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1.  Autosomal recessive postlingual hearing loss (DFNB8): compound heterozygosity for two novel TMPRSS3 mutations in German siblings 
Journal of Medical Genetics  2007;44(6):e81.
Mutations in the transmembrane protease, serine 3 (TMPRSS3) gene, encoding a transmembrane serine protease, cause autosomal recessive deafness childhood (DFNB8) or congenital onset (DFNB10). TMPRSS3 mutations have been mainly identified in patients from Asian and Mediterranean countries and seem to be a rare finding in the Northern European population so far. The identification of two novel pathogenic TMPRSS3 mutations (c.646C→T − R216C; c.916G→A − A306T) is described in four affected siblings of German origin with postlingual hearing loss, treated by bilateral cochlear implantation with good results. Although TMPRSS3 mutations are supposed to be a rare cause of autosomal recessive hearing loss, in families with postlingual disease onset TMPRSS3 is the most favourable candidate gene after exclusion of GJB2 mutations.
doi:10.1136/jmg.2007.049122
PMCID: PMC2752172  PMID: 17551081
2.  Novel TMPRSS3 variants in Pakistani families with autosomal recessive nonsyndromic hearing impairment 
Clinical Genetics  2011;82(1):56-63.
Mutations in the TMPRSS3 gene are known to cause autosomal recessive nonsyndromic hearing impairment (ARNSHI). After undergoing a genome scan, ten consanguineous Pakistani families with ARNSHI were found to have significant or suggestive evidence of linkage to the TMPRSS3 region. In order to elucidate if the TMPRSS3 gene is responsible for ARNSHI in these families, the gene was sequenced using DNA samples from these families. Six TMPRSS3 variants were found to co-segregate in ten families. None of these variants were detected in 500 control chromosomes. Four novel variants, three of which are missense [c.310G>A (p.Glu104Lys), c.767C>T (p.Ala256Val) and c.1273T>C (p.Cys425Arg)] and one nonsense [c.310G>T (p.Glu104Stop)], were identified. The pathogenicity of novel missense variants was investigated through bioinformatics analyses. Additionally, the previously reported deletion c.208delC (p.His70ThrfsX19) was identified in one family and the known mutation c.1219T>C (p.Cys407Arg) was found in five families, which makes c.1219T>C (p.Cys407Arg) the most common TMPRSS3 mutation within the Pakistani population. Identification of these novel variants lends support to the importance of elements within the low-density lipoprotein receptor A (LDLRA) and serine protease domains in structural stability, ligand binding and proteolytic activity for proper TMPRSS3 function within the inner ear.
doi:10.1111/j.1399-0004.2011.01695.x
PMCID: PMC3374056  PMID: 21534946
autosomal recessive nonsyndromic hearing impairment; LDLRA domain; Pakistan; serine protease domain; TMPRSS3
3.  Identification of a Novel Homozygous Mutation, TMPRSS3: c.535G>A, in a Tibetan Family with Autosomal Recessive Non-Syndromic Hearing Loss 
PLoS ONE  2014;9(12):e114136.
Different ethnic groups have distinct mutation spectrums associated with inheritable deafness. In order to identify the mutations responsible for congenital hearing loss in the Tibetan population, mutation screening for 98 deafness-related genes by microarray and massively parallel sequencing of captured target exons was conducted in one Tibetan family with familiar hearing loss. A homozygous mutation, TMPRSS3: c.535G>A, was identified in two affected brothers. Both parents are heterozygotes and an unaffected sister carries wild type alleles. The same mutation was not detected in 101 control Tibetan individuals. This missense mutation results in an amino acid change (p.Ala179Thr) at a highly conserved site in the scavenger receptor cysteine rich (SRCR) domain of the TMPRSS3 protein, which is essential for protein-protein interactions. Thus, this mutation likely affects the interactions of this transmembrane protein with extracellular molecules. According to our bioinformatic analyses, the TMPRSS3: c.535G>A mutation might damage protein function and lead to hearing loss. These data suggest that the homozygous mutation TMPRSS3: c.535G>A causes prelingual hearing loss in this Tibetan family. This is the first TMPRSS3 mutation found in the Chinese Tibetan population.
