:-

 

 

 

 

 

 

..3. .
. Es...
duh... :53.
.1 . . {‘1‘
3352...:
r

r

 

:5
I.

. IQL. \Qll

‘5! .0 will... ‘

“0...ng lofz...

.5525. {geisha

.zi...o.hfld.h
52.: ,f

In"
( .13.“..47‘
Sol. pkg! I...
livid-1‘!!!
‘1. is If
‘ ‘ ’OFlnnl
. hut. ‘ 5.0.5.00

r. .f‘hlfuuf

Illljlllllllllllllllllllllllllll

1293 01771 1122

 

LIBRARY
Michigan State
University

 

 

 

This is to certify that the
dissertation entitled
Mappt‘na . m “15+?“ iJe/n‘i'i‘fim‘h‘o" G" 6’ apt?“ to n ‘3‘.“ J),
of DF~33 , o jene 7501' Au mam feaxn‘ve dwyrm?“

presented by

VOnj Lna'j

has been accepted towards fulfillment
of the requirements for

Pb-D' Hum“ ”70/eca/M Gene/15%

degree in

 

 

%@ fix a 42/24 cu

Major professor

Date ’4 '5’ 9‘7

 

MS U is an Affirmatiw Action/Equal Opportunity Institution 0-12771

 

PLACE IN RETURN Box to remove this checkout from your record.
TO AVOID FINE return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE
10§lb§ 802fl02

 

 

Am 1 22m

 

 

 

 

 

 

 

 

 

 

 

 

1M m.m14

MAPPING, MUTATION IDENTIFICATION AND EXPRESSION STUDY OF
DFNB3, A GENE FOR HUMAN RECESSIVE DEAFNES

By

Yong Liang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
the Graduate Program in Genetics

1999

ABSTRACT

MAPPING, MUTATION IDENTIFICATION AND EXPRESSION STUDY OF
DFNB3, A GENE FOR HUMAN RECESSIVE DEAFNESS

By

Yong Liang

DFNB3 is a form of profound, nonsyndromic, autosomal, recessive
deafness originally identified in the Balinese village of Bengkala. Among the
2200 villagers, 48 individuals are deaf. A genome wide screen was used to
identify an interval of excess homozygosity among affected individuals. This
placed DFNB3 in the pericentromeric region of chromosome 17 between the
marker D178122 on the short arm and D17S783 on theglong arm. This critical
region was further refined to a 3 cM interval. Also, autosomal recessive deafness
segregating in three unrelated consanguineous Indian families were mapped to the
DFNB3 region.

The murine deafness mutation shaker-2 was proposed as the homologue of
DFNB3 on the basis of similar phenotype and conserved synteny. A research
group at the University of Michigan showed that a bacterial artificial chromosome
(BAC) containing a genomic fragment from the shaker-2 critical region was able
to rescue the skaker-Z phenotype. This BAC was analyzed and an unconventional
myosin was revealed, which we later designated My015. To clone the human

MY015, primer pairs synthesized according to the My015 coding sequence were A

used to isolate overlapping cosmids that contain the complete MY 01 5 . MY015 is
a comparatively large gene with 66 exons and has a 10.6 kb coding sequence. The
predicted protein is about 395 kD in molecular weight. Three missense mutations
and one nonsense mutation were identified in the four DFNB3 families, one from
Bali, Indonesia and three from India. A point mutation of MY015 was also found
in one Smith-Magenis syndrome (SMS) patient with moderate to severe hearing
loss. Smith-Magenis syndrome is a multiple congenital anomalies/mental
retardation disorder caused by an interstitial deletion of chromosome 17 band

pl 1.2 with approximately 65% of SMS individuals having a mild conductive,
sensorineural or mixed hearing impairment.

In humans, MY015 can be easily reverse transcription PCR (RT-PCR)
amplified from the inner ear but northern analysis has not yet been possible.
Mon5 is expressed in the developing cochlea and vestibular apparatus in the 15.5
days mouse embryo, as detected by in situ hybridization. Northern analysis also .
showed My015 expression in pituitary and testis. Dot blot analysis with RNAs
from 76 different tissues and cancer cell lines showed very strong hybridization
signals from the pituitary and weak signals from testis.

Data presented in my Ph.D. dissertation show that DFNB3 maps to
17p11.2, that MY015 plays an important role in the human inner ear and
mutations of this gene cause autosomal recessive deafness in four unrelated

families.

This dissertation is dedicated to:

My beloved grandfather, Yusheng Liang
whose death from cancer led to my exploration of human molecular genetics '
and my parents, Renyi Liang and Cuibin Nian

for their love, understanding and support

iv

ACKNOWLEDGEMENTS

I am most grateful to Tom Friedman for his guidance, enthusiasm in this _
study and help both in and outside the lab. I would like to thank Chia-Cheng
Chang, Karen Friderici, Pat Venta, Rachel Fisher, the late Jim Asher, J r., Lenny
Robbins and Rob Morell, members of my Ph.D. guidance committee for their
support, suggestions and interest in my research. I want to also thank Chia-Cheng
Chang for his help with the tissue culture and Susan Sullivan for her help with my
in situ hybridization experiment. I also thank Bob Fridell, Tom Barber, Jeff Kim,
and David Anderson for many helpful discussions.

I sincerely thank National Institute on Deafness and other Communications
Disorders (NIDCD) for fund and support of this research and thank Dr. James
Snow and Dr. James Battey for awarding me the NIDCD pre-doctoral fellowship
which enabled me to continue working on this project. A

The history and complete genealogy of Bengkala was worked out by Dr.
John Hinnant. Blood samples were collected by Dr. Friedman, Dr. Asher and Tom
Barber using personal funds from Dr. Friedman and Dr. Asher.

Lastly, I would like to thank my wife and best friend Aihui for her love and

support and thank my parents-in-law for their understanding and encouragement.

TABLE OF CONTENTS

List of Tables ..................................................................

List of Figures ..................................................................

Introduction ....................................................................

I.

Chapter one: Genetic linkage mapping of DFNB3 to the
pericentromeric region of chromosome 17 ........................
A. Introduction ....................................................
B. Results ..........................................................
1. Mapping Strategy .....................................
2. Linkage of DFNB3 to chromosome 17 and
linkage disequilibrium between DFNB3 and 17p

STRs ....................................................

3. Comparison of allele frequency distributions between
Bengkala and western populations .....................

C. Discussion ..........................................................

Chapter two: Refinement of DFNB3 critical interval
A. Introduction ...................................................
B. Results .........................................................
1. Refining the DFNB3 critical region to ~3 cM
2. DFNB3 segregating in two unrelated families
from India .............................................

C. Discussion .....................................................

Chapter three: MY015 mutation detection in family J -1 and in a

SMS patient ............................................................
A. Introduction ...................................................

B. Results .........................................................

1. Mutation identification in family J -1 ..............

2. Mild to severe hearing loss in a SMS patient is
possibly to be caused by a mutation in MY015
3. Simple Tandem Repeats and Single Nucleotide
Polymorphisms in MY015 ..........................
C. Discussion ....................................................

vi

viii

ix

‘©©G@

14

21
26

29
31
31

48
55

58
58
62
62

67

72
75

IV. Chapter four: The study of expression pattern of myosin 15 in

human and mouse ....................................................
A. Introduction ...................................................
B. Results .........................................................
C. Discussion .....................................................
V. Appendix A: Publications from this work ........................
VI. Appendix B: The mapping of human inner ear 0CP2 cDNA to
human chromosomes 4, 5, 7, 10, and 12 ..........................
VII. Appendix C: Materials and Methods ..............................
1. Typing STR markers by Polymerase Chain
Reaction (PCR) ......................................
2. Linkage analysis .....................................
3. Isolation of human genomic DNA ................
4. Southern blot analysis ...............................
5. Mutation detection in MY 0] 5 by direct
sequencing ............................................
6. PCR amplification of part of myosin 15
sequences from monkey, cow, Chinese hamster,
hyena and mouse .....................................
7. In situ hybridizaan .................................

VIII. Appendix D: Table of primers used to PCR amplify 65 exons

of MY015

IX. Appendix B: List of references .....................................

vii

78
78
81
88

91

93
102
102
103
103
104
105
106
108
131

134

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

Table 10.

LIST OF TABLES

x2 probabilities of detecting disequilibrium among 21
STRs from chromosome 17 typed for 13 unrelated deaf
Individuals .....................................................

Genotypes of 13 unrelated deaf individuals homozygous
for DFNB3 typed for 14 STRs. from chromosome 17

Comparison of allele distributions between CEPH and
Bengkala populations .......................................

Genotypes of 48C, 46C and deaf individuals from
Bengkala for the DFNB3 linked markers .................

Genotypes comparison between deaf individuals from
Anturan, Tamblang, Sinabun, Suwug and Bengkala for
DFNB3 linked markers ......................................
Two-point LOD scores that exclude linkage between
deafness and STRs closely linked to DFNB3 in four
villages, Anturan, Tamblang, Sinabun and Suwug

Two point LOD score for linkage between deafness and
STRs on chromosome 17 in I-1924, M21 and Bila

Single Nucleotide Polymorphism (cSNP) in MY015
Simple Tandem Repeat polymorphism (STR) in MY015

List of primers used for PCR amplifying the 65 exons of
MY015 .........................................................

viii

17

19

23

35

46

47

49

73

74

131

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.
Figure 12.

Figure 13.

LIST OF FIGURES

Diagram of human auditory system ........................

Two of the six kindreds in Bengkala with profound
nonsyndromic autosomal recessive deafness ..............

Log of the contingency x2 probabilities of observing deaf
individuals with a specific homozygous marker genotype
assuming that DFNB3 and the marker alleles are in
Hardy-Weinberg and allelic equilibrium ..................

Allele frequency distributions for 9 tetranucleotide STRs
used in the study .............................................

Pedigree of most of the deaf individuals living in
Bengkala .......................................................

Map of genetic markers flanking DFNB3 and historical
recombinations that define the DFNB3 region at 17p11.2

DFNB3 region integrated map of human/rodent somatic
cell hybrids, STRs and YACs ...............................

Pedigrees of a representative Bengkala family and a Bila
family with the most likely haplotypes for the DFNB3
markers .........................................................

Pedigrees of two unrelated consanguineous Indian
families, M21 and I-l924 ....................................

Pedigree of family J -1 with the most likely haplotype for
DFNB3 markers ...............................................

Audiogram of a deaf individual from J -l .................
Audiogram of SMS patient 1123 ...........................

ClustalW analysis of part of myosin 15 peptide
sequences in different species ' ..............................

ix

10

15

24

32

38

4O

52

63
65

68

70

Figure 14. Diagram of MY015 structure and mutations ............... 76
Figure 15. Human RNA master blot probed with MY015 ............ 81
Figure 16. Northern blot probed with MY 01 5 .......................... 84

Figure 17. In situ hybridization of MY 015 sense and antisense
probes to 15.5 day mouse embryos ......................... 86

Figure 18. . Location of human 0CP2 sequences on chromosomes 4,
5, 7, 10 and 12 by Southern blot analysis of PstI-digested
genomic DNA .................................................. 96

Figure 19. Determination of the chromosomal location of the gene
from which CMl 0CP2 cDNA was transcribed .......... 100

INTRODUCTION

Hearing loss of a degree sufficient to interfere with normal communication
is among the most common neural impairments in the US. population (N adol,
1993; Nance and McConnell, 1973; Nance and Sweeney, 1975). A survey of 2000
representative individuals showed that over 300 people have a significant hearing
impairment (Davis, 1989). More than 25% of people suffer from hearing loss by
the age of 65 and nearly 50% by the age of 80. Hearing impairment affects more
than 25 million Americans and costs over 50 billion dollars each year, surpassing
the combined financial impact of multiple sclerosis, stroke, epilepsy, spinal
injury, Huntington’s, and Parkinson’s disease (Morton, 1991).

The human ear is a remarkably complex structure (Figure 1. William T.
Keeton et al. 5'” edition 1993) consisting of three distinct compartments, the outer,
the middle and the inner car. In the outer ear, the sound, a mechanical vibration, is
collected by the pinna and transmitted through the external auditory canal to the
tympanic membrane. The middle ear contains three small bones or ossicles
(malleus, incus and stapes) which transfer the vibration of the tympanic
membrane to the footplate, the oval window of the inner ear. The inner ear is a
bony cavity that comprises the vestibular apparatus and the cochlea. The
vestibular apparatus responds to gravity and acceleration and the cochlea
processes the auditory signal. The cochlear duct contains the organ of Corti, a

sensory transduction apparatus, which rests on the elastic basilar membrane.

   
    

cochlca with
seclion removed
to show canals

  

tympanic
membrane V
w

    

cochlear ‘-
canal .

     
 
 
   

tectorial

hair cell
’ cochlear nerve

membrane

Figure 1. The structure of human ear (outer, middle and inner ear). Also
shown is the enlarged cross section of the organ of Corti. (Included with
permission of author and publisher. William Keeton et al. 1993).

The organ of Corti is composed of about 15,000 sensory cells (the inner and outer
hair cells) and 30,000 neurons and a large variety of supporting cells. Hair cells
possess a bundle of actin-filled stereocilia, which are bathed by the cndolymph.
At the tip of the stereocilia is located the cationic transduction channel and
filamentous connection, the tip link, which attaches the tip of a stereocilium to an
upper insertion in the nearest taller stereocilium. The tip link has been proposed to
be the gating-spring of the transduction channel (Hudspeth and Gillespie, 1994).
An acellular membrane, the tectorial membrane, covers the hair cells. The
compression of the oval window generates a compression travelling wave in the _
perilymph, which fills the cochlea duct, and creates a movement of the basilar
membrane with the respect to the tectorial membrane. This leads to a deflection of
the hair cell stereocilia bundle that in turn leads to an increase in the tension of the
tip link, thereby opening the transduction channels. This mechanostimulation
leads to an influx of endolymphatic K” into the sensory hair cells resulting in cell
depolarization. Cell depolarization activates the voltage-sensitive Ca++ channels,
leading to a Ca++ inflow that triggers neurotransmitter release. In this way,
auditory information is transduced from mechanical signal to neurally conducted
electrical impulse, and is passed to the brain to be deciphered through the
Auditory nerve VIII (Nadol, 1993).

Inherited deafness affects approximately one child in 2000, and equal
numbers of children are born with significant loss of hearing from other causes

(Morton, 1991). Of those with genetic deafness, two-thirds are due to autosomal

recessive mutations, nearly one-third are due to autosomal dominant inheritance,
and 1-2% have X-linked deafness (Keats and Berlin, 1999; Marres and Cremers,
1989; Steele, 1981). Most individuals with autosomal recessive deafness have no
noticeable anomalies other than hearing impairment. Non-syndromic autosomal
recessive deafness is particularly difficult to study directly because it is known
that there are many genes involved (Brownstein et al., 1991; Chung and Brown,
1970; Van Camp et al., 1997). However, for this reason, before this study begin in
1993, no gene for non-syndromic autosomal recessive deafness had been mapped.
We hypothesized that the key to mapping a non-syndromic autosomal recessive
deafness gene was to find a large isolated population where there is likely to be
only one deafness mutation segregating, or ascertain a large consanguineous
family with three or more affected individuals (Campbell et al., 1997; Guilford et
al., 1994; Jain et al., 1995; Scott et al., 1996; Veske et al., 1996). By using DNA
samples from Bengkala, an isolated Balinese village, where 48 individuals were
segregating an autosomal recessive deafness, we mapped DFNB3, a gene for
human autosomal recessive deafness to a 12 cM pericentromeric region of human
chromosome 17. To facilitate the cloning process, the DFNB3 critical region was
further narrowed to about 3 cM by the development of new polymorphic markers;
by evaluation of several recently reported STRs; and by procurement of DNA
samples from recently ascertained affected individuals from Bengkala and

neighboring villages (Liang et al., 1998).

Based on the similar phenotype and syntenic relationship, we proposed that
mouse deafness mutant shaker-2 to be the murine homologue of human DFNB3.
Dr. Sally Camper’s group at the University of Michigan found a bacterial
artificial chromosome (BAC) that contains a DNA fragment from mouse
chromosome 11 was able to rescue the shaker-2 phenotype (Probst et al., 1998).
The BAC was sequenced at NIDCD. Sequence analysis lead to the discovery of
the mouse MonS and subsequently to the human orthologue MY 0] 5 (Probst et
al., 1998; Wang et al., 1998). Direct sequencing of a portion of MY015 gene in
four unrelated families showing segregation of DFNB3 revealed three distinct
missense mutations and a nonsense mutation. A point mutation was found in
myosin15 in shaker-2 mouse by our research colleagues at the University of
Michigan based on our sequence analysis of mouse MonS gene. A myosin15
point mutation was also found in a SMS patient with moderate to severe hearing
impairment.

Myosin15 can be easily detected from human inner car by RT-PCR. Dot
blot analysis using RNAs from 76 adult and fetal human tissues and cancer cell
lines but not from the inner ear showed that MY015 is predominant expressed in
the pituitary gland. In mouse, an in situ hybridization study with 15.5 days
embryos showed that Mon5 is abundantly expressed in the cochlea and
vestibular apparatus, with no signals detected in other tissues at this stage of

development.

1. Chapter one: Genetic linkage mapping of DFNB3 to the pericentromeric
region of chromosome 17

A. Introduction

The village of Bengkala, Bali, can be dated back to at least the thirteenth
century as documented by charters inscribed in Sanskrit on metallic plates. A
medical and genetic analysis of deafness in Bengkala began in 1990 with the
ascertainment of deaf individuals living in this village (W inata et al., 1995). In
1993, a more thorough investigation was conducted in Bali by Dr. Thomas
Friedman, Dr. James Asher, Dr. John Hinnant and Thomas Barber (Friedman et
al., 1995; Liang et al., 1998). Of the 2200 individuals living in Bengkala in 1996,
48 had profound congenital nonsyndromic neurosensory deafness. As an
adaptation to the high percentage of deaf individuals in this community, the
people of Bengkala have developed a unique sign language that is used by the
majority of hearing as well as deaf villagers. This unique sign language is being
studied by Dr. John Hinnant, Chairmen of Department of Religious Study at
Michigan State University.

Deaf couples in Bengkala produced all deaf progeny with four exceptions
that will be addressed in Chapter 2. Families, identified through children with
profound congenital deafness having hearing parents, give the expected 25% deaf
progeny for a autosomal recessive mutation when corrected for ascertainment bias

(W inata et al., 1995). Congenitally deaf individuals from Bengkala have no

apparent dysmorphic features. They have sensorineural deafness and show no
response to pure tones up to 90dB by audiological examination. Obligate
heterozygotes for autosomal recessive deafness in Bengkala have normal hearing
as measured by pure tone audiometry. A single fully penetrant autosomal
recessive mutation is the most parsimonious explanation for the pattern of
segregation of congenital non-syndromic deafness in Bengkala. The gene symbol
DFNB3 (MIM 600316) was assigned by the HUGO nomenclature committee (for
the third autosomal recessive, non-syndromic deafness locus to be mapped).

On the basis of historical records, the high incidence of deafness, and the
geographical and cultural isolation of Bengkala, we hypothesized that
nonsyndromic autosomal recessive deafness segregating in this village is the
consequence of a founder effect and the mutated DFNB3 gene would in linkage
disequlibrium with the nearby markers. That is, DFNB3 co-segregate with
specific linked marker alleles. The frequency of new combinations of the DFNB3
mutant allele with particular marker alleles on the same chromosome depends
upon: (i) the number of generations since the DFNB3 mutation first appeared in
the village, (ii) the recombination fraction between DFNB3 and the marker locus,
and (iii) spontaneous mutations of marker loci adjacent to DFNB3. The more
generations there are since the DFNB3 first appeared in the village, the higher the
frequency of new recombinations will be. The closer a marker is to the mutation,

the less likely that a recombination will happen. Spontaneous mutations of marker

loci adjacent to DFNB3 will decrease the degree of linkage disequilibrium

between this marker and the mutation.

B. Results
1. Mapping Strategy

Genomic DNA was obtained from 13 congenitally deaf individuals from
Bengkala who: (i) are apparently unrelated, (ii) are not the progeny of
consanguineous marriages, and (iii) have hearing parents. Interviews by Dr. John
Hinnant with parents, relatives, friends and village officials confirmed that the
thirteen individuals had been deaf since birth. Six of the 13 deaf individuals of
hearing parents (#39, #40, #41, #42, #54 and #58) are shown in Figure 2.

DNA samples were obtained from 48 unrelated and unaffected individuals
from Bengkala who have no first or second-degree relatives with hereditary
deafness. Genomic DNA samples from these 48 individuals in the Bengkala
population are referred to as Representative Bengkala Individuals (RBIs) DNA
samples and were used to determine alleles frequencies of short tandem repeats
(STRs) in this population. STRs are sequences with repeat lengths up to about 6
base pairs long with total lengths usually less than 60 bp (Weber and Wong,
1993). 48 representative individuals are approximately 2% of the entire
population of Bengkala. Typing 48 individuals for a STR is sufficient to detect,
with 95% confidence, all alleles with a population frequency of 0.03 or greater
(Winata et al. 1995).

In Bengkala, there are 6 large kindreds with 48 individuals with profound
deafness. However, many of the Bengkala pedigrees are uninformative for LOD

score analysis due to positive assortative mating among the deaf individuals who

IV

200

 

 

   
  
 

 

 

 

 

142 41 42
40 119113117114 113 37 33
133132131129 130 123123127124 39 123 122 33 l 120
‘141140 133139 133 134
194 193 191I190 92 139

 

 

 

 

 

JF—I T
190 54 132 204 203 207 203 57 257 37 53
O I

201 202 203 l 203

193 211

212

Figure 2. Two of six kindreds in Bengkala. Showing segregation of a mutation
DFNB3 causing profound nonsyndromic autosomal recessive deafness. There is
no evidence for consanguineous marriages in these two or the other Bengkala
kindreds with deaf individuals. Filled symbols indicate congenital deafness.
Individuals in this study are identified by a number below each symbol. (From
Friedman et al. 1995)

10

are homozygous for the DFNB3 mutation and hate all deaf offsprings.
Simulations with SLINK using all of the pedigrees with deaf individuals from
Bengkala indicated that a significant LOD score was unlikely to be obtained (the
highest LOD score would be 1.7). To cope with this problem, Dr. Friedman and
Dr. Asher devised an association mapping strategy, which is derived from
homozygosity mapping (Lander and Botstein, 1987), and an analysis of historical
recombinants to map DFNBB. We named it allele-frequency—dependent
homozygosity mapping (AHM). AHM identifies a segment to which a gene maps
rather than providing a map position. This contrasts with a strategy that uses
conventional linkage analysis followed by disequilibrium mapping. AHM was
derived as an alternative to conventional linkage analysis and was used
successfully in mapping DFNB3 because of linkage disequilibrium between
DFNB3 and polymorphic STRs. Initial data indicate that DFNB3 is closely linked
to three markers in the pericentromeric region of chromosome 17, in which four
other neurological disorders have been mapped. These four disorders are Smith-
Magenis syndrome (SMS, MINI 182290), hereditary neuropathy with liability to
pressure palsies (HNPP, MIM 162500), Charcot-Marie-Tooth disease type 1 A
(CMTIA, MIMI 18220), and Dejerine-Sottas (Chance et al., 1993; Nicholson et
al., 1994; Pentao et al., 1992; Roa et al., 1993; Valentijn et al., 1992).

