Genomic analysis of a heterogeneous Mendelian phenotype: multiple novel alleles for inherited hearing loss in the Palestinian population

Recessively inherited phenotypes are frequent in the Palestinian population, as the result of a historical tradition of marriages within extended kindreds, particularly in isolated villages. In order to characterise the genetics of inherited hearing loss in this population, we worked with West Bank schools for the deaf to identify children with prelingual, bilateral, severe to profound hearing loss not attributable to infection, trauma or other known environmental exposure. Of 156 families enrolled, hearing loss in 17 families (11 per cent) was due to mutations in GJB2 (connexin 26), a smaller fraction of GJB2-associated deafness than in other populations. In order to estimate how many different genes might be responsible for hearing loss in this population, we evaluated ten families for linkage to all 36 known human autosomal deafness-related genes, fully sequencing hearing-related genes at any linked sites in informative relatives. Four families harboured four novel alleles of TMPRSS3 (988ΔA = 352stop), otoancorin (1067A >T = D356V) and pendrin (716T > A = V239D and 1001G > T = 346stop). In each family, all affected individuals were homozygous for the critical mutation. Each allele was specific to one or a few families in the cohort; none were widespread. Since epidemiological tests of association of mutations with deafness were not feasible for such rare alleles, we used functional and bioinformatics approaches to evaluate their consequences. In six other families, hearing loss was not linked to any known gene, suggesting that these families harbour novel genes responsible for this phenotype. We conclude that inherited hearing loss is highly heterogeneous in this population, with most extended families acting as genetic isolates in this context. We also conclude that the same genes are responsible for hearing loss in this population as elsewhere, so that gene discovery in these families informs the genetics of hearing loss worldwide.


Introduction
Extended kindreds from highly endogamous communities are ideally suited for identifying genes responsible for clinically important phenotypes. The unique demographic historyo f the MiddleE ast has led to many such communities. Form ore than 5,000 yearsa nd continuing to the present, the eastern shores of theM editerranean have seen immigration of people from awide variety of cultures. Villages were often established by af ew extended families and, despite their geographical proximity,r emained demographically isolated. For centuries, marriages have been arrangedw ithin extended families in these villages, leading to highl evels of consanguinity and consequently high frequencies of recessive traits. 1,2 The willingness of thesek indreds to participate in research has been of enormous help to geneticists. In order to identify genes responsible for inherited hearing loss, we have worked with consanguineous kindreds from the PRIMARY RESEARCH q HENRYSTEWART PUBLICATIONS 1473-9542. HUMANG ENOMICS .V OL 2. NO 4. 203-211 JANUARY2006 Palestinian population. As expected, we have found recessive alleles responsible for inherited hearing loss in such families. We have also discovered that an extremely highl evel of both allelic and locus heterogeneity characterises inherited hearing loss in this population.
Systematic population-wideg enomic analysis mayb et he most effective wayt oi dentify all critical genes and alleles for heterogeneous phenotypesi nc ommunities with many small isolates. We illustrate such as trategy in this paper.I ns uch populations, most alleles of clinical importance arel ikely to be rare,f ound in only one or af ew affected families, thus effectively precluding case-controla pproaches to test associations of alleles with the trait. Functional and bioinformatics approaches can be applied to these situations, and we illustrate such strategies applied to hearing loss phenotypes. The most important consequence of this heterogeneity is that such populations mayo ffer the opportunity to identify and characterise al arge number of alleles and genes critical to common phenotypes. They may, therefore, be even more valuable than previously recognised.