doi:10.1371/journal.pone.0114136
PMCID: PMC4256404  PMID: 25474651
4.  Autosomal recessive nonsyndromic deafness genes: a review 
More than 50 percent of prelingual hearing loss is genetic in origin, and of these up to 93 percent are monogenic autosomal recessive traits. Some forms of genetic deafness can be recognized by their associated syndromic features, but in most cases, hearing loss is the only finding and is referred to as nonsyndromic deafness. To date, more than 700 different mutations have been identified in one of 42 genes in individuals with autosomal recessive nonsyndromic hearing loss (ARNSHL). Reported mutations in GJB2, encoding connexin 26, makes this gene the most common cause of hearing loss in many populations. Other relatively common deafness genes include SLC26A4, MYO15A, OTOF, TMC1, CDH23, and TMPRSS3. In this report we summarize genes and mutations reported in families with ARNSHL. Founder effects were demonstrated for some recurrent mutations but the most significant findings are the extreme locus and allelic heterogeneity and different spectrum of genes and mutations in each population.
PMCID: PMC3683827  PMID: 22652773
Consanguinity; Deafness; Founder effects; Gene; Inner ear; Non syndromic hearing loss; Recurrent mutations; Allelic heterogeneity; Review
5.  Genetic Spectrum of Autosomal Recessive Non-Syndromic Hearing Loss in Pakistani Families 
PLoS ONE  2014;9(6):e100146.
The frequency of inherited bilateral autosomal recessive non-syndromic hearing loss (ARNSHL) in Pakistan is 1.6/1000 individuals. More than 50% of the families carry mutations in GJB2 while mutations in MYO15A account for about 5% of recessive deafness. In the present study a cohort of 30 ARNSHL families was initially screened for mutations in GJB2 and MYO15A. Homozygosity mapping was performed by employing whole genome single nucleotide polymorphism (SNP) genotyping in the families that did not carry mutations in GJB2 or MYO15A. Mutation analysis was performed for the known ARNSHL genes present in the homozygous regions to determine the causative mutations. This allowed the identification of a causative mutation in all the 30 families including 9 novel mutations, which were identified in 9 different families (GJB2 (c.598G>A, p.Gly200Arg); MYO15A (c.9948G>A, p.Gln3316Gln; c.3866+1G>A; c.8767C>T, p.Arg2923* and c.8222T>C, p.Phe2741Ser), TMC1 (c.362+18A>G), BSND (c.97G>C, p.Val33Leu), TMPRSS3 (c.726C>G, p.Cys242Trp) and MSRB3 (c.20T>G, p.Leu7Arg)). Furthermore, 12 recurrent mutations were detected in 21 other families. The 21 identified mutations included 10 (48%) missense changes, 4 (19%) nonsense mutations, 3 (14%) intronic mutations, 2 (9%) splice site mutations and 2 (9%) frameshift mutations. GJB2 accounted for 53% of the families, while mutations in MYO15A were the second most frequent (13%) cause of ARNSHL in these 30 families. The identification of novel as well as recurrent mutations in the present study increases the spectrum of mutations in known deafness genes which could lead to the identification of novel founder mutations and population specific mutated deafness genes causative of ARNSHL. These results provide detailed genetic information that has potential diagnostic implication in the establishment of cost-efficient allele-specific analysis of frequently occurring variants in combination with other reported mutations in Pakistani populations.
doi:10.1371/journal.pone.0100146
PMCID: PMC4065008  PMID: 24949729
6.  Whole-Exome Sequencing Efficiently Detects Rare Mutations in Autosomal Recessive Nonsyndromic Hearing Loss 
PLoS ONE  2012;7(11):e50628.
