Identification of a novel homozygous mutation in transmembrane channel like 1 (TMC1) gene, one of the second-tier hearing loss genes after GJB2 in India.

BACKGROUND & OBJECTIVES: Hearing impairment is a common and heterogeneous sensory disorder in humans. Among about 90 genes, which are known to be associated with hearing impairment, mutations in the GJB2 (gap junction protein beta 2) gene are the most prevalent in individuals with hereditary hearing loss. Contribution of the other deafness-causing genes is relatively poorly understood. Here, we present our findings on two families with transmembrane channel like 1 (TMC1) gene variants of the 47 families with nonsyndromic hearing loss (NSHL) studied. METHODS: Forty seven families including 26 consanguineous families with at least two hearing impaired children and one normal hearing child and 21 non-consanguineous families having at least three hearing impaired children and one normal hearing child were enrolled for this study. Genetic linkage studies were carried out in 41 families that were GJB2 (Connexin 26) negative. Seven polymorphic short tandem repeat markers at the DFNB7/11 locus were studied employing fluorescently labelled markers. RESULTS: A novel homozygous missense mutation c.1283C>A (p.Ala428Asp) was identified co-segregating with hearing loss. This change results in substitution of a highly conserved polar alanine to a charged aspartic acid and is predicted to be deleterious. In addition, a previously reported nonsense mutation, p.R34X in TMC1, was found. INTERPRETATION & CONCLUSIONS: While mutations in TMC1 are not as common a cause of NSHL as those in GJB2, TMC1 should be considered for diagnostic investigations in cases of NSHL in GJB2-negative families.

Williams-Beuren Syndrome: Experience of 43 Patients and a Report of an Atypical Case from a Tertiary Care Center in India.

Williams-Beuren syndrome (WBS) or Williams syndrome (OMIM 194050) is a multisystem disorder manifested by neurodevelopmental delay and is caused by a hemizygous deletion of ∼ 1.5-1.8 Mb in the 7q11.23 region. Clinical features include cardiovascular anomalies (mainly supravalvular aortic stenosis), peripheral pulmonary stenosis, distinctive facies, intellectual disability (usually mild), unique personality characteristics, and growth and endocrine abnormalities. Clinical diagnostic criteria are available for WBS; however, the mainstay of diagnosis is the detection of the contiguous gene deletion. Although FISH remains the most widely used laboratory test, the diagnosis can also be established by means of qPCR, MLPA, microsatellite marker analysis, and chromosomal microarray (CMA). We evaluated the utility of MLPA to detect deletion/duplication in the 7q11.23 region in 43 patients suspected to have WBS using MLPA kits for microdeletion syndromes. A hemizygous deletion in the 7q11.23 region was found in 41 (95.3%) patients using MLPA. One patient had an atypical deletion detected by CMA. During the initial period of this study, the results of 12 patients tested by MLPA were also confirmed by FISH. Compared to FISH and CMA, MLPA is a cheaper, high-throughput, less labor-intensive and less time-consuming technique for the diagnosis of WBS. Although CMA is expensive and labor-intensive, its effectiveness is demonstrated to detect an atypical deletion and to delineate the breakpoints.

Sequential occurrence of preneoplastic lesions and accumulation of loss of heterozygosity in patients with gallbladder stones suggest causal association with gallbladder cancer.

BACKGROUND: Causal association of gallbladder stones with gallbladder cancer (GBC) is not yet well established. OBJECTIVE: To study the frequency of occurrence of preneoplastic histological lesions and loss of heterozygosity (LOH) of tumor suppressor genes in patients with gallstones. METHODS: All consecutive patients with gallstones undergoing cholecystectomy from 2007-2011 were included prospectively. Histological examination of the gallbladder specimens was done for preneoplastic lesions. LOH at 8 loci, that is 3p12, 3p14.2, 5q21, 9p21, 9q, 13q, 17p13, and 18q for tumor suppressor genes (DUTT1, FHIT, APC, p16, FCMD, RB1, p53, and DCC genes) that are associated with GBC was tested from microdissected preneoplastic lesions using microsatellite markers. These LOH were also tested in 30 GBC specimens. RESULTS: Of the 350 gallbladder specimens from gallstone patients, hyperplasia was found in 32%, metaplasia in 47.8%, dysplasia in 15.7%, and carcinoma in situ in 0.6%. Hyperplasia, metaplasia, and dysplasia alone were found in 11.7%, 24.6%, and 1.4% of patients, respectively. A combination of hyperplasia and dysplasia, metaplasia and dysplasia, and hyperplasia, metaplasia, and dysplasia was found in 3.4%, 6.3%, and 4.3% of patients, respectively. LOH was present in 2.1% to 47.8% of all the preneoplastic lesions at different loci. Fractional allelic loss was significantly higher in those with dysplasia compared with other preneoplastic lesions (0.31 vs 0.22; P = 0.042). No preneoplastic lesion or LOH was found in normal gallbladders. CONCLUSIONS: Patients with gallstones had a high frequency of preneoplastic lesions and accumulation of LOH at various tumor suppressor genes, suggesting a possible causal association of gallstones with GBC.

Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.

Positional cloning of hereditary deafness genes is a direct approach to identify molecules and mechanisms underlying auditory function. Here we report a locus for dominant deafness, DFNA36, which maps to human chromosome 9q13-21 in a region overlapping the DFNB7/B11 locus for recessive deafness. We identified eight mutations in a new gene, transmembrane cochlear-expressed gene 1 (TMC1), in a DFNA36 family and eleven DFNB7/B11 families. We detected a 1.6-kb genomic deletion encompassing exon 14 of Tmc1 in the recessive deafness (dn) mouse mutant, which lacks auditory responses and has hair-cell degeneration. TMC1 and TMC2 on chromosome 20p13 are members of a gene family predicted to encode transmembrane proteins. Tmc1 mRNA is expressed in hair cells of the postnatal mouse cochlea and vestibular end organs and is required for normal function of cochlear hair cells.

Noncoding mutations of HGF are associated with nonsyndromic hearing loss, DFNB39.

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3' untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia.

Domino ring-opening metathesis-ring-closing metathesis of bicyclo[2.2.2]octene derivatives: scope and limitations.

Domino metathesis involving ring-opening metathesis-ring-closing metathesis (ROM-RCM) of a bicyclo[2.2.2]octene derivative having an appropriate alkene chain, expected to produce a 7/6 fused bicyclic system, provided a decalin system in contrast to ROM-RCM of the corresponding bicyclo[2.2.1]heptene analogues, which as expected produced the 7/5 fused bicycles. The expected 6/7 bicyclic system could, however, be made through RCM of the elusive ROM product prepared from the same bicyclo[2.2.2]octene analogue by a nonmetathetic route. A rationale to explain the difference in reactivity pattern between these two systems toward ROM-RCM has been forwarded.

Allelic hierarchy of CDH23 mutations causing non-syndromic deafness DFNB12 or Usher syndrome USH1D in compound heterozygotes.

BACKGROUND: Recessive mutant alleles of MYO7A, USH1C, CDH23, and PCDH15 cause non-syndromic deafness or type 1 Usher syndrome (USH1) characterised by deafness, vestibular areflexia, and vision loss due to retinitis pigmentosa. For CDH23, encoding cadherin 23, non-syndromic DFNB12 deafness is associated primarily with missense mutations hypothesised to have residual function. In contrast, homozygous nonsense, frame shift, splice site, and some missense mutations of CDH23, all of which are presumably functional null alleles, cause USH1D. The phenotype of a CDH23 compound heterozygote for a DFNB12 allele in trans configuration to an USH1D allele is not known and cannot be predicted from current understanding of cadherin 23 function in the retina and vestibular labyrinth. METHODS AND RESULTS: To address this issue, this study sought CDH23 compound heterozygotes by sequencing this gene in USH1 probands, and families segregating USH1D or DFNB12. Five non-syndromic deaf individuals were identified with normal retinal and vestibular phenotypes that segregate compound heterozygous mutations of CDH23, where one mutation is a known or predicted USH1 allele. CONCLUSIONS: One DFNB12 allele in trans configuration to an USH1D allele of CDH23 preserves vision and balance in deaf individuals, indicating that the DFNB12 allele is phenotypically dominant to an USH1D allele. This finding has implications for genetic counselling and the development of therapies for retinitis pigmentosa in Usher syndrome. ACCESSION NUMBERS: The cDNA and protein Genbank accession numbers for CDH23 and cadherin 23 used in this paper are AY010111.2 and AAG27034.2, respectively.

Transethnic analysis of psoriasis susceptibility in South Asians and Europeans enhances fine-mapping in the MHC and genomewide.

