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.     

Identities and frequencies of mutations of the otoferlin gene (OTOF) causing DFNB9 deafness in Pakistan

Mutations in OTOF , encoding otoferlin, cause non-syndromic recessive hearing loss. The goal of our study was to define the identities and frequencies of OTOF mutations in a model population. We screened a cohort of 557 large consanguineous Pakistani families segregating recessive, severe-to-profound, prelingual-onset deafness for linkage to DFNB9 . There were 13 families segregating deafness consistent with linkage to markers for DFNB9 . We analyzed the genomic nucleotide sequence of OTOF and detected probable pathogenic sequence variants among all 13 families. These include the previously reported nonsense mutation p.R708X and 10 novel variants: 3 nonsense mutations (p.R425X, p.W536X, and p.Y1603X), 1 frameshift (c.1103_1104delinsC), 1 single amino acid deletion (p.E766del) and 5 missense substitutions of conserved residues (p.L573R, p.A1090E, p.E1733K, p.R1856Q and p.R1939W). OTOF mutations thus account for deafness in 13 (2.3%) of 557 Pakistani families. This overall prevalence is similar, but the mutation spectrum is different from those for Western populations. In addition, we demonstrate the existence of an alternative splice isoform of OTOF expressed in the human cochlea. This isoform must be required for human hearing because it encodes a unique alternative C-terminus affected by some DFNB9 mutations.     

DFNB74, a novel autosomal recessive nonsyndromic hearing impairment locus on chromosome 12q14.2-q15

Autosomal recessive nonsyndromic hearing impairment (ARNSHI) segregating in three unrelated, large consanguineous Pakistani families (PKDF528, PKDF859 and PKDF326) is linked to markers on chromosome 12q14.2-q15. This novel locus is designated DFNB74 . Maximum two-point limit of detection (LOD) scores of 5.6, 5.7 and 2.6 were estimated for markers D 12 S 313, D 12 S 83 and D 12 S 75 at θ = 0 for recessive deafness segregating in these three families. Haplotype analyses identified a critical linkage interval of 5.35 cM (5.36 Mb) defined by D 12 S 329 at 74.58 cM and D 12 S 313 at 79.93 cM. DFNB74 is the second ARNSHI locus mapped to chromosome 12, but the physical intervals do not overlap with one another. A locus contributing to the early onset, rapidly progressing hearing loss of A/J mice ( ahl4 , age-related hearing loss 4) was reported to map to chromosome 10 in a region of conserved synteny to DFNB74 , suggesting that ahl4 and DFNB74 may be due to mutations of the same gene in these two species.     

Mutational spectrum of MYO15A: the large N-terminal extension of myosin XVA is required for hearing

Human MYO15A is located on chromosome 17p11.2, has 66 exons and encodes unconventional myosin XVA. Recessive mutations of MYO15A are associated with profound, nonsyndromic hearing loss DFNB3 in humans, and deafness and circling behavior in shaker 2 mice. In the inner ear, this motor protein is necessary for the development of hair cell stereocilia, which are actin-filled projections on the apical surface and the site of mechanotransduction of sound. The longest isoform of myosin XVA has 3,530 amino acid residues. Two isoform classes of MYO15A are distinguished by the presence or absence of 1,203 residues preceding the motor domain encoded by alternatively-spliced exon 2. It is not known whether this large N-terminal extension of myosin XVA is functionally necessary for hearing. We ascertained approximately 600 consanguineous families segregating hereditary hearing loss as a recessive trait and found evidence of linkage of markers at the DFNB3 locus to hearing loss in 38 of these families ascertained in Pakistan (n=30), India (n=6), and Turkey (n=2). In this study, we describe 16 novel recessive mutations of MYO15A associated with severe to profound hearing loss segregating in 20 of these DFNB3-linked families. Importantly, two homozygous mutant alleles-c.3313G>T (p.E1105X) and c.3334delG (p.G1112fsX1124) of MYO15A-located in exon 2 are associated with severe to profound hearing loss segregating in two families. These data demonstrate that isoform 1, containing the large N-terminal extension, is also necessary for normal hearing.     

