Shahin, Walsh, et al.
Supplementary Information:
Novel mutations in known deafness genes
In 16 of the 20 families evaluated by homozygosity mapping, the longest region of
homozygosity included a gene known to be associated with hearing loss (text Table 1).
For each of these 16 families, the relevant gene was fully sequenced from genomic DNA
of the proband, revealing in 14 families alleles very likely to be deleterious. These alleles
included truncating mutations of Otoferlin, TECTA, Pejvakin, and TMPRSS3, a splice site
mutation leading to a large in-frame deletion of Pendrin/SLC26A4, and nonsynonymous
mutations in TMHS, MYO7A, MYO15A, and CDH23, which were further evaluated by
splicing analysis and molecular modeling. Detailed analyses of these point mutations are
presented here.
OTOF. Family AM was the smallest family analysed by homozygosity mapping
(text Figure 1), so not unexpectedly yielded the most deafness-associated homozygous
regions. The homozygosity profile revealed five peaks of sizes 2.2, 2.8, 2.9, 3.3, and 19.4
MB (text Figure 2). The only peak spanning a known deafness gene was the largest
region at chromosome 2p which includes OTOF, the gene responsible for DFNB9i.
Sequence analysis of OTOF in the proband of family AM revealed a novel variant (c4157
C>T) corresponding to a nonsense allele at codon 577. To date, 56 different pathogenic
mutations of OTOF have been reportedii,iii.
The vast majority of reported alleles are
private, described in only one family. The R577X allele was not present in any of the
other 218 NSHL probands or in 288 Palestinian hearing controls. In a large study of
families with mutations in OTOFiv, 57% of cases with two inactivating alleles had
1
Shahin, Walsh, et al.
preserved transient-evoked otoacoustic emissions (TEOAEs), which is consistent with the
nonsyndromic hearing loss phenotype of family AM.
TECTA. In family BQ, homozygosity mapping yielded a single peak at
chromosome 11q23, including TECTA, the gene responsible for dominant hearing loss
DFNA8/12 and recessive hearing loss DFNB21.
Interestingly, the largest peak of
homozygosity in Family BI overlapped the BQ region. Comparison of the SNP haplotypes
of families BQ and BI revealed perfect identity across 4MB around the TECTA locus,
suggesting a common ancestor, despite ascertainment from different parts of the West
Bank and the families being unaware of any relationship.
TECTA mutations causing
dominant deafness are substitutions of highly conserved amino acid residuesv and an inframe deletion of 37 residuesvi. In contrast, TECTA mutations causing recessive hearing
loss are predicted to cause non-functional proteins through truncation or nonsensemediated decayvii,viii,ix. Sequencing genomic DNA from probands of families BQ and BI
revealed a TECTA nonsense allele C1619X. This allele was subsequently found to be
homozygous in two other Palestinian probands. The audiograms of deaf relatives of
these families revealed the ‘U’ shaped curve previously described in DFNB21 families v
(Supplementary figure 1). The heterozygous carriers of the allele did not exhibit any
hearing loss, an observation consistent with C1619X being a null allele.
Pejvakin. Family A had a single homozygous peak spanning Pejvakin (DFNB59).
Sequencing revealed a single nucleotide substitution, c762C>T, predicted to alter Arg136
(CGA) to a STOP codon (TGA) in exon 3. To date four premature truncations and six
missense alleles of Pejvakin have been described in Iranian, Turkish, and Dutch
familiesx’xi,xii. No otoacoustic emission (OAE) distortion products were present in deaf
relatives of family A, indicating that auditory neuropathy is highly unlikely to be present.
2
Shahin, Walsh, et al.
TMPRSS3. A nonsense allele, TMPRSS3_C194X was identified in family IB,
indicated by the largest homozygous region spanning the DFNB8/10 locus. The predicted
truncation is within the scavenger receptor cysteine-rich domain, which is N-terminal of
the serine protease domain, and hence likely to abolish protease activity. This mutation
represents the third different TMPRSS3 truncating allele discovered in Palestinian
familiesxiii,xiv whereas missense alleles represent approximately two thirds of the
pathogenic alleles in other populationsxv.