We hypothesized that deaf individuals in Bengkala who are homozygous
for a mutant allele of DFNB3 would also be homozygous for a particular allele of

each closely linked marker. Our first step was to screen the human genome with

11

STRs to identify a closely linked STR with a significantly higher number of
allele-specific homozygotes than expected under Hardy-Weinberg and linkage
equilibrium. The second step was to calculate the probability of observing n
homozygotes for a specific marker allele among the 13 deaf individuals of hearing
parents. The method of evaluation was a contingency x2 (with Yates correction
factor), in which the deaf individuals that are homozygous for a specific allele of
the marker versus all other genotypes are compared to representative Bengkala
individuals homozygous for the same allele of the marker versus all other
genotypes. We are, therefore, testing for deviation from the null hypothesis that a
polymorphic marker is in complete Hardy-Weinberg and allelic equilibrium with
the DFNB3 allele.

Whenever more than four homozygotes for a particular STR allele were
observed in the sample of 13 deaf individuals, genomic DNA sample from the 48
RBIs were screened to determine the frequency of each STR in the Bengkala
population. Comparisons between the representative Bengkala individuals and a
sample from a western population (CEPH families) gave similar allele frequencies
for 52% of the STR3 in this study (Morell et al., 1995). Only a few allele
frequencies of some of the STRs were widely discordant. As an extreme example,
D17S801 is polymorphic in western populations, but monomorphic in the
representative sample of 48 individuals from Bengkala. Except for D17S801,

Bengkala villagers were highly polymorphic for the STRs used in this study.

12

When P _<_ 10", we rejected the null hypothesis of Hardy-Weinberg and
allelic equilibrium and suspected linkage disequilibrium and hence linkage
between the marker and DFNB3. This alpha level was chosen to reduce type 1
errors due to multiple evaluations needed to survey the genome and to
approximate the more conservative criterion commonly used to accept linkage.
Because random process might yield a contingency 36 probability of P S 10'4 for a
single marker giving a false positive location for a gene, our criterion for
localizing DFNB3 was the identification of several linked STRs, each with P S 10'
4. The third step was, therefore, to type the 13 deaf individuals with additional
STRs known to be closely linked to the first STR that showed a P5 104 and

identify a cluster of linked STRs each with a P s 10“.

The fourth step requires a fine structure conventional genetic or physical
map of the markers in the DFNB3 region to identify flanking markers. We
constructed a detailed genetic map of the 17p region by a conventional linkage
analysis of CEPH families and then positioned DFNB3 by analyzing historical
recombinants between the STRs and DFNB3 that occurred in either the hearing

parents or in a prior generation.

13

2. Linkage of DFNB3 to chromosome 17 and linkage disequilibrium between

DFNB3 and 17p STRs

Dr. Friedman and I did the majority of the genotyping to map DFNB3 in
Dr. James Weber’s laboratory at Marshfield Medical Research Foundation in
Marshfield, Wisconsin. For nearly two months Dr. Weber gave us free access to
all the resources in his laboratory. We typed genomic DNA samples from the 13
unrelated deaf progeny of hearing parents for 127 (CA)u STRs and 21
tetranucleotide STRs using PCR amplification and denaturing polyacrylarnide gel
electrophoresis (See Appendix C: Typing STR markers by polymerase chain
reaction). The STRs were distributed at approximately 39 cM intervals along the
22 human autosomes. Only STRs on chromosome 17p showed evidence for
significant allelic disequilibrium indicating possible linkage to DFNB3 (Figure 3a
and 3b; Table 1). STRs on all the other autosomes gave probabilities greater than
104 (see Figure 3). Staff of Dr. Weber’s laboratory then constructed a refined
genetic linkage map by conventional linkage analysis for 15 STRs from 17p to
approximately 17q12, using 267 individuals from 18 of the largest CEPH
families. The relative order of STRs labeled with asterisks (Figure 3b) was
supported with odds in all cases > 1000:1. The linkage map (Figure 3b) of the
chromosome 17 pericentromeric STRs was constructed in order to analyze the

historical recombinants between the STRs and DFNB3.

l4

Figure 3. Log of the contingency x2 probabilities of observing deaf
individuals with a specific homozygous marker genotype assuming the null
hypothesis that DFNB3 and the marker alleles are in Hardy-Weinberg and
allelic equilibrium. When p S 10", the null hypothesis was rejected and nearby
markers were investigated. a, Graphical representation of the log of the
probabilities for 50 STRs on 20 of the human autosomes not linked to DFNB3
that showed five or more allele-specific homozygotes among the 13 deaf
individuals. STR8 in (a) were typed for the 48 representative Bengkala individuals
and the contingency 12 probabilities were calculated as described in text. No STRs
with more than four homozygotes with identical genotypes were observed on
these 20 chromosomes. b, A conventional genetic linkage of chromosome 17p to
approximately 17q12 and the lod of the x2 probability of observing n:13 = R:N
STR homozygotes with identical genotypes. The region to which DFNB3 was
located by AHM and by an analysis of historical recombinants is indicated on the
X-axis as a filled rectangle. 15 STRs on chromosome 17 with placement odds in
excess of 1000: 1 from conventional linkage analysis are indicated by asterisks.
The centromere is located between D17S805 and D17S783. The genetic linkage
map in b overlapped and is consistent with genetic, cytogenetic and radiation
hybrid maps of 17q. c, The position of five STRs, D17S799, D178122, D17S261,
pRM7-GT and D17S805, are shown along a YAC contig and confirmed in this
study using the same CEPH YAC clones purchased from Research Genetics.
pRM7-GT was localized to YAC 828B9 which also contains D17S805. (From
Friedman et al. 1995)

15

 

 

7 age

 

     

._.W

3 $232.92.
fl :3“?

 

{1+
‘\\
17¢:

a .3232.
a 3. «an?

 

2.353.;

 

3 v3“:
3 :3- a

 

c 7 Eng.-

 

” 23:32..

 

A a
u n 3828.. can—fl
1 n 23832.. ......

13
I

a .323...
u a 55.... .

 

It]:
. g F';- 3390 0A
a Biases...

11

 

m

 

'1

 

s 33:59;
..
o I 33:56.. .1.

3
0..
4.1

 

 

é
3.8032 n o

 

7

s 33.3... m
a 23832..
a 1353...:
0

 

«up;

 

13.

 

 

wnwnMu-aan-aa-aamau '
I
4I------O-----------------------------------------------------‘---

:
Dru-.-

 

1-4 3-3 ”-11 13-14

o1».
b

 

 

 

 

a

O
‘-

’11.“.
C

16

Figure 3

 

Tabie 1 xi probabilities of detecting
disequilibrium among 21 STFis from

 

chromosome 17 typed for 13
unrelated deaf individuals from
Bengkala, Bail
CM 7 markers Contingency xi
probability
Mfd 152 1.00
AFM051xd10 9.1 x 10"
Mfd144 1.3 x 10'“
AFM2252c1 1.1 x 10'1
GATA10H07 2.2 x 10'"
AFM192yh2 4.5 x 10"
VAW409 2.1 x 10"
0178261b 5.0 x10"
pRM7-GT" <1.0 x 10‘“
0178805” 4.0 x 10'“
AFM026vh7 7.8 x 10"
GGAT2007 4.1 x 10"
AFM179xg11 1.0 x 10'2
GGAA7D11 <1.0 x10"
Mfd15 4.4 x 10"
AFM2002f4 7.5 x 10"
AFM155xd12 1.00 .
Mfd188 7.3 x 10"
AFM0952d11 2.3 x 10"
AFM151xa11 1.2x10“
AFM238y18 3.7 x 10"

 

'P51 0" is considered significant.
I’indicates that the 13 unrelated deaf
individuals are all homozygous for a
specific marker allele of the STR.

l7

The null hypothesis of linkage equilibrium was tested by a contingency x2 ’
comparison of the frequency of STR individuals. Allelic disequilibrium was
detected between DFNB3 and STR8 in the pericentromeric region of chromosome
17 (Figure 3b). All 13 deaf progeny of two hearing parents were homozygous for
a specific allele of each of three 17p markers, D17S261, pRM7GT and D178805
that are approximately 1 magabase apart (Figure 3c). The probabilities of
observing 13 homozygotes for these three markers are 5.0 X 106, < 1.0 X 10‘ and
4.0X106, respectively. I concluded that DFNB3 was in linkage disequilibrium
with three closely linked STRs, D178261, pTM7-GT and D17S805, and,

therefore, DFNB3 maps to the pericentromeric region of chromosome 17.

An analysis of historical recombinants was used to refine the map position
by identifying DFNB3 flanking markers. The three markers that are homozygous
in all 13 deaf individuals, D17S26l, pRM7-GT and D17 S805, must lie close by
DFNB3. However, only with this information, DFNB3 cannot be ordered with
respect to the markers. Two of the 13 deaf individuals were heterozygous for
D17 S7 83, the closest centromere proximal DFNB3 flanking marker. One of the
13 deaf individuals was heterozygous for D178122 (Table 2). D17 S122 is 3.3 cM
but only 1.2 Mb from D17S805 (Figure 3b and 3c) and represented, at that time,

the closest DFNB3 telomere flanking marker.

Seven of the 13 deaf individuals were homozygous for a particular allele of

tetranucleotide STR, GGAA7D11, that has an estimated frequency of 7.4% in the

18

  
                

F: #5 nm— .8. . . . new New
cum .6“ «cm .Nvu 5w .5— 00— .09. SN Gem 0: .0: . N1

         

  

      

  

     

 

    

      

 

 
 
 
 

@333 33.33 8...... 2.8.. 3
83.33 33.33 8...... 8
8.8. 83.83 33.33 33.33 5...... No
.23. 8. .8. “.333 83.83 «3.93 33.33 33.33 5...... 3.
5.... 8.8. 33.33 83.83 0363 33.33 3333 5...... .e
| 83.83 33.33 83.83 0383 33.33 33.33 .e...e. 3
5.... 8. .8. 83.83 33.33 83.83 0383 33.33 33.33 .e...e. , , .m
, 33.33 83.83 .383 33.33 33.33 8...... 3.6.. 8
.. 8. .8. 83.83 .8333 8363 e363 33.33 33.33 5...... 8.8. 83.83 2...... 8.8. 8.8. 3
.2... 8.8. 83.83 33.33 83.83 83.83 33.03 33.33 5.8. 8.8. 83.83 8...... 8.8.. 8.8. 8
.23. 8.8. 83.83 33.33 83.83 03.83 33.33 33.33 5...... 8.8. 83.83 8...... 8.8. 8.8. 3
B... 8»... es... I»... who 3.8 mt... 8.... «.88 83... m3... 338
K. 8. 83 33 83 .3 33 33 .e. 8. 83 2. 8. 8. 8.3.3:
o o a o. o. .. n. n. n. 3. a e n m 883.5:
88.5 839.5 852.5 8525 88.5 .335 3.35 88.25 38.5 33:5 .83
383202 85...: .5928 2962:": 803200 553:"? 2532“? 512% $9.: moss; 3:323... 8:958 .0832“? 35“.: see:

 

n. oEomoEoEo Ea: £95 3 .6. comb 82km. .2 500.3050: £522.85 Eon 3.52:: m. .0 8532.3 N 033.

19

population living in Bengkala and give a probability of < 1.0 X106.
Assuming that the chromosome on which the DFNB3 mutation first occurred
carried this particular rare allele of GGAA7D11, the six recombinant haplotypes
for GGAA7D11 among the 13 deaf individuals (Table 2) imply that GGAA7D11
is more distant from DFNB3 than are D17S261, pTM7-GT and D17S805. Had I
first genotyped GGAA7D11, these data would have given me a strong indication

that DFNB3 was nearby, either up or downstream from this marker.

There is a caveat to the foregoing interpretation. Mutation is also a possible
source of marker heterozygosity among the 13 deaf individuals. Most
spontaneous mutations in STRs occur with changes of one or two repeats (Weber
and Wong, 1993). The two heterozygotes, individuals 23 and 40, at D1787 83 with
a 249 bp allele and three of the five heterozygotes, individuals 22, 41 and 51, at
D17S799 with a 192 bp allele do not fit the likely pattern of a new mutation since
they represent changes of three repeats. They probably represent historical
recombination events between the STR and DFNB3. The single heterozygote for
D17 8 122, in individual 51, could be due to a mutation of STR or a historical
recombinant. To distinguish between these possibilities, 13 deaf individuals were
typed for a second STR, KA52F1, which is on the same cosmid clone as

D17S 122. This marker was identified by Dr. Ken-Shiung Chen in Dr. Lupski’s
lab in their study of Smith-Magenis Syndrome. Only individual 51 is

heterozygous for KA52F1. In addition, arguing strongly for a recombinant event

20

as the cause of heterozygosity at D178122 for this individual, is the observation
that individual 51 is also recombinant for the four markers distal to D17S122.
Markers that are more centromere proximal or distal to D17S805 anle7 S783
showed a trend toward larger numbers of heterozygotes among the 13 deaf

progeny (Table 2).

3. Comparison of allele frequency distributions between Balinese population and
the western population

Short tandem repeat (STR) polymorphisms are the most extensively used
genetic markers in the mapping of disease genes (Buetow et al., 1994; Gyapay et
al., 1994; Matise et al., 1994; Weber and May, 1989) due to their abundance, the
ease of handling and the reliability of scoring (Budowle and Allen, 1998;
Budowle et al., 1991; Butler, 1998; Pena and Chakraborty, 1994; Phillips et al.,
1998; Watts, 1998). More recently, they are being utilized in the construction of
evolutionary trees (Bowcock et al., 1994) and in forensic identification
(Desmarais et al., 1998; Entrala et al., 1998; Fregeau and Foumey, 1993; Gill et
al., 1994). It is important to address the issue of allele distribution between
different populations because in all of these applications the interpretation of the
results would be affected by, and in some case depends on, differences in the
allele frequency distributions between populations. Only a few studies comparing
allele frequencies of STRs among human populations has been published

(Edwards et al., 1992; Wall et al., 1993) before our study (Morell et al., 1995). To

21

this end, in collaboration with Dr. Morell, we randomly picked 44 dinucleotide
STRs and 9 tetranucleotide STRs spaced about 50 cM intervals throughout the
genome that I typed at Marshfield and compared the allele distribution between
Bengkala 48 representative individuals and the representatives from the western
population. These 48 individuals are 2% of the Bengkala p0pulation. Allele
frequency distributions in those individuals were compared with the CEPH
database. CEPH individuals 1331-01 and 1331-02 were used as standard in typing
these markers to ensure the consistency of the allele scoring between the
Bengkala and public CEPH database.

The frequency distribution of STR alleles that I typed was tested using the
Kolrnogorov-Smimov two-sample method (Campbell, RC, 1989), and the results
of the comparison are shown in Table 3. Of the 53 loci tested, 28 showed
significant differences in distributions between the two populations at the 95 % or
greater confidence level. It was noticed that there are significant differences in
distributions observed in 26 of the 44 dinucleotide STRs but only 2 of the 9
tetranucleotide STRs. A contingency x2 test shows that the tendency for a STR
marker to exhibit different allele frequency distributions is dependent on STR
type (p<0.043). The allele frequency distributions for the nine tetranucleotide
STRs in the two populations are illustrated in Figure 4.

In our study of allele frequency distributions in Bengkala and Western

population (CEPH), significant differences were observed in 28 of 53 STR

22

Table 3. Comparison oi allele distribution between CEPH and Bengkala

 

 

populations
Chromosome Locus Marker go. oi chmosomes Bengkala Test static Significance level
EPi-i

1 018233 aim1992d2 58 82 0.159 -
018399 midi88 98 90 0.298 0.001
018549 GATMHOO 98 80 0.15 -

2 02872 «11838 120 88 0.148 -
028104 midi49 72 90 0.128 -
028423 GMT1A5 58 74 0.122 -

3 0381237 mid124 58 92 0.209 -
0381784 GATMM O 58 90 0.178 -

4 048193 11110142 78 90 0.245 0.05
048194 midi48 82 88 0.149 -
0481825 GATA107 88 88 0.114 -

5 058208 "11888 78 92 0.281 0.01
058210 mid122 80 90 0.278 0.01
058409 airn184yb8 54 88 0.519 «0.001

8 088250 midi 18 238 90 0 289 <0.001
088255 mid228 128 92 0 354 «0.001

7 078440 mid50 112 88 0.221 0.05
078559 mid285 98 52 0.098 -

8 08888 mid45 120 90 0.187 -
088199 midi 77 138 82 0.183 -
088283 aimi4ixa5 58 90 0.325 0.01

9 098104 midi 21 82 88 0.135 -
098105 midi78 70 82 0.182 -

10 0108107 mid078 40 90 0.431 <0.001
0108189 midi 87 34 88 0.381 0.01
0108254 mid249 118 88 0.187 -

1 1 0118874 midi 81 190 92 0.448 «0.001
0118897 mid231 252 90 0.238 0.01

12 012880 midi 09 70 90 0.503 «0.001
012881 midi 14 74 92 0.324 0.01
012883 mid133 72 90 0.487 «0.001

13 0138225 mid250 1 18 80 0.127 -

14 014852 mid187 250 90 0.279 0.001
014853 midi 90 123 88 0.187 -

15 015887 mid49 120 90 0.317 0.01
0158107 mid87 70 92 0.88 «0.001

18 0188281 mid249 98 88 0.134 -
0188398 mid188 78 88 0.185 -
0188888 mid180 120 90 0.203 0.05

17 0178989 GATAi 01-107 124 88 0.039 -
0178281 mid41 120 90 0.364 <0.001
0178975 GGAT2007 42 88 0.127 -

18 018834 mid028 108 90 0.182 -
018849 mid245 122 92 0.584 «0.001
0188538 GATABEOS 92 88 0.277 0.001

19 0198244 mid238 254 78 0.409 «0.001
0198245 mid235 200 90 0.129 -

20 020888 mid138 40 84 0.33 0.01

21 0218158 midss 102 90 0.158 -
021821 1 midi 83 40 90 0.539 «0.001

22 0228270 111111204 128 84 0.592 «0.001

X 0X8458 mid79 103 85 0.091 -

 

23

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m 127 1a m m m '210 200 m m m 100 100 in
Autumn) uni-(up)
M GATMW 04' ' ammo
0.0 0.0 »
0.4 0.4
0.: ”r
0.2 02»
0.1 I l 0.1 m
0‘ 1 1a m 1a 1a 120 m 0'31 manta-140241201200 as-
MM) MM)
~°~5 mm °-° om
0.4 0.5
M}
0.:
E ..
L 0.2, E“
0.1 I I 0.1 »
o 100 m m m 117 in 1.3004101? is? 0‘ 110 100 1a m m 100 140
Ala-(0p) Aleieilp
M130072007
0.0
0.5 loan
°~‘ [jam
0.:
“-0.2
0.1
o as an

 

 

 

Figure 4. Allele frequency distributions for the 9 tetranucleotide STRs used
in this study. The distributions of markers D19S244 and GATASEOS are
significantly different between the Bengkala and CEPH populations in a
Kolmogorov-Smirnov two sample test. (From Morel] et al. 1995)

24

markers. The tetranucleotide STRs were significantly more likely to display the
same allele frequency distribution between the two populations than were
dinucleotide STRs. However, more markers need to be typed to confirm this
observation. If true, this may be due to differences in the mutation rates, with that
of tetranucleotide STRs being four times higher that of dinucleotide STRs (Weber
and Wong, 1993). A higher mutation rate combined with selection on repeat
length would tend to recreate similar allele frequency profiles for loci in separated
populations. Natural selection on repeat length in STR loci has been postulated
elsewhere (Stephan and Cho, 1994) with mutation rate positively correlated with
the repeat length. Our data suggests that it is important to use the allele frequency
from the matching population in mapping studies because there may be
significant differences in allele frequency distributions between populations. If
allele frequency from no-matching population has to be used, tetranucleotide
STRs would be better markers to use both due to their ease in scoring and the

similarity of allele frequency distributions in diverse and isolated populations.

25

C. Discussion

A gene for human congenital neurosensory nonsyndromic autosomal
recessive deafness, DFNB3, is segregating in Bengkala, a village in northern Bali.
Our observations support linkage of DFNB3 to the pericentromeric region of
chromosome 17. DFNB3 is in allelic disequilibrium, and there were no historical
recombinants, with three STRs, D17826l, pTM7-GT and D17SSOS, spanning
about 1 Mb. Based on an analysis of historical recombinants, the closest flanking
markers to this 1 Mb region are D178122 and D17S783. These two markers
define a 12 cM interval to which DFNB3 maps. This region of chromosome 17
has been studied extensively by other research groups. Three hereditary
neurological disorders, Smith-Magenis syndrome (SMS, MINI 182290),
hereditary neuropathy with liability to pressure palsies (HNPP, MIM 162500) and
Charcot-Marie-Tooth disease type 1 A (CMTIA, MIM118220), are due to
chromosomal aberrations of 17p] 1.2-p12 (Chance et al., 1993; Nicholson et al.,

1994; Pentao et al., 1992; Roa et al., 1993; Valentijn et al., 1992).

SMS is caused by large chromosomal deletion of chromosome l7pl 1.2,
most likely due to uneven crossover during meiosis, while I-INPP is the reciprocal
of SMS with the duplication of the same chromosome l7p11.2 fragments (Chen
et al., 1997; Wilgenbus et al., 1997). Point mutations in the PMP22 gene can also
cause CMTlA disease as well as another neurological disorder, Dejerine-Sottas

syndrome (Roa et al., 1993). However, DFNB3 is unlikely to be a mutation of

26

PMP22 since PMP22 is on the telomere proximal side of D17 S 122 and, therefore,

outside the region to which DFNB3 most likely maps.

In large consanguineous Tunisian families, Guilford and coworkers
(Guilford et al., 1994) mapped the first autosomal non-syndromic recessive
deafness locus DFNBI to chromosome 13q. The majority of DFNBI
homozygotes are profoundly deaf and a few are severely hearing impaired. A
second locus for nonsyndromic recessive deafness, DFNBZ, for which there is
variable age of onset, maps to chromosome llq13.5. The shake-1 mutation, with
deafness and vestibular defects, was predicted correctly to be the murine

homologue (Gibson et al., 1995; Guilford et al., 1994; Wei] et al., 1997).

In contrast to the highly variable ages of onset among some individuals
with mutant alleles of the DFNBI and DFNB2 loci (Brown et al., 1996; Cohn et
al., 1999; Gasparini et al., 1997; Kelley et al., 1998; Maw et al., 1995; Scott et al.,
1995; Zelante et al., 1997), the fully penetrant mutant allele of DFNB3 causes
profound, congenital, nonsyndromic recessive deafness and maps to the
pericentromeric region of chromosome 17. YAC contigs for part of l7p11.2-p12

and cDNA clones from this region should expedite the cloning of DFNB3.