Identification of the pathogenic mutations underlying autosomal recessive nonsyndromic hearing loss (ARNSHL) is difficult, since causative mutations in 39 different genes have so far been reported. After excluding mutations in the most common ARNSHL gene, GJB2, via Sanger sequencing, we performed whole-exome sequencing (WES) in 30 individuals from 20 unrelated multiplex consanguineous families with ARNSHL. Agilent SureSelect Human All Exon 50 Mb kits and an Illumina Hiseq2000 instrument were used. An average of 93%, 84% and 73% of bases were covered to 1X, 10X and 20X within the ARNSHL-related coding RefSeq exons, respectively. Uncovered regions with WES included those that are not targeted by the exome capture kit and regions with high GC content. Twelve homozygous mutations in known deafness genes, of which eight are novel, were identified in 12 families: MYO15A-p.Q1425X, -p.S1481P, -p.A1551D; LOXHD1-p.R1494X, -p.E955X; GIPC3-p.H170N; ILDR1-p.Q274X; MYO7A-p.G2163S; TECTA-p.Y1737C; TMC1-p.S530X; TMPRSS3-p.F13Lfs*10; TRIOBP-p.R785Sfs*50. Each mutation was within a homozygous run documented via WES. Sanger sequencing confirmed co-segregation of the mutation with deafness in each family. Four rare heterozygous variants, predicted to be pathogenic, in known deafness genes were detected in 12 families where homozygous causative variants were already identified. Six heterozygous variants that had similar characteristics to those abovementioned variants were present in 15 ethnically-matched individuals with normal hearing. Our results show that rare causative mutations in known ARNSHL genes can be reliably identified via WES. The excess of heterozygous variants should be considered during search for causative mutations in ARNSHL genes, especially in small-sized families.
doi:10.1371/journal.pone.0050628
PMCID: PMC3511533  PMID: 23226338
7.  Five novel loci for inherited hearing loss mapped by SNP-based homozygosity profiles in Palestinian families 
In communities with high rates of consanguinity and consequently high prevalence of recessive phenotypes, homozygosity mapping with SNP arrays is an effective approach for gene discovery. In 20 Palestinian kindreds with prelingual nonsyndromic hearing loss, we generated homozygosity profiles reflecting linkage to the phenotype. Family sizes ranged from small nuclear families with two affected children, one unaffected sibling, and parents to multigenerational kindreds with 12 affected relatives. By including unaffected parents and siblings and screening 250 K SNP arrays, even small nuclear families yielded informative profiles. In 14 families, we identified the allele responsible for hearing loss by screening a single candidate gene in the longest homozygous region. Novel alleles included missense, nonsense, and splice site mutations of CDH23, MYO7A, MYO15A, OTOF, PJVK, Pendrin/SLC26A4, TECTA, TMHS, and TMPRSS3, and a large genomic deletion of Otoancorin (OTOA). All point mutations were rare in the Palestinian population (zero carriers in 288 unrelated controls); the carrier frequency of the OTOA genomic deletion was 1%. In six families, we identified five genomic regions likely to harbor novel genes for human hearing loss on chromosomes 1p13.3 (DFNB82), 9p23–p21.2/p13.3–q21.13 (DFNB83), 12q14.3–q21.2 (DFNB84; two families), 14q23.1–q31.1, and 17p12–q11.2 (DFNB85).
doi:10.1038/ejhg.2009.190
PMCID: PMC2987250  PMID: 19888295
hearing; deafness; DFNB; homozygosity mapping; mutation; protein modeling
8.  Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome 
Human genetics  2008;124(3):215-223.
Mutations of PCDH15, encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be comprised of 35 exons and encodes a variety of isoforms with 3 to 11 ectodomains (EC), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1 we identified homozygous mutant alleles (1 missense, 1 splice site, 3 nonsense and 2 deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F, which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15, mutations in the introns or regulatory elements of PCDH15, or an additional locus for type I USH at chromosome 10q21.1.
doi:10.1007/s00439-008-0543-3
PMCID: PMC2716558  PMID: 18719945
DFNB23; Usher syndrome; protocadherin 15; PCDH15; deafness; retinitis pigmentosa
9.  Genomic analysis of a heterogeneous Mendelian phenotype: multiple novel alleles for inherited hearing loss in the Palestinian population 
Human Genomics  2006;2(4):203-211.
