Identities, frequencies and origins of TMC1 mutations causing DFNB7/B11 deafness in Pakistan

Non-syndromic deafness is genetically heterogeneous. We previously reported that mutations of transmembrane channel-like gene 1 (TMC1) cause non-syndromic recessive deafness at the DFNB7/B11 locus on chromosome 9q13-q21 in nine Pakistani families. The goal of this study was to define the identities, origins and frequencies of TMC1 mutations in an expanded cohort of 557 large Pakistani families segregating recessive deafness. We screened affected family members for homozygosity at short-tandem repeats flanking known autosomal recessive (DFNB) deafness loci, followed by TMC1 sequence analysis in families segregating deafness linked to DFNB7/B11. We identified 10 new families segregating DFNB7/B11 deafness and TMC1 mutations, including three novel alleles. Overall, 9 different TMC1 mutations account for deafness in 19 (3.4%) of the 557 Pakistani families. A single mutation, p.R34X, causes deafness in 10 (1.8%) of the families. Genotype analysis of p.R34X-linked markers indicates that it arose from a common founder. We also detected p.R34X among normal control samples of African-American and northern European origins, raising the possibility that p.R34X and other mutations of TMC1 are prevalent contributors to the genetic load of deafness across a variety of populations and continents.     

Mutation analysis of TMC1 identifies four new mutations and suggests an additional deafness gene at loci DFNA36 and DFNB7/11

Hearing loss is the most frequent sensorineural disorder affecting 1 in 1000 newborns. In more than half of these babies, the hearing loss is inherited. Hereditary hearing loss is a very heterogeneous trait with about 100 gene localizations and 44 gene identifications for non-syndromic hearing loss. Transmembrane channel-like gene 1 ( TMC1 ) has been identified as the disease-causing gene for autosomal dominant and autosomal recessive non-syndromic hearing loss at the DFNA36 and DFNB7/11 loci, respectively. To date, 2 dominant and 18 recessive TMC1 mutations have been reported as the cause of hearing loss in 34 families. In this report, we describe linkage to DFNA36 and DFNB7/11 in 1 family with dominant and 10 families with recessive non-syndromic sensorineural hearing loss. In addition, mutation analysis of TMC1 was performed in 51 familial Turkish patients with autosomal recessive hearing loss. TMC1 mutations were identified in seven of the families segregating recessive hearing loss. The pathogenic variants we found included two known mutations, c.100C>T and c.1165C>T, and four new mutations, c.2350C>T, c.776+1G>A, c.767delT and c.1166G>A. The absence of TMC1 mutations in the remaining six linked families implies the presence of mutations outside the coding region of this gene or alternatively at least one additional deafness-causing gene in this region. The analysis of copy number variations in TMC1 as well as DNA sequencing of 15 additional candidate genes did not reveal any proven pathogenic changes, leaving both hypotheses open.     

Four novel TMC1 (DFNB7/DFNB11) mutations in Turkish patients with congenital autosomal recessive nonsyndromic hearing loss

Mutations in the transmembrane channel-like gene 1 (TMC1) cause prelingual autosomal recessive (DFNB7/11) and postlingual progressive autosomal dominant (DFNA36) nonsyndromic hearing loss. To determine the genetic causes of autosomal recessive nonsyndromic hearing loss (ARNSHL) in the northeast and east of Turkey, 65 unrelated families without mutations in the protein coding region of the GJB2 (GJB2-negative) were analyzed. A genomewide scan for homozygosity and linkage analysis in one of these families revealed a 13.2 cM critical region between D9S273 and D9S153 at chromosome 9p13.2-q21.31 with a maximum two-point lod score of 4.00 at theta=0.0 for marker D9S175. TMC1 is in this critical region. Homozygosity screening with intragenic markers for TMC1 in the remaining 64 families suggested involvement of this gene in three additional families. Subsequent sequencing of TMC1 in these four families revealed four novel homozygous mutations, c.776A>G [p.Tyr259Cys], c.821C>T [p.Pro274Leu], c.1334G>A [p.Arg445His], and c.1083_1087delCAGAT [p.Arg362ProfrX6]. Our results indicate that TMC1 mutations account for at least 6% (4/65) of ARNSHL in GJB2-negative Turkish families from the northeast and east of Turkey.     

