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A STUDY ON GJB2 AND GJB6 GENE MUTATIONS AMONG MALAYS WITH
NON-SYNDROMIC HEARING LOSS
by
SITI AISHAH ZAINAL
Thesis submitted in fulfillment of the requirements
for the degree of
Master of Science
MAY 2010
ii
DEDICATION
First of all, thank you very much to Allah s.w.t for giving me such a wonderful parents,
Mr. Zainal Mahamood and Mrs. Normah Bador and husband, Mr. Mohd Rusydi Mohd.
Noor. Special thanks to them for their love, prayer and support to me. For my parents-
in-law, Mr. Mohd. Noor bin Kasim and Mrs. Chempawan Ismail thank you for being a
supportive parents. Also for my sweet charming daughter, Nur ‘Iffah, thanks for always
being there at my side.
iii
ACKNOWLEDGMENT
My deepest appreciation is dedicated to my main supervisor, Assoc. Prof. Dr. Zilfalil
bin Alwi for his support, advice and motivation throughout my study especially in thesis
writing. Thank you very much to my co-supervisors, Assoc. Prof. Dr. Mohd. Khairi Md.
Daud and Dr. Zafarina Zainuddin for their guidance and help until I have completed my
MSc project.
My acknowledgment also goes to Assoc. Prof. Dr. Normastura Ab. Rahman for helping
me a lot in sample calculation and statistical analysis. My special thanks to all members
of the Human Genome Centre; Prof. Dr. Ravindran Ankathil, Dr. TP Kannan, Dr.
Sarina Sulong, Che Na, Kak Ann, Kak Cma, Kak Yati, Kak Finie, Kak Que, Kak Ijan,
Nizam, Kakcik, Sha, Mareen, Kak Nita, Siti, Sathiya and the students and staffs for
being a helpful and supportive friends.
Not forgetting this appreciation also goes to headmasters, teachers, parents and subjects
who had contributed to my sample collection and Syuhada who had help and teach me
in hearing assessment.
I would like to thank USM for providing USM Short Term Grant (304/PPSP/6131446)
for the financial support. Last but not least, thanks a lot for those who have contributed
to this study either directly or indirectly.
iv
LIST OF CONTENTS
CONTENTS PAGE
TITLE
DEDICATION
ACKNOWLEDGEMENTS
LIST OF CONTENTS
LIST OF APPENDICES
LIST OF TABLES
LIST OF FIGURES
LIST OF PLATES
LIST OF ABBREVIATIONS
ABSTRAK
ABSTRACT
CHAPTER 1 INTRODUCTION
i
ii
iii
iv
ix
x
xi
xiii
xiv
xvii
xix
1
1.1 Hearing loss
1
1.2 Hearing-How it works
2
1.3 Degree of hearing loss 5
1.3.1 Pure Tone Audiometry (PTA)
5
1.4 Types of hearing loss
7
1.5 Non-syndromic hearing loss (NSHL)
10
1.6 Gap junction proteins in cochlea
14
1.7 Gap junction protein genes 22
1.7.1 Gap junction protein beta-2 (GJB2) gene 23
1.7.1 (a) GJB2 gene mutation
29
v
1.7.2 Gap junction protein beta-6 (GJB6) gene 31
1.7.2 (a) GJB6 gene mutation
34
1.8 Denaturing Performance Liquid Chromatography (dHPLC) 36
1.8.1 The principle of dHPLC
37
1.8.2 Sensitivity and specificity of dHPLC
41
1.9 Screening for sequence variations in GJB2 gene
42
1.10 Objective of the study 44
1.10.1 General objective
44
1.10.2 Specific objectives
44
CHAPTER 2 MATERIALS AND METHODS
45
2.1 Materials 45
2.1.1 Selection of patients
45
2.1.2 Sample size
46
2.1.3 Inclusion and exclusion criteria
47
2.1.4 Data collection procedures 47
2.1.4 (a) Pure Tone Audiometry (PTA)
48
2.1.5 Collection of buccal swab samples
49
2.1.6 Genomic DNA extraction 49
2.1.6 (a) Reagents 49
2.1.6 (a) (i) Phosphate Buffered Saline
(PBS) (1X)
49
2.1.6 (a) (ii) Absolute ethanol 49
2.1.6 (a) (iii) DNA extraction kit
(GeneAll Blood SV Mini kit)
50
2.1.7 Polymerase chain reaction (PCR) 50
2.1.7 (a) Reagents 50
2.1.7 (b) Thermocycler
51
2.1.8 Gel electrophoresis 51
2.1.8 (a) Electrophoresis buffer 51
2.1.8 (a) (i) Preparation of 10X TBE (Tris
Boric EDTA) pH 8.3 solution
51
vi
2.1.8 (a) (ii) Preparation of 1X TBE (Tris
Boric EDTA) solution
52
2.1.8 (b) Agarose gel 2% 52
2.1.8 (c) SYBR®
Green 1 52
2.1.8 (d) Loading dye (6X) 52
2.1.8 (e) DNA ladder (100 bp)
53
2.1.9 Denaturing High Performance Liquid Chromatography
(DHPLC)
53
2.1.9 (a) DHPLC reagents 53
2.1.9 (b) DHPLC column 54
2.1.9 (c) DHPLC modules
54
2.1.10 PCR purification
54
2.2 Methods 55
2.2.1 DNA extraction from buccal swab
55
2.2.2 DNA quantification
56
2.2.3 PCR amplification of exon 2 GJB2 gene coding region
57
2.2.4 PCR amplification of exon 6 of GJB6 gene coding region-
multiplex PCR
60
2.2.5 Agarose gel electrophoresis
63
2.2.6 DHPLC analysis 63
2.2.6 (a) Calculation of melting temperature 64
2.2.6 (b) Procedures of running dHPLC system 66