Because transethnic analysis may facilitate prioritization of causal genetic variants, we performed a genomewide association study (GWAS) of psoriasis in South Asians (SAS), consisting of 2,590 cases and 1,720 controls. Comparison with our existing European-origin (EUR) GWAS showed that effect sizes of known psoriasis signals were highly correlated in SAS and EUR (Spearman ρ = 0.78; p < 2 × 10-14). Transethnic meta-analysis identified two non-MHC psoriasis loci (1p36.22 and 1q24.2) not previously identified in EUR, which may have regulatory roles. For these two loci, the transethnic GWAS provided higher genetic resolution and reduced the number of potential causal variants compared to using the EUR sample alone. We then explored multiple strategies to develop reference panels for accurately imputing MHC genotypes in both SAS and EUR populations and conducted a fine-mapping of MHC psoriasis associations in SAS and the largest such effort for EUR. HLA-C*06 was the top-ranking MHC locus in both populations but was even more prominent in SAS based on odds ratio, disease liability, model fit and predictive power. Transethnic modeling also substantially boosted the probability that the HLA-C*06 protein variant is causal. Secondary MHC signals included coding variants of HLA-C and HLA-B, but also potential regulatory variants of these two genes as well as HLA-A and several HLA class II genes, with effects on both chromatin accessibility and gene expression. This study highlights the shared genetic basis of psoriasis in SAS and EUR populations and the value of transethnic meta-analysis for discovery and fine-mapping of susceptibility loci.

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.

Application of chromosomal microarrays in the evaluation of intellectual disability/global developmental delay patients - A study from a tertiary care genetic centre in India.

Intellectual disability (ID)/Global developmental delay (GDD) is a diverse group of disorders in terms of cognitive and non-cognitive functions and can occur with or without associated co-morbidities. It affects 1-3% of individuals globally and in at least 30-50% of cases the etiology remains unexplained. The widespread use of chromosomal microarray analysis (CMA) in a clinical setting has allowed the identification of submicroscopic copy number variations (CNVs), throughout the genome, associated with neurodevelopmental phenotypes including ID/GDD. In this study we investigated the utility of CMA in the detection of CNVs in 106 patients with unexplained ID/DD, dysmorphism with or without multiple congenital anomalies (MCA). CMA study was carried out using Agilent 8×60K chips and Illumina Human CytoSNP-12 chips. Pathogenic CNVs were found in 15 (14.2%) patients. In these patients, CNVs on single chromosome were detected in 10 patients while 5 patients showed co-occurrence CNVs on two chromosomes. The size of these CNVs ranged between 322kb to 13Mb. The yield of pathogenic CNVs was similar for both mild and severe ID/GDD cases. One patient described in this paper is considered to harbour a likely pathogenic CNV with deletion in 17q22 region. Only few cases have been described in literature for 17q22 deletion and patient reported here was found to have an atypical deletion in 17q22 region (Case 90). This study re-affirms the view point that CMA is a powerful diagnostic tool in the evaluation of idiopathic ID/GDD patients irrespective of the degree of severity. Identifying pathogenic CNVs helps in counseling and prenatal diagnosis if desired.

Genetics of deafness in India.

Linkage analysis in families with hereditary hearing loss have revealed a plethora of chromosomal locations linked to deafness reflecting the extreme heterogeneity of the disorder. 40 of the genes contained within these loci have been mapped lending an insight into the diverse molecules operating in the inner ear and the remarkable complexity of the cellular and molecular processes involved in the transucdation of sound in the auditory system. Among this diversity, Connexin 26 has been found to be the most common cause of deafness the world around. The authors review here the prevalence of this gene in the Indian population as found in their study, together with other deafness genes segregating non-syndromic deafness, accounting for approximately 40% of all cases. This indicates there are several more to be identified yet. Knowledge of the genetic cause of deafness in our families is important for accurate genetic counseling and early diagnosis for timely intervention and treatment options.

Novel non-identical MECP2 mutations in Rett syndrome family: a rare presentation.