Involvement of DFNB59 mutations in autosomal recessive nonsyndromic hearing impairment

In a consanguineous Turkish family, a locus for autosomal recessive nonsyndromic hearing impairment (ARNSHI) was mapped to chromosome 2q31.1-2q33.1. Microsatellite marker analysis in the complete family determined the critical linkage interval that overlapped with DFNB27, for which the causative gene has not yet been identified, and DFNB59, a recently described auditory neuropathy caused by missense mutations in the DFNB59 gene. The 352-amino acid (aa) DFNB59 gene product pejvakin is present in hair cells, supporting cells, spiral ganglion cells, and the first three relays of the afferent auditory pathway. A novel homozygous nonsense mutation (c.499C>T; p.R167X) was detected in the DFNB59 gene, segregating with the deafness in the family. The mRNA derived from the mutant allele was found not to be degraded in lymphocytes, indicating that a truncated pejvakin protein of 166 aa may be present in the affected individuals. Screening of 67 index patients from additional consanguineous Turkish families with autosomal recessive hearing impairment revealed a homozygous missense mutation (c.547C>T; p.R183W) that segregates with the hearing impairment in one family. Furthermore, in a panel of 83 Dutch patients, two additional novel mutations (c.509_512delCACT; p.S170CfsX35 and c.731T>G; p.L244R), which were not present in ethnically matched controls, were found heterozygously. Together, our data indicate that also nonsense mutations in DFNB59 cause nonsyndromic hearing loss, but that mutations in DFNB59 are not a major cause of nonsyndromic hearing impairment in the Turkish and Dutch population.     

Identification of TMC1 as a relatively common cause for nonsyndromic hearing loss in the Saudi population

Hearing loss (HL) is the most common sensory disorder worldwide and genetic factors contribute to approximately half of congenital HL cases. HL is subject to extensive genetic heterogeneity, rendering molecular diagnosis difficult. Mutations of the transmembrane channel‐like 1 (TMC1) gene cause hearing defects in humans and mice. The precise function of TMC1 protein in the inner ear is unknown, although it is predicted to be involved in functional maturation of cochlear hair cells. TMC1 mutations result in autosomal recessive (DFNB7/11) and sometimes dominant (DFNA36) nonsyndromic HL. Mutations in TMC1 are responsible for a significant portion of HL, particularly in consanguineous populations. To evaluate the importance of TMC1 mutations in the Saudi population, we used a combination of autozygome‐guided candidate gene mutation analysis and targeted next generation sequencing in 366 families with HL previously shown to lack mutations in GJB2. We identified 12 families that carried five causative TMC1 mutations; including three novel (c.362+3A > G; c.758C > T [p.Ser253Phe]; c.1396_1398delACC [p.Asn466del]) and two reported mutations (c.100C > T [p.Arg34Ter]; c.1714G > A [p.Asp572Asn]). Each of the identified recessive mutation was classified as severe, by both age of onset and severity of HL. Similarly, consistent with the previously reported dominant variant p.Asp572Asn, the HL phenotype was progressive. Eight families in our cohort were found to share the pathogenic p.Arg34Ter mutation and linkage disequilibrium was observed between p.Arg34Ter and SNPs investigated. Our results indicate that TMC1 mutations account for about 3.3% (12/366) of Saudi HL cases and that the recurrent TMC1 mutation p.Arg34Ter is likely to be a founder mutation.     