Allele-frequency-dependent homozygosity mapping uses a direct genome-
wide analysis of disequilibrium. AHM is best suited for rapidly determining the

chromosomal location of recessive genes in situations where a founder effect is

27

expected, such as in isolated populations. AHM will not work for mapping very
old mutations where the decay of allelic disequilibrium is nearly complete or for
genes where there are no closely linked markers. However, since the density of
highly informative marker on human genetic maps is rapidly increasing and the
typing of STRs is becoming automated, AHM should be useful for directly

mapping even very old recessive mutations.

28

11. Chapter two: Genetic mapping refines DFNB3 to a 3cM region in l7pl 1.2

multiple alleles of DFNB3

A. Introduction

In chapter one, I described my contributions to the mapping of DFNB3, a
gene for human nonsyndromic profound autosomal recessive deafness (Friedman
et al., 1995). DFNB3 was initially identified in a population of 2200 villagers of
Bengkala, Bali, in which 48 individuals are deaf (Winata et al., 1995). We
hypothesized that the gene causing deafness should exhibit a founder effect.
Accordingly, a genome-wide screen to identify an interval of excess
homozygosity among affected individuals placed the DFNB3 locus between
D178122, a marker on the short arm, and D17S783, a marker on the long arm, of
chromosome 17 (Friedman et al., 1995). On the basis of genetic maps available,

the size of this interval was estimated to be about 12 cM (Dib et al., 1996).

The data I presented in this chapter defines the DFNB3 region to a ~3 cM
interval that falls within the Smith-Magenis syndrome (SMS [MIM 182290])
region of l7pl 1.2. This was accomplished by the development of new
polymorphic markers in the DFNBB region; by evaluation of several recently
reported STRs on chromosome 17; and by procurement of additional DNA

samples from recently ascertained affected individuals from Bengkala and

29

neighboring villages, one of which, Bila, is segregating DFNB3. In addition, in
two unrelated consanguineous families from India ascertained by Dr. Edward
Wilcox, Dr. Anil Lalwani, and their colleagues in India, Dr. Dilip Deshmukh and
Dr. Pawan Jain, nonsyndromic hereditary deafness was likely to be due to DFNB3
mutations. Each family had a unique l7p1 1.2 haplotype for markers closely
linked to DFNB3. These data suggest there maybe at least three independently

arising mutations of DFNB3 (Liang et al., 1998).

30

B. Results
1. Refining the DFNB3 critical region to a 3 cM internal

At the time we mapped DFNB3 (Friedman et al., 1995), we reported that
there were six kindreds in Bengkala that contain deaf individuals. At that time we
were unaware of ancestors who connected five of the six kindreds. By 1995 the
genealogy of the about 2200 living and 834 deceased Bengkala villagers was
completed by Dr. John T. Hinnant. A portion of this genealogy, including seven
generations with 45 living and 19 deceased deaf Bengkala villagers, is shown in
Figure 5. Relatives have not yet been identified who link 2 affected individuals
(individuals 22 and 32) in kindred 4 with the 43 other extant deaf individuals from
Bengkala.

It did not surprise me that not all of the deafness in Bengkala has a genetic
etiology. Several relatives of individuals 46C and 48C indicated that they had
been able to hear at birth, as evidenced, they said, by a startle response to loud
noise. Supposedly 46C and 48C lost their hearing in childhood after intractable
high fevers, and are now profoundly deaf. Suspecting that 46C and 48C were
DFNB3 phenocopies, we excluded them from our original mapping strategy. In
this study, I genotyped 46C and 48C using the markers that are closely linked to
DFN33 and found that 46C carries the DFN33 haplotype (Table 4). After we
cloned MY015 a few months later, I sequenced exon 30, and found 46C is
homozygous for the A to T transversion that associates with deafness in

Bengkala. If the story of this child being able to hear at birth is accurate,

31

Figure 5. Genealogy of families with deaf individuals from Bengkala, Bali.
filled symbols indicate the subject is profoundly deaf. Hearing individuals are
indicated by open symbols. Hatched symbols for Bengkala villagers 46C and
48C indicate deafness was likely acquired and not genetic. A dot below a symbol
indicates that genomic DNA was obtained from the subject. An asterisk to the
right of the subject’s identification number indicates that the individual or his/her
parents are not from Bengkala. With the exception of individuals 22 and 32 who
are homozygous for the DFNB3 haplotype (fig. 9) and deaf individuals from other
villages in Bali (subjects 179*, 205* and 5494*), there are familial connections
between all the other deaf individuals in Bengkala. (From Liang et a1. 1998)

32

 

 

m2 8:

         

        

 

 

 

      
      

 

 

O
on. 2:

on. ”2
=>

0

N: E a: .33. n. as 402 am eon N: .3 as an
_>

“V E. o: w: n: «can 3: On

 

 

0 _mMN Ene— _
3: S: 8: 3: E: _m 3. 42 SN .84 .3~ 08 SN
3: fl _2

mm 83% .mM...8~ .3” 8. an

      

 

 

 

I . HAW We”. >
. O C O O 0
mm #3 mm 09— var 8N mm nNN Nmn No. ; ohm 3% N: mm

2. N: m: 22 won

.2. sols. ___

nVNm 02.

 

 

 

=>
0 o o o o
.2 Q 1: n2 8: no; 8:. 8. $2 9.2 33 u:
_>
O 3: O
S.— :3 m2... 3 my no. oM_nm_um_¢__~ N00 new mnu
Mfl — %o >
o
3. n: I; ask. >2 32 82 S:
>—
o: «.2
E
o e 0
2n mmvm 83 m2 o: _I 02 Na: v2

Figure 5

33

:3..— 2 man—

 

=>

5

Figure 5 (cont’d)

34

Table 4 Genotypes of deaf individuals 48C, 46C, suspected of being
Phenocopies, and a deaf individual from Bengkala
homozygous for the DFNB3 linked markers

 

 

marker 48c 48c 42(deaf)
We 1 5
0178122 2 3
0178281 4 4
0178953 2 5
0178740 4 4
017871 2 2
0178820 3 5
01782202 2 3
0178805 1 1
01782201 2 3
0178842 1 2

 

0178783 7 7

 

35

this is the only case of late onset deafness that we have found in Bengkala. As
regards 48C, she does not carry the A to T transversion and is likely became deaf
by other cause. There are many possible infections that could have caused her
profound hearing impairment (Estrada, 1997).

With regards to the statement that two deaf parents in Bengkala always
have deaf children, there are four exceptions in Bengkala. The first two
exceptions are cases of non- paternity, as revealed by marker genotypes, and are
therefore not shown on Figure 5. The remaining two exceptions appear to be
instances of genetic complementation, since they involve marriages between deaf
people from Bengkala and deaf individuals who have recently moved to Bengkala
from other areas in Bali. As shown in Figure 5, marriage of individual #125 from
Bengkala and #5494 from Banjar Jawa (22 km from Bengkala) have produced
children who were able to hear at birth. The same is true for marriage between
individuals #206 from Bengkala and #205 from another village. These married in
deaf individuals carry complete different haplotype from that of Bengkala deaf
individuals for the DFNB3 linked markers. Some of the deaf people of Bengkala
have indicated their desire to have hearing children and have discovered that
marrying deaf individuals from other villages usually produces hearing children.

A complete genealogy of the population of Bengkala village was
constructed and additional deaf individuals from Bengkala were ascertained with

the goal of revealing historical recombinants of more closely flanking markers to

36

DFNBB. DNA from 24 affected individuals from Bengkala were typed for two
new STRs discovered by Aihui Wang (D17S2201 and D17 82202), five STR5 in
the databases (D178952, D17S740, D17S71, D17S620 and D17S842), and five
markers that have been typed previously (D17 S799, D178122, D17826l ,
D17S805 and D17 S7 83). The most likely disease haplotype for these 12 STRs
was constructed. As shown in Figure 6, there are two historical recombinations,
between markers D17S953 and D17S740, represented in individuals 32 and 51
and there are five historical recombinations, between markers D17S805 and
D1752201, in individuals 23, 40, 42, 126 and 127. Individual 42 is also
heterozygous for D17S842, which is likely to be more proximal than D17S2201.
Both D17 S2201 and D178842 are in somatic cell-hybrid bin IX (Figure 7), but
they are found on different YACs, with 797h1 being more distal than 714a10.
We considered the possibility that congenital recessive deafness in families
in other Balinese villages might be due to mutations in DFNB3. Innorthern Bali,
Drs. Friedman, Asher, Hinnant and Thomas Barber surveyed five villages:
Anturan, Bila, Sinabun, Suwug and Tamblang and discovered families with
apparent nonsyndromic recessive deafness. Unlike Bengkala where 2.2% of the
population is profoundly deaf, in five other Balinese villages the total number of
deaf individuals was less than 0.2% of the population. The center of Bila is 2 km
from Bengkala and has a long history of close ties with Bengkala. The vital
Subak system of rice irrigation channels and water allocation (Lansing 1991) has

its administrative and religious headquarters at the higher elevation of Bila, but

37

Figure 6. Map of genetic markers flanking DFNB3 and historical
recombinations that define the DFNB3 region. The DFNB3 critical region
between D17S953 and D17S2201 is indicated by a rectangle. A likely historical
recombination between D17S7I and D17S2201 is indicated by the filled
rectangle. Horizontal demarcations on the right represent STRs for which a
genetic distance has not been reported. The placement of these markers is based
on their position on the physical map of 17p (fig. 7 and Chen et al. 1997).
Horizontal bars that span the vertical line are markers for which a genetic distance
(CM) have been reported. Chromosomes with historical recombinations are
depicted with arrows indicating the location of the DFNB3 interval. Filled circles
represent the region that contains DFNB3. Open circles represent the region from
which DFNB3 has been excluded. The number(s) next to each circle indicates the
marker allele. When a haplotype can not be determined, both alleles are shown.
Alleles assumed to be identical by state are indicated by dotted open circles. The
identification number of Bengkala individuals with recombinant chromosomes are
also shown in figure 6. Individuals from Bengkala with the DFNB3 haplotype are
24, 54, 58, 60, 125, 201, 202 and 247 (fig. 6). Bila individual 21 is shown in the
pedigree in figure 9. (From Liang et al. 1998)

38

 

 

 

R; 322%:
can mm :8 En Ea «ma «6qu
98.9.8 £9.98 £8.33

0N8 N3“ ova
Smxmcom Smxmcom maxmcom 98.9.3 .25

_. n N? h)» h h u.

NV NNNLDCON

v-ON’

 

etc

 

N
\

NVNNNION

PM

NV NNNLOOON

PM

 

NV NNNIOC‘DN

FM

 

Cm:

NVMNNID¢N

 

Co:

LO? NNNIDVN

mm

 

10V NNNIDV‘N

NM

 

cm:

a $52.5

NVNNNIDVN

PM

 

29:03:00

vamh _b
Fommms —O
mowmn PD
NONth FD

owomh FD
{mu FD

ovuwh FD
mmmwx. _b

Pomwh PD
mm as _b
>4-Nmn.<v_

8nmn FD
29:28.

ontL

llllllll

Jl

lol

6'9

6'11

 

Figure 6

39

Figure 7. DFNB3 region integrated map of human/rodent somatic cell
hybrids, STRs and YACs. The black vertical lines represent those regions of
chromosome 17 that remain in each somatic cell hybrid and the horizontal dashed
lines indicate the breakpoints in each hybrid demarcating eleven bins. STRs were
assigned to bins by PCR assay. The short vertical lines represent YAC clones
mapped within or adjacent to the DFNB3 critical region. YAC clones indicated by
dashed lines are either chimeric or have rearranged. The open circle represents a
STR hit and the filled circle represents an EST hit on a YAC clone (ESTs are not
shown). The dot adjacent to an open or filled circle indicates that the data was
obtained from the physical map published by Chen et al. (1997) or from the
Whitehead Institute for Biomedical Research IMIT Center for Genome Research
database (http://www-genome.wi.mit.edul). Note that there are two copies of STR
MSU3, one on 567a12 in bin VI and the other on 797hl in bin VII. An asterisk
indicates that a YAC is not available that includes the STR. (Modified from Liang
et al. 1998)

40

 

____________________l.1_
111
IV

BUN

YACs

DI 73799
DI 75122
DI 7S)6I
DI 7SI356
DI 78135 7
DI 757358

 

_ v _ v _ v _ v _ m _x __ x

_ _ _ _ _ _ __
_ _ _ _ . _ _ _
_ _ _ _ _ _ __
_ _ _ _ _ _ _ _
_ _ _ _ _ _ __
_ _ _ _ _ _ _ _
_ _ _ _ _ _ _ _
_ _ _ _ _ _ __
“a..." 5.. 5 5:551 .5. x
.485... 5%.“... mum... sag»- ea. .1193. ._
_ L _ E San _ ills. _ _
_Nfiwn—o. ”.m—fim—ura ti uYn—uuuficuucmmmmhui—rlod _ _
18.5.1 31.3-5.7... _ __
. _. _ .m _ _ _

mg m mm m w m "a w "m 2»_m "_umm

5mm. mnw m m mmmm "mmm _mmm

mmm mom m m mm...” m pmma __mmm
— b _ _ _

J<>¢HFZ~ MNZKQ

 

 

 

 

Chromosome 17

 

 

——_q——Ip_————————
—_—q——1-L—-————

 

_
_
_
_
_
_
_
_
_
_
.
_
_
_
_
_
_
N.

 

:8:

41

Figure7

approximately 75% of the Subak members are from Bengkala. Rice fields and
gardens of members of both villages are interspersed suggestive of past maniages
between communities, since most land is obtained through inheritance. One of
the twelve Bengkala clans segregating for DFNB3 traces its origin to Bila.
Taking into consideration the historical and geographical relations between these
two villages, it is reasonable to hypothesize that deafness segregating in Bila is
due to DFNB3.

Tamblang (population 5009 as of May 1995) is about 4 km from Bengkala
and has two families with a total of four congenitally deaf individuals that were
typed for STRs in the DFNB3 region. Two other villages were surveyed because
of their historical connection to Bengkala which was chartered in the twelfth
century and has moved twice, once from an area now occupied by Sinabun
(population 4980 as of May 1997) and then from the region where Suwug
(population 6075 as of May 1997) is now located. There are still ancestral
Bengkala temples in Sinabun and Suwug attended by people from Bengkala.
Sinabun has three families with five congenitally deaf individuals and Suwug has
three families with four congenitally deaf individuals, all of whom were typed for
the DFNB3 markers. We also typed 5 deaf members of 3 families from Anturan, a
large village with no obvious historical connection to Bengkala.

Pure tone audiological exams were conducted in each village with a
portable Belltone Model 120 with headphones that had ambient sound-

attenuating audiocups that did not entirely remove the sound of the ubiquitous

42

Balinese crowing game cocks. With only a few exceptions, the affected
individuals had sound pressure level thresholds greater than 90 dB uniformly
from 250 Hz to 8000 Hz. A few individuals were able to hear at low frequency at
80 dB but were profoundly deaf at higher frequencies.

In Bila there is a family with 11 siblings- four of whom are congenitally
deaf and 7 are hearing. The parents are deceased but were said to have been able
to hear. The hearing grandmother was also genotyped. For five STRs in the
l7p11.2 region (D178260, D17S740, D1782202, D17S805 and D17S783) a LOD
score of 1.58 at a recombination fraction (0) of 0 was obtained for this small
family, by means of two-point linkage analysis. Although this LOD score is not
significant, this family appears to have the l7p11.2 Bengkala DFNB3 haplotype,
with the exception of the allele at D17S7l and four distal STRs for which the
three affected individuals are heterozygous (Figure 8). In contrast, deaf
individuals in Anturan, Tamblang, Sinabun, and Suwug have, for 17p11 markers,
genotypes that are entirely different from the Bengkala haplotype (Table 5).
Deafness in individuals from these four villages, if genetic in origin, is likely not
to be due to DFNB3 (Table 6). This conclusion is confirmed by sequencing the
exon 30 of MY015 of deaf individuals from those five villages after we cloned the
gene for DFNB3 and identified the A to T transversion among the deaf
individuals in Bengkala. All four deaf individuals in Bila are homozygous for this
mutation but none of the deaf individuals from the other villages carry this

mutation.

43

Figure 8. Pedigrees of a representative Bengkala family and a Bila family.
The most likely haplotypes for the DFNB3 region markers are shown. The
DFNB3 linked haplotype is boxed and highlighted in red.

44

 

 

 

37
0178799 11
0178122
0178261
0178953
0178740
017871
0178620
01782202
0178805
01782201,
0178842
0178783

N mNNfohloh
d

umhmwmwbmmmao
return to

2
4,
~2
4
2
2
2
5
4
2.

 

 

 

 

 

 

Bila

Mic! 111.111.

814 BIS 316 817 318 B4 819 820 821 822‘ 32; 825 327

 

 

 

 

 

 

0178799

0178122 2 2
0178261 5 6
0178740 2 7
017871 3 3
0178620 2 2
01782202 3 3
0178805 ‘13
01782201 4 4
0178842 2 2
0178783 5 8

 

 

 

 

 

Figure 8

45

Table 5 Genotypes comparison between deaf individuals from Anturan,
Tamblang, Sinabun, Suwug and Bengkala for DFNB3 linked

 

 

markers
marker A23 T79 85 SW35 B20 42
(Antum) (T amblang) (Sinabun) (Suwug) (Bila) (Bengkala)
0178799 1 5 2 2 2 3 3 4
0178122 3 4 2 3 3 3 1 3
0178281 2 3 4 5 3 7 2 4
0178953 3 5 1 3 2 3 3 3
0178740 2 4 2 4 1 3 1 3
017871 1 3 2 3 4 4 2 4
0178820 3 3 3 5 1 3 3 5
01782202 1 2 2 3 2 4 1 3
0178805 3 5 1 1 2 3 1 2
01782201 3 4 2 3 4 4 3 5
0178842 2 3 1 2 3 4 1 3
01 78783 7 8 3 7 1 5 4 4

 

 

Table 6 Two-point LOD Scores that exclude linkage between deafness
and STRs closely linked to DFNB3 in four surrounding villages,
Anturan, Tamblang, Sinabun and Suwug

 

LODscoreat8=

 

Family Locus 0.00 0.01 0.05 0.10 0.20 0.30 0.40

 

Antum 017871 -4.03 -3.39 -2.30 -2.1 1 -2.03 -l .81 -l.73
0178805 -7.27 -7.75 -4.03 -3.39 -2.88 -2.42 -2.21
D17S2201 -5.46 -5.23 -4. 15 -3.67 -2.33 - l .90 -0.04
Tamblang 017871 -0.87 -l .48 - l .78 -2.67 -2.89 -3.22 -4.55
0178805 -2.31 -2.37 -3.45 -3.79 -4.56 -4.87 -5.13
D17S2201 -3.21 -3.46 -5.68 -6.56 -2.45 -0.89 0.23
Sinabun 017871 -0.87 -0.72 -0.27 0.13 0.46 0.78 0.80
D178805 -2.65 -2.13 - l . l7 - l .02 -0.71 -0.42 -0.18
D1782201 -1.19 -0.89 -O.75 —0.67 0.39 -015 -0.07
Suwug 017871 -1 .83 -l .58 -1.32 -0.99 -0.76 -0.34 -0.10
D178805 -4.12 -3.67 -3.15 -1.64 -0.71 -0.11 0.28

01782201 -l.39 -0.89 -0.46 -0.19 0.46 0.79 0.83

 

47

2. DFNB3 segregating in two unrelated families from India

Two large consanguineous families, M-2l and I-1924, with probable
hereditary hearing impairment were first ascertained by Dr. Edward Wilcox and
Dr. Anil Lalwani in schools for the deaf from the district of Kolhapur,
Maharashtra State in western India. A medical history was obtained to exclude
environmental causes of hearing impairment. Physical examinations were
performed to eliminate syndromic causes of hearing impairment. There were no
sign of syndromic hearing impairment in family M21 or family I-l924. Audiology
was performed with a pure tone, portable audiometer and bone conductance was
also tested. Affected individuals in these two families demonstrated profound
sensorial hearing impairment. Obligate carriers had normal hearing. We
concluded that the deafness segregating in these two families was nonsyndromic
congenital recessive deafness. A family history showed that M21 and I-1924 are
unrelated. Genomic DNA was isolated from venous blood samples (Grimberg et
al. 1989).

M21 and I-1924 were screened for linkage to 10 DFNB3 loci (DFNBI-
DFNB8, DFNBIO, and DFNBII), by means of STRs from the Hereditary Hearing
Loss Screening Panel and some additional markers. With the exception of
DFNB3, linkage to all of the reported DFNB loci at the time was excluded in
these two Indian families. For family M21 and I-1924, positive LOD scores of

4.45 and 4.32, at 0 = 0, for markers D17S805 and D17S71, respectively, were

48

Table 7 Two-point LOD Score for Linkage between Deafness and STRs
on Chromosome 17 in three Different Families

 

 

 

LOD score at 0 =
Family Locus 0.00 0.01 0.05 0.10 0.20 0.30 0.40
1-1924 D17S799 «an -0.68 0.47 0.73 0.87 0.63 0.43
0178122 2.15 1.99 1.87 1.65 1.34 0.75 0.33
0178261 2.77 2.67 2.43 2.05 1.34 0.66 0.17
0178953 1.53 1.48 1.27 0.99 0.46 0.04 -0.10
0178740 1.43 1.38 1.19 0.95 0.51 0.18 0.03
017871 4.32 4.14 3.67 3.23 2.51 1.39 0.57
Dl7S620 2.86 2.63 2.37 2.01 1.30 0.79 0.28
D17S2202 1.64 1.30 1.17 ' 1.02 0.71 0.42 0.18
0178805 0.93 0.89 0.76 0.58 0.26 0.04 -0.04
01782201 3.53 3.28 2.99 2.74 1.99 1.29 0.53
Dl7S842 2.83 2.65 2.43 2.14 1.73 0.91 0.38
0178783 3.68 3.57 3.26 2.86 2.13 1.38 0.66
M-21 Dl7S799 -oo -1.06 0.08 0.46 0.37 0.31 0.09
0178122 1.13 1.04 0.94 0.80 0.53 0.29 0.11
D17S261 2.03 1.97 1.71 1.51 1.08 0.67 0.30
D17S953 -oo 2.32 2.87 2.62 1.78 1.25 0.35
0178740 2.71 2.62 2.48 2.31 1.53 1.09 0.28
017871 3.68 3.36 3.10 2.66 1.78 0.94 0.29
Dl7S620 2.89 2.80 2.54 2.20 1.50 0.83 0.29
D17S2202 2.52 2.43 2.22 1.95 1.40 0.86 0.38
Dl7S805 4.45 4.13 3.67 3.38 2.11 1.45 0.57

49

Bila

Table 7 (cont’d)

Dl7S2201

0178842

0178783

Dl7S799

0178122

0178261

Dl7S953

0178740

017871

0178620

01782202

D17S805

01782201

Dl7S842

0178783

1.25

4.14

0.01

0.62

1.58

0.00

1.58

0.62

0.01

1.58

1.58

0.01

0.01

1.58

l .05

3.92

-0.49

0.01

0.61

1.54

0.00

l .54

0.61

0.01

1.54

l .54

0.01

0.01

l .54

1.01

3.65

0.64

0.01

0.5 8

l .40

0.00

l .40

0.57

0.01

l .40

1 .40

0.01

0.01

1.40

0.97

3.18

0.90

0.01

0.52

1.22

0.00

l .22

0.51

0.01

1.22

1.22

0.01

0.01

1 .22

0.68

2.23

0.81

0.01

0.36

0.83

0.00

0.83

0.36

0.01

0.83

0.83

0.01

0.01

0.83

0.49

1.25

0.50

0.01

0.20

0.45

0.00

0.45

0.19

0.01

0.45

0.45

0.01

0.01

0.45

0.23

0.57

0.20

0.01

0.06

0.13

0.00

0.13

0.06

0.01

0.13

0.13

0.01

0.01

0.13

 

50

found at the DFNBB locus (Table 7). Twelve DFNB3 markers in the DFNB3
region were then typed, and haplotypes were constructed (Figure 9). The 17p11.2
haplotypes were different in the two Indian families and were distinct from the
Bengkala DFNB3 haplotype.

