGAAATAACTTTGCAAG
************************************************************
C GAGAGCCAAAGCTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCA
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
CLUSTAL 2.1 multiple sequence alignment
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.
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
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
CLUSTAL 2.1 multiple sequence alignment
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
S.No Sample ID DNA Concentration Unit
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
S.No Sample ID DNA Concentration Unit
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
S.No Sample ID DNA Concentration Unit
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
S.No Sample ID DNA Concentration Unit
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
S.No Sample ID DNA Concentration Unit
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
218
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