Ascertainment of subjects
Probands arec hildren withp relingual bilateralh earing loss who attend one of the following schools for deaf children: Eftah School for the Deaf in Bethlehem; Al Amal School for the Deaf in Hebron; the Association for the Deaf in Nablus; and the Princess Basma RehabilitationC enter for Deaf Children in Jerusalem. To gether,t hese schools serve nearly all hearing-impaired Palestinian children living on the We st Bank. Children were referred to one of us (M.K.) by their teachers. Families were contacted through teachersa nd socialw orkers. If the family expressed an interest, they were visiteda th ome (byM .K. and A.A.R.) to discuss the project in detail, explaining that the first steps in participationw ould be physical and audiological examinationst oe xclude hearing loss due to infection or trauma and to evaluate severity and laterality of the hearing loss. Forf amilies who agreed, informed consent waso btained from parents and assent from older children. Controls comprised 100 Palestinian adults with normal hearing, ascertainedw hen bloods amples were drawn at We st Bank clinics for preventiveh ealth services. Controls agreed that their DNA samples could be used anonymously for this project. Thep roject is approved by the Human Subjects Division of the University of Wa shington, by the Helsinki Committee of Te lA viv University and by the Human SubjectsC ommittee of Bethlehem University. Single Project Assurances for Te lA viv University and for Bethlehem University have been obtained from the US Office for Human Research Protection (OHRP). Consenting deaf adults and children, hearing adults and hearing children older than the oldest age of onset of inherited deafness in their family were enrolled.

Genotyping and sequencing
Microsatellite markerswithin and flanking deafness genes were selected from the Genome Browsero rd esigned usingt he simpler epeat track of the Genome Browser. 3 DNA samples were genotyped and lod scores calculated as previously described. 4 For sequencing candidate genes, primer pairswere designed to amplify and sequencet he entirec odingr egionsa nd splice sites of all isoforms of GJB2 (connexin 26), 4 TMPRSS3, 5 pendrin 6 and otoancorin. 7 Sequencing wasc arried out using BigDyet erminator 3.1 sequencing chemistry( Applied Biosystems) and wast hen visualisedo nA BI 3100 sequencers. Sequences were aligned and compared using SeqHelp. 8 Mutations described in the text were genotypedi na ll probands and controls by sequencing or by polymerase chain reaction (PCR) and restriction enzyme digest. Restriction enzyme digests were made with Mbo II for TMPRSS3.988delA, Hph Ifor pendrin 1001G . T and Fok I for otoancorin 1067A . T .P endrin 716T . A wasscreened by DNA sequencing in probands and controls. All probands and controls were also screened by PCR and restriction enzyme digest for previously published Palestinian and Lebanese deafness alleles of otoferlin, 9 TECTA, 10 otoancorin 7 and whirlin. 11 Restriction enzyme digests were madew ith Dde I for otoferlin 2416T . A( Y730X), PfiMI for TECTA IVS9(þ 1)G . A , Cac81 for otoancorin IVS12(þ 2)T . C and BvbCI for whirlin 2332C . T( R778X). The bsatellite insertion in TMPRSS3 wasa nalysed by PCR,a s published. 5

Results
The cohortpresently includes 156 probands and their families. Probands were characterised as having prelingual, bilateral hearing loss that could not be attributed to infection or trauma. No vision problems were present. More subtle syndromic signs in some individuals were detected subsequently, as described below.
Becausemutations in the gap junction GJB2 (connexin 26) are responsible for as ubstantial fraction of recessive inherited hearing loss worldwide, 13 we first sequenced both exonso f this gene in genomic DNA from all probands. 4 We also screened for the neighbouring deletion of GJB6. 14 Of the 156 families in the project, inherited hearing loss in 17 families (11 per cent) wasd ue to mutations in GJB2.F ived ifferent mutant alleles of GJB2 were present. 4 Thef raction of inherited hearing loss due to GJB2 in this population is somewhat lowert han in other populations.
In ordert oa ssess genetic heterogeneity of inheritedd eafness in the population, we chose ten families, which each included at least four relativeswith prelingual bilateral hearing loss whowere wild-type for GJB2.( An average of six affected individuals per family were sampled.) All living relativesw ith hearing loss were evaluatedb yp hysical examination and audiology to distinguish inherited prelingual hearing loss from age-related hearing loss. Prelingual deafness wasc onsistent with recessivei nheritance in each family.
We next carried out ah earing-loss-targeted linkage scan of the ten families, using6 8m icrosatellite markersw ithin and flankingt he 36 autosomal genes known to be responsible for inheritedh earing loss ( Ta ble 1). 15 We tested for linkage to genes for either recessive or dominant hearing loss,b ecause different alleles of the sameg ene mayl eadt oh earing loss by different modes of inheritance.Ifmarkersw ereuninformative, other markersw eres ubstituted in that family. 3 We noted whether affected individuals were homozygous for al inked haplotype,b ut did not exclude families with more than one linked haplotype that mightr eflect more than one deafnessassociated allele.