It is possible that there may be more than one DFNB gene in this region
since 17p11 is a gene rich chromosomal interval (Saccone et al. 1996). However,
a parsimonious explanation for our data is that nonsyndromic congenital recessive
deafness in Bengkala and in the two unrelated families from India are caused by
independently arising allelic mutations of DFNB3 suggesting that DFNB3 may
make a contribution to hereditary deafness worldwide. Moreover, three
independently arising and possibly different molecular defects of DFNB3 should

make it easier to causally connect a candidate gene with DFNB3.

51

Figure 9. Pedigrees of two unrelated consanguineous Indian families. The
most likely haplotypes for the DFNB3 region markers are shown. The DFNB3
linked haplotype is boxed. The haplotypes for M21 and I-1924 affected
individuals are different from each other and are distinct from Bengkala

haplotype.

52

 

 

 

 

Kindred M21

.1 202.. ff

000000

 

 

 

Ill

 

g, a

Neaanohmmn
a

0178799
0178122
0178261
0178953
0178740
017871
0178620
01782202
0178805
0178842
0178783!

. .12

0178799
0178122
0178261
0178953
0178740
017871
0178620
01782202
0178805
0178842
0178783

GONWdNW-‘UINH

 

 

 

 

 

 

NW
0)

 

VOVG-‘MQ-‘(flfld

D WW

0)
(a)
‘1“UM045MOIN

 

 

 

 

 

 

 

 

 

 

 

O__ «endeavours-recover»

V1

m
(a)
(0)

0173799
0173122
0178281
0173953
0173740
017371
0178820
01732202 33
0178805 11
0178842 44
0178783 21

nwhnwj
mmhmmm
Vudwmwhmmm

w

w

n

N

d
‘1
lb
03
_A

 

 

 

 

 

53

Kindred [-1924

 

 

11 $1

 

 

513.333

 

 

. .E 1.

01 78799

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 4
0173122 2 2 2 2 2
0173261 4 4 4 5 4
0173953 2 5 3 3 5 3
0173740 3 4 7 4 7 7
017871 1 3 3 i 2 3 1
0173620 2 3 3 2 4
01732202 2 3 3 2 3 3
0178805 1 7 4 4 4
01732201 J 3 6 3 2 6
0173342 2 4 4 2 4 4
0173763 7 7 7 6 6 7
V (W
0173799 6 8
0173122 2 2
0173261 4 4
0173953 3 3
0173740 7 7
017371 1 1
0173620 4 4
01732202 3 3
0173605 4 4
01732201 6 6
0173342 4 4
0173763 7 7
V1 5 . i 6
0173799 6 6 6 6 6 6 311
0173122 22 2'2 '27 2 2
0173261 4 4 4 4 4 4 4 4
0173953 3 3 3 3 3 3 3 3
0173740 7 7 7 7 7 7 7 7
017371 1 1 1 1 1 1 1 1
0178820 4 4 4 4 4 4 4 4
01732202 33 33 33 33
0173605 4 4 4 4 4 4 4 4
01732201 66 66 66 88
0178842 4 4 4 4 4 4 4 4
0178789 2.7 11 L7_7 a
Figure 9 (cont’d)

54

3

 

$M$ONMN§WM§OI

 

 

DJ

01

ISO

 

 

 

V15011hw-h-‘VQ-hn

C. Discussion

DFNB3, a gene for non-syndromic, congenital, profound deafness, was
mapped previously to ~12 cM region near the centromere of chromosome 17
(Friedman et al. 1995). To further refine the DFNB3 region we obtained DNA
samples from additional Bengkala affecteds not ascertained in 1993 and also
surveyed five villages in northern Bali for families with hereditary deafness.
Seven historical recombinations occurring among affected individuals from
Bengkala refined the DFNB3 region to an ~6 cM interval flanked by marker
D17S953 on the distal side of 17p11.2 and marker D1752201 on the proximal side
of 17p11.2 within the SMS critical deletion region (Figure. 7). In the village of
Bila the DFNB3 Bengkala haplotype from D17S783 to D17S620 was associated
with deafness. The three deaf individuals in this Bila family are heterozygous for
D1757] with a 167 bp/ 171 bp genotype while Bengkala affecteds are
homozygous for the 171 hp allele. A historical mutation of D1757] from 171 bp
to 167 bp (2 CA repeat changes) is an unlikely explanation given that eighty-six
percent of mutations of STRs result in one CA repeat change (Weber and Wong,
1993). Heterozygosity for D1757] is more likely to be the result of a historical
recombination event since alleles of 3 of the 5 flanking markers distal to the 167
bp allele of D1 7S7] are also different from the Bengkala haplotype (Figures 6 and
8). Recombination with marker D17S71 limits the DFNB3 region to ~ 3cM within

the interval between DI7S71 and DI 7S2201 (Figure 6).

55

The DFNB3 critical region, defined by D] 7S7] on the distal side and
D17S2201 on the proximal side of 17p11, is included within the Smith-Magenis
syndrome (SMS) common deletion (del(17)pl 1.2) which is estimated to be about
5 Mb (Chen et al., 1997). SMS is a highly pleiotropic contiguous gene deletion
syndrome characterized by mental retardation, sleep abnormalities, minor
craniofacial and skeletal dysmorphology as well as short stature, behavioral
abnormalities and cardiac and renal malformations (Greenberg et al., 1996).
Audiological evaluations of 78 SMS patients indicated that 49 (63%) have mild
conductive, sensorineural or mixed hearing impairment (Chen et al., 1997).
Sensorineural hearing impairment of some SMS individuals may be due to a
mutation of DFNB3 in trans to the SMS deletion. Such SMS individuals might
allow for a more comprehensive analysis of the relationship between the deafness
phenotype and the molecular genetics of DFNB3.

Clusters of repeated genes in l7p11 appear to be responsible for
homologous but unequal recombination events that give rise to l7pl 1.2
microdeletions (Chen et al., 1997). We considered the possibility that DFNB3
might be a null mutation caused by a deletion of part of 17p11. Prophase spreads
of chromosomes from an obligate DFNB3 heterozygote and two DFNB3
homozygotes from Bengkala were examined at the 700+ band resolution level and
were normal (data not shown). Moreover, no submicroscopic deletions of 17p11

in affected individuals from Bengkala, Bila or the two Indian families (MZl and I-

56

1924) segregating for DFNB3 were detected by PCR analysis of 28 ESTs and 8
STR3 in the DFNB3 interval.

Without additional large DFNB3 families, further refinement of the
DFNBB critical region to less than ~3 cM seemed unlikely. Two interrelated
strategies to clone DFNB3 in a 3 cM region were pursued. One relied on the
identification of shaker-2 in the mouse by positional cloning and BAC rescue
(Probst et al., 1998). The other involved screening candidate genes in the DFNB3
and shaker-2 intervals. To this end we and others (Beeson et al., 1990; Bloch et
al., 1995; Campbell et al., 1997; Chen et al., 1995; Chen et al., 1997; Chevillard et
al., 1993; De Laurenzi et al., 1996; Elsea et al., 1998; Elsea et al., 1995; Greco et
al., 1996; Hua et al., 1995; Hugnot et al., 1997 ; Liang et al., 1998; Seranski et al.,
1999; Townsend-Nicholson et al., 1995; Webb et al., 1990; Wilgenbus et al.,
1997) have assigned at least 53 ESTs to 17p11. These genes were characterized

by Aihui Wang and are described in detail in her doctoral dissertation.

57

III. Chapter three: Mutation detection of MY015 in family J-l and SMS

patients

A. Introduction

On the basis of conserved synteny and similar phenotypes, we proposed
that the autosomal recessive mouse mutation shaker-2 was the murine orthologue
of DFNB3 (Friedman et al., 1995). Our collaborators, Dr. Sally Camper and Frank
Probst in the Department of Human Genetics at the University of Michigan,
generated a complete l-Mb yeast artificial chromosome and BAC contigs that
span the shaker-2 critical region. BAC clones from the contig were injected
individually into fertilized eggs from sh2/sh2 parents, and a BAC clone that
rescues the sh2 phenotype was identified. A transgenic founder that responded to
sound and did not circle was shown to contain the BAC 425p24 clone (Probst et
al., 1998). This 140-kb BAC was sequenced and examined for potential coding
regions using GENSCAN, GRAIL, and BLAST(Altschul et al., 1994; Altschul
and Gish, 1996; Altschul et al., 1990; Burge and Karlin, 1997; Burge and Karlin,
1998). This approach identified a gene encoding a novel unconventional myosin
that we have designated MonS.

Myosins are a family of actin-based molecular motors that use energy from
hydrolysis of adenosine triphosphate (ATP) to generate mechanical force. Prior to
our discovery, the superfamily of myosin consisted of conventional myosin and

13 structurally distinct unconventional myosins (Hasson and Mooseker, 1997;

58

Hasson and Mooseker, 1994; Hasson and Mooseker, 1996; Hasson et al., 1996;
Heintzelman et al., 1994; Mermall et al., 1998; Mooseker and Cheney, 1995;
Sellers et al., 1996; Wang et al., 1998). The classic, two headed filament-forming
myosins that provide the basis for muscle contractions are referred to as
conventional myosins (Class II). Other members of the myosin super-family
(Class I, II-XV), the unconventional myosins, have functions that are less well
understood but in some cases are thought to mediate intracellular transport of
cargoes such as specific proteins, RNA, Organelles and are involved in the
processes of endocytosis, regulating ion channels, localizing calmedan and
cross-linking extensions of filopodia. (Hasson and Mooseker, 1995; Mermall et
al., 1998; Warrick and Spudich, 1987). All myosins share a common structure
organization consisting of a conserved N-terminal motor domain followed by a
variable number of light-chain binding motifs (IQ motif, which conform to the
consensus IQxxxRGxxxRK ) and a highly divergent tail (Cope et al., 1996;
Espreafico et al., 1992; Houdusse and Cohen, 1996; Houdusse and Cohen, 1995;
Houdusse et al., 1997; Houdusse et al., 1996; Mermall et al., 1998). In the shaker-
2 mouse, an amino acid substitution was found in a highly conserved residue in
the motor domain of Mon5 (Probst et al., 1998).

Primers were synthesized according to the predicted coding sequences of
mouse My015 and were used to PCR amplify the human genomic DNA. One
primer pair (YL12, YL13 from mouse exon 32) was able to amplify human

genomic DNA and gave the expected size product. Sequence of this product was

59

virtually same as the mouse sequence and predicted an amino acid sequence that
was 100% identical to the mouse. I used this PCR product as probe to screen a
human chromosome 17 specific cosmid library. Seven cosmids (72B 12, 155002,
58F6, 131G], 125A8, 24H2 and 57A8) were isolated. Cosmid 155002 was
sequenced and contig was assembled by the National Institute of Health
Sequencing Center (NISC). I then developed primer pairs (YL40:
CCAGACI‘CCTCATTGCACAGAGGG,
YL41 :GCAGAGGCI‘GAGACGCACCTGC;
YL42:CCAGGAGTCCI'I'CTGGAACC; YL43:
GGCI'GGCI‘GCCI‘GCI‘CCGCC) to PCR amplify the 5’ and 3’ end of cosmid
155002. The PCR products were used to screen the human chromosome 17
specific cosmid library again. Two more cosmids, 145G11 and 3H3, were isolated
that were centromere distal and proximal to 155002. These two additional
cosmids (145G11 and 3H3) were also sequenced by NISC. GEN SCAN, GRAIL
and BLAST analysis of these sequences predicted a series of exons of an
unconventional myosin and a G-protein (0RG2) (Schenker and Trueb, 1997). RT-
PCR analysis, 5’ RACE and 3’ RACE by Aihui Wang eventually identified and
confirmed a total of 66 exons of MY015.

Exons of human MY015 were PCR amplified using genomic DNA from
deaf individuals in M21, I-1924 and Bengkala. A nonsense mutation that creates a
premature stop codon was found in all the affected individuals in family I-1924.

An 116 to Phe missense mutation was found in Bengkala affected individuals in

exon 30. It is present in all 29 deaf individuals from Bengkala for whom we have
DNA samples. 25% of the 48 representative Bengkala individuals are carrier of
this mutation. An Asn to Tyr missense mutation was found to be associated with
deafness in family M21 also in exon 30 (Wang et al., 1998). This exon is part of
the coding sequence that encodes a Myth4 domain in MY015.

Smith-Magenis syndrome is a multiple congenital anomalies/mental
retardation disorder caused by an interstitial deletion of chromosome 17 band
pl 1.2 that removes one or more dosage sensitive genes (Elsea et al., 1997;
Greenberg et al., 1991; Greenberg et al., 1996; Juyal et al., 1995 ; Wilgenbus et al.,
1997; Zhao et al., 1995). Most SMS patients (>95%) have the same 5 Mb
genomic region deleted. Approximately 65% of SMS individuals have a mild
conductive, sensorineural or mixed hearing impairment (Chen et al. 1996 Mental
Retard Dev Disabil Res Rev 2:122).

We hypothesized that sensorineural deafness in some SMS patients might
be caused by hemizygosity for a mutant allele of MY015 in trans to the SMS
deletion (Liang et al., 1998). To explore this idea, I sequenced the 65 exons of
MY015 from seven SMS patients with sensorineural hearing loss that range from
threshold increases of 10 dB to 50 dB between 4000 Hz and 8000 Hz as revealed
by pure tone air and bone conduction testing. This chapter describes the detection
of additional mutations in MY015 from DFNB3 individuals in an Indian family

and one SMS patients with mild to moderate severe hearing loss.

61

B. Results
1. Mutation identification in family J -1

J -1 is a family from India with 5 deaf individuals (Figure 10). A medical
history was obtained to exclude environmental causes of hearing impairment.
Physical exams were performed by Dr. Lalwani, MD and Indian collaborators to
eliminate syndromic causes of hearing impairment. Audiology was performed
with a pure-tone, portable audiometer, and bone conductance was also tested.
Affected individuals demonstrated severe-to-profound sensory hearing
impairment (Figure 11). Obligate carriers had normal hearing. Genomic DNA was
isolated from venous blood samples. As shown in Figure 9 all five children in the
generation III are deaf with two hearing parents. Although the number of deaf
individuals deviates from the expected 25% affected progeny, considering the fact
that none of the parents and grandparents are deaf, the best explanation of
inheritance pattern is autosomal recessive.

All of the known autosomal dominant and recessive deafness loci were
checked for linkage to the deafness segregating in J-l. A positive LOD score of
2.41 was obtained for marker 0178953 in the DFNB3 critical region. Four
additional DFNB3 markers were typed and the most likely haplotypes was
constructed. Markers 01782206 and 01782207, which are located within the
transcribed region of MY015 gene, each also has a two point LOD score of 2.41.
The haplotype indicates that the deafness in this family is most likely caused by

compound heterozygous mutations of MY015 since each of the DFNB3 markers

62

Figure 10. Pedigree of Indian family J -l. The most likely haplotypes for the
DFNB3 region markers are shown. The DFNB3 linked haplotype from paternal
side is shown in open box, while the haplotype from maternal side is shown in
grayed box, respectively, to show that they are different from each other and the
mutations are compound heterzygous for M Y 01 5.

63

 

 

 

Family J-i

 

 

 

 

 

 

 

 

 

 

 

01732196
0173953
01732206
01732207
0173606
u [:1—
01732196 [3 4 5.1 1
0173953 1 3 4 2
01732206 1 4 .2 4
01732207 1 3 2 1
0173305 _ZJ 1 g4
Ill l
01732196 ‘ 3
0173953 1
01732206 1 ’
01732207 1
0173605 2

 

 

 

 

 

 

 

 

 

 

 

 

Figure 10

FIGURE 11

PURETONE AUDIOGRAM OF A DEAF INDIVIDUAL IN J-l

Frequency in Hertz

500

1000 2000 4000 8000

250

125

 

 

IIJUIaTllvllf'l 'l'llll

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.Tlu.r.l.#In-lu.¢lultl.lllllull III-
.lnll|:ll.II.Llr|l|ll.llll.l.L "
ommmmwmmwmmm

$3 5.33.5 2.96.5.

1500 3000 6000 12000

750

65

in the deaf individuals are heterozygous for two identical alleles (Figure 10). This
explanation is consistent with the pedigree since all five deaf individuals in J-1
are the descendents of marriage between two large consanguineous families, each
presumably contribute a different MY015 mutant allele to the deaf individuals.

Primer pairs were synthesized according to the intronic sequences flanking
the 65 exons of MY015 so that both coding sequences and splice junction
sequences can be obtained (Appendix VIII). PCR products were sequenced using
Thermo Sequenase radiolabled terminator cycle sequencing kit from Amersham
Life Science. A “G” to “A” change in exon 9 was found in one copy of the
MY015 genes, which results in 3 Gly to Ser substitution. This mutation was
inherited from the maternal side of the family as revealed by sequencing all
available DNA samples. The mother, the grandfather and an uncle are
heterozygous carriers of this Gly to Ser substitution.

The Gly residue is in the motor region of MY015. A ClustalW analysis of
all known myosins showed that this Gly residue is conserved in 83 out of 84
myosins (see ht_tp://www.mrc-lmb.cam.ac.uk/myosin/myosinhtml for the list of
myosins). The only exception is an alanine. This suggests that the Gly residue
must play a very important role in maintaining the function of myosins.

No sequence change was found on the homologue of MY015 gene in the
65 coding exons. Since the mutation detection method consists of PCR
amplification of each exon and then direct sequencing, a deletion that includes

one or both primers will result in a failure to amplify that a deletion, and hence

only the normal allele is sequenced and mutation will not be found. Also, the
mutation could reside in the regulatory region of MY 0] 5 that has not yet been

identified.

2. Mild to severe hearing loss in 3 SMS patient is likely to be caused by a
mutation in MY 0] 5

Genomic DNAs from seven SMS patients who have mild to moderate
severe hearing loss were subjected to PCR amplification and the products
sequenced. A C to T transition which results in a substitution of isoleucine for
threonine was found in exon 31 of MY015 of one SMS patient (#1123) who has
moderately severe high frequency hearing loss (Figure 12). This SMS patient had
the most significant hearing loss of the seven SMS individuals, who were
ascertained by Dr. James R. Lupski and Dr. Lori Potacki.

Audograms of #1123’s parents showed that they both have normal hearing.
Genotypes of polymorphic markers from the DFNB3 region are heterozygous
indicating that none of them carry the 5 MB deletion. Exon 31 was PCR amplified
from genomic DNA of his parents and sequenced. The mother was heterozygous
for the C to T mutation.

An earlier study by Dr. K-S Chen has shown that patient #1123 has the
common 5 Mb deletion (Chen et al., 1997). To further demonstrate that patient

#1123 has only one copy of MY015, Dr. Lupski and colleagues at Baylor College

67

Sound Intensity (dB)
8 8 8 3 8 8 8 8 8 8 o

110

 

FIGURE 12 PURETONE AUDIOGRAM OF PATIENT #1123

Frequency in Hertz
125 250 500 1000 2000 4000 8000

68

of Medicine performed fluorescence in situ hybridization analysis to interphase
lymphoblast cell from patient #1123. Cosmid, 15502, containing exon 5 to exon
55 of MY015 was used as probe. Only one hybridization signal per cell was
found. A positive control probe containing PMP22 (cosmids 103B11, 132G8 and
77F4) was used and gives two hybridization signals per cell indicating both
chromosome 17s are present. PMP22 is located telomere proximal to the SMS
common deletion and hence marked chromosome 17 and served as a positive
control for the in situ hybridization efficiency.

A primer pair was developed from the human exon 31 and used to PCR
amplify genomic DNA from monkey, hyena, cow, Chinese hamster and mouse.
The PCR product was sequenced. ClustalW analysis showed the threonine residue
that was mutated to isoleucine in SMS patient #1123 was conserved among the
five species.

Fluorescence in situ hybridization shows that one copy of MY015 in SMS
patient 1123 is deleted. Moreover, to exclude the possibility that this isoleucine is
a common polymorphism, I have demonstrated this missense mutation is not
present in 720 chromosomes from random individuals from a wide variety of
ethnic backgrounds (include northern European, Chinese, Indo-Pakistani, African
American, American Indians, Japanese, Mexican and Puerto Rican). Moreover,
the threonine residue is conserved in orthologues of MY015 from mouse, Chinese
hamster, Rhesus monkey, hyena and cow suggesting that it is probably important

for MY015 function (Figure 13).

69

Figure 13. ClustalW formatted alignments of exon 29 peptide sequences in
myolS from Hyena, bovine, mouse, Chinese hamster, rhesus monkey and
human. The threonine residue that is mutated in SMS patient #1123 is

highlighted in gray.

70

 

 

20.55.
80028.: 03093
Hmumfimm 0002.430
00:02

mugom

0804mm

 

$58—$23. 60338.8..— 3.355

Figure 13

71

3. Simple Tandem Repeats and Single Nucleotide Polymorphisms in MY 01 5
Sequence analysis revealed two long (CA), repeats in the MY015 genomic
sequence, one located between exon 1 and 2, the other is upstream of exon 8.
Primers were designed from the sequences flanking these two repeats and used to
type DNA from 100 random individuals with different ethnic background (Table
8). These two repeats are highly polymorphic, one (01782206) has 10 alleles with
a heterozygosity of 0.9 and the other (01782207) has 9 alleles with a
heterozygosity of 0.89. These two markers are ideal polymorphic markers for
checking linkage between deafness segregating in a family and the DFNB3 locus.
Six single nucleotide polymorphisms (cSNPs) were found in 6 of the 65
MY015 coding exons when I sequenced l6 deaf individuals for mutations (Table
9). The cSNPs did not change the amino acid sequence of the MY015. These
cSNPs will be ideal SNP markers for determining linkage between hereditary

deafness segregating in a family and DFNB3.