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.
doi:10.1186/1479-7364-2-4-203
PMCID: PMC3525152  PMID: 16460646
genetics; genomics; inherited; deafness; hearing loss; mutation; Palestinian; TMPRSS3; pendrin; otoancorin
10.  Profound, prelingual nonsyndromic deafness maps to chromosome 10q21 and is caused by a novel missense mutation in the Usher syndrome type IF gene PCDH15 
We studied a consanguineous family (Family A) from the island of Newfoundland with an autosomal recessive form of prelingual, profound, nonsyndromic sensorineural hearing loss. A genome-wide scan mapped the deafness trait to 10q21-22 (max LOD score of 4.0; D10S196) and fine mapping revealed a 16 Mb ancestral haplotype in deaf relatives. The PCDH15 gene was mapped within the critical region and was an interesting candidate because truncating mutations cause Usher syndrome type IF (USH1F) and two missense mutations have been previously associated with isolated deafness (DFNB23). Sequencing of the PCDH15 gene revealed 33 sequencing variants. Three of these variants were homozygous exclusively in deaf siblings but only one of them was not seen in ethnically matched controls. This novel c.1583 T>A transversion predicts an amino-acid substitution of a valine with an aspartic acid at codon 528 (V528D). Like the two DFNB23 mutations, the V528D mutation in Family A occurs in a highly conserved extracellular cadherin (EC) domain of PCDH15 and is predicted to be more deleterious than the previously identified DFNB23 missense mutations (R134G and G262D). Physical assessment, vestibular and visual function testing in deaf adults ruled out syndromic deafness because of Usher syndrome. This study validates the DFNB23 designation and supports the hypothesis that missense mutations in conserved motifs of PCDH15 cause nonsyndromic hearing loss. This emerging genotype–phenotype correlation in USH1F is similar to that in several other USH1 genes and cautions against a prognosis of a dual sensory loss in deaf children found to be homozygous for hypomorphic mutations at the USH1F locus.
doi:10.1038/ejhg.2008.231
PMCID: PMC2986256  PMID: 19107147
PCDH15; isolated deafness; usher syndrome type IF; hypomorphic alleles
11.  Identification of a novel in-frame deletion in KCNQ4 (DFNA2A) and evidence of multiple phenocopies of unknown origin in a family with ADSNHL 
European Journal of Human Genetics  2013;21(10):1112-1119.
Autosomal dominant sensorineural hearing loss (ADSNHL) is extremely genetically heterogeneous, making it difficult to molecularly diagnose. We identified a multiplex (n=28 affected) family from the genetic isolate of Newfoundland, Canada with variable SNHL and used a targeted sequencing approach based on population-specific alleles in WFS1, TMPRSS3 and PCDH15; recurrent mutations in GJB2 and GJB6; and frequently mutated exons of KCNQ4, COCH and TECTA. We identified a novel, in-frame deletion (c.806_808delCCT: p.S269del) in the voltage-gated potassium channel KCNQ4 (DFNA2), which in silico modeling predicts to disrupt multimerization of KCNQ4 subunits. Surprisingly, 10/23 deaf relatives are non-carriers of p.S269del. Further molecular characterization of the DFNA2 locus in deletion carriers ruled out the possibility of a pathogenic mutation other than p.S269del at the DFNA2A/B locus and linkage analysis showed significant linkage to DFNA2 (maximum LOD=3.3). Further support of genetic heterogeneity in family 2071 was revealed by comparisons of audio profiles between p.S269del carriers and non-carriers suggesting additional and as yet unknown etiologies. We discuss the serious implications that genetic heterogeneity, in this case observed within a single family, has on molecular diagnostics and genetic counseling.
doi:10.1038/ejhg.2013.5
PMCID: PMC3778362  PMID: 23443030
deafness; KCNQ4; DFNA2; ADSNHL; audio profiles; novel mutation
12.  Non-Syndromic Hearing Impairment in India: High Allelic Heterogeneity among Mutations in TMPRSS3, TMC1, USHIC, CDH23 and TMIE 
PLoS ONE  2014;9(1):e84773.
Mutations in the autosomal genes TMPRSS3, TMC1, USHIC, CDH23 and TMIE are known to cause hereditary hearing loss. To study the contribution of these genes to autosomal recessive, non-syndromic hearing loss (ARNSHL) in India, we examined 374 families with the disorder to identify potential mutations. We found four mutations in TMPRSS3, eight in TMC1, ten in USHIC, eight in CDH23 and three in TMIE. Of the 33 potentially pathogenic variants identified in these genes, 23 were new and the remaining have been previously reported. Collectively, mutations in these five genes contribute to about one-tenth of ARNSHL among the families examined. New mutations detected in this study extend the allelic heterogeneity of the genes and provide several additional variants for structure-function correlation studies. These findings have implications for early DNA-based detection of deafness and genetic counseling of affected families in the Indian subcontinent.
doi:10.1371/journal.pone.0084773
PMCID: PMC3885616  PMID: 24416283
13.  Phenotypic variability of CLDN14 mutations causing DFNB29 hearing loss in the Pakistani population 
Journal of human genetics  2012;58(2):102-108.
