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Human hereditary deafness at the DFNB29 locus on chromosome 21q22.1 is caused by recessive mutations of CLDN14, encoding claudin 14. This tight junction protein is tetramembrane 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 prelingual, bilateral, 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), whereas 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 ~67% of the mutant alleles of CLDN14 in our cohort. Combined with the 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 (for example drug and noise exposure) and/or genetic modifiers.

Correction to: Journal of Human Genetics (2013) 58, 102–108; doi:10.1038/jhg.2012.143; published online 13 December 2012 The authors of the above paper noticed an error in publication (AOP and in February issue) in the list of authors. The fifth author Sheikh Amer Riazuddin should have read S Amer Riazuddin.

It has been previously demonstrated that the λ O protein initiates viral replication by binding to each of four repeating sequences present in the origin of replication to form a nucleoprotein complex termed as O-some. While it has been shown that an N-terminal domain of λ O has the capacity of wild type λ O, both to dimerize and bind to origin of replication, nothing was known about the specific amino acid residues involved in these events. We devised a two-plasmid system to identify the O residues that play a critical role in origin recognition. In this system, the O polypeptides expressed in a regulated manner from one plasmid bind to an oriλ sequence on the second plasmid and blocks binding of RNA polymerase to a promoter sequence, embedded with in oriλ which controls the expression of the chloramphenicol resistance gene. Thus cells that express the wild type λ O are chloramphenicol sensitive (Cm S). PCR mutagenesis was used to introduce random sequence changes into a DNA segment encoding the N-terminal domain of λ O. The modified DNA was fused to a GFP gene present on the first plasmid. Plasmids that encode a stable λ O protein fragment with an oriλ binding defect were recovered in the two-plasmid system, which are Cm R and fluoresced green when irradiated by UV light. In each of these cases where the λ O coding sequence contained a unique single nucleotide substitution, the mutation was inserted back into the full-length λ O gene. Subsequently the mutant proteins were purified and their physical and functional properties were characterized. The mutations that affected the DNA-binding activity of λ O protein clustered into two regions, one comprising amino acids 41–55 and the other comprising residues 81–94. Electrophoretic mobility shift assays demonstrated that the affinities of the mutant O proteins for the DNA-binding fragments carrying a single iteron repeat were reduced 10 to 100 fold relative to the wild type λ O protein. Analysis of the quaternary structure of the mutant O proteins by gel filtration chromatography, sedimentation equilibrium centrifugation and protein cross-linking assays indicated that most existed in a monomeric state in solution when they contained a mutation between residues 41 and 55. In contrast, all mutant proteins remained dimeric if the mutation was located between residues 81 and 94. We conclude that the region of λ O comprising residues 81–94 participate directly in specific recognition of the oriλ sequence. The DNA-binding deficiency of λ O proteins mutated between residues 41–55 is most likely an indirect effect caused by an inability to dimerize effectively.

To investigate the genetic basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous Pakistani family. All participating members of family, PKCC074 underwent an ophthalmic examination. Slit-lamp photographs were ascertained for affected individuals that have not been operated for the removal of the cataractous lens. A small aliquot of the blood sample was collected from all participating individuals and genomic DNAs were extracted. A genome-wide scan was performed with polymorphic short tandem repeat (STR) markers and the logarithm of odds (LOD) scores were calculated. All coding exons and exon-intron boundaries of HSF4 were sequenced and expression of Hsf4 in mouse ocular lens was investigated. The C-terminal FLAG-tagged wild-type and mutant HSF4b constructs were prepared to examine the nuclear localization pattern of the mutant protein. The ophthalmological examinations suggested that nuclear cataracts are present in affected individuals. Genome-wide linkage analyses localized the critical interval to a 10.95 cM (14.17 Mb) interval on chromosome 16q with a maximum two-point LOD score of 4.51 at θ = 0. Sanger sequencing identified a novel missense mutation: c.433G>C (p.Ala145Pro) that segregated with the disease phenotype in the family and was not present in ethnically matched controls. Real-time PCR analysis identified the expression of HSF4 in mouse lens as early as embryonic day 15 with a steady level of expression thereafter. The immunofluorescence tracking confirmed that both wild-type and mutant HSF4 (p.Ala145Pro) proteins localized to the nucleus. Here, we report a novel missense mutation in HSF4 associated with arCC in a familial case of Pakistani descent.