Pendrin/SLC26A4. Homozygosity mapping of family CB yielded four peaks >2MB,
the longest of which includes the gene Pendrin/SLC26A4. Sequencing of SLC26A4
revealed a single base deletion at the last base of exon 11 (c1341G). This mutation has
been previously reported in an Arab-Israeli family with Pendred syndromexvi and
subsequently in a Turkish family with deafness and goiterxvii, although the consequence of
the mutation on SLC26A4 message was not described in these studies.
The consequence of SLC26A4.1341G on the SLC26A4 message was evaluated
in cDNA of CB1, a hearing parent, and CB3, a deaf child. Total RNA was prepared from
whole blood with a Qiagen RNeasy mini kit according to the manufacturer's protocol. Five
micrograms of RNA was reversed transcribed with an SLC26A4-specific primer located in
exon 14 at nucleotides 1835-1811 (5' TTTGTAATTCTTGGTACTTTTGTAG 3') of the
SLC26A4 RefSeq cDNA, accession NM_000411. The resulting cDNA was amplified with
a forward primer in exon 8, nt 1232-1251 (5' GCCTCCTGAACTTCCACCTG 3') and a
reverse primer in exon 12, nt 1709-1690 (5' ATCCAGCCCCAGAATGATGG 3'). PCR
products were electrophoresed on 2% agarose, and splice variants were gel extracted
and sequenced in both directions with Big Dye V3.1 on an Applied Biosystems 3130xl
instrument. cDNA sequence revealed that SLC26A4.1341G leads to skipping of exon
3
Shahin, Walsh, et al.
11. The predicted effect on the Pendrin protein is deletion of residues 422-447 which
encode the tenth transmembrane domain. The deletion of a transmembrane domain is
very likely to abrogate transport function.
Inner ear computerized tomography (CT) images were performed on individual
CB5. The vestibular aqueduct was considered enlarged when the diameter between the
common crus and external aperture was 1.5mm or more on thin CT sections. CT analysis
of CB5 revealed an EVA of 3.5 mm. Both deaf individuals of family CB were evaluated for
goiter. Tri-iodothyronine (T3), tetra-iodothyronine (T4) and thyroid stimulating hormone
(TSH) were determined by radioimmunoassay kits. CB3 and CB5 did not exhibit signs of
goiter and their thyroid function tests were in the normal range (T3 = 1.3, T4 = 1.6, TSH =
1.15 mlU/ml).
TMHS. The longest deafness-associated homozygous region in family DD
included the gene encoding TMHS (Tetraspan Membrane protein of Hair Stereocilia).
Mutations in TMHS (also known as LHFPL5) are responsible for DFNB66/67xviii,xix.
Sequencing TMHS in genomic DNA from the proband of family DD revealed a substitution
c1A>G leading to a Met to Val change at codon 1. Translation initiation codon mutations
may prevent protein productionxx or translation of a mutant allele initiating from a
downstream methioninexxi. The nearest downstream methionine in TMHS is located at
residue 28.
TMHS is predicted to encode four transmembrane helices with two
extracellular loopsxxii. We modeled transmembrane topology of the wildtype protein and
the mutant protein initiating from Met 28 using TMpredxxiii. The model strongly favours
flipping the orientation of the N terminal domain from intracellular to extracellular in the
predicted mutant protein thereby drastically altering function. Four other mutations have
4
Shahin, Walsh, et al.
been described in humans with congenital bilateral profound hearing lossxviii,xix and one
mutation responsible for the hurry-scurry mousexxii.
MYO7A. Homozygosity mapping of family J revealed a 26.4MB peak on
chromosome 11. This region contains three known deafness genes: FGF3, responsible
for
congenital
sensorineural
deafness
with
microtia
and
microdontiaxxiv,xxv;
COMT2/LRTOMT, which is responsible for non-syndromic hearing loss DFNB63xxvi,xxvii;
and MYO7A, at which recessive alleles cause Usher syndrome type 1Bxxviii and
nonsyndromic hearing loss DFNB2xxix,xxx and dominant alleles are associated with
DFNA11xxix. Sequence analysis of FGF3 and LRTOMT did not reveal any rare variants.