2.2.6 (c) Mapping of temperature 67
2.2.6 (d) Samples screening
69
2.2.7 PCR purification
69
2.2.8 DNA sequencing
70
2.2.9 Statistical analysis
71
vii
CHAPTER 3 RESULTS 72
3.1 Recruitment of patients
72
3.2 Clinical data (severity and laterality)
72
3.3 Genomic DNA extraction
75
3.4 Amplification of GJB2 gene coding region
75
3.5 Detection of deletion of GJB6 gene coding region
78
3.6 DHPLC mutational analysis
80
3.7 Analysis of DNA sequencing 85
3.8 DNA and amino acid sequence variations in GJB2 among non-
syndromic hearing loss (NSHL) cases
94
3.9 Association of mutation and polymorphism in GJB2 gene with non-
syndromic hearing loss patients
95
3.10 Association of homozygous and heterozygous variations in GJB2
gene with severity and laterality of hearing loss
98
CHAPTER 4 DISCUSSION
101
4.1 Severity of hearing loss
101
4.2 DHPLC as an effective screening method for sequence variations in
GJB2 gene among Malay patients with non-syndromic hearing loss
102
4.3 GJB2 gene mutations and polymorphisms
106
4.4 GJB6 gene deletion
114
4.5 Digenic effect on non-syndromic hearing loss
116
4.6 Limitation of the study
117
4.7 Future prospect of non-syndromic hearing loss study in Malaysia 119
4.7.1 Screening for mutations or SNPs in the whole region of
GJB2 gene
119
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4.7.2 Screening for mutations or SNPs in the whole region of
GJB6 gene
120
CHAPTER 5 CONCLUSION
123
REFERENCES
125
APPENDICES
138
LIST OF PUBLICATIONS AND PRESENTATIONS 171
ix
LIST OF APPENDICES
Page
Appendix A
Appendix B
Appendix C
Appendix D
Appendix E
Appendix F
Patient information and consent form
Borang maklumat dan keizinan pesakit
Sociodemographic sheet
Audiograms of non-syndromic hearing loss patients
List of non-syndromic hearing loss patients
Publications and proceedings
139
143
147
151
155
158
x
LIST OF TABLES
Page
Table 1.1
Table 1.2
Table 2.1
Table 2.2
Table 2.3
Table 2.4
Table 3.1
Table 3.2
Table 3.3
Table 3.4
Table 3.5
Table 3.6
Table 3.7
The effect of the different degrees of hearing loss
Causes of hearing loss
Primer sequences for the amplification of three regions of
exon 2 of GJB2 gene (Zelante et al., 1997) and its sizes of
amplicons
Total volume of reagents used in PCR amplification of
GJB2 gene (exon 2; region 1)
Primer sequences for the multiplex PCR of exon 6 of GJB6
gene and β-globin gene and its sizes of amplicons
Total volume of reagents used in multiplex PCR
amplification of GJB6 gene and β-globin gene
Distribution of the severity of hearing loss based on Pure
Tone Audiometry (PTA) test
Variations in GJB2 identified by DNA sequencing of 32
patients with non-syndromic hearing loss and 37 normal
hearing control cases
Mutations and polymorphisms in GJB2 gene in non-
syndromic hearing loss patients in present study and other
studies
Association of homozygous mutations and polymorphism
with NSHL patients
Association of heterozygous mutations and polymorphism
with NSHL patients
Association of homozygous and heterozygous variations
with severity at right ear of non-syndromic hearing loss
patients
Association of homozygous and heterozygous variations
with severity at left ear of non-syndromic hearing loss
patients
9
13
58
59
61
62
74
91
92
96
97
99
100
xi
LIST OF FIGURES
Page
Figure 1.1
Figure 1.2
Figure 1.3
Figure 1.4
Figure 1.5
Figure 1.6
Figure 1.7
Figure 1.8
Figure 1.9
Figure 1.10
Figure 1.11
Figure 2.1
Figure 2.2
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Cross-section of the outer, middle and inner human ear
Examples of pure tone audiogram
Schematic representation of connexins and gap junctions
Schematic representation of basic connexin
Flow diagram of human auditory pathway
Schematic illustration indicating possible pathway for the
transport of K+ in the cochlea
Location of GJB2 and GJB6 genes
Schematic diagram of Cx26 with sequence of amino acids
Location of connexin 26 (GJB2) in the potassium
recycling pathway of the cochlea
Schematic representation of the genomic structure of
GJB6 on chromosome 13
Basic principles of dHPLC in mutation detection
Result for GJB2 exon 2 region 2 melting temperature
calculation from dHPLC Melt Program website
Temperature mapping for region 2 exon 2 GJB2 gene
which indicated 61.5oC as the optimum temperature
Distribution of hearing loss patients based on gender.