A novel missense mutation in a C2 domain of OTOF results in autosomal recessive auditory neuropathy

Screening of 12 Turkish families with apparently autosomal recessive nonsyndromic sensorineural deafness without GJB2 and mtDNA m.1555A > G mutations for 11 previously mapped recessive deafness loci showed a family in which hearing loss cosegregated with the DFNB9 (OTOF) locus. Three affected children were later found to carry a novel homozygous c.3032T > C (p.Leu1011Pro) mutation in the OTOF gene. Both parents were heterozygous for the mutation. p.Leu1011Pro alters a conserved leucine residue in the C2D domain of otoferlin. Pure tone audiometry of the family showed severe to profound sensorineural hearing loss (with U-shape audiograms) in children, and normal hearing in the parents. Otoacoustic emissions and auditory brainstem response (ABR) suggested the presence of auditory neuropathy in affected individuals.     

Searching for evidence of DFNB2

Deafness is the most common form of sensory impairment in humans, affecting about 1 in 1,000 births in the United States. Of those cases with genetic etiology, approximately 80% are nonsyndromic and recessively inherited. Mutations in several unconventional myosins, members of a large superfamily of actin-associated molecular motors, have been found to cause hearing loss in both humans and mice. Mutations in the human unconventional Myosin VIIa (MYO7A), located at 11q13.5, are reported to be responsible for both syndromic and nonsyndromic deafness. MYO7A mutations are responsible for Usher syndrome type Ib, the most common genetic subtype of Usher I. Usher I is clinically characterized by congenital profound deafness, progressive retinal degeneration called retinitis pigmentosa (RP), and vestibular areflexia. Although a wide spectrum of MYO7A mutations have been identified in Usher Ib patients, four mutations have been reported to cause DFNB2, a recessive deafness without retinal degeneration, and one mutation has been implicated in a single case of dominant nonsyndromic hearing loss (DFNA11). Our study attempts to ascertain additional DFNB2 families to investigate the disparate nonsyndromic phenotype and alleged causative mutations. Data from both linkage and heterogeneity analyses on 36 selected autosomal recessive nonsyndromic deafness (RNSD) families, all previously excluded by mutational analysis from GJB2 (Cx26), the leading cause of nonsyndromic deafness, showed no evidence of DFNB2 within the sample. These negative results and the isolated reports of DFNB2 bring into question whether certain MYO7A mutations produce nonsyndromic recessive hearing loss.     

Mutations in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene cause autosomal recessive nonsyndromic hearing loss

In two large Turkish consanguineous families, a locus for autosomal recessive nonsyndromic hearing loss (ARNSHL) was mapped to chromosome 6p21.3 by genome-wide linkage analysis in an interval overlapping with the loci DFNB53 (COL11A2), DFNB66, and DFNB67. Fine mapping excluded DFNB53 and subsequently homozygous mutations were identified in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene, also named tetraspan membrane protein of hair cell stereocilia (TMHS) gene, which was recently shown to be mutated in the "hurry scurry" mouse and in two DFNB67-linked families from Pakistan. In one family, we found a homozygous one-base pair deletion, c.649delG (p.Glu216ArgfsX26) and in the other family we identified a homozygous transition c.494C>T (p.Thr165Met). Further screening of index patients from 96 Turkish ARNSHL families and 90 Dutch ARNSHL patients identified one additional Turkish family carrying the c.649delG mutation. Haplotype analysis revealed that the c.649delG mutation was located on a common haplotype in both families. Mutation screening of the LHFPL5 homologs LHFPL3 and LHFPL4 did not reveal any disease causing mutation. Our findings indicate that LHFPL5 is essential for normal function of the human cochlea.     