In six families, hearing loss wasn ot linked to any known hearing-related gene.The model for linkage wasrecessive fully penetrant hearing loss withn os poradics; marker allele frequencies were estimated from the families in the sample.B y genotyping all flankingm arkerss hown in Ta ble 1, linkage could be excluded for all known genes by negativel od scores; however, no one marker wasc onsistently sufficiently informativet oe xclude linkage.G enes for hearing loss in these families will be identifiedb yg enome-wide linkage analysis and positional cloning.Inf our other families, hearing loss was linked to locales of known deafness genes: TMPRSS3 on chromosome 21q23, pendrin on 7q31 and otoancorino n 16p12( Figure 1).

TMPRSS3
FamilyWincluded 11 individuals with prelingual, bilateral, severe to profoundh earing loss,w ith thresholds poorert han 85 decibels (dB) at all frequencies. All ten living affected individuals were homozygous at markersD 21S1225a nd GT(42.677), which flank TMPRSS3 ( Figure 1A). Thel od score for linkage of hearing loss to the D21S1225-GT(42.67) haplotype was9 .26. We sequenced TMPRSS3 in an affected child from Family Wand identified 988delA,aframeshift mutation in exon 10 leading to as top at codon3 57. All relativesofFamily Ww ere genotyped for 988delA:all affected individuals were homozygous for the mutation and all hearing individuals were heterozygous carrierso rw ild-type.
TMPRSS3 is at ransmembrane serine protease,w ith a trypsin-likep rotease domaine xtendingf romr esidues 217 -444 and conserveda ctives ites at histidine 257, aspartic acid 304 and serine 401. 5 Tr uncation at residue 357 would eliminate the activesite at serine 401 and much of the domain, abrogating TMPRSS3 serine protease activity.Asignalling pathway in the inner ear controlled by proteolytic cleavage includes activation by TMPRSS3 of the epithelials odium channel (ENaC). 16 Tr uncated TMPRSS3 of Family Wc ould not activate ENaC.
Disruptions of proteolysis areassociated with awide variety of genetic disorders, 17 but TMPRSS3 wast he first protease associated with hearing loss, demonstrated by mutations that defined DFNB8 and DFNB10. 5 The DFNB10 mutation, found in aP alestinian family,w as remarkable,consisting of the insertion of 18 complete b -satellite repeat monomers, which are normally present in mobile tandem arrays of up to several hundred kilobases (kb) on the shorta rmso fa crocentric chromosomes. The b -satellitemutation wasnot present in any probands in our series, nor in any hearing controls in our series. In additiont oa ll affected memberso fF amily W, three other probands in our series were homozygous for 988delA ¼ 357stop,o ne hearing control wasa lso heterozygous for this mutation.

Otoancorin
FamilyB Ri ncluded six individuals with prelingual, bilateral, moderate to severe hearing loss. Fivea ffected individuals were homozygous for markersD 16S3045 and TTA(21.603), which flank otoancorin. The sixth affected individual was homozygous at TTA(21.603) and heterozygous at D16S3045, possibly reflecting an ancestral recombination event ( Figure  1B). The lod score for linkage of hearing loss to the D16S3045 -TTA(21.603) haplotype was3 .49. We sequenced otoancorini nadeaf individual and identifiedm is-sense mutation 1067A . T, which leads to substitution of valine for aspartic acid at residue 356 (D356V). All six affected individuals were homozygous for D356Vand all hearing individuals were heterozygous for the mutationo rw ild-type.
Otoancorin wass hown to be associated with hearing loss as the result of as ingle mutation, ap resumptive splice variantI VS12(þ 2)T . C, identifiedi naPalestinian family. 7 Otoancorin IVS12(þ 2)T . Cd id not appear among any probands in our series, nor among any Palestinian hearing controls. Similarly,t he FamilyB R mutation D356V did not appeara mong any other probands in our series, but one control wasaheterozygous carrier of the allele.