Human hereditary deafness at the DFNB29 autosomal locus on chromosome 21q22.1 is caused by recessive mutations of CLDN14, encoding claudin 14. This tight junction protein is tetra-membrane spanning that localizes to the apical tight junctions of organ of Corti hair cells and in many other tissues. Typically, the DFNB29 phenotype is characterized by pre-lingual, bi-lateral, sensorineural hearing loss. The goal of this study was to define the identity and frequency of CLDN14 mutations and associated inner ear phenotypes in a cohort of 800 Pakistani families segregating deafness. Hearing loss in 15 multi-generational families was found to co-segregate with CLDN14-linked STR markers. The sequence of the six exons and regions flanking the introns of CLDN14 in these 15 families revealed five likely pathogenic alleles. Two are novel missense substitutions (p.Ser87Ile and p.Ala94Val) while p.Arg81His, p.Val85Asp and p.Met133ArgfsX23 have been reported previously. Haplotype analyses indicate that p.Val85Asp and p.Met133ArgfsX23 are founder mutations. The p.Val85Asp accounts for approximately 67% of the mutant alleles of CLDN14 in our cohort. Combined with previously reported data, CLDN14 mutations were identified in 18 of 800 Pakistani families (2.25%; 95% CI, 1.4-3.5%). Hearing loss in the affected individuals homozygous for CLDN14 mutations varied from moderate to profound. This phenotypic variability may be due to environmental factors (e.g. drug and noise exposure) and/or genetic modifiers.
doi:10.1038/jhg.2012.143
PMCID: PMC3596117  PMID: 23235333
CLDN14; claudin 14; DFNB29; mild hearing loss; profound deafness; Pakistan
14.  Loss of the NKX3.1 tumorsuppressor promotes the TMPRSS2-ERG fusion gene expression in prostate cancer 
BMC Cancer  2014;14:16.
Background
In normal prostate epithelium the TMPRSS2 gene encoding a type II serine protease is directly regulated by male hormones through the androgen receptor. In prostate cancer ERG protooncogene frequently gains hormonal control by seizing gene regulatory elements of TMPRSS2 through genomic fusion events. Although, the androgenic activation of TMPRSS2 gene has been established, little is known about other elements that may interact with TMPRSS2 promoter sequences to modulate ERG expression in TMPRSS2-ERG gene fusion context.
Methods
Comparative genomic analyses of the TMPRSS2 promoter upstream sequences and pathway analyses were performed by the Genomatix Software. NKX3.1 and ERG genes expressions were evaluated by immunoblot or by quantitative Real-Time PCR (qRT-PCR) assays in response to siRNA knockdown or heterologous expression. QRT-PCR assay was used for monitoring the gene expression levels of NKX3.1-regulated genes. Transcriptional regulatory function of NKX3.1 was assessed by luciferase assay. Recruitment of NKX3.1 to its cognate elements was monitored by Chromatin Immunoprecipitation assay.
Results
Comparative analysis of the TMPRSS2 promoter upstream sequences among different species revealed the conservation of binding sites for the androgen inducible NKX3.1 tumor suppressor. Defects of NKX3.1, such as, allelic loss, haploinsufficiency, attenuated expression or decreased protein stability represent established pathways in prostate tumorigenesis. We found that NKX3.1 directly binds to TMPRSS2 upstream sequences and negatively regulates the expression of the ERG protooncogene through the TMPRSS2-ERG gene fusion.
Conclusions
These observations imply that the frequently noted loss-of-function of NKX3.1 cooperates with the activation of TMPRSS2-ERG fusions in prostate tumorigenesis.
doi:10.1186/1471-2407-14-16
PMCID: PMC3897978  PMID: 24418414
Tumor suppressor; NKX3.1; Prostate; ERG; NFкB; Oncogene
15.  An Alteration in ELMOD3, an Arl2 GTPase-Activating Protein, Is Associated with Hearing Impairment in Humans 
PLoS Genetics  2013;9(9):e1003774.
Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.
Author Summary
Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound mixed hearing loss locus, DFNB88 on chromosome 2p12-p11.2. Exome enrichment followed by massive parallel sequencing revealed a c.794T>C transition mutation in ELMOD3 that segregated with DFNB88-associated hearing loss in a large Pakistani family. This transition mutation is predicted to substitute a highly invariant leucine residue with serine (p.Leu265Ser) in the engulfment and cell motility (ELMO) domain of the protein. No biological activity has been described previously for the ELMOD3 protein. We investigated the biochemical properties and ELMOD3 expression to gain mechanistic insights into the function of ELMOD3 in the inner ear. In rodent inner ears, ELMOD3 immunoreactivity was observed in the cochlear and vestibular hair cells and supporting cells. However, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Ex vivo, ELMOD3 is associated with actin-based structures, and this link is impaired by the DFNB88 mutation. ELMOD3 exhibited GAP activity against Arl2, a small GTPase, providing a potential functional link between Arf family signaling and stereocilia actin-based cytoskeletal architecture. Our study provides new insights into the molecules that are necessary for the development and/or function of inner ear sensory cells.
doi:10.1371/journal.pgen.1003774
PMCID: PMC3764207  PMID: 24039609
16.  A Strong Anti-Inflammatory Signature Revealed by Liver Transcription Profiling of Tmprss6−/− Mice 
PLoS ONE  2013;8(7):e69694.
Control of systemic iron homeostasis is interconnected with the inflammatory response through the key iron regulator, the antimicrobial peptide hepcidin. We have previously shown that mice with iron deficiency anemia (IDA)-low hepcidin show a pro-inflammatory response that is blunted in iron deficient-high hepcidin Tmprss6 KO mice. The transcriptional response associated with chronic hepcidin overexpression due to genetic inactivation of Tmprss6 is unknown. By using whole genome transcription profiling of the liver and analysis of spleen immune-related genes we identified several functional pathways differentially expressed in Tmprss6 KO mice, compared to IDA animals and thus irrespective of the iron status. In the effort of defining genes potentially targets of Tmprss6 we analyzed liver gene expression changes according to the genotype and independently of treatment. Tmprss6 inactivation causes down-regulation of liver pathways connected to immune and inflammatory response as well as spleen genes related to macrophage activation and inflammatory cytokines production. The anti-inflammatory status of Tmprss6 KO animals was confirmed by the down-regulation of pathways related to immunity, stress response and intracellular signaling in both liver and spleen after LPS treatment. Opposite to Tmprss6 KO mice, Hfe−/− mice are characterized by iron overload with inappropriately low hepcidin levels. Liver expression profiling of Hfe−/− deficient versus iron loaded mice show the opposite expression of some of the genes modulated by the loss of Tmprss6. Altogether our results confirm the anti-inflammatory status of Tmprss6 KO mice and identify new potential target pathways/genes of Tmprss6.
doi:10.1371/journal.pone.0069694
PMCID: PMC3726786  PMID: 23922777
17.  Identities and frequencies of mutations of the otoferlin gene (OTOF) causing DFNB9 deafness in Pakistan 
Clinical genetics  2009;75(3):237-243.
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.
doi:10.1111/j.1399-0004.2008.01128.x
PMCID: PMC3461579  PMID: 19250381
DFNB9; hearing; OTOF; otoferlin
18.  A p.C343S Missense Mutation in PJVK causes Progressive Hearing Loss 
Gene  2012;504(1):98-101.
Mutations in PJVK, encoding Pejvakin, cause autosomal recessive nonsyndromic hearing loss in humans at the DFNB59 locus on chromosome 2q31.2. Pejvakin is involved in generating auditory and neural signals in the inner ear. We have identified a consanguineous Pakistani family segregating sensorineural progressive hearing loss as a recessive trait, consistent with linkage to DFNB59. We sequenced PJVK and identified a novel missense mutation, c.1028 G>C in exon 7 (p.C343S) co-segregating with the phenotype in the family. The p.C343 residue is fully conserved among orthologs from different vertebrate species. We have also determined that mutations in PJVK are not a common cause of hearing loss in families with moderate to severe hearing loss in Pakistan. This is the first report of PJVK mutation in a Pakistani family and pinpoints an important residue for PJVK function.
doi:10.1016/j.gene.2012.05.013
PMCID: PMC3534776  PMID: 22617256
Pejvakin; Pakistan; Progressive hearing loss; DFNB59; Deafness
19.  Molecular and Clinical Studies of X-linked Deafness Among Pakistani Families 
Journal of human genetics  2011;56(7):534-540.