Allelic heterogeneity of SMARD1 at the IGHMBP2 locus

Spinal Muscular Atrophy with Respiratory Distress (SMARD) is an autosomal recessive disorder characterized by neurogenic muscular atrophy due to progressive anterior horn cell degeneration and early life-threatening respiratory failure ascribed to diaphragmatic dysfunction. SMARD is clinically and genetically heterogeneous. SMARD type 1 is characterized by onset of respiratory failure within the first weeks of life and has been ascribed to mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene on chromosome 11q13-q21. We report here the identification of nine novel IGHMBP2 mutations in five SMARD1 patients, including seven missense [ c.587A>G (p.Gln196Arg), c.647C>T (p.Pro216Leu), c.752T>C (p.Leu251Pro), c.1693G>A (p.Asp565Asn), c.1730T>C (p.Leu577Pro), c.1807C>T (p.Arg603Cys), c.1909C>T (p.Arg637Cys)] and two nonsense mutations [ c.1488C>A (p.Cys496X), c.2368C>T (p.Arg790X)]. Interestingly, 7 of 9 mutations occurred at highly conserved residues of the putative DNA helicase domain. The identification of novel IGHMBP2 variants will hopefully help diagnosing SMARD1 and contribute to a better functional characterization of IGHMBP2 gene product.     

Mutation spectrum of MYO7A and evaluation of a novel nonsyndromic deafness DFNB2 allele with residual function

Recessive mutations of MYO7A, encoding unconventional myosin VIIA, can cause either a deaf-blindness syndrome (type 1 Usher syndrome; USH1B) or nonsyndromic deafness (DFNB2). In our study, deafness segregating as a recessive trait in 24 consanguineous families showed linkage to markers for the DFNB2/USH1B locus on chromosome 11q13.5. A total of 23 of these families segregate USH1 due to 17 homozygous mutant MYO7A alleles, of which 14 are novel. One family segregated nonsyndromic hearing loss DFNB2 due to a novel three-nucleotide deletion in an exon of MYO7A (p.E1716del) encoding a region of the tail domain. We hypothesized that DFNB2 alleles of MYO7A have residual myosin VIIA. To address this question we investigated the effects of several mutant alleles by making green fluorescent protein (GFP) tagged cDNA expression constructs containing engineered mutations of mouse Myo7a at codons equivalent to pathogenic USH1B and DFNB2 alleles of human MYO7A. We show that in transfected mouse hair cells an USH1B mutant GFP-myosin VIIa does not localize properly to inner ear hair cell stereocilia. However, a GFP-myosin VIIa protein engineered to have an equivalent DFNB2 mutation to p.E1716del localizes correctly in transfected mouse hair cells. This finding is consistent with the hypothesis that p.E1716del causes a less severe phenotype (DFNB2) than the USH1B-associated alleles because the resulting protein retains some degree of normal function.     

Low frequency of deafness-associated GJB2 variants in Kenya and Sudan and novel GJB2 variants

A large proportion of non-syndromic autosomal recessive deafness (NSARD) in many populations is caused by variants of the GJB2 gene. Here, the frequency of GJB2 variants was studied in 406 and 183 apparently unrelated children from Kenya and Sudan, respectively, with mostly severe to profound non-syndromic deafness. Nine (2.2 %) Kenyan and 12 (6.6 %) of the Sudanese children only were carriers of variants within the coding sequence of the GJB2 gene. Variants in the 5'-adjacent region were detected in further 115 individuals. A total of 10 novel variants was recognized, among them four variants in the adjacent 5'-region of the GJB2 coding exon 2 (g.3318-6T>A, g.3318-15C>T, g.3318-34C>T, g.3318-35T>G), a 6 base-pair deletion (g.3455_3460del [p.Asp46_Gln48delinsGlu]), a variant leading to a stop codon (g.3512C>A [p.Tyr65X]), synonymous variants (g.3395C>T [p.Thr26], g.3503C>T [p.Asn62], g.3627A>C [p.Arg104]), and one non-synonymous variant (g.3816C>A [p.Val167Met]). In addition, the previously described variants g.3352delG (commonly designated 30delG or 35 delG), g.3426G>A [p.Val37Ile], g.3697G>A [p.Arg127His], g.3774G>A [p.Val153Ile], and g.3795G>A [p.Gly160Ser] were identified. With the exception of g.3318-34C>T and g.3352delG, all variants occurred heterozygously. For most of the variants identified in the Kenyan and Sudanese study population, a causative association with NSARD appears to be unlikely. Compared to many other ethnic groups, deafness-associated variants of the coding region of GJB2 are rare in Sudan and Kenya, suggesting a role of other genetic, or epigenetic factors as a cause for deafness in these countries.     