Otoancorin is located at the interface between the apical surface of the sensoryepithelia and its overlying acellular gels, and is expressed only in the inner ear. 7 It remains attached to

Pendrin (SLC26A4)
FamilyYincluded four individuals withp relingual, bilateral, severe to profoundh earing loss.A ll were homozygous at markersD 7S496 and D7S2459, which flank pendrin ( Figure  1C). In FamilyY ,t he lod score for linkage of hearing loss to the D7S496-D7S2459 haplotype was2 .11, close to the maximum lod score possible in this family.W es equenced pendrin in an affected child from family Yand identified 716T . A in exon 6, which causes as ubstitution of aspartic acid for valine at amino acid 239 (V239D). All relativesw ere genotyped for this mutation: all four affected individuals were homozygous for V239D and all hearing individuals were either heterozygous or wild-type. Mutations in pendrin can cause either non-syndromic hearing loss or Pendred syndrome, 6 which, in addition to sensorineuralh earing loss,i ncludes thyroid enlargement (goitre) and temporal bone abnormalities of the inner ear, ranging from isolated enlargement of thev estibular aqueduct (EVA) to Mondini dysplasia-a complex malformation of the cochlears piral. 21 Therefore, we evaluatedt he temporalb ones of the affected children of Family Ybycomputed tomography (CT) scan. As Figure 2A indicates, the vestibular aqueduct is enlarged. There wasn oc linical evidence of thyroid abnormalities and serum thyroid-stimulating hormone and thyroxine concentrations in the four affected individuals were normal.
Pendrin is ac hloride -iodide transporter;m utations associated with Pendred syndromec an cause its retention in the endoplasmic reticulum. 22 In order to test the consequences of the V239D mis-sense mutation on intracellular localisation of pendrin, we evaluated trafficking of wild-type pendrin and of the V239D mutant protein in living cells using GFP chimeras. 22,23 As shown in Figure 2B,t he V239D mutant pendrin is retained in the endoplasmic reticulum, whereas the wild-type protein targets the plasma membrane. Retention of pendrin in the endoplasmic reticulum is a major mechanism for Pendred syndrome, 12 suggestingt hat V239Di sp robably thep athogenic mutationi nF amilyY .
We also developed am ethod for evaluating the severity of mis-sense mutations in pendrin, based on conservation across the large SLC26A family of solute carrier proteins and the growing number of known disease-associated pendrin missense mutations. 6,24 -28 We compared the severity of mis-sense changesi np endrin withn aturally occurring variationa cross all proteinsi nt he family.F igure 2C illustrates these comparisons as a' diversity plot', calibrated againstwild-type pendrin. For each amino acid site at which a pendrin mis-sense mutationh as been reported, we plotted the amino acid difference based on Grantham'sf ormula 29 for all naturally occurring amino acid differences in any SLC26A transporter.T hen we compared scores of pendrin mis-sensest os coreso fn aturally occurring variants. The missense mutationV 239Di saless conservatives ubstitution than anyn aturally occurring varianta tt he homologous site, and is among the least conservativec hangeso bserveda ta ny site in the SLC26A genef amily. Pendrin V239D did not occur in other Palestinian probands with hearing loss, nor among Palestinian controls; however, pendrin 716T . A (V239D) appeared in aT urkish brother and sister with prelingual hearing loss, hypothyroid and EVA. 30 The Palestinian and Tu rkish haplotypes harbouring 716A . T were identical for microsatellite markerss panning 262 kb,w hereas other haplotypes in this region in these families were diverse ( Figure 2D). Them utation mayh avea common ancestor,a lthough the families aren ot awaret hat they arer elated. The difference betweenn ormal thyroid function in thea ffected individuals in Family Yv ersus hypothyroidism in the affected individuals in the Tu rkish family is not surprising, givent he intra-familialv ariability of Pendred syndrome described for other mis-sense mutations. 26,31 In FamilyB F, the lod score for linkage of hearing loss to the D7S496-D7S2459 haplotype was1 .66, which wasn ot statistically significant but wasc lose to the maximum possible for this family.S equencing genomic DNA from Family BF revealed ad ifferent mutation in pendrin. Four children with prelingual, bilateral, severe to profoundh earing loss were homozygous at markersD 7S496 and D7S2459 but for different alleles than in Family Y ( Fig.1 D). We sequenced pendrin in an affected child from Family BF and identified 1001G . T ,asplice mutation in thel ast base pair of exon 8. All affected individuals in Family BF were homozygous for 1001G . T and all hearing individuals were either heterozygous for the mutationo rw ild-type.