INTRODUCTION: Rett syndrome (RS), an X-linked neurodevelopmental disorder and the common cause of mental retardation in females, is caused by methyl CpG binding protein 2 (MECP2) gene mutations with a frequency of more than 95% in classical Rett patients. Majority of RS cases are sporadic but few familial cases caused by either skewed X-chromosome inactivation in healthy female carriers or mosaicism in male carriers are also reported. Most of the times, the mutation carried in a family is the same as found in affected child. METHODS AND RESULTS: Here we report a unique family carrying non-identical MECP2 mutations in exon 2 wherein the proband with classical RS was carrying a de-novo early truncating frameshift mutation while her asymptomatic mother was carrying a missense mutation, both predicted as pathogenic mutations. CONCLUSIONS: These findings further validate the importance of MECP2 mutation screening in parents of all mutation positive patients and careful evaluation of the pathogenicity of the mutation found in asymptomatic carriers before providing genetic counseling to the family. The results also propose the role of other factors including other gene mutations, environmental and epigenetics factors in modifying the expression of MECP2 mutations.

Brachytelephalangic chondrodysplasia punctata: a case series to further delineate the phenotype.

Chondrodysplasia punctata (CDP) is a heterogenous group of skeletal dysplasias characterized by aberrant bone mineralization, manifesting radiologically as epiphyseal stippling. Among this group, brachytelephalangic dysplasia, a benign form of CDP (CDPX1), is probably under-reported. It is an X-linked recessive disorder and is characterized by a flat nasal tip, short columella and maxillary hypoplasia, involvement of terminal phalanges, and stippled chondrodystrophy. This paper presents a clinical series of 13 patients with brachytelephalangic dysplasia. These patients enrolled during 2002-2006 were re-evaluated and their dysmorphic features were compiled in a predesigned proforma. Skeletal survey, karyotype, cardiac evaluation, and ophthalmic evaluation were planned for all the cases. Out of 13 patients, 10 were males and three were females. All patients had flat facies, a depressed nasal bridge, a hypoplastic nose, a short philtrum, notched alae nasi, brachydactyly, and hypoplastic terminal phalanges. In addition, congenital heart disease, optic nerve hypoplasia, and developmental delay were found in a few patients. Radiography showed hypoplastic terminal phalanges, delayed bone age (1/13), epiphyseal stippling in carpal (3/13) and tarsal bones (2/13), sacral bone (1/13), and bullet-shaped lumbar vertebra (1/13). Cranial neuroimaging, thyroid profile, and karyotype carried out in a few were normal. The present paper discusses various clinical features and associated abnormalities in patients with brachytelephalangic dysplasia (CDPX1) to further delineate the phenotype. The presence of a similar phenotype in females suggests the possibility of another locus or manifestation of disease in heterozygous females. Arylsulfatase E gene analysis would further help in establishing the genotype-phenotype correlation.

Norrie disease: first mutation report and prenatal diagnosis in an Indian family.

Norrie Disease (ND) is a rare X-linked recessive disorder characterised by congenital blindness due to severe retinal dysgenesis. Hearing loss and intellectual disability is present in 30-50 % cases. ND is caused by mutations in the NDP gene, located at Xp11.3. The authors describe mutation analysis of a proband with ND and subsequently prenatal diagnosis. Sequence analysis of the NDP gene revealed a hemizygous missense mutation arginine to serine in codon 41 (p.Arg41Ser) in the affected child. Mother was carrier for the mutation. In a subsequent di-chorionic di-amniotic pregnancy, the authors performed prenatal diagnosis by mutation analysis on chorionic villi sample at 11 wk of gestation. The fetuses were unaffected. This is a first mutation report and prenatal diagnosis of a familial case of Norrie disease from India. The importance of genetic testing of Norrie disease for confirmation, carrier testing, prenatal diagnosis and genetic counseling is emphasized.

A novel MECP2 change in an indian boy with variant rett phenotype and congenital blindness: implications for genetic counseling and prenatal diagnosis.

Mutations in MECP2 gene are the primary cause of Rett syndrome, a neurodevelopmental disorder that primarily affects girls, and affect 90% to 95% patients with classical Rett syndrome. MECP2 mutations, once thought to be lethal in males, now present a broad spectrum of clinical manifestations in males. This article reports a family with a 9-year-old boy with Rett-like phenotype and congenital blindness, who inherited a novel MECP2 variant (p.P430S) from his asymptomatic mother. The variant was also identified in the asymptomatic maternal grandfather and maternal aunts of the proband, ruling out the possibility that the p.P430S was involved in the phenotype. Findings of the study suggest that a careful evaluation of the pathogenic nature of MECP2 variants identified in males be conducted before proposing genetic counseling or prenatal diagnosis to the family and that the interference of other factors like modifier genes, environment, epigenetics, and mosaicism be taken into account.