However, in MYO7A, the variant Gly2163Ser segregated with hearing loss. This variant is
located in the FERM domain of the myosin tail, and a glycine residue is highly conserved
at this site. Gly2163Ser was previously reported in a family with Usher syndrome type 1B
as a compound heterozygote in trans with an intronic variantxxxi. Deaf individual Jn17 (age
25) was evaluated by fundoscopy. Visual acuity, color test, and fundus were normal,
ruling out retinitis pigmentosa and Usher syndrome.
Hearing loss of affected relatives in family J is severe to profound, with thresholds
>90dB at all frequencies.
The role of MYO7A missense mutations in nonsyndromic
hearing loss DFNB2 has been controversial, because homozygosity for all MYO7A
truncating
mutations
syndromexxxii,xxxiii.
and
most
MYO7A
missense
mutations
leads
to
Usher
However, homozygosity for another mutation in the MYO7A tail,
Glu1716del, leads to nonsyndromic hearing lossxxxiii. In an assay of the mouse allele
homologous to MYO7A_Glu1716del, this mutant protein retained some normal function,
localizing along the length of stereocilia in a manner similar to wild-type MYO7A. It is
possible that Gly2163Ser leads to a mutant protein that also has some residual activity
5
Shahin, Walsh, et al.
enabling normal retinal function, but when co-inherited with a second deleterious allele
leads to Usher syndrome
MYO15A. Hearing loss in families AN and P mapped to overlapping regions of
chromosome 17. Deaf individuals in the two families shared an identical haplotype across
a region of 1.54MB around the MYO15A locus (chr17:17,936,632-19,478,062).
Sequencing MYO15A in genomic DNA of deaf relatives from both families revealed a
novel variant (c7545G>T in exon 35 of NM_016239), segregating with deafness. If it were
a missense, this mutation would lead to Asp2403Tyr, in the myosin tail domain between
the MyTH4 and FERM domains.
However, aspartic acid is not evolutionarily well
conserved at this site.
MYO15A splicing was evaluated in RNA from whole blood from AN2, a deaf child,
using RT-PCR. Total RNA was reversed transcribed with an oligo dT primer and cDNA
amplified
with
primers
located
GCTGGGACTCGGATGAGGAC-3)'
in
exon
34,
nucleotides
7308-7328
(5'-
and
exon
36,
nucleotides
7662-7643
(5'-
GCTTTGGCTCTGGGGGTCTC-3'). Coordinates refer to RefSeq MYO15A cDNA,
accession NM_016239. PCR products were sequenced as described for SLC26A4.
Sequencing revealed a message splicing from c7544 to the first base of exon 36 (c7551).
The c7545G>T mutation led to the creation of an alternate GT donor splice site and
deletion of 7 nucleotides (c7544-c7550) from the message. The mis-spliced message
alters the reading frame and is predicted to produce 11 novel amino acids prior to the
introduction of a nonsense codon. Sequence analysis of the cDNA demonstrated that the
nonsense codon is stable and evades nonsense-mediated decay.
This allele was
homozygous in five of 218 (2.3%) of deaf probands, making MYO15A the third most
6
Shahin, Walsh, et al.
common cause of non-syndromic deafness in the West Bank after connexin 26 and
TRIOBP.
CDH23. Mapping of families AB, DA, and G revealed extended homozygosity in
each family on chromosome 10q, in regions including the CDH23 locus. Haplotypes were
not shared by deaf relatives from different families, and sequencing revealed three
different missense mutations, each homozygous in the deaf individuals in one family:
Pro346Ser in family G, Pro346Leu in family DA, and Pro559Ser in family AB.