NDP gene mutations in 14 French families with Norrie disease

Norrie disease is a rare X-inked recessive condition characterized by congenital blindness and occasionally deafness and mental retardation in males. This disease has been ascribed to mutations in the NDP gene on chromosome Xp11.1. Previous investigations of the NDP gene have identified largely sixty disease-causing sequence variants. Here, we report on ten different NDP gene allelic variants in fourteen of a series of 21 families fulfilling inclusion criteria. Two alterations were intragenic deletions and eight were nucleotide substitutions or splicing variants, six of them being hitherto unreported, namely c.112C>T (p.Arg38Cys), c.129C>G (p.His43Gln), c.133G>A (p.Val45Met), c.268C>T (p.Arg90Cys), c.382T>C (p.Cys128Arg), c.23479-1G>C (unknown). No NDP gene sequence variant was found in seven of the 21 families. This observation raises the issue of misdiagnosis, phenocopies, or existence of other X-linked or autosomal genes, the mutations of which would mimic the Norrie disease phenotype.     

Novel SLC4A11 mutations in patients with recessive congenital hereditary endothelial dystrophy (CHED2). Mutation in brief #958. Online

Autosomal recessive congenital hereditary endothelial dystrophy (CHED2) is a severe and rare corneal disorder that presents at birth or shortly thereafter, characterized by corneal opacification and nystagmus. Recently the gene for CHED2 was identified and seven different mutations in the SLC4A11 gene were reported. Here, we report seven novel mutations and two previously identified mutations in families from India and the United Kingdom with recessive CHED. The novel changes include two nonsense (p.Trp240X; p.Gln800X) three missense (p.Glu143Lys; p.Cys386Arg; p.Arg755Trp) and two splice site mutations (c.2240+1G>A; c.2437-1G>A). Interestingly, the c.2398C>T (p.Gln800X) and c.2437-1G>A identified in two affected siblings represent the first compound heterozygous mutations in the SLC4A11 gene.     

Identification of three novel hearing loss mouse strains with mutations in the Tmc1 gene

We report the identification of three new mouse models, baringo, nice, and stitch, with recessively inherited sensorineural deafness due to novel mutations in the transmembrane channel-like gene 1 (Tmc1). These strains were generated by N-ethyl-N-nitrosourea mutagenesis. DNA sequence analysis revealed changes in c.545A>G, c.1345T>C, and c.1661G>T, causing p.Y182C, p.Y449H, and p.W554L amino acid substitutions in baringo, nice, and stitch mutants, respectively. The mutations affect amino acid residues that are evolutionarily conserved across species. Similar to the previously reported Beethoven Tmc1 mutant, both p.Y182C and p.W554L are located outside a predicted transmembrane domain, whereas the p.Y449H mutation resides in the predicted transmembrane domain 4. Homozygous stitch-mutant mice have severe hearing loss at the age of 4 weeks and are deaf by the age of 8 weeks, whereas both baringo and nice mutants are profoundly deaf at the age of 4 weeks. None of the strains displays signs of vestibular dysfunction. Scanning electron microscopy revealed degeneration of outer hair cells in the basal region of baringo, nice, and stitch mutants. Immunolocalization studies revealed expression of TMC1 protein in the hair cells, spiral ganglion neurons, supporting cells, and stria ligament in the inner ear. Reduced levels of TMC1 protein were observed in the spiral ligament of mutants when compared with wild-type animals. These three allelic mutants provide valuable models for studying nonsyndromic recessive sensorineural hearing loss (DFNB7/11) in humans.     