72

Table 8. Short Tandem Repeats (STR) in MY015

 

 

Start 0 number No. of Het. Product Primers
Position" ’ Alleles Size (bp)
21672 01782207 9 0.89 135-155 F: TATTCI'I‘ACCACCI‘CCCCI‘G
R: CAGGACCTGCTAGTGCAGG
38680 01782206 10 0.90 141-165 F'

CTGCC‘I‘GTCCCI‘CCACCCACAC
R: ccrcccrcchoAcocrcrro

Allele sizes and frequency distributions for 01782207

 

 

Allele Size (bp) number oi allele irequency
number allele

1 155 8 0.025
2 153 18 0.075
3 151 24 0.1

4 149 30 0.125
5 147 51 0.2125
6 143 39 0.163
7 141 21 0.0875
8 137 45 0.188
9 135 6 0.025

 

Allele sizes and frequency distributions for 01782206

 

 

Allele Size (bp) number of allele frequency '

number allele
1 185 3 0.0125
2 181 24 0.1
3 159 24 0.1
4 157 30 0.125
5 155 54 0.225
6 153 36 0.15
7 151 39 0.163
8 149 21 0.0875
9 147 0.0125
10 141 0.025

 

"' GenBank accession no. AF051976

. Position of the first base of forward primer

73

Table 9. Single Nucleotide Polymorphisms (cSNP) in MY 01 5

 

Position" ‘

46051

47519

59772

63064

64037

67394

0 number

01782208

01782209

01782210

01782211

01782212

01782213

Exon Variant

15 C/T

17 CIA

39 G/A

44 CIT

46 C/T

53 T/G

"' GenBank accession no. AF051976
. Position of the first base of forward primer

74

Frequency Product

059/041

059/041

059/041

0.62/0.38

0.62l0.38

0.97/0.03

Size (bp)
257

284

279

247

297

249

Primer pair

F:CTCCITCAGA
TI‘CAACATGG
RzTCI'GTGACCI‘
GACI'AGGCI‘C
F:AGAAGTGAG
GAGGATCCAGT
R:TGTCTGGCC
AAAGCTAAG
F:TCl‘CCCTGGA
'I'I‘CI'CAT'ITA
RzTC'l'I'I‘GTC'I'I’
CI‘G'ITCCACC

F: GTATCAGCC
ACCT CCCI' C

R: TGAATCCTCT
GGACAGGTAG
FzATCAGAAGC
AGCACAATAG
R:GTCATAT'I'I‘C
CCTGGAACAG
F:AGAGAATAG
AAGCI'CCCAGC
RzGAACCACAG
AGCAGGAAAG

3. Discussion

Four independently arising mutations in MY015 have been associated with
autosomal recessive deafness DFNB3 (Figure 14). One mutation was found
among deaf individuals in a Balinese village. Three were identified in three
unrelated consanguineous families from India. Three mutations are described in
Aihui Wang’s dissertation entitled “Molecular cloning of an unconventional
myosin MY 01 5 and the identification of its mutations responsible for human
nonsyndromic deafness DFNB3”. Two of the mutations are found in the first
Myth4 domain encoded by exons 29 to 31, one was found in the talin like domain
in exons 40 to 48. The newly identified Gly to Ser mutation of the J -1 family is
the only human mutation of MY 01 5 found in the myosin motor region. The motor
domain of characterized myosin binds actin and ATP and generates force through
the hydrolysis of the ATP. In contrast to the motor domain, the function of the
myosin tail domains is largely unknown. However, a common assumption is that
the tail directs the interaction of a given myosin with its cargo (Cheney and
Mooseker, 1992; Hasson et al., 1997; Hasson ct al., 1995; Hasson and Mooseker,
1997; Hasson and Mooseker, 1995; Hasson and Mooseker, 1996; Houdusse et al.,
1996; Mooseker and Cheney, 1995). Yeast two hybrid system is designed to
screen for proteins that directly interact with the bait (B31 and Elledge, 1997;
Luban and Goff, 1995; Staudinger et al., 1993; White, 1996) and has been used

successfully to identify a microtubule-based transport motor, thU, that interact

75

62-22.84»... 20 8.2.6 E92: ucm 932:6 .3 05m."—

mz 2.53...
2.411095 Nam
«_mchom 23.5150
2 2.216..
23. “a.-. 2:628 2% 3 2.532
9m . m 5 2.19.2 3m 26
a. 1_

 

 

 

mcdm hw1mm _mumv wvév gram vm VN1m 1|.
Tll. TI. .ITIIII. Tl. . u ..
36.652.52.92 $.52 Em 36.682.62.32 4:22.). a. 662 38.

 

76

with MyoVA (Huang et al., 1999). This system should be helpful in identifying
the partner(s) of MY 015 and understanding its function in the human inner ear.
Although our previous findings in four DFNB3 families show that mutant
alleles of MY 015 cosegregate only with profound nonsyndromic recessive
deafness, these data are biased because only profoundly deaf individuals were
ascertained and screened for MY 01 5 mutations. Although limited to only one
SMS patient, my data suggest that at least one “mild” mutant allele of MY015 in a
Smith-Magenis syndrome individual may be associated with moderate high
frequency hearing impairment. The four mutations found in DFNB3 individuals
are all located in domain structures such as the motor, Myth4 and talin like
domains. It’s not hard to imagine that mutations in these domains would cause
more severe hearing loss than mutation(s) that are found outside these domains,
such as the one in SMS patients. Examples where different mutations result in
different phenotype can also be found in another unconventional myosin, MY 07A.
Some M Y 07A mutations cause Usher syndrome type 1B. The phenotype of Usher
syndrome 1B includes congenital balance, hearing defects and progressive
retinitis pigmentosa (Levy et al., 1997; Weil et al., 1995; Weston et al., 1996).
Mutations of MYO7A also cause autosomal recessive, non-syndromic deafness
DFNB2 (Liu et al., 1997 ) and autosomal dominant, non-syndromic deafness

DFNAII (Liu et al., 1997; Weil et al., 1997).

77

 

A.l

rec
Liz
gel
ac:

ICE

in.

IV. Chapter four: The study of expression pattern of myosin 15 in human and

mouse embryo

A. Introduction

In chapters one and two I presented the data that indicate human autosomal
recessive deafness DFNB3 maps to chromosome 17p11.2 (Friedman et al., 1995;
Liang et al., 1998). An unconventional myosin, MY015 was then identified as the
gene for DFNB3 (Wang et al., 1998), while mutation in the homologue, MonS,
accounted for shaker-2 phenotype (Probst et al., 1998). Mutations of MY015
result in profound hearing loss in DFNB3 individuals in four unrelated families
and possibly mild to severe hearing loss in at least one SMS patient. A point
mutation in the motor region results in deafness and vestibular defects such as
head-toss and circling behavior in shaker-2 (Probst et al., 1998).

Work by Aihui Wang has shown that the longest MY015 transcript
consists of at least 66 exons. The MY015 open reading frame is 10.6 kb and
encodes a predicted 3530 amino acid peptide with a calculated molecular weight
of 395.2 kDa. Unlike other known unconventional myosins, MY015 has an
unusual 1200 amino acid extension 5’ to the conserved motor domain. Following
the motor domain, there are two light chain binding motifs, a 1586 amino acid tail
with two myosin tail homology 4 (MyTH4) domain, two talin like domain, and a
8H3 like domain (Figure 14). Among the four mutation identified in DFNB3

individuals, one was found in the motor domain, two were identified in the first

78

MyTH4 domain, and one was detected in the talin-like domain. This chapter
describes the expression pattern of myosin 15 in human and developing mouse

embryo.

79

B. Results

To learn more about the expression pattern of M Y 01 5 , human RNA master
blot(Clontech #7770-1) with polyA RNA from 50 different human tissues (whole
brain, amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe,
hippocampus, medulla oblongata, occipital lobe, putamen, substantia nigra,
temporal lobe, thalamus, nucleus accumbeus, spinal cord, heart, aorta, skeletal
muscle, colon, bladder, uterus, prostate, stomach, testis, ovary, pancreas, pituitary
gland, adrenal gland, thyroid gland, salivary gland, mammary gland, kidney, liver,
small intestine, spleen, thymus, peripheral leukocyte, lymph node, bone marrow,
appendix, lung, trachea, placenta, fetal brain, fetal heart, fetal kidney, fetal liver,
fetal spleen, fetal thymus, fetal lung) was probed with MY015 cDNA sequence
from exons 31 to 41 (primers BF62/YL 802). The blot was then striped and re-
probed with MY015 cDNA sequence from exon 2 (LMG188/LMG191).

Strong hybridization signal was observed with RNA from the pituitary
gland (Figure 15). Another strong signal was observed from kidney but this result
could not be repeated in two separate dot blot analyses and by northern analysis.
Weak hybridization signals were detected in RNAs from testis and ovary. This
experiment was repeated three times using three separate human RNA master
blots purchased from Clontech. The same results were obtained with the
exception of the first blot that had a strong signal from kidney RNA. I then

purchased a Human Multiple Tissue Expression (MTE) Array (Clontech # 777 5-

80

Figure 15. Human RNA master blot probed with sequences from MY 015
tail and head region and DRGZ. Human RNA master blot from Clonetech with
polyA RNAs from 50 tissues are used. (A) Blot was first probed with sequences
from MY015 exons 31 to 41. (B) Blot was striped and then probed with MY015
exon 2 sequence. (C) The Blot was then striped and sequenced with DRGZ, a G-
protein coding gene upstream of MY 01 5 .

81

 

 

54. 5 9.98 29»:

 

....... tflil.

 

' i

N coxm m 2 9»:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O O 0 e

I... u cl. 1min. I.- I... I. I... Iqlfll II I... I. II.
o O 0 e

11.1... 1 100.01.01.01... 1 1013.10.18.01 1 1
fl. 0 e o 0 e

i i 3 i I! ' i 3'“ 3 I ll ' i 3 3 i
e e o e .

PHI.“ 01 1.0.10.1“ 04101041111.”
.0 C O C

Imiuluurrm. “41884.01 0 iirfifd i. u.

 

 

 

 

 

 

 

 

 

Figure 15

82

1) which contains polyA RNAs from 26 cardiovascular and digestive tissues and
cancer cell lines in addition to the RNAs from 50 different tissues that were
included in human RNA master blot. These additional tissues include the pons,
left atrium, right atrium, left ventricle, right ventricle, interventricular septum,
apex of the heart, esphagus, duodenum, jejunum, ileum, ilocecum, ascending
colon, transverse colon, descending colon, rectum, peripheral blood leukocyte,
lymph node, leukemia HL-60, Hela S3, leukemia K-562, leukemia MOLT-4,
Burkitt’s lymphoma Raji, Burkitt’s lymphoma Daudi, colorectal adeno-carcinoma
SW480, lung carcinoma A549. Again, only RNA from pituitary gland gave strong
hybridization signal, with weak hybridization signals from testis and ovary
(Figure 15).

Northern blots with polyA RN As from human pituitary gland, kidney, lung
and heart were probed with the same MY015 head and tail probes as used in the
dot blot analysis. Strong hybridization signals were only obtained from the
pituitary gland RNA using the exons 31 to 41 as probe (Figure 16). These
hybridization signals include a smear ranging from ~5 to 9 kb and a distinct ~12
kb band. The blot was striped and probed again with the exon 2 sequence. A
strong, distinct ~12 kb hybridization signal was obtained from pituitary gland. A
much weaker smear was also observed in the lane with pituitary gland RNA
(Figure 16). The blot was again striped and probed with the upstream DRGZ
sequence. Two hybridization signals of 1.8 kb and 2.2 kb as published (Schenker

and Trueb, 1997) were detected in RNA from all four tissues (Figure 16). These

83

lung
brain
lung
brain
lung
brain

.t' .t' .t“
D. O. O.

     

MY015 MY015 DRGZ
exon 2 exons 31-41

 

Figure 16. Human northern blot probed with sequences from MY015 tail
region, head reglon and DRGZ. Five pg of PolyA RNA from human pituitary
gland, kidney and lung were used. (A) Blot was first probed with sequences from
MY015 exons 31 to 41. (B) Blot was striped and then probed with MY015 exon 2
sequence. (C) The Blot was then striped and probed with sequence from DRGZ, a
G-protein coding gene upstream of MY015.

same results were observed in four independently prepared and probed northern
blots.

Two 15.5 days mouse embryos were used to study the expression of
MYOlS in the developing mouse embryo (Figure 17). One embryo was sectioned
perisagitally and the other sectioned transversely. (See appendix for the in situ
hybridization protocol). PCR products (primers BF56/BF59) that contain mouse
exons 48 to 51 were subcloned into pGEM-T easy vectors in both orientations.
Slides were probed with 35S-UTP and 3’SS-CTP labeled antisense RNA transcribed
from this construct. After a ten day exposure, strong hybridization signals were
observed from the mouse inner ear in both vastibular apparatus and cochlea. No
hybridization signal was observed in any other tissues. A sense probe transcribed

from the construct was used as a negative control and gave no hybridization

signal (Figure 17).

85

Figure 17. In situ hybridization of MY 015 tail sequence to 15.5 day mouse
embryos. The mouse embryo was sectioned perisagitally. Strong hybridization
signal was observed in the developing vestibular apparatus (top left) and cochlea
(top right) at 50 X magnification. The two panels on the bottom show a higher
magnification (400 X) of signals from the cochlea (dark field plus fluorescence
image on the bottom left and bright field image on the bottom right).

86

 

 

Figure 17

C. Discussion

My in Situ hybridization experiments with 15.5 days mouse embryo
(Figure 17) showed that MY 01 5 is exclusively expressed in the mouse inner ear.
Although the resolution of the in situ is not fine enough to tell which cells exactly
MY 01 5 express, the hybridization signals are close to the regions where hair cells
are going to develop in the cochlea. Hybridization signals were observed in both
the developing vestibular system and cochlea. This finding is consistent with the
phenotype of the shaker-2 mouse, which is deafness and circling and head
. tossing, signs of cochlear and vastibular malfunction in those mutants.

In human, MY 01 5 is most abundantly expressed in pituitary gland with
trace amount found in testis and ovary. Since large quantities of RNA can not be
obtained from human inner ears, a northern and dot blot analysis data on the
expression of MY 01 5 in the human inner ear has not been possible. However, the
expression of MY015 in human inner ear can be easily detected by reverse
transcription PCR (RT-PCR). The fetal human inner ear RNA was obtained from
Dr. Cynthia Morton.

There are indications that there are alternative splice isoforrns of MY015.
Northern blots probed with MY 015 exons 31 to 41 (Figure 16) showed
hybridization signal of ~12 kb and a smear from ~5 kb to 9 kb. The only strong
signal seen on the same blot when probed with MY 0] 5 exon 2 was the 12 kb band
with a light smear. In situ hybridizations completed by Mr. Thomas Barber

showed that the MY015 isoform with exon 2 was also present in the mouse inner

88

ear, but the signal is much weaker compared to signals observed from slides
probed with the tail region sequence. This is not surprising since northern analysis
(Figure 16) has shown the tail probe can recognize both the 12 kb band and the 5
kb to 9 kb smear. What was observed in the in situ hybridization probed with the
tail sequence is the sum of the signals from all isoforms. The smear from the 5 kb
to 9 kb could be due to RNA degradation, or alternative splicing of other exons of
MY015. It’ s not known which of these forms are functional important in human
and mouse.

Although the strongest MY015 signals was observed in human pituitary by
dot blot and northern analysis, mutations of MY 0] 5 do not result in an obvious
clinical phenotype in DFNB3 individuals and in the shaker-2 mouse. This
suggests that either loss of MY015 function in the pituitary is compensated by
some other unconventional myosins, or a pituitary defect in DFNB3 individuals is
so subtle that it was not detected. Another possibility is that there is still residual
function in the mutant MY 01 5 protein, and the inner ear is more sensitive to the
defect.

Antibodies to both exon 2 and the tail regions are being prepared. They
will help us locate the expression of MY015 in specific cell types in both the
inner ear and pituitary and should give clues as to what MY015 is doing in these

tissues and organs.

89

APPENDICES

90

APPENDIX A

List of publications from This Work:

Liang Y., Wang A., Probst F., Arhya N., Barber T., Chen K-S, Deshmukh D.,
Dolan D., Hinnant J., Moeljopawiro S., Morell R., Negrini C., Wilcox E.,
Winata S., Camper S., and Friedman T..B. Genetic mapping refines DFNB3 to
l7p11.2, suggests multiple alleles of DFNBB, and supports homology to the
mouse model shaker-2. The American Journal of Human Genetics 1998, 62:904-
915

Wang A., Liang Y., Fridell R.A., Probst F.J., Wilcox E.R., Touchman J .W.,
Morton C.C., Morell R.J., Noben-Trauth K., Camper S.A., and Friedman T.B.
Association of unconventional myosin MY 01 5 mutations with human
nonsyndromic deafness DFNB3. Science 1998, 280: 1447-1451

Probst F.J., Fridell R.A., Raphael Y., Saunders T.L., Wang A., Liang Y., Morell
R.J., Touchman J.W., Lyons R.H., Noben-Trauth K., Friedman TB. and Camper

S.A. Correction of deafness in shaker-2 mice by an unconventional myosin in a
BAC transgene. Science 1998, 280:1444-1147

Liang Y., Chen K—S, Potocki L., Wang A., Fridell R.A., Lupski JR. and
Friedman T.B. High frequency hearing loss in an individual with Smith-Magenis
syndrome is likely due to a missense mutation in MY015 uncovered by
del(l7)(p11.2p11.2). (Manuscript in preparation)

Wang A.*, Liang Y.*, Probst FJ., Wilcox E.R., Sellers J.R., Camper S.A.,
Friedman TB. and Fridell R.A. Complete genomic and cDNA structures of the
human and mouse Myosin-IS genes associated with hereditary deafness.
(Manuscript in preparation)

* These two authors contribute equally to this paper

Liang Y., Chen H., Asher J.H. Jr., Chang CC. and Friedman T.B. Human inner
ear OCP2 maps to 5q22-5q35.2 with the related sequences on chromosomes
4p16.2-4p14, 5p13-5q22, 7pter-qZZ, 10 and 12pl3-12qter. Gene 1997, 184: 163-
167

Friedman T.B., Liang Y., Weber J .L., Hinnant J.T., Barber T.D., Winata S.,
Arhya IN. and Asher J.H. Jr. A gene for congenital, recessive deafness DFNB3

maps to the pericentromeric region of chromosome 17. Nature Genetics 1995,
Vol. 9:86-91.

91

Morell R., Liang Y., Asher J.H. Jr, Weber J ., Hinnant J .T., Winata S., Arhya IN.
and

Friedman T.B. Analysis of short tandem repeats (STR) allele frequency
distribution in a Balinese population. Human Molecular Genetics 1995, Vol. 4,
No. 1:85-91

Winata S., Arhya I.N., Moeljopawiro S., Hinnant J.T., Liang Y., Friedman TB.
and Asher J.H. Jr. Congenital non-syndromal autosomal recessive deafness in
Bengkala, an isolated Balinese village. Journal of Medical Genetics 1995,
32:336-343

Carey M.L., Liang Y., Barber T.D., Morell R., Johnson D.H., Cox 8., Asher J .H.
Jr. and

Friedman T.B. Dinucleotide repeat polymorphism at D14SS42. Human
Molecular Genetics 1994; Vol. 3, No. 9:1712

Additional publications by Yong Liang:

Innis, J.W., Asher, J .H. Jr., Liang Y., Wang, A., Wilke, C.M., Dierick, H.A.,
Kazen-Gillespie, K., Sheldon, S., Glover, T.W. and Friedman, T.B. Exclusion of
BMP6 as a candidate gene for cleidocranial dysplasia. The American Journal of
Medicine 1997, 71:292-297

Fong D, Chan MMY, Rodriguez R, Chen C.C., Liang Y., Littlewood TJ. and
Ford S.E. Small subunit ribosomal RNA gene sequence of the parasitic protozoan
Haploporidium nelsoni provides a molecular probe for the oyster MSX disease.
Molecular and Biochemical Parasitology 1993, 62: 139-142

92

APPENDIX B

Mapping of human inner ear OCP2 cDNA to human chromosomes 4, 5, 7, 10 and
12
A. Introduction

The identification of genetic and physical map position of genes expressed
abundantly or exclusively in the inner ear should expedite cloning of loci
responsible for hereditary hearing loss (Harter et al., 1999; Morton, 1991;
Robertson et al., 1994; Skvorak et al., 1999). To this end, Ocp2-r112 was brought
to our attention as a candidate for DFNB3 by Dr. G. Duyk. Ocp2-r112 maps to the
mouse chromosome 11 in a region that has syntenic relationship with either
human chromosome 5q23-q3l or chromosome 17p to which the DFNB3 has been
mapped.

Ocp2-r32 encodes OCP-II, an 18.6 kDa protein abundantly expressed in
the organ of Corti in Guinea pig. OCP-II is also found at lower concentration in
the vestibular apparatus and a few other tissues (Chen et al., 1995; Thalmann et
al., 1980; Thalmann et al., 1993). Murine Ocp2-r32 was cloned from mouse
cochlear cDNA by PCR using degenerate primers based on the guinea pig Ocp2
sequence. We used mouse Ocp2-r32 to identify cDNA clones for a human OCP2
gene expressed in the inner ear. Five chromosomal loci for human OCP2 were
found by Southern analysis and PCR amplification. The experiments to isolate

and sequence human OCP2 cDNA clones from a human fetal inner ear library

93

were performed by Dr. Thomas Friedman. They are included in this chapter as
part of story of OCP2 initiated by me. I tested the hypothesis that OCP2 was a

candidate for DFNB3 by first mapping OCP2.

94

 

B. Results
1. Using human/rodent somatic cell hybrids to map human OCP2

To determine the map locations of human OCP2 loci, a 254 bp mouse
Ocp2 cDNA emcompassing a portion of the 5’-UTR up to codon 50 was used to
probe a Southern blot of human genomic DNA and human/rodent somatic cell
hybrids. Each hybrid cell line contained one of 22 human autosomes. Genomic
DNAs from each host parental cell line (mouse and hamster) were included as
controls. The data indicated that human homologues of murine Ocp2 are located
on chromosomes 4, 5, 7, 10 and 12 (Figure 18). To refine the location of human
OCP2-like sequences, genomic DNA samples from a subsets of the human-rodent
somatic cell hybrid mapping panel and hybrids with human chromosomes
containing translocations and deletions with known breakpoints that allow for
regional localizations were used.

Human genomic DNA digested with PstI restriction enzyme showed six
strong hybridization signals to OCP2 and one faint signal. These PstI restriction
fragments are derived from human chromosome 4 (2.0 kb), chromosome 5 (7.0
kb, 3.9 kb and 2.6 kb), chromosome 7 (1.7 kb), chromosome 10 (3.7 kb) and
chromosome 12 (1.8 kb) (Figure 18). These data suggest that there are at least five
independently assorting human OCP2-like sequences, possibly a family of OCP2
gene in human. But none of the OCP2 gene mapped to human chromosome

17p11.2, hence they are not a candidate for DFNB3.