Total number of cases is 91 (n=91)
DHPLC results for region 1 of exon 2 for patients‟
samples
DHPLC results for region 1 of exon 2 for controls‟
samples
DHPLC results for region 1 of exon 2 for a patient sample
4
6
15
17
19
20
24
25
28
32
39
65
68
73
81
82
83
xii
Figure 3.5
Figure 3.6
Figure 3.7
Figure 3.8
Figure 3.9
Figure 3.10
DHPLC results for region 1 of exon 2 for a patient sample
Electropherogram showing the sequencing result for
heterozygous polymorphism from G to A in exon 2 at
position 79 using forward primer
Electropherogram showing the sequencing result for
homozygous polymorphism from G to A in exon 2 at
position 109 using forward primer
Electropherogram showing the sequencing result for
heterozygous deletion of CGC bases in exon 2 at position
257-259 using forward primer
The BLASTn result obtained from program for forward
primer for heterozygous G to A at position 79
The BLASTn result obtained from program for forward
primer for homozygous G to A at position 109
84
86
87
88
89
90
xiii
LIST OF PLATES
Page
Plate 3.1
Plate 3.2
Plate 3.3
Electrophoresis of genomic DNA extracted from buccal
swab on 1% agarose gel
Gel electrophoresis result of PCR products
PCR products of multiplex PCR for GJB6 coding region
(872 bp) and β–globin gene (240 bp)
76
77
79
xiv
LIST OF ABBREVIATIONS
oC : degree celcius
µl : microlitre
µM : micromolar
A260/A280 : ratio of 260 absorbance over 280 absorbance
bp : base pair
Buffer BL : Lysis Buffer
Buffer BW : Wash Buffer I
Buffer TW : Wash Buffer II
Buffer AE : Elution Buffer
Buffer PB : Bind Buffer
Buffer NW : Wash Buffer
Buffer EB : Elution Buffer
Cx : connexin
ddH2O : deionized distilled water
dsDNA : double strand DNA
DGGE : denaturing gradient gel electrophoresis
dHPLC : denaturing High Performance Liquid Chromatography
DNA : deoxyribonucleic acid
dNTPs : dinucleotide triphospate
EDTA : ethylenediamine tetraacetic acid
e.g. : example
xv
GJB2 : Gap Junction Beta-2
GJB3 : Gap Junction Beta-3
GJB6 : Gap Junction Beta-6
LOH : loss of heterozygosity
K+
: potassium ion
MgCl2 : magnesium chloride
min : minute
ml : mililitre
mM : milimolar
Na+ : sodium ion
NCBI : National Center for Biotechnology Information
ng/µl : nanogram per microliter
NSHL : non-syndromic hearing loss
PBS : Phosphate buffered saline
PCR : Polymerase chain reaction
rpm : round per minute
SNP : single nucleotide polymorphism
SSCP : single-strand conformation polymorphism
Taq : Thermophilus aquaticus
TBE : Tris Boric EDTA
U : unit
UV : ultra violet
V : voltage
xvi
vs : versus
xvii
KAJIAN TERHADAP MUTASI GEN GJB2 DAN GJB6 DI KALANGAN
PESAKIT MELAYU YANG MENGALAMI KECACATAN PENDENGARAN
JENIS TIDAK SINDROMIK
ABSTRAK
Kecacatan pendengaran merupakan kecacatan deria yang paling kerap berlaku pada
manusia. Lebih kurang satu daripada seribu bayi yang baru dilahirkan di seluruh dunia
lahir dengan kecacatan pendengaran samada dengan darjah kecacatan pendengaran yang
ringan atau tiada pendengaran langsung. Kecacatan ini boleh disebabkan oleh dua faktor
iaitu faktor genetik dan persekitaran dengan lebih 50% daripada kecacatan tersebut
disebabkan oleh faktor genetik. Telah terbukti bahawa terdapat pelbagai gen yang
terlibat dalam penyakit kecacatan pendengaran tidak sindromik (NSHL) iaitu sejenis
kecacatan pendengaran tanpa gejala lain dan gen tersebut dikaji dalam penyelidikan ini.