A novel nonsense mutation in MYO6 is associated with progressive nonsyndromic hearing loss in a Danish DFNA22 family

Autosomal dominant inheritance is described in about 20% of all nonsyndromic hearing loss with currently 54 distinct loci (DFNA1-54), and >20 different genes identified. Seven different unconventional myosin genes are involved in ten different types of syndromic and nonsyndromic hearing loss with different patterns of inheritance: MYO7A in DFNA11/DFNB2/USH1B, MYH9 in DFNA17, MYH14 in DFNA4, MYO6 in DFNA22/DFNB37, MYO3A in DFNB30, MYO1A in DFNA48, and MYO15A in DFNB3. Two missense mutations in MYO6 (p.C442Y and p.H246R) have been characterized in families of Italian and American Caucasian extraction with autosomal dominant hearing loss, respectively, and the latter was associated with cardiomyopathy in some patients. Three Pakistani families had homozygosity for three MYO6 mutations (c.36insT, p.R1166X, and p.E216V, respectively), and was in one instance associated with retinal degeneration. In the present study, we linked autosomal dominant hearing loss in a large Danish family to a 38.9 Mb interval overlapping with the DFNA22/DFNB37 locus on chromosome 6q13. A novel nonsense mutation in MYO6 exon 25 (c.2545C > T; p.R849X) was identified in the family. The mutation co-segregated with the disease and the mutant allele is predicted to encode a truncated protein lacking the coiled-coil and globular tail domains. These domains are hypothesized to be essential for targeting myosin VI to its cellular compartments. No other system was involved indicating nonsyndromic loss. In conclusion, a novel nonsense MYO6 mutation causes post-lingual, slowly progressive autosomal dominant nonsyndromic moderate to severe hearing loss in a Danish family.     

Mutations in the TMPRSS3 gene are a rare cause of childhood nonsyndromic deafness in Caucasian patients

Two loci for nonsyndromic recessive deafness located on chromosome 21q22.3 have previously been reported, DFNB8 and DFNB10. Recently a gene which encodes a transmembrane serine protease, TMPRSS3 or ECHOS1, was found to be responsible for both the DFNB8 and DFNB10 phenotypes. To determine the contribution of TMPRSS3 mutations in the general congenital/childhood nonsyndromic deaf population we performed mutation analysis of the TMPRSS3 gene in 448 unrelated deaf patients from Spain, Italy, Greece, and Australia who did not have the common 35delG GJB2 mutation. From the 896 chromosomes studied we identified two novel pathogenic mutations accounting for four mutant alleles and at least 16 nonpathogenic sequence variants. The pathogenic mutations were a 1-bp deletion resulting in a frameshift and an amino acid substitution in the LDLRA domain of TMPRSS3. From this and another study we estimate the frequency of TMPRSS3 mutations in our sample as 0.45%, and approximately 0.38% in the general Caucasian childhood deaf population. However, TMPRSS3 is still an important contributor to genetic deafness in populations with large consanguineous families.     

Mapping of a new autosomal recessive nonsyndromic hearing loss locus (DFNB32) to chromosome 1p13.3-22.1

Approximately 80% of the hereditary hearing loss is nonsyndromic. Isolated deafness is the most genetically heterogeneous trait. We have ascertained 10 individuals from a large consanguineous Tunisian family with congenital profound autosomal recessive deafness. All affected individuals are otherwise healthy. Genotype analysis excluded linkage to known recessive deafness loci in this family. Following a genome wide screening, a linkage was detected only with locus D1S206 on chromosome 1, thereby defining a novel deafness locus, DFNB32. In order to confirm linkage and for fine mapping the genetic interval, 12 individuals belonging to this family were added and 19 microsatellite markers were tested. A maximum two-point lodscore of 4.96 was obtained at a new polymorphic marker D1S21401. Haplotype analysis defined a 16 Mb critical region between D1S2868 and afmb014zb9. The interval of DFNB32 locus overlap with DFNA37 locus and the Marshall and Stickler syndromes locus. The entire coding region of COL11A1, responsible of the later syndromes, was screened and no mutation was observed. Towards the identification of the DFNB32 gene, a search on the Human Cochlear cDNA Library and EST Database was done. The genes corresponding to the ESTs found in the DFNB32 interval are being screened for deafness-causing mutations.     