95

Figure 18. Locations of human 0CP2 sequences on chromosomes 4, 5, 7, 10
and 12 by Southern blot analyses of PstI restricted genomic DNA. (A) Mouse
genomic DNA (M) showing a PstI polymorphism between our C3H murine (Lane
1) and 3T6 used to construct the somatic cell hybrids in lanes 4 and 9, lane (2)
Chinese hamster genomic DNA (C), lanes (3 and 10) human male genomic DNA
designated 46,XY, lane (4) human/mouse somatic cell hybrid retaining human
chromosomes 1 and X (NA 07299), lane (5) to lane (8) human/Chinese hamster
somatic cell hybrids retaining human chromosomes 4 (NA10115), 5 (NA10114),
7 (NA10791), and 10 (NA10926), respectively and lane (9) human/mouse
somatic cell hybrid retaining human chromosome 17 (NA10498). (B)
Localization of the human 0CP2L5 on chromosome 12 and sub-localization of
the human 0CP2L1, 0CP2L2 and 0CP2L3 on chromosomes 4, 5 and 7 by
Southern blot analysis. Lane (1) human genomic DNA, lane (2) human/Chinese
hamster somatic cell hybrid retaining a human der(5)t(5;7)
(5pter>5q35.2::7q22>7qter) (GM 11446), lane (3) human/Chinese hamster hybrid
retaining a human der(5)t(5: l3)(5qter>5pl3::13q13>13qter) and human
chromosome 6 (GM11437), lane (4) human/Chinese hamster somatic cell hybrid
retaining human der(2)t(2;5)(pl 1.2;q22) and human chromosomes 4, 6, 10, 12,
16, 18, and 21 (GM11444), lane (5) human/Chinese hamster somatic cell hybrid
retaining a human del(4)(4qter>4p14::4p16.2>4pter), and human chromosomes 5
and 13 (GM11447), lane (6) human/Chinese hamster somatic cell hybrid retaining
a human der(12)t(4;12)(12qter>12p13::4q25>4qter) (GM11449), lane (7) Chinese
hamster genomic DNA and lane (8) mouse genomic DNA. Human chromosomes
with PstI restriction fragments hybrydizing to Ocp2 are indicated between panels
A and B. (From Liang et al. 1997)

96

 

Figure 18

-MCCCCM

Rodent IIC

12345678

“1012345678910

 

2. Isolation and sequence of human 0CP2 cDNA clones from a human fetal

inner ear library

To determine which of the 0CP2-like sequences are expressed in the

human inner ear, a human fetal inner ear-specific A. Uni-ZAP XR cDNA library
(Robertson et al., 1994) was screened with murine 0CP2-r32 and two 1 clones
were plaque purified. The clone with larger cDNA insert, CMl , was sequenced
and the data archived (GenBank accession No. U37558). Based on similarity to
the murine gene, the sequences of CMl begins with murine Ocp2—r32 codon 14,
includes the remainder of the coding region with a translation stop codon at nt
451-453 and 258 nt of 3’-UTR. The protein coding region of human CMl showed
94.7% nt sequence identity with murine Ocp2-r52. With the exception of the first
14 codons missing in cDNA clone GM], has an identical deduced amino acid
sequence.

The chromosomal location of the 0CP2 gene from which CMl cDNA was
derived was determined. PCR primers (TF 174, 5’-ATG CAG CAG CAA GTC
AAT TG and TF 186, 5’-GAA ACT TGG CCT AAG G'IT TGG) were
synthesized based on the sequence in the 3’-UTR corresponding to nt 551-639 of
CM] (U 37558), respectively. For PCR amplification of the expected 89 bp
product, a 25 Ill reaction mixture included 40 ng of genomic DNA, 200 M
dNTPs, 1.5 mM MgC12, 50 mM KCl, 10 mM Tris pH 8.3 and 1 unit of thermal

stable DNA polymerase. The reaction was cycled 32 times at 94°C (1 min), 62°C

98

(l min) and 72°C (15 s). Human, mouse, Chinese hamster and human/rodent
somatic cell hybrid line genomic DNA samples for the PCR reactions are
described in the legends to Figures 19 and 20. For each somatic cell hybrid,
control PCR reactions using primer pairs for one to three STRs for each human
chromosomal region tested gave a product of the expected size confirming the
presence in the human/rodent somatic cell hybrid DNA of the appropriate human
chromosomal region.

Employing an annealing temperature of 62°C for the PCR reaction
described above, PCR primers TF174 and TF186 amplified a predicted 89 bp
product only from human chromosome 5q22-5q35.2 present in human/rodent
somatic cell lines GM11444 and GM11446 (Figure 19, lanes 1 and 4). Therefore
the human 0CP2 gene expressed abundantly in the human inner ear is most likely
on chromosome 5q22-5q35.2, not chromosome 17.

When the primer annealing stringency was reduced from 62°C to 55°C for
TF174 and TF186 in the PCR reaction described above, a 89 bp product was
amplified from DNA isolated from hybrids containing human chromosomes 4, 7,
and 12 (Figure 19, lanes 5, 6 and 8), in addition to chromosome 5q22-5q35.2.
Except for the chromosome 10 location of an 0CP2-like sequence detected by
Southern analysis, the PCR data are consistent with the locations of other 0CP2
like genes and/or pseudogenes detected by Southern analysis of human/rodent
monochromosomal somatic cell hybrids using a murine 0cp2-rs2 probe from the

5’ coding region (Figure 18 A and B).

99

1234567891011

 

Figure 19. To determine the chromosomal location of the gene from which CMl
was transcribed, PCR primers (TF174 and TF186) corresponding to sequence in
the 3’UTR were used to amplify a predicted 89 bp product. Methods: A 25 pl
PCR reaction includes 40 ng of genomic DNA, 200pM dNTP, 1.5 mM MgCl; 50
mM KCl, 10 mM Tris pH 8.3, 1 unit of thermal stable DNA polymerase which
was cycled 32 times at 94°C (1 min), 62°C or 55°C (1 min) and 72°C (15 s).
Mouse and hamster DNA did not amplify under these conditions (data not
shown). Gel electrophoreses of PCR products are shown. Genomic DNA for PCR
reaction are: human monochromosomal 5 (GM10114) (lane 1), human
der(2)t(2;5)(p11.2;q22) and human chromosomes 4, 6, 10, 12, 16, 18, and 21
(GM11444) (lane 2), human der(5)t(5;l3)(5qter>5p13::l3q13>l3qter) and human
chromosome 6 (GM11437) (lane 3), human
der(5)t(5;7)(5pter>5q35.2::7q22>7qter) (GM 11446) (lane 4), human
monochromosomal 4 (GM10115) (lane 5), human monochromosomal 7
(GM10791) (lane 6), human monochromosomal 10 (GM10926) (lane 7), human
monochromosomal 12 (GM10868), Chinese hamster (lane 9), and 46, XY human
DNA '(lane 10). A control PCR reaction without template is in lane 11. (From
Liang et al. 1997)

1AA

C. Discussion

Using human/rodent somatic cell hybrids and Southern and PCR analyses,
an 0CP2 gene expressed in the human fetal inner ear maps to 5q22-5q35.2. Other
0CP2-like sequences are located on human chromosomes 4p16.2-4p14, 5p13-
5q22, 7pter-qZZ, 10 and 12pl3-12qter and may comprise an 0CP2 gene family.

It is noteworthy that 0CP2 sequences on chromosomes 4, 5, 7 and 10 are
detected with probes specific to both 5’ coding sequences and 3’-UTR by
Southern blot and PCR, respectively. This suggests that there may be intact copies
of 0CP2 genes at these map positions. We do not know if or when these 0CP2-
like sequences are transcribed and translated. No 0CP2 like sequences map to
human chromosome 17, thus 0CP2 was excluded as a candidate gene for DFNB3

(Liang et al., 1997).

101

' APPENDIX C

Materials and Methods
1. Typing STR markers by Polymerase Chain Reaction (PCR)

PCR fragments were amplified from 50 ng of genomic DNA in 10 pl
reactions containing 0.25 M of each primer, 200 11M dATP, dTI‘P, dGTP, 25
M dCTP, 0.07 ul of 3°P-dC1'P(10mCi/ml), 2 units of thermal stable polymerase,
Boehringer Mannheim’s PCR reaction buffer (10 mM Tris-HCl, pH 8.3; 50 mM
KCl; 1.5 mM MgClz), and 30 11.1 overlay of mineral oil. All genotyping reactions
were performed in a FTC-100 thermocycler (MJ Research).

All PCR amplifications were performed using conditions described above with
one exception. The PCR reaction for D17S2201 (95°C for 1 min, 55°C for l min,
and 72°C for l min for 30 cycles) was performed using a buffer that contained 75 .
mM KCl; 10 mM Tris-HCl; 1.5 mM MgC12; pH 8.8.

For DNA samples isolated from Isocode cards that were difficult to PCR,
the following primers were developed: DI 7S805: forward 5’ GAGGCAGGAGA
ATCAC'I'I‘GAAC 3’ , reverse 5’CCAAATGTGGTGTGTCCI‘AAAC 3 ’;
D1752202: forward 5’ ATC'l'l‘GCTCAGGCTGGTAAAGC 3’, reverse 5’
CCI‘GCAC'ITAAATTCAAATGGTI‘C 3’; D1752201: forward 5’ CAGAC
TGGGTGACAGGGTGAGAC 3 ’ , reverse 5’ AGTGGAGGAGATGGCCA

TGGAG 3’.

102

Genomic DNAs from CEPH individuals 1331-01 and 1331-02 were
always used as standards to size the alleles for the markers been genotyped.
Reactions were performed for 32 cycles at 95°C for 1 minute, 55 °C for 1 minute
and 72°C for 1 minute for each cycle.

Approximately 4 Ill of the reaction were loaded on to 6% denaturing
acrylamide gel and electrophorized at 55 0C for two to five hours depending on
the size of the PCR products. The gels were then dried on 3M blotting paper and

autoradiographed using Kodak X-AR film.

2. Linkage analysis

Linkage analyses were performed using Fastlink version 3.0P (Cottingham
et al. 1993). The frequency and penetrance of DFNB3 were estimated to be
1/ 1,000 and 100%, respectively. Genetic distances are based on CHLC and

Genethon maps (http://www.chlc.org/chlcmarkers.html; Dibb et al. 1996).

3. Isolation of human genomic DNA
Two methods of DNA isolation were used in this study.

Method 1: This method was used for the majority of DNA samples
collected from Bengkala. 20 ml of venous blood was collected from each
individuals and genomic DNA was extracted at the blood bank of Sangla Bali

Hospital, Denpasar, Bali, by Dr. Thomas Friedman and Tom Barber, using

103

reagents from a DNA-Quick Kit (Analytical Genetic Testing Center, Denver,
Colorado).

Method 2: A few drops of blood from a finger puncture from
individuals in the villages of Anturan, Bila, Sinabun, Suwug and Tamblang were
collected on Isocode cards (Schleicher & Schuell). DNA was isolated according
to the manufacturer’s instructions and used as template to amplify markers
DI 7S799, D1 73122, D] 7S26I , D] 75740, D] 78620, D] 7S2202, DI 78783.

A 1/16 inch circle of the Isocode filter paper from which some but not all
DNA was previously extracted according to Schleicher & Schuell was used as
template to amplify markers D17S953, D1 7871 , D1 7S805, DI 7S2201 and
D17S842 since DNA in the supernatant from blood collected on the Isocode card
did not permit amplification of these five markers. Nested PCR was performed
for markers D17S805, D1 7S2202 and D1 732201. None of the five STRs that
required solid state amplification from the Isocode card were problematic when

using DNA isolated from venous blood cells.

4. Southern blot analysis

In each reaction, 5 pg of high molecular weight human genomic DNA was
digested with 40 units of restriction enzyme for two hours. The restricted genomic
DNA samples were electrophorized at 50 volts overnight in 4°C cold room using
0.5 X TBE buffer. A peristaltic pump was used to circulate buffer from the anode

to cathode. The gels were stained in Ethidium Bromide for 30 minutes.

104

DNA sample in the gel were denatured by soaking the gel for 45 minutes
in several volumes of 1.5 M NaCl, 0.5 N NaOH with constant and gentle
agitation. When the fragments of interest are larger than 15 kb, the gels were
soaked for 10 minutes in several volumes of 0.2 N HCl before denaturation. The
gels were rinsed briefly in deionized water, and then neutralized by soaking for 30
minutes in several volumes of a solution of l M Tris (pH 7.4), 1.5 M N aCl at
room temperature with gentle, constant agitation. DNA was capillary transferred
from agarose gel to Nylon membrane over night in 10 X SSC. The Nylon
membranes were rinsed in 6 X SSC briefly and let air dry. DNA was UV cross-
linked to membrane using the “Auto Cross Link” setting on the Stratagene UV
Stratalinker.

The blots were pre-hybridized in 20 ml Hybrisol I (Oncor) at 42 °C for 2
hours. The Rediprime DNA labeling system from Amersham Life Science was
used for probe labeling. Blots were then hybridized in 20 ml fresh Hybrisol I with
1 million CPM probe per ml hybridization solution at 42°C overnight. They were
wash in 0.1 X SSC, 0.5 % SDS at 55 °C for 1 hour, then sealed in a plastic bag
and expose it to Kodak BioMax MS film with Kodak BioMax MS intensifying

screen at -70 °C overnight before the film was developed and the results analyzed.

5. Mutation detection in MY015 by direct sequencing
65 exons of MY015 were PCR amplified using primer pairs synthesized

according to the intronic sequences (Table 10). All reactions were done on

105

Stratagene Robocycler Gradient 96. PCR reactions (except for exon 2 and 51)
were performed in 25 111 using 1 unit of Taq DNA polymerase with 0.4 M of
each primer, 200 11M each of dATP, dTTP, dGTP and dCTP, and 1 x PCR buffer
(10 mM Tris-HCl, pH 8.3; 50 mM

KCl; 1.5 mM MgC12) in a cycle condition of 94°C for 1 minute then 35 cycles

of 94°C for 30 second, 55°C for 30 second and 72°C for 30 second followed by
72°C

for 6 minutes. ‘

MY015 exon 2 and exon 51 are GC rich and have to be amplified using the
CloneTech Advantage GC Genomic Polymerase mix kit (cat. No. 8420-1) and the
following condition: one cycle of 95°C 1 min; 5 cycles of 94°C for 30 seconds,
70°C for 6 minutes; 6 cycles of 94°C for 30 seconds, 68°C for 6 minutes; and 25
cycles of 94°C for 30 seconds, 65°C for 6 minutes.

After amplification, PCR products were purified using Qiaquick 96 PCR
purification kit (cat. No. 28181) and sequenced using Amersham Thermo
Sequenase Radiolabeled Terminator Cycle Sequencing kit (U 879760) following

the manufacturer’s suggested protocols.

6. PCR amplification of part of myosin 15 sequences from monkey, cow,
Chinese hamster, hyena and mouse
Primers YL 900: GTI‘ TGT GTC TGA 'ITA TG and YL903: GGTI‘ CGC

TGT CCA CTC GAG C were synthesized according to the human exon 31 cDNA

106

sequence and used to amplify genomic DNA of Rhesus monkey, cow, Chinese
hamster, hyena and mouse. PCR was performed in Strategene Opti-Prime 10X
PCR Buffer #1(cat. No. 200423) for 35 cycles of 95°C for 30 seconds, 45°C for
30 seconds and 72°C for 30 seconds. PCR products were then gel purified and

sequenced according to protocols mentioned above.

107

1'11

0

£110.09”?

In Situ Hybridization (a protocol from Dr. Susan Sullivan)
Tissue Preparation
Slide Preparation and Tissue Sectioning

Prehybridization

. Preparation of Probe

Hybridization

Washing

. Autoradiography

. List of Reagents

108

A. Tissue Preparation:

1. Dissect in cold 1x PBS (or 4% paraformaldehyde in 1x PBS)

2. Fix the tissue overnight at 4 °C in 4% paraformaldehyde! 1x PBS
4% Paraforrnaldehyde/ 1x PBS
weigh 4.0 g of paraformaldehyde
add sterile H20 to a volume of 50.0 ml
add 10.0 [.11 of 10 N NaOH to the solution
heat the solution to 65°C and mix until crystals dissolve
filter through Whatrnan filter paper to remove particulates
add 10.0 ml 10x PBS
adjust the volume to 100 ml
The solution can be stored at 4 °C and used for up to 24 hours.

3. Dehydrate the tissue through several changes of solutions

a. 1x PBS 4°C 60 minutes
b. 0.85% NaCl 4°C 60 minutes
c. 50% ETOH/0.425% NaCl RT 30 minutes
(1. 70% ETOH (2x) RT 30 minutes
e. 85% ETOH RT 60 minutes
f. 95% ETOH RT 60 minutes
g. 100% ETOH (2x) RT 60 minutes
h. Xylene (3x) RT 30 minutes

109

1. 1:1 Xylenelparafin 60°C 45 minutes

j. Paraf'm (3x) 60°C 20 minutes

110

B. Slide Preparation and Tissue Sectioning:

1. Slide Preparation:

Slides must be free of grease and gelatin coated. The slides used for this

procedure were Corning Single Frosted Micro Slides cat# 2948 (75mm x 25mm x

1mm)

a.

Place slides in metal racks and soak in hot tap water with detergent (Alconox).
Keep the original slide boxes.

Rinse the slides for 1 hour under running tap water.

Rinse the slides under running distilled water for 15 minutes or longer

Air dry or place in an oven at 40°C for 1 hour

Boil 1 liter of RNase free water. Keep water stirring.

When the temperature drops to 60°C, add 2 g of Gelatin type A (300 bloom;
Sigma cat# G-2500)

When the temperature reaches 50°C, add 0.1 g potassium Chrome [[1 Sulfate
KCr(III)SO4 (EM Science cat# CX1605-2)

Allow the solution to cool to 35°C

Dip each rack in the gelatin solution for 1 minute then allow to dry on its side
with the frosted portion down for 5 minutes.

Repeat this procedure then dry the slides overnight at 35°C.

Store the slides in the original boxes.

2. Tissue Sectioning:

111

. Make sure the microtome blade is clean and that the cutting edge is in good
shape.

. Securely fasten the parafm block in the jaws of the microtome.

. Align the face of the block to get as close to a parallel cut as is possible.

. Trim the block so that there is a trapazoid shape around the tissue with the
wide edge closest to the blade.

. Prelabel slides in sets of at least 4 for alternating sections

. Preheat the slides on a warming tray to approximately 37°C

. Cover the clear area of the slide with a 0.2% ethanol solution

. Cut sections to a thickness of 10p.

Float the sections on the ethanol solution and allow them to flatten out.
Allow the liquid to evaporate and leave the sections to dry overnight on the
warming tray.

. Place the slides in clean slide boxes and store until ready to use.

112

C. Prehybridization:

1. Prepare the solutions in advance. Remember that everything has to be RN ase

free.
a. 4% paraformaldehyde! 1x PBS (made the same day as it is used) 800
ml
b. 1x PBS 2-3 liters
c. 0.85% NaCl 2-3 liters
d. Proteinase K
e. PK Buffer 200 ml
10mM Tris pH 7.5
SmM EDTA pH 8.0

f. 0.1 M triethanolamine (6.7 mll500ml H20) 500 ml/slide rack
g. Acetic Anhydride

h. Ethanol Solutions

30% 400 ml
50% 400 ml
70% 400 ml
85% 400 ml
95% 400 ml
100% 600 ml

Xylene ‘ 400 ml/2 slide racks

113

The solutions are set up so that racks can be processed sequentially. It is
important to have enough containers to hold all the solutions because this process
will take more than 2 hours for each rack of slides. The process will go faster if
multiple racks can be processed at the same time. The bottleneck in this process is
the 30 minute incubation time in paraformaldehyde which is performed twice.

2. Process the tissue sections

Start in the fume hood:

Xylene #1 10 minutes
Xylene #2 10 minutes
100% Ethanol 2 minutes
Move to a lab bench

100% Ethanol 2 minutes
95% Ethanol 2 minutes

85% Ethanol 2 minutes

70% Ethanol 2 minutes

50% Ethanol 2 minutes

30% Ethanol 2 minutes
0.85% NaCl 5 minutes
1xPBS 5 minutes

4% Paraformaldehyde 30 minutes
1xPBS 5 minutes

1xPBS 5 minutes

114

Proteinase K 7.5 minutes

1xPBS 5 minutes

4% Paraformaldehyde 30 minutes

H20 Dip

0.1 M TriethanolaminelO minutes with stirring
1xPBS (fresh) 5 minutes

0.85 % NaCl (fresh) 5 minutes

Dehydrate the slides through ethanol

30% ethanol 2 minutes
50% ethanol 2 minutes
70% ethanol 2 minutes
85% ethanol 2 minutes
95 % ethanol 2 minutes
100% ethanol 2 minutes
100% ethanol 2 minutes

Air dry the slides for 30 minutes. Slides may be used immediately, or stored at

room temperature for several weeks.

115

D. Probe Preparation:

The probes for in-situ hybridization are riboprobes that are synthesized
from a linearized plasmid template from the T7, T3 or SP6 RNA polymerase
promotors. The template is linearized with an appropriate restriction endonuclease
then treated with Proteinase K and phenol-chloroform extraction to ensure that the
DNA is RNase free. The probes are continuously labeled with 3SS-UTP or a
combination of 35S-CI'P and 35S-UTP. The isotope is dried in advance of the
reactions and resuspended in the reagents from the riboprobe synthesis kit.

1. Template Preparation:
a) Digest the plasmid with an appropriate restriction endonuclease.
Incubate the reaction long enough to ensure complete digestion
b) Proteinase K treatment
to 50 111 DNA add:
20 pl PK buffer
5 111 10% SDS
2 pl PK (20 mg/ml)
123 11.1 dH20
incubate at 37 °C for 1 hour
c) Phenol-Chloroform extract
(1) Ethanol precipitate

e) Resuspend in dHZO and adjust concentration to 14 ng/ pl

116

2. Probe Labeling:

Labeling can be performed with either 35 S or 33P UTP. Standard reaction
will use 2.5 111 of labeled nucleotide or 7 .5 [1.1 which has been dried down. High
specificity probes can be synthesized by using a double label of 35 S ATP and
UTP 7.5 ul of each (3000 pCi/ml) which have been dried down.