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.     

DFNB79: reincarnation of a nonsyndromic deafness locus on chromosome 9q34.3

Genetic analysis of an inbred Pakistani family PKDF280, segregating prelingual severe to profound sensorineural hearing loss, provided evidence for a DFNB locus on human chromosome 9q34.3. Co-segregation of the deafness trait with marker D9SH159 was determined by a two-point linkage analysis (LOD score 9.43 at θ=0). Two additional large families, PKDF517 and PKDF741, co-segregate recessive deafness with markers linked to the same interval. Haplotype analyses of these three families refined the interval to 3.84 Mb defined by D9S1818 (centromeric) and D9SH6 (telomeric). This interval overlaps with the previously reported DFNB33 locus whose chromosomal map position has been recently revised and assigned to a new position on chromosome 10p11.23–q21.1. The nonsyndromic deafness locus on chromosome 9q segregating in family PKDF280 was designated DFNB79. We are currently screening the 113 candidate DFNB79 genes for mutations and have excluded CACNA1B, EDF1, PTGDS, EHMT1, QSOX2, NOTCH1, MIR126 and MIR602.     

Novel TMC1 structural and splice variants associated with congenital nonsyndromic deafness in a Sudanese pedigree

Mutations of the transmembrane channel-like gene 1 (TMC1) have been shown to cause autosomal dominant and recessive forms of congenital nonsyndromic deafness linked to the loci DFNA36 and DFNB7/B11, respectively. In a Sudanese pedigree affected by an apparently recessive form of nonsyndromic deafness, we performed a linkage analysis using markers covering the deafness loci DFNB1 - DFNB30. A two-point LOD score of 3.08 was obtained at marker position D9S1876, located within the first intron of the TMC1 gene at DFNB7/B11. By DNA sequencing of TMC1 exons 3-22, we identified the structural variant c.1165C>T in exon 13, leading to the stop codon p.Arg389X, and the splice-site variant c.19+5G>A, independently segregating with the deafness phenotype. The c.1165C>T [p.Arg389X] mutation was also observed in four out of 243 unrelated deaf Sudanese individuals, but none of the mutations was found among 292 normal hearing controls. The finding of TMC1 mutations contributing to deafness in Sudan confirms and extends previous reports on the role of TMC1 in recessive nonsyndromic deafness and shows that deafness-causing TMC1 mutations may occur in various ethnic groups.     

Novel missense mutations of TMPRSS3 in two consanguineous Tunisian families with non‐syndromic autosomal recessive deafness

Recently the TMPRSS3 gene, which encodes a transmembrane serine protease, was found to be responsible for two non‐syndromic recessive deafness loci located on human chromosome 21q22.3, DFNB8 and DFNB10. We found evidence for linkage to the DFNB8/10 locus in two unrelated consanguineous Tunisian families segregating congenital autosomal recessive sensorineural deafness. The audiometric tests showed a loss of hearing greater than 70 dB, in all affected individuals of both families. Mutation screening of TMPRSS3 revealed two novel missense mutations, W251C and P404L, altering highly conserved amino acids of the serine protease domain. Both mutations were not found in 200 control Tunisian chromosomes. The detection of naturally‐occurring TMPRSS3 missense mutations in deafness families identifies functionally important amino acids. Comparative protein modeling of the TMPRSS3 protease domain predicted that W251C might lead to a structural rearrangement affecting the active site H257 and that P404L might alter the geometry of the active site loop and therefore affect the serine protease activity. Hum Mutat 18:101–108, 2001. © 2001 Wiley‐Liss, Inc.     