Mutasi pada gen GJB2 telah dikenal pasti sebagai gen utama yang terlibat dengan
kecacatan pendengaran tidak sindromik (NSHL). Satu gen lain yang berkaitan iaitu gen
GJB6 yang terletak berhampiran dengan gen GJB2 pada kromosom mungkin berkaitan
dengan kecacatan pendengaran jenis NSHL. Tujuan kajian ini adalah untuk
mengenalpasti mutasi pada dua gen tersebut dan perkaitannya dengan kecacatan
pendengaran jenis NSHL. Sebanyak 91 sampel sel tisu pipi pesakit NSHL dan 91
sampel sel tisu pipi subjek kawalan di Kelantan diambil untuk kajian ini. Tindak balas
rantaian polimerase (PCR) digunakan untuk menggandakan gen GJB2. Produk PCR gen
GJB2 seterusnya disaring untuk mutasi dengan menggunakan teknik DHPLC dan
xviii
mutasi yang berjaya dikesan kemudiannya disah tentukan dengan penjujukan DNA.
Sebelas mutasi dan polimorfisma dikenal pasti pada 32 pesakit dan 37 subjek kawalan
setelah sebanyak 182 pesakit NSHL dan kawalan disaring. Walaubagaimanapun, semua
mutasi dan polimorfisma tidak menunjukkan hubungan statistik secara signifikan
dengan NSHL dan darjah kecacatannnya. Bagi gen GJB6, pemotongan gen dikenal
pasti dengan menggunakan teknik pelbagai PCR yang mana gen β-globin digunakan
sebagai kawalan dalaman. Semua 182 pesakit dan kawalan telah dikenal pasti tidak
mempunyai pemotongan gen GJB6 walaupun di antara mereka mempunyai mutasi gen
GJB2 pada satu alel, dua alel atau tidak. Kami percaya bahawa pengambilan sampel
yang lebih banyak dan menyaring seluruh gen GJB2 dan GJB6 serta gen lain yang
berkaitan akan membantu mengenal pasti dan mengesahkan hubung kait antara mutasi
dan polimorfisma dengan darjah kecacatan pendengaran.
xix
A STUDY ON GJB2 AND GJB6 GENE MUTATIONS AMONG MALAYS WITH
NON-SYNDROMIC HEARING LOSS
ABSTRACT
Hearing loss is the most common congenital sensory defects in human. About one in a
thousand newborn in the world is born with the abnormality, which may vary from mild
level of hearing loss to profound loss. This loss can be caused by two factors, genetic
and environmental factors and more than 50% of the defect is due to genetic causes. It
has been proven that multi genes are involved in non-syndromic hearing loss (NSHL), a
type of hearing loss without other symptoms and covered in this study. Mutations in
GJB2 gene have been shown to be a major role for congenital NSHL. A related gene,
GJB6 which is located adjacent to GJB2 might be related and associated with NSHL.
The objectives of this study are to identify the mutations in the two genes and study the
association with NSHL. A total of 91 buccal cell samples of NSHL patients and 91
normal volunteer buccal cells samples in Kelantan were taken for this study.
Polymerase chain reaction (PCR) was used to amplify the coding region of GJB2 gene.
The PCR product of GJB2 coding region was preceded with screening for mutations
using denaturing High Performance Liquid Chromatography (dHPLC) and mutations
detected were confirmed by DNA sequencing. Eleven sequence variations including
mutations and polymorphisms were found in 32 patients and 37 control subjects after
182 NSHL patients and controls were screened. However, all the variations did not
show any statistically significant association with NSHL and the severity. For GJB6
xx
gene coding region, the deletion was identified by multiplex PCR assay whereby β-
globin gene was used as internal control. All 182 patients and controls were found to
have no deletion of GJB6 coding region irrespective of whether they have genetic
variation in GJB2 or not. Consequently, it is believed that a larger sample size and
screening all regions in GJB2 and GJB6 and other related genes are necessary to verify
the possible association between the mutations and polymorphisms and the severity of
hearing loss in patients.
