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