Identification of three novel TECTA mutations in Iranian families with autosomal recessive nonsyndromic hearing impairment at the DFNB21 locus

Forty-five consanguineous Iranian families segregating autosomal recessive nonsyndromic hearing loss (ARNSHL) and negative for mutations at the DFNB1 locus were screened for allele segregation consistent with homozygosity by descent (HBD) at the DFNB21 locus. In three families demonstrating HBD at this locus, mutation screening of TECTA led to the identification of three novel homozygous mutations: one frameshift mutation (266delT), a transversion of a cytosine to an adenine (5,211C > A) leading to a stop codon, and a 9.6 kb deletion removing exon 10. In total, six mutations in TECTA have now been described in families segregating ARNSHL. All of these mutations are inactivating and produce a similar phenotype that is characterized by moderate-to-severe hearing loss across frequencies with a mid frequency dip. The truncating nature of these mutations is consistent with loss-of-function, and therefore the existing TECTA knockout mouse mutant represents a good model in which to study DFNB21-related deafness.     

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.     

Borate transporter SLC4A11 mutations cause both Harboyan syndrome and non-syndromic corneal endothelial dystrophy

Harboyan syndrome, or corneal dystrophy and perceptive deafness (CDPD), consists of congenital corneal endothelial dystrophy and progressive perceptive deafness, and is transmitted as an autosomal recessive trait. CDPD and autosomal recessive, non-syndromic congenital hereditary endothelial corneal dystrophy (CHED2) both map at overlapping loci at 20p13, and mutations of SLC4A11 were reported recently in CHED2. A genotype study on six families with CDPD and on one family with either CHED or CDPD, from various ethnic backgrounds (in the seventh family, hearing loss could not be assessed because of the proband's young age), is reported here. Novel SLC4A11 mutations were found in all patients. Why some mutations cause hearing loss in addition to corneal dystrophy is presently unclear. These findings extend the implication of the SLC4A11 borate transporter beyond corneal dystrophy to perceptive deafness.     

Screening of families with autosomal recessive non-syndromic hearing impairment (ARNSHI) for mutations in GJB2 gene: Indian scenario

Several studies have reported that mutations in the GJB2 gene (coding for connexin26) are a common cause of recessive non-syndromic hearing impairment. A GJB2 mutant allele, 35delG, has been found to have a high prevalence in most ethnic groups. Though mutations in the GJB2 gene have been shown to cause autosomal recessive deafness in Indian families, the frequencies of the various mutations are still unknown. In the present study, we analyzed 45 Indian families belonging to three different states, namely, Karnataka, Tamil Nadu, and Delhi with non-syndromic hearing impairment and an apparently autosomal recessive mode of inheritance. All the families were initially screened for three mutations (W24X, W77X, and Q124X) by using allele-specific PCR primers; mutations were confirmed by DNA sequencing. Families that were heterozygous or negative for tested mutations of the GJB2 gene were sequenced directly to identify the complementary mutation and other mutations in GJB2. Four families were homozygous for W24X, constituting around 8.8%. In two families, the affected individuals were compound heterozygotes for W24X; one family (DKB16) carried 35delG with W24X while the other family (DKB7) carried R143W with W24X. We suggest that W24X is a common allele among the mutations screened, causing autosomal recessive non-syndromic hearing impairment (ARNSHI) in the Indian population.     

Mutations in connexin31 underlie recessive as well as dominant non-syndromic hearing loss

Mutations in the GJB3 gene encoding connexin31 (Cx31) can cause a dominant non-syndromic form of hearing loss (DFNA2). To determine whether mutations at this locus can also cause recessive non-syndromic deafness, we screened 25 Chinese families with recessive deafness and identified in two families affected individuals who were compound heterozygotes for Cx31 mutations. The three affected individuals in the two families were born to non-consanguineous parents and had an early onset bilateral sensorineural hearing loss. In both families, differing SSCP patterns were observed in affected and unaffected individuals. Sequence analysis in both families demonstrated an in-frame 3 bp deletion (423-425delATT) in one allele, which leads to the loss of an isoleucine residue at codon 141, and a 423A-->G transversion in the other allele, which creates an Ile-->Val substitution at codon 141 (I141V). Neither of these two mutations was detected in DNA from 100 unrelated control subjects. The altered isoleucine residue lies within the third conserved alpha-helical transmembrane domain (M3), which is critical for the formation of the wall of the gap junction pore. Both the deletion of the isoleucine residue 141 and its substitution to valine in the two families could alter the structure of M3, and impair the function of the gap junction. The present data demonstrate that, like mutations in connexin26, mutations in Cx31 can lead to both recessive and dominant forms of non-syndromic deafness.     