CHAPTER 1
INTRODUCTION
1.1 Hearing loss
Hearing loss happens when someone could not hear voices and sounds due to problems
with one or more parts of the ear or ears. It is also called hearing impairment or
deafness. Since they are unable to hear their own voices when they do try to speak, they
therefore cannot imitate themselves (Martin and Grover, 1986).
One in a thousand babies is born with hearing loss, making it the most common sensory
type of birth defect (Watkin, 1996; Watkin and Baldwin, 1999). A study done by
Abdullah et al., (2006) in Hospital Universiti Kebangsaan Malaysia (HUKM), Malaysia
showed that the prevalence of hearing loss is 0.42%. Hearing loss can be due to
environmental factors and genetic defects. Fifty one percent of hearing loss cases are
caused by genetic factors and 77% are inherited in an autosomal recessive pattern
(Morton, 1991). To date, it is believed that more than a hundred genes may be involved
in hearing loss with several of these genes have been identified; two of them are gap
junction protein beta-2 (GJB2) gene and gap junction protein beta-6 (GJB6) gene (Van
Camp and Smith, 1999).
2
1.2 Hearing – How it works
Our ear is made up of three different sections; outer ear, middle ear and inner ear. Each
section has a specific function. The outer ear collects sound waves and the middle ear
converts the sound energy into a mechanical force, then it is transmitted to the inner ear
thus converting it to electrical energy and sent via the auditory nerve to the brain
(Freeland, 1989).
The outer ear comprises of pinna, which is shaped like a shell and its function is to pick
up sound waves. The pinna leads to the ear-canal at the bottom of which the eardrum
(tympanic membrane) is located. The ear drum is the division between the outer and
middle ears (Freeland, 1989).
The middle ear is an air-containing space which contains three unusually shaped
ossicles; the malleus (hammer), incus (anvil) and stapes (stirrup). The sound waves
received by the eardrum will cause the vibration and this vibration will pass through the
malleus, incus and stapes, thus converting sound energy into mechanical energy. This
energy is directed on to a membrane (oval window) to which the stapes is connected
and which is 22 times smaller than the size of the eardrum. The chain of ossicles thus
acts as much as a hydraulic press; it magnifies 22-fold the small pressures on the
surface of the eardrum and transmits them to the oval window, then sends the
mechanical energy into the inner ear for conversion into electrical activity (Freeland,
1989).
3
The inner ear is an extraordinarily complicated mechanism, comprises a coiled structure
called the cochlea. One end of the cochlea is connected to the oval window; the other
end is also in contact with another part of the middle ear through another membrane
called the round window. The inner ear filled with fluid and when the stapes moves, a
ripple is sent right through the fluid of the inner ear to the round window. The
movement of the inner ear fluid deflects a thin membrane in the centre of the cochlea
called basilar membrane. Delicate cells like tiny hairs balance on this membrane (the
organ of Corti) and connect directly to a multitude of nerve endings, which join together
to form the auditory nerve (Freeland, 1989) (Figure 1.1).
4
Figure 1.1: Cross-section of the outer, middle and inner human ear
(http://www.dizziness-and-balance.com/disorders/hearing/hearing.html)
5
1.3 Degree of hearing loss
Hearing loss or deafness (and sound in general) is measured in decibels (dB). Hearing
loss can be classified in different degrees according to the intensity: mild (30 to 45 dB),
moderate (50 to 65 dB), severe (70 to 90 dB) and profound (>95 dB). Table 1.1 shows
the relationship between the decibel hearing loss and degree of difficulty it may cause
(Martin and Grover, 1986).
1.3.1 Pure Tone Audiometry (PTA)
Pure tone audiometry is pure tone test to determine the type and degree of hearing loss
for both right and left ear. PTA is a behavioral test used to measure hearing sensitivity
(Kurtz Jr, 1994) (Figure 1.2). A calibrated machine called an audiometer is used to
present tones at different frequencies (pitches) and at different intensity (loudness)
level. The responses from the emitted sounds are recorded and plotted on a graph called
an audiogram. The frequency of the sound is referred to in Hertz (Hz) while the
intensity is measured in decibels (dB). The PTA testing is done between frequency 125
and 8000 Hz. Once the audiogram is completed, the audiologist computes the pure tone
average for each ear. It is the average of hearing thresholds at 500, 1000, and 2000 Hz,
which are considered to be the major frequencies for speech. The pure-tone average
represents the degree of hearing loss in decibels, not a percentage
(http://www.asha.org/public/hearing/testing/assess.htm).