Labeling is performed with a Stratagene RNA Synthesis Kit (cat# 200340).
A reaction cocktail is assembled from Kit reagents with the exception of the
enzyme. If a double lable is used, replace the unlabeled nucleotide with dHZO.
Rnasin is available from Promega. RNase Block from Stratagene may be
substituted.

a. Reaction Mix (1x)

5x Buffer 1.0 Ill

0.1 M DTT 0.5 111

rCI'P 0.25 111

rGTP 0.25 111

rATP 0.25 111

Rnasin 0.25 111 (20 U/ul)

b. Resuspend isotope in 2.5 m1 of reaction mix
c. Add 2.15 1.11 of the linearized template (30 ng)

(1. Add 0.35 11.1 T7/T3/SP6 Polymerase (depends on the vector used)

e. Incubate at 37°C for 1 hour (in air incubator to avoid evaporation)

117

 

f. Add an additional 1.0 pl polymerase and incubate at 37°C for at least

30 minutes
g. Add 0.5 pl Dnase RQl and incubate at 37 °C for 15 minutes
h. Add 3.0 pl of Yeast RNA (10 mg/ml)
i. Bring the volume to 50.0 pl with dH20

j. Add 50.0 [.11 of hydrolysis solution:

1 M DTI‘ 10.0 111
1 M NaHCO3 80.0 111
1 M Na2CO3 120.0 pl
H20 790.0 111

k. Digest at 60°C by the following criterion:

_Siz_e_(l_)p) Time (min)
300 15
400 20
600 25
800 45
1300 60

1. Add 50 pl of neutralization buffer:
1M DTT 10.0 p1
1M NaAc 200.0 pl

Acetic Acid 10.0 pl

118

H20 780.0 pl
4. Probe Purification:

Prepare a slurry of G50 Sephadex

a. Swell a mixture of 5.0 g each of medium and of fine G50 in 160
ml
RNase Free dHZO.

b. Spin down resin in the clinical centrifuge.

c. Wash 3x in RNase Free dH20.

d. Equilibrate the resin in 160 ml 1x TE pH 7.5

Prepare a Saturated Phenol Red Solution in RNase Free dHZO.

Prepare fresh running buffer:
1 M Tris pH 7.5 0.5 ml

0.5 M EDTA pH 8.0 0.5 ml

10% SDS 0.5 ml
1 M DTT 0.5 ml
RNase Free deO 48.0 ml

The DTT is added just prior to use.
G50 Columns:
a. Break the tip off of a Pasteur pipette. The tip should be broken where

the glass wools into the narrow tip.

119

 

b. Plug the pipette with glass wool. Do not ball the glass wool or pack it
too

tight. Use a long Pasteur pipette to push the glass wool into bottom of

the
column.

0. Pass 500 pl of running buffer down the column to wet the glass

surfaces.

(1. With an individually wrapped sterile transfer pipette, load GSO resin
into the
column until there is a head space of a little more than 500 pl.

e. Pass 2 x 500 pl of running buffer through the column.

f. Add 50 pl of Yeast RNA (10 mglml)to the column

g. Run 500 p1 of running buffer through the column.

b. Plug the column with dental wax (Parafilrn works well; make sure it
has not
been used for other common lab activities)

i. Add 2.0 pl saturated Phenol Red to the labeled probe.

i. Remove the seal from the column and slowly add the probe to the

column. Do

not disturb the resin bed.

120

k. When the resin becomes visible through the red dye, add 200 pl running

buffer to the column.

m. Use a survey meter to monitor the activity coming off the end of the

section).

column.

When the counts increase, switch to the collection tube.

Collect eluent until the red dye reaches the bottom of the Be_sig_(not the
glass

wool).

Add 1/10th volume 3M Kac and 2x volume 100% ethanol. Vortex and
precipitate at -70°C overnight.

Spin 30 minutes at 4°C.

Resuspend in 100 pl 100 mM DTT. Resuspend very carefully and pool
into a

single tube for each probe.

Count 1.0 pl in the scintilation counter.

Dilute to 25,000 to 50,000 CPM/ pl in hybridization buffer (see next

121

D. Hybridization:

There are a number of reagents that need to be prepared in advance for the

hybridization procedure. Coverslips must be silanized prior to starting this step.

These can be done well in advance and stored at room temperature indefinitely.

The reagents for the hybridization buffer must be on hand well before starting.

1. Coverslips:

a. Prepare a work area in the fume hood by fan folding a piece of Whatrnan

paper to act as a drying rack. This needs to hold between 50 and 120

cover slips (1 or 2 boxes).

b. Dip the cover slips individually into 5% Silane in Chloroform.

c. Let slips air dry on the Whatrnan paper rack. Make sure the slides do not

overlap.

(1. Dip cover slips into 5% Silane in Chloroform

e. Dip in 100% ethanol 2x (separate containers of ethanol)

f. Allow the slips to air dry on the Whatrnan paper rack.

g. Store cover slips in the original boxes.

2. Hybridization Solution:

Deionized formamide 15 ml
4 M NaCl 2.25 ml
1 M Tris pH 7.5 0.600 ml

0.5 M EDTA pH 8.0 0.300 ml

1 M Na112P04 0.300 ml

122

50.0%

0.3 M

20mM

SmM

10mM

50% Dextran Sulfate 6.0 ml 10.0%
50x Denhardt’s 0.600 ml 1 x
10 mg/ml Yeast RNA 1.5 ml 0.5 mg/ml
Adjust the solution to 10 mM DTT with a 1M stock solution prior to
adding the probe. This represents 9/10 of the final solution. The last 1/10th is made
up of the probe plus water.
3. Hybridization:
a. Dilute probe to 25,000 to 50,000 CPM/pl in enough quantity to apply
80-100 pl of hybridization mix to each slide.
b. Heat the hybridization mix to 80°C for 2 minutes
c. Vortex to mix thoroughly.
(1. Carefully add solution to the slide
e. Place cover slip over tissue. Put one end of slip down and carefully
lower the other end.
f. Place slides horizontally in slide boxes with the cover slips facing up
g. Make sure there is a paper towel soaked in 10 ml humidity buffer (50%
formamide/4x SSC) in each slide box.
h. Seal boxes with electrical tape. Sequencing gel sealing tape will work as
well. Make sure the tape forms a good seal.
i. Incubate the slides at 52°C for 16 hours with the boxes standing on end

to keep the slides horizontal.

123

F. Washing:

Three water baths will be needed for this procedure. They need to be
turned on and equilibrated to 50°C, 65°C, and 37°C well before you are ready to
start. The washing solutions must be prepared in advance without DTT and
equilibrated to the designated temperatures before beginning the procedure.
Weigh out the DTT and have it ready in individual containers. DTT is labile and
should be added just prior to the incubation. Fresh DTI‘ is added for each rack of
slides, and each solution is used for no more that 2 racks of slides.

1. First Wash:

5x SSC/10 mM DTT 50°C 30 minutes

a. Equilibrate the 5x SSC solution at 50°C.

b. Add 0.32 g DTT / 200 ml solution (have extra prepared)

c. In a separate container, soak the cover slips off of the slides in 5x
SSC/10mM DTT at 50°C. The cover slips may fall off, or they can be
carefully loosened with a razor blade. Do Not force the cover slip off; it
may disturb the tissue.

d. Place the slides in a rack in 200 ml of wash solution.

e. Start timing for 30 minutes when the rack is full.

2. Second Wash:
50% Formamide/2x SSC/ 10 mM DTT 65°C 20 minutes

a. Equilibrate 50% Formamide/Zx SSC at 65°C

124

b. Add 0.32 g DTT just before adding the rack of slides
c. Incubate for 20 minutes
3. Washing Solution:
10x Stock:
NaCl 233.8 g
l M Tris pH 7.5 100 ml
0.5 M EDTA pH 8.0100 ml
H20 to 1000 ml
a. Make at least 2 liters of 1x washing solution
b. Equilibrate the solution at 37 °C
c. Wash the slides for 10 minutes at 37°C
(1. Transfer to a new container of 1x wash solution and wash for 10 minutes
at 37°C
4. RNase A Treatment
a. Add 400 pl of a 10 mg/ml stock of RNase A to 200 ml of 1x washing

solution at 37 °C.

b. Incubate the slides for 30 minutes at 37°C

5. 1x Washing solution 5 minutes 37°C
6. 2x SSC 15 minutes 37°C
7. 0.1x SSC 15 minutes 37°C

8. Dehydrate through increasing concentrations of ethanol solutions

125

a. 30% ethanol/0.3M NH4Ac
b. 60% ethanol/0.3M NH4Ac
c. 80% ethanol/0.3M We
(1. 95% ethanol/0.3M NH4Ac

e. 100% ethanol

2 minutes

2 minutes

2 minutes

2 minutes

2 minutes RT

126

RT

RT

RT

RT

G. Autoradiography:

This procedure must be performed in the darln-oom under extremely
stringent conditions. No extraneous light can be allowed into the room. Leave all
glow in the dark and illuminated watches and timers outside. The only light
source allowed is a 15 watt bulb with a Kodak #2 Safelight filter.

1. Warm the Kodak NTB-2 emulsion in a 45°C water bath in the dark room. The

emulsion is stored at 4°C and is gelatinous at room temperature.

2. Prepare a container in which the slides will be dipped. A Coplin Jar with a
block of clean glass slides to take up the excess space works well. The idea is
to have enough emulsion to coat the entire clear portion of the slide without
wasting a lot of the emulsion.

3. Prepare slide boxes with Dri-Rite “cigars” (Dri-Rite wrapped in paper to absorb
moisture).

3. Pour the warmed emulsion into the dipping container. Pass a clean slide
through the emulsion to remove any air bubbles.

4. Dip the slides into the emulsion one at a time, and allow the excess to drip back
into the container. Refill the container with emulsion as needed.

5. Place the slides in a clean slide box and allow them to dry for at least 1 hour.

6. Carefully place the slides into clean slide boxes with a Dri-Rite Cigar and triple
wrap them in aluminum foil.

7. Place the Slide boxes at 4°C where they will not be subjected to shocks.

127

8. Before developing, allow the boxes to warm to room temperature.
9. Develop with Kodak D-l9 and fix with Rapid Fix
a. D-19 3.5 minutes 16°C
31.3 g D-l9 powder/ 200 ml H2O
add powder to stirring 52°C H2O
use below 16°C; store 4°C in brown or foil covered glass
make fresh weekly or after 34 uses
b. H20 Dip
c. Fix 5 minutes RT
35.8 g fixer powder/ 200 ml H20
add powder to stirring RT H2O
store at RT in brown or foil covered bottle

can be used for up to 1 month

(1. Fix 5 minutes RT
e. H20 (cold) 5 changes RT
f. Hoechts 5 minutes RT (200 pl 0.5% / 200ml)
g. H20 (cold) 5 changes RT

10. Dehydrate through a series of ethanol solutions for 2 minutes each.
50%, 70%, 85%, 95%, 100%, 100%.

ll. Dip in Xylene twice 10 minutes each

128

12. Apply cover slips with Cryoseal Mounting Media 60 (VWR cat. # 48212-154)

and allow to dry while laying fiat.

129

H. Reagents and Suppliers:

Reagent

Acetic Anhydride
Ammonium Acetate

Beeswax Impression Wax
24x60 Cover Glass (Corning)
Cytoseal Mounting Media

50x Denhardt’s
Dextran Sulfate

1,4 Dithiothreitol (DTI‘)

Formamide

Gelatin Type A (300 bloom)

Kodak D-l9
Kodak Fixer

Micro Slide, Frosted (Corning)

NTB-2
Paraformaldehyde
Paraplast

Phenol Red
KCr(III)SO4
Proteinase K

RNase A

RQl RNase free Dnase

Sephadex G-50 Medium

Sephadex G-50 Fine

Silane (Dichlorodimethylsilane)

Siliconized Glass Wool
Triethanolamine
Yeast RNA

Supplier

Sigma

Sigma

The Hygenic Company
VWR

VWR

Sigma

Intergen Company
Boehringer-Mannheim
Fluka

Sigma

Eastman Kodak
Eastman Kodak

VWR

Eastman Kodak

J .T. Baxter

VWR

Sigma

E.M. Science
Boehringer-Mannheim
Boehringer-Mannheim
Promega

Amersham Pharmacia
Biotech, Inc.
Amersham Pharmacia
Biotech, Inc.
Mallinckrodt OR
Supelco

Sigma
Boehringer-Mannheim

130

Catalog #

A-6404

A- 1542
00833
48396- 160
48212- 154
D2532
S403 1

197 777
47670
G-2500
146-4593
197-1746
483 12-036
165-4433
S 898-07
15 159-409
P-4758
CX1605-2
745-723
109-169
M6101
17-0043-01

17-0042-01

75-78-5
2-041 1M
T-1377
109 223

APPENDD( D: Table of primers used to PCR amplify the 65 exons of MY015

I1
12

11/1
1

A 0211
W1
AW1
AW1

AW170I171

174/1
1

AW182/1
W1

AW188/1

AW188I1
1

1
AW196/197
W1
A
A
AW206/207

AW210/211
A 12/21
AW214/21
AW21 17
A 1

A

A

AW238/239 MWACCCCTATAAGC
AW2 1 AMAACGAGGTGCCTI’CT

 

131

Table 10 (cont’d)

A

A

AW248/249
1

A

0
1
LMGZ13I214
5/21

LM6221l222

 

132

BIBLOGRAPHY

133

APPENDIX E

List of references

Altschul, S. F., Boguski, M. S., Gish, W., and Wootton, J. C. (1994). Issues in
searching molecular sequence databases. Nat Genet 6, 119-29.

Altschul, S. F., and Gish, W. (1996). Local alignment statistics. Methods
Enzymo1266, 460-80.

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990).
Basic local alignment search tool. J Mol Biol 215, 403-10.

Bai, C., and Elledge, S. J. (1997). Gene identification using the yeast two-hybrid
system. Methods Enzym01283, 141-56.

Beeson, D., Jeremiah, S., West, L. F., Povey, S., and Newsom-Davis, J. (1990).
Assignment of the human nicotinic acetylcholine receptor genes: the alpha and

delta subunit genes to chromosome 2 and the beta subunit gene to chromosome
17. Ann Hum Genet 54, 199-208.

Bloch, K. D., Wolfram, J. R., Brown, D. M., Roberts, J. D., Jr., Zapol, D. G.,
Lepore, J. J., Filippov, G., Thomas, J. E., Jacob, H. J ., and Bloch, D. B. (1995).
Three members of the nitric oxide synthase II gene family (N 082A, NOS2B, and
NOSZC) colocalize to human chromosome 17. Genomics 27, 526-30.

Bowcock, A. M., Ruiz-Linares, A., Tomfohrde, J., Minch, E., Kidd, J. R., and
Cavalli-Sforza, L. L. (1994). High resolution of human evolutionary trees with
polymorphic microsatellites. Nature 368, 455-7.

Brown, K. A., Janjua, A. H., Karbani, G., Parry, G., Noble, A., Crockford, 6.,
Bishop, D. T., Newton, V. E., Markham, A. F., and Mueller, R. F. (1996).
Linkage studies of non-syndromic recessive deafness (NSRD) in a family
originating from the Mirpur region of Pakistan maps DFNBI centromeric to
D13S175 [published erratum appears in Hum Mol Genet 1996 May;5(5):710].
Hum Mol Genet 5, 169-73.

Brownstein, Z., Friedlander, Y., Peritz, E., and Cohen, T. (1991). Estimated
number of loci for autosomal recessive severe nerve deafness within the Israeli

Jewish population, with implications for genetic counseling. Am J Med Genet 41,
306-12.

134

Budowle, B., and Allen, R. C. (1998). Analysis of amplified fragment-length
polymorphisms (VNTR/STR loci) for human identity testing. Methods Mol Biol
98, 155-71.

Budowle, B., Giusti, A. M., Waye, J. S., Baechtel, F. S., Foumey, R. M., Adams,
D. B., Presley, L. A., Deadman, H. A., and Monson, K. L. (1991). Fixed-bin
analysis for statistical evaluation of continuous distributions of allelic data from
VNTR loci, for use in forensic comparisons. Am J Hum Genet 48, 841-55.

Buetow, K. H., Weber, J. L., Ludwigsen, S., Scherpbier-Heddema, T., Duyk, G.
M., Sheffield, V. C., Wang, Z., and Murray, J. C. (1994). Integrated human
genome-wide maps constructed using the CEPH reference panel. Nat Genet 6,

391-3.

Burge, C., and Karlin, S. (1997). Prediction of complete gene structures in human
genomic DNA. J Mol Biol 268, 78-94.

Burge, C. B., and Karlin, S. (1998). Finding the genes in genomic DNA. Curr
Opin Struct Biol 8, 346-54.

Butler, J. M. (1998). The use of capillary electrophoresis in genotyping STR loci.
Methods Mol Biol 98, 279-89.

Campbell, D. A., McHale, D. P., Brown, K. A., Moynihan, L. M., Houseman, M.,
Karbani, G., Parry, G., Janjua, A. H., Newton, V., al-Gazali, L., Markham, A. F.,
Lench, N. J ., and Mueller, R. F. (1997). A new locus for non-syndromal,
autosomal recessive, sensorineural hearing loss (DFNB16) maps to human
chromosome 15q2l-q22. J Med Genet 34, 1015-7.

Campbell, H. D., Fountain, 8., Young, 1. G., Claudianos, C., Hoheisel, J. D.,
Chen, K. S., and Lupski, J. R. (1997). Genomic structure, evolution, and
expression of human FLII, a gelsolin and leucine-rich-repeat family member:
overlap with LLGL. Genomics 42, 46-54.

Chance, P. F., Alderson, M. K., Leppig, K. A., Lensch, M. W., Matsunami, N.,
Smith, B., Swanson, P. D., Odelberg, S. J ., Disteche, C. M., and Bird, T. D.
(1993). DNA deletion associated with hereditary neuropathy with liability to
pressure palsies. Cell 72, 143-51.

Chen, H., Thalmann, 1., Adams, J. C., Avraham, K. B., Copeland, N. G., Jenkins,

N. A., Beier, D. R., Corey, D. P., Thalmann, R., and Duyk, G. M. (1995). cDNA
cloning, tissue distribution, and chromosomal localization of Ocp2, a gene

135

encoding a putative transcription-associated factor predominantly expressed in the
auditory organs. Genomics 27, 389-98.

Chen, K. S., Gunaratne, P. H., Hoheisel, J. D., Young, I. G., Miklos, G. L.,
Greenberg, F., Shaffer, L. G., Campbell, H. D., and Lupski, J. R. (1995). The
human homologue of the Drosophila melanogaster flightless-I gene (flil) maps
within the Smith-Magenis microdeletion critical region in 17p11.2. Am J Hum
Genet 56, 175-82.

Chen, K. S., Manian, P., Koeuth, T., Potocki, L., Zhao, Q., Chinault, A. C., Lee,
C. C., and Lupski, J. R. (1997). Homologous recombination of a flanking repeat

gene cluster is a mechanism for a common contiguous gene deletion syndrome.
Nat Genet 1 7, 154-63.

Cheney, R. B., and Mooseker, M. S. (1992). Unconventional myosins. Curr Opin
Cell Biol 4, 27-35.

Chevillard, C., Le Paslier, D., Passage, B., Ougen, P., Billault, A., Boyer, S.,
Mazan, S., Bachellerie, J. P., Vignal, A., Cohen, D., and et al. (1993).
Relationship between Charcot-Marie-Tooth 1A and Smith-Magenis regions. an3
may be a candidate gene for the Smith-Magenis syndrome. Hum Mol Genet 2,
1235-43.

Chung, C. S., and Brown, K. S. (1970). Family studies of early childhood
deafness ascertained through the Clarke School for the Deaf. Am J Hum Genet
22, 630-44.

Cohn, E. S., Kelley, P. M., Fowler, T. W., Gorga, M. P., Lefkowitz, D. M.,
Kuehn, H. J., Schaefer, G. B., Gobar, L. S., Hahn, F. J ., Harris, D. J., and
Kimberling, W. J. (1999). Clinical studies of families with hearing loss
attributable to mutations in the Connexin 26 gene (GJB2/DFNB1). Pediatrics 103,
546-550.

Cope, M. J. T., Whisstock, J ., Rayment, I., and Kendrick-Jones, J. (1996).
Conservation within the myosin motor domain: implications for structure and
function. Structure 4, 969-87.

Davis, A. C. (1989). The prevalence of hearing impairment and reported hearing
disability among adults in Great Britain. Int J Epidemiol 18, 911-7.

De Laurenzi, V., Rogers, G. R., Hamrock, D. J ., Marekov, L. N., Steinert, P. M.,
Compton, J. G., Markova, N., and Rizzo, W. B. (1996). Sjogren-Larsson

136

syndrome is caused by mutations in the fatty aldehyde dehydrogenase gene. Nat
Genet 12, 52-7.

Desmarais, D., Zhong, Y., Chakraborty, R., Perreault, C., and Busque, L. (1998).
Development of a highly polymorphic STR marker for identity testing purposes at
the human androgen receptor gene (HUMARA). J Forensic Sci 43, 1046-9.

Dib, C., Faure, S., Fizames, C., Samson, D., Drouot, N., Vignal, A., Millasseau,
P., Marc, S., Hazan, J., Seboun, E., Lathrop, M., Gyapay, G., Morissette, J ., and
Weissenbach, J. (1996). A comprehensive genetic map of the human genome
based on 5,264 microsatellites. Nature 380, 152-4.

Edwards, A., Hammond, H. A., Jin, L., Caskey, C. T., and Chakraborty, R.
(1992). Genetic variation at five trimeric and tetrameric tandem repeat loci in four
human population groups. Genomics 12, 241-53.

Elsea, S. H., Fritz, E., Schoener-Scott, R., Meyn, M. S., and Patel, P. I. (1998).
Gene for topoisomerase [[1 maps within the Smith-Magenis syndrome critical
region: analysis of cell-cycle distribution and radiation sensitivity. Am J Med
Genet 75, 104-8.

Elsea, S. H., Juyal, R. C., Jiralerspong, S., Finucane, B. M., Pandolfo, M.,
Greenberg, F., Baldini, A., Stover, P., and Patel, P. I. (1995). Haploinsufficiency

of cytosolic serine hydroxymethyltransferase in the Smith-Magenis syndrome.
Am J Hum Genet 57, 1342-50.

Elsea, S. H., Purandare, S. M., Adell, R. A., Juyal, R. C., Davis, J. G., Finucane,
B., Magenis, R. B., and Patel, P. I. (1997). Definition of the critical interval for
Smith-Magenis syndrome. Cytogenet Cell Genet 79, 276-81.

Entrala, C., Lorente, M., Lorente, J. A., Alvarez, J. C., Moretti, T., Budowle, B.,
and Villanueva, E. (1998). Fluorescent multiplex analysis of nine STR loci:
Spanish population data. Forensic Sci Int 98, 179-83.

Espreafico, E. M., Cheney, R. B., Matteoli, M., Nascimento, A. A., De Carnilli, P.
V., Larson, R. E., and Mooseker, M. S. (1992). Primary structure and cellular
localization of chicken brain myosin-V (p190), an unconventional myosin with
calmodan light chains. J Cell Biol 119, 1541-57.

Fregeau, C. J ., and Foumey, R. M. (1993). DNA typing with fluorescently tagged
short tandem repeats: a sensitive and accurate approach to human identification.
Biotechniques 15, 100-19.