In order to predict changes in the macromolecular structure of CDH23 due to these
mutations, we applied molecular modeling tools. A partial CDH23 model was generated
by threading the individually aligned CDH23 dimers onto the N terminal domains of the
mouse CDH8xxxiv using HHSearchxxxv. Unaligned regions in the CDH23 dimer sequences
were rebuilt using fragments from known protein structures and a low-resolution force
fieldxxxvi. Positions of calcium ions and calcium-coordinating backbone and side-chain
atoms, on the full-sequence model, were inferred by homology to the structure of mouse
CDH8. Structures were then refined using the Rosetta full-atom energy function with
spatial restraints applied to the starting coordinates in order to prevent inappropriate
divergence from the CDH8 structure, following previously published protocolsxxxvii,xxxviii.
We used the Rosetta programxxxviii to predict changes in macromolecular
structure due to the missense mutations. Cadherin proteins form extended linear
structures with interactions only between adjacent domainsxxxiv,xxxix. Every dimer of two
cadherin domains contains a full calcium-binding site. Models of CDH23 cadherin dimers
constructed using the Rosetta “rebuild and refine” protocol suggest that Pro346 lies
between calcium binding domains 3 and 4 and that Pro559 is in the analogous position
7
Shahin, Walsh, et al.
between calcium binding domains 5 and 6 (Supplementary figure 2a). All three mutations
alter a proline adjacent to the alanine residue whose backbone oxygen is critical to the
binding of calcium to cadherin. In the wildtype sequence, these prolines have restricted
angular motion and strongly constrain the conformations of the adjacent calcium-binding
alanine residues. These angles are predicted to be less strongly constrained in the mutant
proteins (Supplementary figure 2b).
Structural modeling of calcium binding in cadherin repeats suggests a more
general association of amino acid position with functional consequence of missense
mutations in CDH23. The published literature on CDH23 includes 22 mutations reported
to cause DFNB12, a form of nonsymdromic hearing loss, and 20 nonsynonymous variants
reported to be benignxl,xli. The minimum distance from each residue in a cadherin domain
to its nearest calcium ion can be predicted with Rosetta tools (Supplementary table 2).
Mutations associated with NSHL occur in residues closer to their nearest calcium ion that
do mutations reported to be benign: P = 0.009 by 2-tailed Mann-Whitney U-test.
8
Shahin, Walsh, et al.
Supplementary table 1.
Palestinian families with inherited hearing loss
Number of individuals genotyped
Nuclear
families
Deaf
Hearing
siblings
Parents
Total
A
AB
AM
AN
BG
BI
BQ
C
CB
CG
CN
DA
DD
DE
DO
DP
GN
IB
JN
P
2
1
1
1
1
1
2
5
1
4
1
1
2
1
1
2
3
1
1
1
4
4
2
3
3
3
4
11
2
7
3
3
5
3
4
2
5
3
3
4
4
0
1
1
2
0
1
4
1
3
1
0
1
4
1
1
0
0
3
0
4
2
1
2
2
2
4
5
2
6
1
2
3
2
1
1
4
2
2
1
12
6
4
6
7
5
9
20
5
16
5
5
9
9
6
4
9
5
8
5
Total
33
78
28
49
155
Kindred
9
Shahin, Walsh, et al.
Supplementary table 2.
Variants in cadherin 23 (CDH23)
CD1
1
3
3
4
5
5
5
5
6
9
10
10
11
12
13
13
13
14
15
15
15
15
16
16
17
17
18
18
19
19
19
19
20
21
21
22
22
22
23
24
24
25
1
2
AA WT
124 D
346 P
346 P
452 N
480 L
490 G
496 S
559 P
582 R
990 D
1060 R
1061 E
1186 G
1222 A
1341 D
1349 R
1351 D
1437 R
1575 T
1586 A
1595 E
1620 V
1671 T
1675 V
1804 R
1846 D
1887 T
1888 F
1999 T
2044 E
2045 D
2066 R
2148 D
2202 D
2283 V
2358 R
2376 D
2380 P
2465 R
2588 E
2608 R
2635 V
V Distance2
G
3.81
L
5.11
S
5.11
S
2.61
Q
13.37
A
6.99
N
10.39
S
5.11
Q
10.90
N
3.01
W
3.58
K
3.76
D
8.88
T
11.56
N
2.33
C
4.25
N
6.69
Q
14.77
A
13.34
P
18.57
K
3.76
M
15.68
S
7.41
I
15.33
Q
17.15
N
3.01
I
7.38
S
7.94
S
10.39
S
13.43
N
11.85
Q
4.22
N
9.66
N
2.33
I
6.07
Q
10.66
N
2.61
L
11.76
W
3.58
Q
13.43
H
3.01
F
14.73
Consequence
DFNB12
DFNB12
DFNB12
DFNB12
DFNB12
Benign
Benign
DFNB12
DFNB12
DFNB12
DFNB12
DFNB12
DFNB12
Benign
DFNB12
Benign
Benign
Benign
Benign
DFNB12
DFNB12
Benign
Benign
Benign
Benign
DFNB12
Benign
DFNB12
Benign
Benign
DFNB12
Benign
DFNB12
DFNB12
Benign
Benign
Benign
Benign
DFNB12
Benign
DFNB12
DFNB12
CD: cadherin domain
Predicted distance in angstroms to nearest calcium
10
Shahin, Walsh, et al.