6
Figure 1.2: Examples of pure tone audiogram. (a) Scale of degrees of hearing
level (http://www.springvalleyhearingctr.com/hearingassessment) (b) The
audiogram in the illustration reveals a hearing loss for tones above 500 Hz
(http://content.answers.com/main/content/img/McGrawHill/Encyclopedia/image
s/CE062100FG0010.gif)
a
b
7
1.4 Types of hearing loss
Classification of hearing loss is based on the localization of the point at which the
auditory pathway has broken down. It determines whether the patients‟s hearing loss is
conductive, sensorineural, central, and functional or a combination of these (Sataloff
and Sataloff, 2005).
Conductive hearing loss is caused by any condition that interferes in the transmission of
sound through the external and middle to the inner ear. If the damage is in the middle
ear, it may involve the stapes, as in otosclerosis or the mobility of the eardrum and
ossicles caused by fluid (Sataloff and Sataloff, 2005). Hearing tests on patient with
conductive hearing loss show the inner ear or nerve function to be normal but air
conduction to be reduced (Freeland, 1989). Most patients with this type of loss have a
mild degree of loss, correctable and can be improved by medical treatment (i.e.
surgical) (Ballantyne, 1993; Sataloff and Sataloff, 2005).
Sensorineural hearing loss happens when inner ear or auditory nerve or both is/are
damaged. The term „sensory‟ hearing loss is applied when the damage is localized in
the inner ear while „neural‟ implies the damage is in auditory nerve. The cochlea has
~30 000 hair cells that connect with nerve endings. The tiny hair cells could be
damaged from a variety of causes. In the majority of cases, the loss is not curable
(Sataloff and Sataloff, 2005).
8
In central hearing loss the damage is situated in central nervous system. The damage
may be at any point from auditory nuclei in the medulla oblongata to the cortex.
Formerly, central hearing loss was described as a type of „perceptive deafness‟ (Sataloff
and Sataloff, 2005).
Functional hearing loss is described when there is no detectable organic damage to
auditory pathways but some underlying psychological or emotional problem is at fault
(Sataloff and Sataloff, 2005).
Patients, who experienced two or more types of hearing impairment, are described as
having mixed hearing loss. However, for practical purposes this term is used only when
conductive hearing loss accompanied by a sensory or a neural (or a sensorineural) loss
in same ear (Sataloff and Sataloff, 2005).
9
Table 1.1: The effect of the different degrees of hearing loss (Martin and Grover, 1986)
Decibels of hearing loss Degree of impairment Practical effect on hearing
Up to 25
30 – 45
50 – 65
70 – 90
Over 95
Within normal range
Mild
Moderate
Severe
Profound
Little effect
Difficulty with quiet voices
Difficulty with many sounds
Cannot hear speech without a
hearing aid
Can hear only a little even
with a powerful hearing aid
10
1.5 Non-syndromic hearing loss (NSHL)
Hearing loss is a symptom of many injuries and diseases. Therefore, it may expressed at
any age and classified in many ways, such as genetic vs. acquired, syndromic vs. non-
syndromic, prelingual vs. postlingual, and conductive vs. sensorineural (Morton, 1991).
Hearing loss can be due to environmental factors, genetic defects or a combination of
these factors. Approximately 25% of children with hearing loss in the United States
(U.S) are caused by environmental factors, such as prematurity, infections, exposure to
ototoxic medications and trauma. It is estimated that at least 50% of prelingual hearing
loss is caused by genetic changes, whereas the etiology remains obscure in the
remaining 25%. Most of the cases, however, are assumed to be of genetic origin. Thus,
genetic causes account for the largest proportion of all cases of prelingual hearing loss
(Avraham, 2001).
Clinically, hearing loss may be associated with other disorders in the form of syndromic
hearing loss or as an isolated finding, non-syndromic hearing loss. However, both
syndromic and non-syndromic phenotypes can result from the mutation in the same
gene (e.g MYO7A, PDS). Syndromic hearing loss tends to be less genetically
heterogenous than non-syndromic, but more than one locus has been identified for
several syndromes (Avraham, 2001; Keats and Berlin, 1999).
11
Hearing loss can follow a pattern of autosomal recessive, autosomal dominant, X-linked
and mitochondrial inheritance. Allelic mutations in some genes can cause recessive and
dominant hearing loss and recessive hearing loss may be caused by a combination of
two mutations in different genes from the same functional group (Schrijver, 2004).
Autosomal dominant deafness loci are designated DFNA, autosomal recessive loci
designated DFNB and X-linked loci, DFN. The loci are numbered according to the
order in which they were mapped, DFNA1 being the first autosomal gene mapped in
1992 (Van Camp and Smith, 1999).