137

Friedman, T. B., Liang, Y., Weber, J. L., Hinnant, J. T., Barber, T. D., Winata, S.,
Arhya, I. N., and Asher, J. H., Jr. (1995). A gene for congenital, recessive
deafness DFNB3 maps to the pericentromeric region of chromosome 17 . Nat
Genet 9, 86-91.

Gasparini, P., Estivill, X., Volpini, V., Totaro, A., Castellvi-Bel, S., Govea, N.,
Mila, M., Della Monica, M., Ventruto, V., De Benedetto, M., Stanziale, P.,
Zelante, L., Mansfield, E. S., Sandkuijl, L., Surrey, S., and Fortina, P. (1997).
Linkage of DFNBI to non-syndromic neurosensory autosomal-recessive deafness
in Mediterranean families. Eur J Hum Genet 5, 83—8.

Gibson, F., Walsh, J., Mburu, P., Varela, A., Brown, K. A., Antonio, M., Beisel,
K. W., Steel, K. P., and Brown, S. D. (1995). A type VII myosin encoded by the
mouse deafness gene shaker-1. Nature 374, 62-4.

Gill, P., Ivanov, P. L., Kimpton, C., Piercy, R., Benson, N., Tully, G., Evett, I.,
Hagelberg, E., and Sullivan, K. (1994). Identification of the remains of the
Romanov family by DNA analysis. Nat Genet 6, 130-5.

Greco, T. L., Takada, 8., Newhouse, M. M., McMahon, J. A., McMahon, A. P.,
and Camper, S. A. (1996). Analysis of the vestigial tail mutation demonstrates

that Wnt-3a gene dosage regulates mouse axial development. Genes Dev 10, 313-
24.

Greenberg, F., Guzzetta, V., Montes de Oca-Luna, R., Magenis, R. B., Smith, A.

C., Richter, S. F., Kondo, I., Dobyns, W. B., Patel, P. I., and Lupski, J. R. (1991).
Molecular analysis of the Smith-Magenis syndrome: a possible contiguous- gene
syndrome associated with del(17)(p11.2). Am J Hum Genet 49, 1207-18.

Greenberg, F., Lewis, R. A., Potocki, L., Glaze, D., Parke, J ., Killian, J ., Murphy,
M. A., Williamson, D., Brown, R, Dutton, R., McCluggage, C., Friedman, E.,
Sulek, M., and Lupski, J. R. (1996). Multi-disciplinary clinical study of Smith-
Magenis syndrome (deletion 17pl 1.2). Am J Med Genet 62, 247-54.

Guilford, P., Ayadi, H., Blanchard, S., Chaib, H., Le Paslier, D., Weissenbach, J.,
Drira, M., and Petit, C. (1994). A human gene responsible for neurosensory, non-

syndromic recessive deafness is a candidate homologue of the mouse sh-l gene.
Hum Mol Genet 3, 989-93.

Guilford, P., Ben Arab, S., Blanchard, S., Levilliers, J., Weissenbach, J.,
Belkahia, A., and Petit, C. (1994). A non-syndrome form of neurosensory,

recessive deafness maps to the pericentromeric region of chromosome 13q. Nat
Genet 6, 24-8.

138

Gyapay, G, Morissette, J., Vignal, A., Dib, C., Fizames, C., Millasseau, P., Marc,
S., Bemardi, G., Lathrop, M., and Weissenbach, J. (1994). The 1993-94 Genethon
human genetic linkage map. Nat Genet 7, 246-339.

Harter, C., Ripoll, C., Lenoir, M., Hamel, C. P., and Rebillard, G. (1999).
Expression pattern of mammalian cochlea outer hair cell (OHC) mRNA:
screening of a rat OHC cDNA library. DNA Cell Biol 18, 1-10.

Hasson, T., Gillespie, P. G., Garcia, J. A., MacDonald, R. B., Zhao, Y., Yee, A.
G., Mooseker, M. S., and Corey, D. P. (1997). Unconventional myosins in inner-
ear sensory epithelia. J Cell Biol 137, 1287-307.

Hasson, T., Heintzehnan, M. B., Santos-Sacchi, J ., Corey, D. P., and Mooseker,
M. S. (1995). Expression in cochlea and retina of myosin VIIa, the gene product
defective in Usher syndrome type 1B. Proc Natl Acad Sci U S A 92, 9815-9.

Hasson, T., and Mooseker, M. S. (1997 ). The growing family of myosin motors
and their role in neurons and sensory cells. Curr Opin Neurobiol 7, 615-23.

Hasson, T., and Mooseker, M. S. (1995). Molecular motors, membrane
movements and physiology: emerging roles for myosins. Curr Opin Cell Biol 7,
587-94.

Hasson, T., and Mooseker, M. S. (1994). Porcine myosin-VI: characterization of a
new mammalian unconventional myosin. J Cell Biol 127, 425-40.

Hasson, T., and Mooseker, M. S. (1996). Vertebrate unconventional myosins. J
Biol Chem 271, 16434.

Hasson, T., Skowron, J. F., Gilbert, D. J., Avraham, K. B., Perry, W. L., Bement,
W. M., Anderson, B. L., Sherr, E. H., Chen, Z. Y., Greene, L. A., Ward, D. C.,
Corey, D. P., Mooseker, M. S., Copeland, N. G., and Jenkins, N. A. (1996).
Mapping of unconventional myosins in mouse and human. Genomics 36, 431-9.

Heintzelman, M. B., Hasson, T., and Mooseker, M. S. (1994). Multiple
unconventional myosin domains of the intestinal brush border cytoskeleton. J Cell
Sci 107, 3535-43.

Houdusse, A., and Cohen, C. (1996). Structure of the regulatory domain of
scallop myosin at 2 A resolution: implications for regulation. Structure 4, 21-32.

139

Houdusse, A., and Cohen, C. (1995). Target sequence recognition by the
calmodulin superfamily: implications from light chain binding to the regulatory
domain of scallop myosin. Proc Natl Acad Sci U S A 92, 10644-7.

Houdusse, A., Love, M. L., Dominguez, R., Grabarek, Z., and Cohen, C. (1997).
Structures of four Ca2,-bound troponin C at 2.0 A resolution: further insights into
the Ca2,-switch in the calmodulin superfamily. Structure 5, 1695-711.

Houdusse, A., Silver, M., and Cohen, C. (1996). A model of Ca(2,)-free
calmoduhn binding to unconventional myosins reveals how calmodan acts as a
regulatory switch. Structure 4, 1475-90.

Hua, X., Wu, J ., Goldstein, J. L., Brown, M. S., and Hobbs, H. H. (1995).
Structure of the human gene encoding sterol regulatory element binding protein-1
(SREBFI) and localization of SREBFl and SREBF2 to chromosomes 17pl 1.2
and 22q13. Genomics 25, 667-73.

Huang, J. D., Brady, S. T., Richards, B. W., Stenolen, D., Resau, J. H., Copeland,
N. G., and Jenkins, N. A. (1999). Direct interaction of microtubule- and actin-
based transport motors. Nature 397, 267-70.

Hudspeth, A. J., and Gillespie, P. G. (1994). Pulling springs to tune transduction:
adaptation by hair cells. Neuron 12, 1-9.

Hugnot, J. P., Pedeutour, F., Le Calvez, C., Grosgeorge, J ., Passage, B., Fontes,
M., and Lazdunski, M. (1997). The human inward rectifying K+ channel Kir 2.2
(KCNJ 12) gene: gene structure, assignment to chromosome 17p11.1, and
identification of a simple tandem repeat polymorphism. Genomics 39, 113-6.

Jain, P. K., Fukushima, K., Deshmukh, D., Ramesh, A., Thomas, B., Lalwani, A.
K., Kumar, S., Plopis, B., Skarka, H., Srisailapathy, C. R., and et al. (1995). A
human recessive neurosensory nonsyndromic hearing impairment locus is
potential homologue of murine deafness (dn) locus. Hum Mol Genet 4, 2391-4.

Juyal, R. C., Greenberg, F., Mengden, G. A., Lupski, J. R., Trask, B. J., van den
Engh, 6., Lindsay, E. A., Christy, H., Chen, K. S., Baldini, A., and et al. (1995).
Smith-Magenis syndrome deletion: a case with equivocal cytogenetic findings
resolved by fluorescence in situ hybridization. Am J Med Genet 58, 286-91.

Keats, B. J. B., and Berlin, C. I. (1999). Genomics and hearing impairment [In
Process Citation]. Genome Res 9, 7-16.

140

Kelley, P. M., Harris, D. J ., Comer, B. C., Askew, J. W., Fowler, T., Smith, S. D.,
and Kimberling, W. J. (1998). Novel mutations in the connexin 26 gene (GJB2)
that cause autosomal recessive (DFNB 1) hearing loss. Am J Hum Genet 62, 7 92-
9.

Lander, E. S., and Botstein, D. (1987). Homozygosity mapping: a way to map
human recessive traits with the DNA of inbred children. Science 236, 1567-70.

Levy, G., Levi-Acobas, F., Blanchard, S., Gerber, S., Larget-Piet, D., Chenal, V.,
Liu, X. Z., Newton, V., Steel, K. P., Brown, S. D., Munnich, A., Kaplan, J., Petit,
C., and Weil, D. ( 1997 ). Myosin VIIA gene: heterogeneity of the mutations
responsible for Usher syndrome type B. Hum Mol Genet 6, 111-6.

Liang, Y., Chen, H., Asher, J. B., Jr., Chang, C. C., and Friedman, T. B. (1997).
Human inner ear OCP2 cDN A maps to 5q22-5q35.2 with related sequences on
chromosomes 4p16.2-4p14, 5p13-5q22, 7pter-q22, 10 and 12p13-12qter. Gene
184, 163-7.

Liang, Y., Wang, A., Probst, F. J., Arhya, I. N., Barber, T. D., Chen, K. S.,
Deshmukh, D., Dolan, D. F., Hinnant, J. T., Carter, L. B., Jain, P. K., Lalwani, A.
K., Li, X. C., Lupski, J. R., Moeljopawiro, S., Morell, R., Negrini, C., Wilcox, E.
R., Winata, S., Camper, S. A., and Friedman, T. B. (1998). Genetic mapping
refines DFNB3 to l7p11.2, suggests multiple alleles of DFNB 3, and supports
homology to the mouse model shaker-2. Am J Hum Genet 62, 904-15.

Liu, X. Z., Walsh, J ., Mburu, P., Kendrick-Jones, J ., Cope, M. J., Steel, K. P., and
Brown, S. D. (1997 ). Mutations in the myosin VIIA gene cause non-syndromic
recessive deafness. Nat Genet 16, 188-90.

Liu, X. Z., Walsh, J., Tamagawa, Y., Kitamura, K., Nishizawa, M., Steel, K. P.,
and Brown, S. D. (1997). Autosomal dominant non-syndromic deafness caused by
a mutation in the myosin VIIA gene. Nat Genet 1 7, 268-9.

Luban, J ., and Goff, S. P. (1995). The yeast two-hybrid system for studying
protein-protein interactions. Curr Opin Biotechnol 6, 59-64.

Marres, H. A., and Cremers, C. W. (1989). Autosomal recessive nonsyndromal
profound childhood deafness in a large pedigree. Audiometric features of the
affected persons and the obligate carriers. Arch Otolaryngol Head Neck Surg 115,
591-5.

Matise, T. C., Perlin, M., and Chakravarti, A. (1994). Automated construction of
genetic linkage maps using an expert system (MultiMap): a human genome

141

linkage map [published erratum appears in Nat Genet 1994 Jun;7(2):215]. Nat
Genet 6, 384—90.

Maw, M. A., Allen-Powell, D. R., Goodey, R. J ., Stewart, I. A., Nancarrow, D. J.,
Hayward, N. K., and Gardner, R. J. (1995). The contribution of the DFNBI locus
to neurosensory deafness in a Caucasian population. Am J Hum Genet 5 7, 629-
35.

Mermall, V., Post, P. L., and Mooseker, M. S. (1998). Unconventional myosins in
cell movement, membrane traffic, and signal transduction. Science 279, 527-33.

Mooseker, M. S., and Cheney, R. E. (1995). Unconventional myosins. Annu Rev
Cell Dev Biol 11, 633-75.

Morell, R., Liang, Y., Asher, J. B., Jr., Weber, J. L., Hinnant, J. T., Winata, S.,
Arhya, I. N ., and Friedman, T. B. (1995). Analysis of short tandem repeat (STR)
allele frequency distributions in a Balinese population. Hum Mol Genet 4, 85-91.

Morton, N. E. (1991). Genetic epidemiology of hearing impairment. Ann N Y
Acad Sci 630, 16-31.

Nadol, J. B., Jr. (1993). Hearing loss. N Engl J Med 329, 1092-102.

Nance, W. E., and McConnell, F. E. (1973). Status and prospects of research in
hereditary deafness. Adv Hum Genet 4, 173-250.

Nance, W. B., and Sweeney, A. (1975). Symposium on sensorineural hearing loss
in children: early detection and intervention. Genetic factors in deafness of early
life. Otolaryngol Clin North Am 8, 19-48.

Nicholson, G. A., Valentijn, L. J., Cherryson, A. K., Kennerson, M. L., Bragg, T.
L., DeKroon, R. M., Ross, D. A., Pollard, J. D., McLeod, J. G., Bolhuis, P. A.,
and et al. (1994). A frame shift mutation in the PMP22 gene in hereditary
neuropathy with liability to pressure palsies. Nat Genet 6, 263-6.

Pena, S. D., and Chakraborty, R. (1994). Paternity testing in the DNA era. Trends
Genet 10, 204-9.

Pentao, L., Wise, C. A., Chinault, A. C., Patel, P. I., and Lupski, J. R. (1992).

Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at
repeat sequences flanking the 1.5 Mb monomer unit. Nat Genet 2, 292-300.

142

Phillips, C. P., Carracedo, A., and Lareu, M. V. (1998). Manual electrophoretic
methods for genotyping amplified STR loci. Methods Mol Biol 98, 181-92.

Probst, F. J., Fridell, R. A., Raphael, Y., Saunders, T. L., Wang, A., Liang, Y.,
Morell, R. J., Touchman, J. W., Lyons, R. H., Noben-Trauth, K., Friedman, T. B.,
and Camper, S. A. (1998). Correction of deafness in shaker-2 mice by an
unconventional myosin in a BAC transgene. Science 280, 1444-7.

Roa, B. B., Garcia, C. A., Suter, U., Kulpa, D. A., Wise, C. A., Mueller, J.,
Welcher, A. A., Snipes, G. J ., Shooter, E. M., Patel, P. I., and et al. (1993).
Charcot-Marie-Tooth disease type 1A. Association with a spontaneous point
mutation in the PMP22 gene. N Engl J Med 329, 96-101.

Robertson, N. G., Khetarpal, U., Gutierrez-Espeleta, G. A., Bieber, F. R., and
Morton, C. C. (1994). Isolation of novel and known genes from a human fetal
cochlear cDNA library using subtractive hybridization and differential screening.
Genomics 23, 42-50.

Schenker, T., and Trueb, B. (1997). Assignment of the gene for a developmentally
regulated GTP-binding protein (DRG2) to human chromosome bands l7p13—-
>p12 by in situ hybridization. Cytogenet Cell Genet 79, 274—5.

Scott, D. A., Carmi, R., Elbedour, K., Duyk, G. M., Stone, E. M., and Sheffield,
V. C. (1995). Nonsyndromic autosomal recessive deafness is linked to the
DFNBI locus in a large inbred Bedouin family from Israel [letter]. Am J Hum
Genet 5 7, 965-8.

Scott, D. A., Carmi, R., Elbedour, K., Yosefsberg, S., Stone, E. M., and Sheffield,
V. C. (1996). An autosomal recessive nonsyndromic-hearing-loss locus identified
by DNA pooling using two inbred Bedouin kindreds. Am J Hum Genet 59, 385-
91.

Sellers, J. R., Goodson, H. V., and Wang, F. (1996). A myosin family reunion. J
Muscle Res Cell Motil 17, 7 -22.

Seranski, P., Heiss, N. S., Dhorne-Pollet, S., Radelof, U., Korn, B., Hennig, S.,
Backes, B., Schmidt, S., Wiemann, S., Schwarz, C. B., Lehrach, H., and Poustka,
A. (1999). Transcription mapping in a medulloblastoma breakpoint interval and
Smith-Magenis syndrome candidate region: identification of 53 transcriptional
units and new candidate genes. Genomics 56, 1-1 1.

143

Skvorak, A. B., Weng, Z., Yee, A. J., Robertson, N. G., and Morton, C. C. (1999).
Human cochlear expressed sequence tags provide insight into cochlear gene
expression and identify candidate genes for deafness. Hum Mol Genet 8, 439-452.

Staudinger, J ., Perry, M., Elledge, S. J., and Olson, E. N. (1993). Interactions
among vertebrate helix-loop-helix proteins in yeast using the two-hybrid system. J
Biol Chem 268, 4608-11.

Steele, M. W. (1981). Genetics of congenital deafness. Pediatr Clin North Am 28,
973-80.

Stephan, W., and Cho, S. (1994). Possible role of natural selection in the
formation of tandem- repetitive noncoding DNA. Genetics 136, 333-41.

Thalmann, I., Rosenthal, H. L., Moore, B. W., and Thalmann, R. (1980). Organ of
Corti-specific polypeptides: OCP-I and OCP-II. Arch Otorhinolaryng01226, 123-
8. ‘

Thalmann, I., Suzuki, H., McCourt, D. W., Comegys, T. H., and Thalmann, R.
(1993). Partial amino acid sequences of organ of Corti proteins OCPl and OCP2:
a progress report. Hear Res 64, 191-8.

Townsend-Nicholson, A., Baker, B., Sutherland, G. R., and Schofield, P. R.
(1995). Localization of the adenosine A2b receptor subtype gene (ADORA2B) to
chromosome l7p1 1.2-p12 by FISH and PCR screening of somatic cell hybrids.
Genomics 25, 605-7.

Valentijn, L. J ., Bolhuis, P. A., Zorn, I., Hoogendijk, J. B., van den Bosch, N.,
Hensels, G. W., Stanton, V. P., Jr., Housman, D. B., Fischbeck, K. H., Ross, D.
A., and et al. (1992). The peripheral myelin gene PMP-22/GAS-3 is duplicated in
Charcot-Marie- Tooth disease type 1A. Nat Genet 1, 166-70.

Van Camp, G., Willems, P. J ., and Smith, R. J. (1997). Nonsyndromic hearing
impairment: unparalleled heterogeneity. Am J Hum Genet 60, 758-64.

Veske, A., Oehlrnann, R., Younus, F ., Mohyuddin, A., Muller-Myhsok, B.,
Mehdi, S. Q., and Gal, A. (1996). Autosomal recessive non-syndromic deafness
locus (DFNB8) maps on chromosome 21q22 in a large consanguineous kindred
from Pakistan. Hum Mol Genet 5, 165-8.

Wall, W. J ., Williamson, R., Petrou, M., Papaioannou, D., and Parkin, B. H.

(1993). Variation of short tandem repeats within and between populations. Hum
Mol Genet 2, 1023-9.

144

Wang, A., Liang, Y., Fridell, R. A., Probst, F. J., Wilcox, E. R., Touchman, J. W.,
Morton, C. C., Morell, R. J ., Noben-Trauth, K., Camper, S. A., and Friedman, T.
B. (1998). Association of unconventional myosin MY015 mutations with human
nonsyndromic deafness DFNB3. Science 280, 1447-51.

Warrick, H. M., and Spudich, J. A. (1987). Myosin structure and function in cell
motility. Annu Rev Cell Biol 3, 379-421.

Watts, D. (1998). Genotyping STR loci using an automated DNA sequencer.
Methods Mol Biol 98, 193-208.

Webb, G. C., Baker, R. T., Pagan, K., and Board, P. G. (1990). Localization of the
human UbB polyubiquitin gene to chromosome band 17p11.1-17p12. Am J Hum
Genet 46, 308-15. -

Weber, J. L., and May, P. E. (1989). Abundant class of human DNA
polymorphisms which can be typed using the polymerase chain reaction. Am J
Hum Genet 44, 388-96.

Weber, J. L., and Wong, C. (1993). Mutation of human short tandem repeats.
Hum Mol Genet 2, 1123-8.

Weil, D., Blanchard, S., Kaplan, J ., Guilford, P., Gibson, F., Walsh, J ., Mburu, P.,
Varela, A., Levilliers, J ., Weston, M. D., and et al. (1995). Defective myosin
VIIA gene responsible for Usher syndrome type 1B. Nature 374, 60-1.

Weil, D., Kussel, P., Blanchard, S., Levy, G., Levi-Acobas, F., Drira, M., Ayadi,
H., and Petit, C. (1997). The autosomal recessive isolated deafness, DFNB2, and
the Usher 1B syndrome are allelic defects of the myosin-VIIA gene. Nat Genet
16, 191-3.

Weston, M. D., Kelley, P. M., Overbeck, L. D., Wagenaar, M., Orten, D. J .,
Hasson, T., Chen, Z. Y., Corey, D., Mooseker, M., Sumegi, J., Cremers, C.,
Moller, C., Jacobson, S. G., Gorin, M. B., and Kimberling, W. J. (1996). Myosin
VIIA mutation screening in 189 Usher syndrome type 1 patients. Am J Hum
Genet 59, 1074-83.

White, M. A. (1996). The yeast twO-hybrid system: forward and reverse
[comment]. Proc Natl Acad Sci U S A 93, 10001-3.

Wilgenbus, K. K., Seranski, P., Brown, A., Leuchs, B., Mincheva, A., Lichter, P.,
and Poustka, A. (1997 ). Molecular characterization of a genetically unstable

145

region containing the SMS critical area and a breakpoint cluster for human
PNETs. Genomics 42, 1-10.

Winata, S., Arhya, I. N., Moeljopawiro, S., Hinnant, J. T., Liang, Y., Friedman, T.
B., and Asher, J. H., Jr. (1995). Congenital non-syndromal autosomal recessive
deafness in Bengkala, an isolated Balinese village. J Med Genet 32, 336-43.

Zelante, L., Gasparini, P., Estivill, X., Melchionda, S., D’Agruma, L., Govea, N.,
Mila, M., Monica, M. D., Lutfi, J ., Shohat, M., Mansfield, B., Delgrosso, K.,
Rappaport, E., Surrey, S., and Fortina, P. (1997). Connexin26 mutations
associated with the most common form of non- syndromic neurosensory
autosomal recessive deafness (DFNB 1) in Mediterraneans. Hum Mol Genet 6,
1605-9.

Zhao, 2., Lee, C. C., Jiralerspong, S., Juyal, R. C., Lu, F., Baldini, A., Greenberg,
F., Caskey, C. T., and Patel, P. I. (1995). The gene for a human microfibril-
associated glycoprotein is commonly deleted in Smith-Magenis syndrome
patients. Hum Mol Genet 4, 589-97.

146

"I1111111]leljllllllullljlijjl“