Reverse transcriptase-PCR (RT-PCR) of the pendrin message from lymphoblast cDNA of ah omozygous child revealed aberrant splicing, leadingt oi nsertion of 41 bp of intron 8i nto the pendrin message and ap rematures topa t codon3 46. The region of pendrin homologoust oo ther sulphatet ransportersi ncludes the site of the truncation, suggesting that this mutation would abrogate sulphate transporter activity.T he Family BF mutation 1001G . T Figure 1. Novel mutations responsible for inherited deafness in four kindreds. In each pedigree,fi lled symbols represent individuals with severe, bilateral, prelingual hearing loss. The mutation found in each family is illustrated by as equence below the appropriate pedigree, with mutations indicated on the sequences by reda rrows. (a) In Family W, hearing loss is linked to markers flanking the serine protease TMPRSS3 on chromosome 21q22.3. Sequence of TMPRSS3 in affected members of the family revealed frameshift 988D A (357stop), which abrogates serine protease activity.A sp redicted by linkage data, all affected relatives in Family Ware homozygous for TMPRSS3.988D A and all unaffected relatives are heterozygous or wild-type.( b) In Family BR, hearing loss is linked to markers flanking otoancorin on chromosome 16p12.2. The sequence of otoancorin in affected members of the family revealed missense mutation 1067A . T (D356V), ah ighly non-conservative change that is likely to disrupt the otoancorin transmembrane structure. All affected relatives in Family BR arehomozygous for otoancorin 1067A . T (D356V) and all unaffected relatives areh eterozygous or wild-type. D356V is the first deafness-associated missense mutation reported in otoancorin. (c) In Family Y, hearing loss is linked to markers flanking pendrin on chromosome 7q31.1. The sequence of pendrin in affected members of the family revealed missense mutation 716T . A (V239D), which leads to cellular mislocalisation of pendrin protein (see Fig. 2). All affected relatives in Family Yare homozygous for pendrin 716T . A (V239D) and all unaffected relatives areheterozygous or wild-type.( d) In Family BF,h earing loss is also linked to markers flanking pendrin, but to different alleles than werelinked to the phenotype in Family Y. The sequence of pendrin in affected members of Family BF revealed 1001G . T .R T-PCR of pendrin from lymphoblasts of affected relatives of Family BF indicates that 1001G . T alters splicing, leading to insertion of 41 intronic base pairs into the pendrin message and ap remature stop at codon 346 (red box). By homology with other sulphate transporters, truncation at this site would abrogate sulphate transporter activity of the protein. All affected relatives in Family BF arehomozygous for pendrin 1001G . T and all unaffected relatives are heterozygous or wild-type.
has not been previously reported. Mutationo ft he adjacent base pair, 1001(þ 1)G . A ,h owever, which also leads to 346stop,i safounderm utationa mongE nglish families withP endred syndrome,p robably including those families in whom the syndrome waso riginally described. 24,32  There was no clinical indication of goitre and serum thyroid-stimulating hormone and thyroxine levels werenormal. (b) Intracellular localisation and trafficking of YFP-tagged mutant (V239D) and wild-type (PDS) pendrin, prepared as previously described. 12,23 Living COS7 cells were transiently transfected with YFP-V239D mutant pendrin (green) and with CFP-GPI (glycosylphosphatidylinositol [GPI] tagged with crimson fluorescent protein [CFP], red), which localises to the Golgi apparatus and the plasma membrane. 12 After incubation, cells werevisualised by confocal microscopy. 12 Merged confocal images (yellow) indicate that pendrin V239D is retained in the endoplasmic reticulum, whereas wild-type pendrin (PDS) colocalises with GPI to the Golgi apparatus and plasma membrane.( c) The predicted severity of V239D in Family Yw as compared with other diseaseassociated mis-sense mutations in pendrin (red diamonds) and to wild-type sequences of other human SLC26A anion transporters at homologous sites (blue symbols) using Grantham'samino acid difference formula. 29 Some,b ut not all, pendrin mis-sense mutations are more diverged from wild-type pendrin than are other SLC26A transporters. By this measure, V239D is among the most divergent mis-sense mutations of pendrin, particularly given the conservation of other SLC26A proteins at this site.( d) Palestinian and Tu rkish individuals with the 716A (239D) mutation share an extended haplotype of 126-260 kilobases flanking pendrin (yellow boxes), based on markers polymorphic among wild-type ( 716T)h aplotypes. Pendrin 716T . A has not been observed in any other Palestinian families with inherited hearing loss or Tu rkish individuals with Pendred syndrome, 30 suggesting that these two families mays harea recent common ancestor.