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, 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.
doi:10.1038/jhg.2011.55
PMCID: PMC3143270  PMID: 21633365
DFN3; Norrie disease; POU3F4; NDP; hearing loss; gushers
20.  Polymorphisms and Mutations of Human TMPRSS6 in Iron Deficiency Anemia 
Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.
doi:10.1016/j.bcmd.2009.09.001
PMCID: PMC2818284  PMID: 19818657
hepcidin; iron deficiency; anemia; TMPRSS6; matriptase
21.  N-myc Downstream Regulated Gene 1 (NDRG1) Is Fused to ERG in Prostate Cancer12 
Neoplasia (New York, N.Y.)  2009;11(8):804-811.
A step toward the molecular classification of prostate cancer was the discovery of recurrent erythroblast transformation-specific rearrangements, most commonly fusing the androgen-regulated TMPRSS2 promoter to ERG. The TMPRSS2-ERG fusion is observed in around 90% of tumors that overexpress the oncogene ERG. The goal of the current study was to complete the characterization of these ERG-overexpressing prostate cancers. Using fluorescence in situ hybridization and reverse transcription-polymerase chain reaction assays, we screened 101 prostate cancers, identifying 34 cases (34%) with the TMPRSS2-ERG fusion. Seven cases demonstrated ERG rearrangement by fluorescence in situ hybridization without the presence of TMPRSS2-ERG fusion messenger RNA transcripts. Screening for known 5′ partners, we determined that three cases harbored the SLC45A3-ERG fusion. To discover novel 5′ partners in these ERG-overexpressing and ERG-rearranged cases, we used paired-end RNA sequencing. We first confirmed the utility of this approach by identifying the TMPRSS2-ERG fusion in a known positive prostate cancer case and then discovered a novel fusion involving the androgen-inducible tumor suppressor, NDRG1 (N-myc downstream regulated gene 1), and ERG in two cases. Unlike TMPRSS2-ERG and SCL45A3-ERG fusions, the NDRG1-ERG fusion is predicted to encode a chimeric protein. Like TMPRSS2, SCL45A3 and NDRG1 are inducible not only by androgen but also by estrogen. This study demonstrates that most ERG-overexpressing prostate cancers harbor hormonally regulated TMPRSS2-ERG, SLC45A3-ERG, or NDRG1-ERG fusions. Broader implications of this study support the use of RNA sequencing to discover novel cancer translocations.
PMCID: PMC2713587  PMID: 19649210
22.  Genetic Analysis through OtoSeq of Pakistani Families Segregating Prelingual Hearing Loss 
Objective
To identify the genetic cause of prelingual sensorineural hearing loss in Pakistani families using a next-generation sequencing (NGS)-based mutation screening test named OtoSeq.
Study Design
Prospective Study
Setting
Research laboratory
Subjects and Methods
We used three fluorescently labeled short tandem repeat (STR) markers for each of the known autosomal recessive nonsyndromic (DFNB) and Usher syndrome (USH) locus to perform a linkage analysis of 243 multi-generational Pakistani families segregating prelingual hearing loss. After genotyping, we focused on 34 families with potential linkage to MYO7A, CDH23 and SLC26A4. We screened affected individuals from a subset of these families using the OtoSeq platform to identify underlying genetic variants. Sanger sequencing was performed to confirm and study the segregation of mutations in other family members. For novel mutations, normal hearing individuals from ethnically matched backgrounds were also tested.
Results
Hearing loss was found to co-segregate with locus-specific STR markers for MYO7A in 32 families, CDH23 in one family and SLC26A4 in one family. Using the OtoSeq platform, a microdroplet PCR-based enrichment followed by NGS, we identified mutations in 28 of the 34 families including 11 novel mutations. Sanger sequencing of these mutations showed 100% concordance with NGS data and co-segregation of the mutant alleles with the hearing loss phenotype in the respective families.