W44C mutation in the connexin 26 gene associated with dominant non-syndromic deafness

Although more than 50% of recessive non-syndromic deafness is attributed to mutations in the connexin 26 (Cx26) gene, only a few reported families have shown dominant transmission of the trait. The W44C mutation was originally reported in two families from the same geographic region of France, which exhibited dominant non-syndromic hearing loss. In this report, we describe a third family with early-onset severe-to-profound non-syndromic hearing loss segregating with the W44C mutation. Our observation places W44C among recurrent mutations in the Cx26 gene and emphasizes the importance of screening for this as well as other Cx26 mutations in autosomal dominant families.     

Two Iranian families with a novel mutation in GJB2 causing autosomal dominant nonsyndromic hearing loss

Mutations in GJB2, encoding connexin 26 (Cx26), cause both autosomal dominant and autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNA3 and DFNB1 loci, respectively. Most of the over 100 described GJB2 mutations cause ARNSHL. Only a minority has been associated with autosomal dominant hearing loss. In this study, we present two families with autosomal dominant nonsyndromic hearing loss caused by a novel mutation in GJB2 (p.Asp46Asn). Both families were ascertained from the same village in northern Iran consistent with a founder effect. This finding implicates the D46N missense mutation in Cx26 as a common cause of deafness in this part of Iran mandating mutation screening of GJB2 for D46N in all persons with hearing loss who originate from this geographic region.     

Novel missense mutations in MYO7A underlying postlingual high- or low-frequency non-syndromic hearing impairment in two large families from China

The myosin VIIA (MYO7A) gene encodes a protein classified as an unconventional myosin. Mutations within MYO7A can lead to both syndromic and non-syndromic hearing impairment in humans. Among different mutations reported in MYO7A, only five led to non-syndromic sensorineural deafness autosomal dominant type 11 (DFNA11). Here, we present the clinical, genetic and molecular characteristics of two large Chinese DFNA11 families with either high- or low-frequency hearing loss. Affected individuals of family DX-J033 have a sloping audiogram at young ages with high frequency are most affected. With increasing age, all test frequencies are affected. Affected members of family HB-S037 present with an ascending audiogram affecting low frequencies at young ages, and then all frequencies are involved with increasing age. Genome-wide linkage analysis mapped the disease loci within the DFNA11 interval in both families. DNA sequencing of MYO7A revealed two novel nucleotide variations, c.652G > A (p.D218N) and c.2011G > A (p.G671S), in the two families. It is for the first time that the mutations identified in MYO7A in the present study are being implicated in DFNA11 in a Chinese population. For the first time, we tested electrocochleography (ECochG) in a DFNA11 family with low-frequency hearing loss. We speculate that the low-frequency sensorineural hearing loss in this DFNA11 family was not associated with endolymphatic hydrops.     

Mutations of ESPN cause autosomal recessive deafness and vestibular dysfunction

We mapped a human deafness locus DFNB36 to chromosome 1p36.3 in two consanguineous families segregating recessively inherited deafness and vestibular areflexia. This phenotype co-segregates with either of two frameshift mutations, 1988delAGAG and 2469delGTCA, in ESPN, which encodes a calcium-insensitive actin-bundling protein called espin. A recessive mutation of ESPN is known to cause hearing loss and vestibular dysfunction in the jerker mouse. Our results establish espin as an essential protein for hearing and vestibular function in humans. The abnormal vestibular phenotype associated with ESPN mutations will be a useful clinical marker for refining the differential diagnosis of non-syndromic deafness.     