Discussion
Inherited hearing loss in Middle Easternp opulations is highly heterogeneous, both in the number of genes involved and in then umber of alleles at each gene. Of the ten families screened for linkage of deafness to all genes known to influence hearing loss, none carried any of the more than 100 previously identifiedalleles. All families with hearing loss who were wild-type for GJB2 harboured novela lleles,e ither in known genes (fourf amilies) or in genes yett ob ei dentified (six families).A lleles responsible for hearing loss in this population are individually very rare.
Allelich eterogeneity of recessive diseases in the Palestinian population is well documented. 2 As pointed out by Zlotogora, 2 the many unique mutations amongp atients with autosomal recessived iseases in this population reflect the high rate of de novo deleterious mutations occurring in all human genomes. 33 De novo recessive alleles are generally not detectable because the associatedp henotypes aren ot expressed in outbred populations. The appearance of multiple novel alleles of known deafness-related genesa lso suggests that hearing loss in this population is genetically similar to hearing loss worldwide,a nd that the phenotype is simply more common in this populationb ecause of consanguinity.
More than 50 yearsb efore the studies of Mendel, Darwin or Galton,J oseph Adams recognised the importance of isolated communities to the study of human disease,n oting that 'endemic peculiarities mayb ef ound in certain sequestered districts'. 34 Av eryl arge number of the discoveries of modern human genetics have been as the result of studies of such 'sequestered' populations. 35 Historical demographyofi solated populations mayl eadt ot he occurrence of population-specific alleles of relatively high frequency,s ucha st he ancient mutations responsible for diseases found in the Ashkenazi Jewish population, 36,37 and also many population-specific alleles that are individually rare,each present in only one afew extended families, as described here.
Given the availability of genomics tools, it is now possible to develop gene discoverys trategies best suited to the demographyo fe ach community.F or phenotypes with ah igh level of allele and locus heterogeneity,i dentifiedi ne xtended consanguineous families, we developed the following strategy.
(1) For anyg enew ithr elatively widespread mutations (eg GJB2), we sequenced all affected individuals in all families. It wasi mportant to fully sequence such genes, rather than only screening known mutations, because both old and new alleles are likely to appear. 4 It wasa lso important to sequence all affected individuals, not only probands, because more than one gene for hearing loss mights egregate in af amily.
(2) For families which are wild-type at the commonly mutant genes, we tested for linkage of the phenotype to all known disease genes by a' subgenome scan' consisting of markers intragenic or immediately flankinga ll genes known to be associated with the phenotype.B ecause there are al arge number of known genes for hearing loss,w efi rstg enotyped twos uch markersa te ach site,t hen added additional markers as necessary. (3) In each potentiallyl inked family,w ef ully sequenced the known genel inked to the phenotype in an affected relative and an obligate carrier.C learly,i tw as not sufficient to genotype only known disease alleles because it is likely that new families will harbour new alleles. Also, although in this project each novela llele wash omozygous in affected individuals, it is certainly possible that deaf individuals in some families will be compound heterozygotes. (4) We screened each mutation in all probands and in a small series (100) of Palestinian controls to determine allele frequencies, but mutations were individually too rare for casecontrol comparisonst oh avea dequate statistical powerf or epidemiological analyses. Therefore, to evaluate the consequenceo fm is-sense mutations, we developed and used both functional and bioinformatics approaches.
In conclusion, extended consanguineousk indreds prove extremely valuable for the identification of novelg enes for otherwise intractable,h ighly heterogeneous phenotypes. Of ten extended kindreds in our series evaluatedthus far, four families carried novelm utations in known genes and six families are likelyt oh arbourn ovel mutations in novelg enes for hearing loss. The rate of discovery of new genes related to anyp henotype will eventually reach an asymptote. The ratio of mutations in known versus unknown genes in the present study (4:6), however, suggests that in this cohorto f1 56 families, al arge number of novelg enes for hearing loss still await discovery.