Conclusion
Using NGS based platforms like OtoSeq in families segregating hearing loss, will contribute to the identification of common and population specific mutations, early diagnosis, genetic counseling and molecular epidemiology.
doi:10.1177/0194599813493075
PMCID: PMC4030297  PMID: 23770805
Hearing loss; Usher syndrome; microdroplet PCR; next generation sequencing; clinical diagnosis; genetic etiology; PDS; MYO7A; CDH23
23.  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.
doi:10.1038/sj.ejhg.5200905
PMCID: PMC2917542  PMID: 12529709
autosomal recessive nonsyndromic hearing loss; DFNA9; DFNA23; DFNB5; DFNB35; 14q24.1—14q24.3 prelingual; Pakistan
24.  Mutations of human TMHS cause recessively inherited non‐syndromic hearing loss 
Journal of Medical Genetics  2006;43(8):634-640.
Background
Approximately half the cases of prelingual hearing loss are caused by genetic factors. Identification of genes causing deafness is a crucial first step in understanding the normal function of these genes in the auditory system. Recently, a mutant allele of Tmhs was reported to be associated with deafness and circling behaviour in the hurry‐scurry mouse. Tmhs encodes a predicted tetraspan protein of unknown function, which is expressed in inner ear hair cells. The human homologue of Tmhs is located on chromosome 6p.
Objective
To determine the cause of deafness in four consanguineous families segregating recessive deafness linked to markers on chromosome 6p21.1‐p22.3 defining a novel DFNB locus.
Results
A novel locus for non‐syndromic deafness DFNB67 was mapped in an interval of approximately 28.51 cM on human chromosome 6p21.1‐p22.3. DNA sequence analysis of TMHS revealed a homozygous frameshift mutation (246delC) and a missense mutation (Y127C) in affected individuals of two families segregating non‐syndromic deafness, one of which showed significant evidence of linkage to markers in the DFNB67 interval. The localisation of mTMHS in developing mouse inner ear hair cells was refined and found to be expressed briefly from E16.5 to P3.
Conclusions
These findings establish the importance of TMHS for normal sound transduction in humans.
doi:10.1136/jmg.2005.039834
PMCID: PMC2564584  PMID: 16459341
25.  Severe Microcytic Anemia but Increased Erythropoiesis in Mice Lacking Hfe or Tfr2 and Tmprss6 
Blood cells, molecules & diseases  2012;48(3):173-178.
Cell surface proteins Hfe, Tfr2, hemojuvelin and Tmprss6 play key roles in iron homeostasis. Hfe and Tfr2 induce transcription of hepcidin, a small peptide that promotes the degradation of the iron transporter ferroportin. Hemojuvelin, a co-receptor for bone morphogenic proteins, induces hepcidin transcription through a Smad signaling pathway. Tmprss6 (also known as matriptase-2), a membrane serine protease that has been found to bind and degrade hemojuvelin in vitro, is a potent suppressor of hepcidin expression. In order to examine if Hfe and Tfr2 are substrates for Tmprss6, we generated mice lacking functional Hfe or Tfr2 and Tmprss6. We found that double mutant mice lacking functional Hfe or Tfr2 and Tmprss6 exhibited a severe iron deficiency microcytic anemia phenotype mimicking the phenotype of single mutant mice lacking functional Tmprss6 (Tmprss6 msk/msk mutant) demonstrating that Hfe and Tfr2 are not substrates for Tmprss6. Nevertheless, the phenotype of the mice lacking Hfe or Tfr2 and Tmprss6 differed from Tmprss6 deficient mice alone, in that the double mutant mice exhibited much greater erythropoiesis. Hfe and Tfr2 have been shown to play important roles in the erythron, independent of their role in regulating liver hepcidin transcription. We demonstrate that lack of functional Tfr2 and Hfe allow for increased erythropoiesis even in the presence of high hepcidin expression, but the high levels of hepcidin levels significantly limit the availability of iron to the erythron, resulting in ineffective erythropoiesis. Furthermore, repression of hepcidin expression was unaffected by loss of functional Hfe, Tfr2 and Tmprss6.
doi:10.1016/j.bcmd.2011.12.005
PMCID: PMC3294186  PMID: 22244935
hepcidin; iron; TMPRSS6; hemochromatosis; anemia; HFE; TFR2; matriptase

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