Approximately 70% of genetic hearing loss is non-syndromic in nature. The largest
proportion (about 80%) is inherited in an autosomal recessive mode, 18% is inherited in
an autosomal dominant mode and 2% is X-linked. Mitochondrial/maternal inheritance
also contributes to a small (1%) proportion of NSHL (Avraham, 2001) (Table 1.2).
Over 100 genes may be involved in non-syndromic hearing loss and the chromosomal
location for over 80 have already been found. In the field of NSHL, 21 genes associated
with autosomal recessive inheritance, 20 associated with autosomal dominant
inheritance and one with X-linked recessive transmission have been identified and
characterized (http://uia.ac.be/dnalab/hhh) with the most dramatic recent discovery is
the high incidence of mutations found in the gap junction protein, connexin 26 (locus
designation, GJB2) (Sobe et al., 2000).
12
Two additional genes have been implicated in hearing loss which are connexin 30
(GJB6) (Xia et al., 1998) and connexin 31 (GJB3) (Liu et al., 2000), both in non-
syndromic hearing loss.
13
Table 1.2: Causes of hearing loss (Bitner-Glindzicz, 2002)
Genetic
(Syndromic and non-syndromic)
Environmental
Autosomal recessive
Autosomal dominant
X-linked
Mitochondrial
Ototoxic medication
Severe neonatal jaundice
Head trauma
Noise exposure
Low birth weight
Infection: prenatal, e.g.:
toxoplasmosis, rubella; postnatal,
e.g. meningitis
Severe neonatal jaundice
Prematurity
14
1.6 Gap junction proteins in cochlea
The gap junction proteins, encodes connexins which is a component of connexons that
allows molecules to pass from cell to cell. This organization requires the membranes of
two neighboring cells to come close to each other (White and Bruzzone, 1996). Six
connexins form a connexon, which then aligns in the extracellular space to complete the
formation of gap junction channels. Two different connexins can interact with each
other to form homomeric (all the connexin are same), heteromeric (two different
connexins within a single connexon) and heterotypic (two different homomeric
connexons) channels, which differ in their content and spatial arrangement of connexins
subunits, while gap junction channels which are made from only one type of connexin
is called homotypic channel (Sosinsky, 1995; Chang et al., 2003) (Figure 1.3).
15
Figure 1.3: Schematic representation of connexins and gap junctions. (a) Blue and
yellow connexins interact with each other to form homomeric, heteromeric and
heterotypic channels, which differ in their content and spatial arrangement of connexins
subunits. Adapted from Meşe et al., (2007) with modification (b) Six connexins form a
connexon. Two connexons of neighbouring cells form pores; allow intercellular
transport of small molecules (Wagner, 2008 - with modification)
a
b
16
Connexin family members share a similar structural topology. Each connexin has four
transmembrane domains (TM1-TM4) that constitute the pore of the channels, with two
intercellular loops (IC1 and IC2) and two extracellular loops (EC1 and EC2). Two
terminal tails, N- (NH2) and C- (COOH) termini are cytoplasmic. The length of the
cytoplasmic C-terminus varies greatly among connexins and the cytoplasmic loop
shows some variation in length. Otherwise the overall structure of the molecules is
highly conserved (Bennett et al., 1991; Simon and Goodenough, 1998) (Figure 1.4).
17
Figure 1.4: Schematic representation of basic connexin (a) Schematic representation of
connexin. TM1-TM4; transmembrane domains, EC1-EC2; extracellular loops, IC1-IC3;
intercellular loops and N- and C-termini; terminal domains. Adapted from Simon and
Goodenough, 1998, with modification (b) Schematic representation of connexin 26, 30
and 31. The length of C-terminus show some variation, also the length of cytoplasmic
loop. Other regions are highly conserved among connexins (Rabionet et al., 2000, with
modification)
b
a
18
The amino terminus plays an important role in voltage gating, dynamically regulating
gap junction properties by noncovalent or covalent modifications in the surrounding
amino acids. The extracellular loops are critical for docking between two connexons
and connexon compatibility. The transmembrane domains form the pore of the gap
channel and it is important in channel permeability (Krutovskikh & Yamasaki, 2000).
Connexin 26 and 30, encoded by GJB2 and GJB6 gene, which are highly expressed in
epithelial supporting cells of the mammalian cochlea, have been speculated to have a
crucial role in the recycling of potassium ions (K+) from the hair cells back to the
endolymph of the cochlear duct. (Kikuchi et al., 1995; Petit et al., 2001).