Autoimmune disease in a DFNA6/14/38 family carrying a novel missense mutation in WFS1

Most familial cases of autosomal dominant low frequency sensorineural hearing loss (LFSNHL) are attributable to mutations in the wolframin syndrome 1 (WFS1) gene at the DFNA6/14/38 locus. WFS1 mutations at this locus were first described in 2001 in six families segregating LFSNHL that was non-progressive below 2,000 Hz; the causative mutations all clustered in the C-terminal domain of the wolframin protein. Mutations in WFS1 also cause Wolfram syndrome (WS), an autosomal recessive neurodegenerative disorder defined by diabetes mellitus, optic atrophy and often deafness, while numerous single nucleotide polymorphisms (SNPs) in WFS1 have been associated with increased risk for diabetes mellitus, psychiatric illnesses and Parkinson disease. This study was conducted in an American family segregating autosomal dominant LFSNHL. Two hearing impaired family members also had autoimmune diseases-Graves disease (GD) and Crohn disease (CD). Based on the low frequency audioprofile, mutation screening of WFS1 was completed and a novel missense mutation (c.2576G --> A) that results in an arginine-to-glutamine substitution (p.R859Q) was identified in the C-terminal domain of the wolframin protein where most LFSNHL-causing mutations cluster. The family member with GD also carried polymorphisms in WFS1 that have been associated with other autoimmune diseases.     

Molecular analysis of the PDS gene in Pendred syndrome

Pendred syndrome is an autosomal recessive disorder characterized by the association between sensorineural hearing loss and thyroid swelling or goitre and is likely to be the most common form of syndromic deafness. Within the thyroid gland of affected individuals, iodide is incompletely organified with variable effects upon thyroid hormone biosynthesis, whilst the molecular basis of the hearing loss is unknown. The PDS gene has been identified by positional cloning of chromosome 7q31, within the Pendred syndrome critical linkage interval and encodes for a putative ion transporter called pendrin. We have investigated a cohort of 56 kindreds, all with features suggestive of a diagnosis of Pendred syndrome. Molecular analysis of the PDS gene identified 47 of the 60 (78%) mutant alleles in 31 families (includes three homozygous consanguineous kindreds and one extended family segregating three mutant alleles). Moreover, four recurrent mutations accounted for 35 (74%) of PDS disease chromosomes detected and haplotype analysis would favour common founders rather than mutational hotspots within the PDS gene. Whilst these findings demonstrate molecular heterogeneity for PDS mutations associated with Pendred syndrome, this study would support the use of molecular analysis of the PDS gene in the assessment of families with congenital hearing loss.      

A founder TMIE mutation is a frequent cause of hearing loss in southeastern Anatolia

Using Affymetrix 10K arrays, we searched for regions of homozygosity in 51 Turkish families including at least three members with either congenital or prelingual autosomal recessive non-syndromic sensorineural hearing loss (ARNSSNHL), and identified four families whose deafness mapped to the DFNB6 locus on 3p21 containing the TMIE gene. Mutation analysis revealed the p.R84W mutation in all four families. Screening of this mutation in 254 families with ARNSSNHL, without GJB2 mutations, revealed four additional affected families. A novel mutation was found in a non-complementary marriage between a deaf couple who were homozygous for p.R84W and p.W57X, respectively with two affected children who were compound heterozygotes. Six of the TMIE families originated from southeastern Anatolia, making p.R84W a common cause of hearing loss in that region with a relative frequency of 10.3% (95% CI is 2.5–18.1%). The overall prevalence of the p.R84W mutation in ARNSSNHL in Turkey is 2.4% (95% CI is 0.7–4.0%). Genotyping of single-nucleotide polymorphisms flanking the TMIE gene revealed a conserved haplotype, suggesting a single origin for p.R84W from a common ancestor 1250 years ago (95% CI is 650–2500 years). We conclude that p.R84W could be a common mutation in other Middle Eastern populations and should be included in mutation screening offered to individuals with ARNSSNHL.