Supplementary figure 1.
Audiogram of 24 year old with nonsense mutation
TECTA.C1619X.
0
10
Left ear
Right ear
Hearing level (dB)
20
30
40
50
60
70
80
90
100
250
500
1000
2000
Frequency Hz
11
4000
8000
Shahin, Walsh, et al.
Supplementary figure 2. Structural modeling of CDH23 mutations. Conserved
molecular architecture of cadherin calcium-binding sites is based on crystal structure of
mouse CDH8 (PDB ID 2a62).
CDH23
cadherin domain
(a) Rosetta homology model of the interface of a pair of cadherin domains in human
CDH23, including three calcium ions (orange). Between cadherin repeats 3 and 4,
mutations at the highly conserved proline346 (yellow star) are predicted to disrupt the
conformation of adjacent alanine345 (red star) whose backbone oxygen coordinates
binding of a calcium ion. Proline559, which is mutated to serine559 in family AB, is at the
analogous site between cadherin repeats 5 and 6.
12
Shahin, Walsh, et al.
0.050
0.045
0.040
0.035
Pro
Leu
Ser
Density
0.030
0.025
0.020
0.015
0.010
0.005
0.000
-150
-100 -50
Phi
0
50
Angle
100
150
200
250
Psi
(b) Predicted changes in strength of constraints on conformation of angles at this site
given wildtype residue proline vs mutant residues leucine (family DA) or serine (families G
and AB).
13
Shahin, Walsh, et al.
Genomic regions harboring novel genes for hearing loss in consanguineous
families.
Supplementary figure 3, on the following pages, illustrates the chromosomal regions
linked to loci for hearing loss in families CG, DE, CN, DP, and C, as revealed by
homozygosity mapping. (A) DFNB82 at chromosome 1p13.3 in family CG. (B) DFNB83
at chromosome 9p23-p21.1 and 9p13.3-q21.12 in family DE. (C) DFNB84 at chromosome
12q14.3-q21.2 in families CN and DP. (D) DFNB85 at chromosome 17p12-q11.2 in family
C. (E) linked region on chromosome 14q23.1-q31.1 in family BG.
14
Shahin, Walsh, et al.
(A) DFNB82
1p13.3
Chr1:108,108,849-111,251,931
15
Shahin, Walsh, et al.
(B) DFNB83: 9p23-p21.1 and 9p13.3-q21.1;
chr9:9,959,023-26,430,090 and chr9:34,401,026-77,904,900
16
Shahin, Walsh, et al.
(C) DFNB84
12q14.3-q21.1
chr12:65,434,594-74,498,486
17
Shahin, Walsh, et al.
(D) DFNB85
17p12-q11.2
chr17:15,212,798-26,490,848
18
Shahin, Walsh, et al.
(E) 14q23.1-q31.1
chr14:57,474,255-80,664,133
19
Shahin, Walsh, et al.
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Tekin M, Akçayöz D, Comak E et al: Screening the SLC26A4 gene in probands
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Alsmadi O, Meyer BF, Alkuraya F et al: Syndromic congenital sensorineural
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