Cochlea, a structure in the inner ear, contains transduction machinery to sense the
vibration transmitted from the middle ear after sound stimulus (Martinez et al., 2009)
(Figure 1.5). Cochlea is formed by three adjacent and paralleled tubular compartments;
scala media, scala tympani and scala vestibule. The epithelial cells, fibrocytes and
receptor cells which are located in the wall of tubular compartments are the principal
cellular components of cochlea. These compartments are filled with two types of fluid,
endolymph and perilymph. The endolymph possesses a high concentration of potassium
ion (K+) and low level of sodium ion (Na
+) fills the scala media. The perilymph contains
high concentration of Na+
and low K+ fills the scala vestibuli and scala tympani
(Wangemann, 2006; Zhao et al., 2006) (Figure 1.6).
19
Figure 1.5: Flow diagram of human auditory pathway (http://eardoctors.org/med_info/images_med_info/earworks.gif and
http://universe-review.ca/I10-85-cochlea.jpg)
19
20
Figure 1.6: Schematic illustration indicating possible pathway for the transport of K+ in
the cochlea (Kikuchi et al., 2000)
21
In the mammalian cochlea, there are two independent gap junction systems, epithelial
cell gap junction system and connective tissue cell gap junction system, which are
necessary for the normal hearing function (Zhao et al., 2006). The first system, the
epithelial cell gap junction system, is mainly composed of all organ of Corti supporting
cells, and also includes interdental cells in the spiral limbus and root cells within the
spiral ligament. The second system, the connective tissue cell gap junction system,
consists of strial intermediate cells, strial basal cells, fibrocytes in the spiral ligament,
mesenchymal cells lining the bony otic capsule facing the scala vestibuli, mesenchymal
dark cells in the supralimbal zone, and fibrocytes in the spiral limbus (Kikuchi et al.,
2000).
Activation of hair cells by acoustic stimuli induces influx of K+ from the endolymph to
sensory hair cells. This K+ is released basolaterally to the extracellular space of the
organ of Corti, from which they enter the cochlear supporting cells. Once inside the
supporting cells, the ions move via the epithelial cell gap junction system laterally to the
lower part of the spiral ligament. The K+
is released into the extracellular space of the
spiral ligament by root cells and taken up by type II fibrocytes. This uptake incorporates
K+ into the connective tissue gap junction system. Within the system, the K
+ passes
through the tight junctional barrier of the stria vascularis and are released within the
intrastrial extracellular space. The marginal cells of the stria vascularis then take up K+
and return it to the endolymphatic space, where it can be used again in sensory
transduction (Kikuchi et al., 2000).
22
It is highly probable that mutations of connexin genes cause dysfunction of cochlear
gap junctions and thereby interrupt K+ recirculation pathway (Kikuchi et al., 2000).
Interruption of the recirculation may be caused by mutation in the connexin 26 (Cx26)
and connexin 30 (Cx30) genes, which both connexins are functioned as epithelial cell
gap junction system.
1.7 Gap junction protein genes
Autosomal recessive non-syndromic hearing loss make up about 80% of hereditary
hearing loss (Avraham, 2001). The DFNB1 locus, which is located on chromosome
13q11-12, was the first deafness recessive locus to be discovered (Avraham, 2001). The
locus contains two genes which are GJB2 and GJB6 (Figure 1.7). The human
connexins are classified by their molecular mass and by extent of sequence identity,
which is indicated in the gene symbols for GJA, B and C subtypes. The connexin genes
are very similar and contain their coding region within a single exon, separated from 5‟-
untranslated region (UTR) by an intron (Schrijver, 2004).
23
1.7.1 Gap junction protein beta-2 (GJB2) gene
Gap junction protein beta-2 or GJB2 gene (Accession no.: M86849) encodes protein
connexin 26 (Cx26), a gap junction protein of the beta group with a moleculer weight of
26 kDa. The size of GJB2 gene is 2.2 kb with 2 exons and 1 intron. The coding
sequence of GJB2 encompassed entirely by exon 2 and consists of 680 bp, which are
translated into a protein with 226 amino acids including the stop codon (Kenneson et
al., 2002) (Figure 1.8). Exon 1 is contained in the 5‟-UTR (del Castillo et al., 2003).
24
Figure 1.7: Location of GJB2 and GJB6 genes. Location of GJB2 gene; at long (q) arm
of chromosome 13 between positions 11 and 12 and location of GJB6 gene; at long (q)
arm of chromosome 13 at position 12 (http://ghr.nlm.nih.gov/gene=gjb2 and
http://ghr.nlm.nih.gov/gene=gjb6).
GJB2 and GJB6 gene
25
Figure 1.8: Schematic diagram of Cx26 with sequence of amino acids. Adapted from
Martinez et al., 2009 with modification