Identification of ten novel mutations in patients with eIF2B-related disorders

Autosomal recessive inherited mutations in each of the five eukaryotic initiation factor 2B (eIF2B) subunits are known to cause white matter abnormalities with a wide continuum of clinical signs and severity leading to the concept of eIF2B-related disorders. The clinical spectrum extends from fatal infantile forms to adult forms with slow or absent neurological deterioration. In this study 15 well-characterised patients with the classical form of leukoencephalopathy with vanishing white matter (VWM) or with phenotypic variants like ovarioleukodystrophy were investigated for mutations in the genes EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5 encoding eIF2B. We identified one novel nonsense mutation (EIF2B4, c.625C>T, p.Arg209X), one novel frameshift mutation (EIF2B5, c.453_454del, p.Tyr152fsX12), eight novel missense muations (EIF2B1, c.547G>T, p.Val183Phe; EIF2B2, c. 586C>T, p.Pro196Ser; EIF2B4, c.806T>G, p.Leu269Arg; EIF2B5, c.203T>C, p.Leu68Ser; EIF2B5, c.220G>A, p.Ala74Thr; EIF2B5, c.805C>G, p.Arg269Gly; EIF2B5, c.929G>T, p.Cys310Phe; EIF2B5, c.1003T>C, p.Cys335Arg), and eight previously described alterations.     

A novel autosomal recessive non-syndromic deafness locus (DFNB35) maps to 14q24.1-14q24.3 in large consanguineous kindred from Pakistan

Autosomal recessive nonsyndromic deafness is one of the most frequent forms of inherited hearing impairment. Over 30 autosomal recessive nonsyndromic hearing loss loci have been mapped, and 15 genes have been isolated. Of the over 30 reported autosomal recessive nonsyndromic hearing loss (NSHL) loci, the typical phenotype is prelingual non-progressive severe to profound hearing loss with the exception of DFNB8, which displays postlingual onset and DFNB13, which is progressive. In this report we describe a large inbred kindred from a remote area of Pakistan, comprising six generations and segregating autosomal recessive nonsyndromic prelingual deafness. DNA samples from 24 individuals were used for genome wide screen and fine mapping. Linkage analysis indicates that in this family the NSHL locus, (DFNB35) maps to a 17.54 cM region on chromosome 14 flanked by markers D14S57 and D14S59. Examination of haplotypes reveals a region that is homozygous for 11.75 cM spanning between markers D14S588 and D14S59. A maximum two-point LOD score of 5.3 and multipoint LOD score of 7.6 was obtained at marker D14S53. The interval for DFNB35 does not overlap with the regions for DFNA9, DFNA23 or DFNB5.     

Molecular and clinical studies of X-linked deafness among Pakistani families

There are 68 sex-linked syndromes that include hearing loss as one feature and five sex-linked nonsyndromic deafness loci listed in the OMIM database. The possibility of additional such sex-linked loci was explored by ascertaining three unrelated Pakistani families (PKDF536, PKDF1132 and PKDF740) segregating X-linked recessive deafness. Sequence analysis of POU3F4 (DFN3) in affected members of families PKDF536 and PKDF1132 revealed two novel nonsense mutations, p.Q136X and p.W114X, respectively. Family PKDF740 is segregating congenital blindness, mild-to-profound progressive hearing loss that is characteristic of Norrie disease (MIM#310600). Sequence analysis of NDP among affected members of this family revealed a novel single nucleotide deletion c.49delG causing a frameshift and premature truncation (p.V17fsX1) of the encoded protein. These mutations were not found in 150 normal DNA samples. Identification of pathogenic alleles causing X-linked recessive deafness will improve molecular diagnosis, genetic counseling and molecular epidemiology of hearing loss among Pakistanis.