Background and purpose Retinitis pigmentosa is an important cause of severe visual dysfunction. This study reports a novel splicing mutation in the lecithin retinol acyltransferase ( LRAT ) gene associated with early onset retinitis pigmentosa and characterizes the effects of this mutation on mRNA splicing and structure. Methods Genome-wide linkage analysis followed by dideoxy sequencing of the linked candidate gene LRAT was performed in a consanguineous Pakistani family with autosomal recessive retinitis pigmentosa. In silico prediction and minigene assays were used to investigate the effects of the presumptive splicing mutation. Results ARRP in this family was linked to chromosome 4q31.21-q32.1 with a maximum LOD score of 5.40. A novel homozygous intronic mutation (NM_004744.4: c.541-15T>G) was detected in LRAT . In silico tools predicted that the AG-creating mutation would activate an intronic cryptic acceptor site, but cloning fragments of wild-type and mutant sequences of LRAT into Exontrap Cloning Vector pET01 and Expression Cloning Vector pCMV-(DYKD 4 K)-C showed that the primary effect of the sequence change was to weaken the nearby authentic acceptor site and cause exon skipping, with only a small fraction of transcripts utilizing the acceptor site producing the reference transcript. Conclusions The c.541-15T>G mutation in LRAT results in aberrant splicing and is therefore predicted to be causal for the early onset retinitis pigmentosa in this family. In addition, this work suggests that minigenes adapted to the specific gene and exon may need to be designed for variants in the first and last exon and intron to mimic the authentic splicing mechanism in vivo.

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generation exome sequencing identified a novel homozygous missense variant (c.653 T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.

To delineate the genetic bases of primary congenital glaucoma (PCG), we ascertained a large cohort consisting of 48 consanguineous families. Of these, we previously reported 26 families with mutations in CYP1B1 and six families with LTBP2 , whereas the genetic bases responsible for PCG in 16 families remained elusive. We employed next-generation whole exome sequencing to delineate the genetic basis of PCG in four of these 16 familial cases. Exclusion of linkage to reported PCG loci was established followed by next-generation whole exome sequencing, which was performed on 10 affected individuals manifesting cardinal systems of PCG belonging to four unresolved families along with four control samples consisting of genomic DNAs of individuals harboring mutations in CYP1B1 and LTBP2 . The analyses of sequencing datasets failed to identify potential causal alleles in the 10 exomes whereas c.1169G > A (p. Arg390His) in CYP1B1 and c.3427delC (p.Gln1143Argfs*35) in LTBP2 were identified in the control samples. Taken together, next-generation whole exome sequencing failed to delineate the genetic basis of PCG in familial cases excluded from mutations in CYP1B1 and LTBP2 . These data strengthen the notion that compound heterozygous coding variants or non-coding variants might contribute to PCG.

FOXE3 is a lens-specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole-exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in a mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, here we report DNAJB1 is a transcriptional target of FOXE3 in a novel pathway that is crucial for the development of the anterior segment of the eye. Peter's Anomaly is a developmental disorder of the eye and has been linked to mutations in a range of genes, including the transcription factor FOXE3. Here the authors use next-generation RNA sequencing and mass spectrometry to identify an autophagy-associated protein, DNAJB1 as the transcriptional target of FOXE3.

This study was initiated to identify causal mutations responsible for autosomal recessive congenital cataracts in consanguineous familial cases. Affected individuals underwent a detailed ophthalmological and clinical examination, and slit-lamp photographs were ascertained for affected individuals who have not yet been operated for the removal of the cataractous lens. Blood samples were obtained, and genomic DNA was extracted from white blood cells. A genome-wide scan was completed with short tandem repeat (STR) markers, and the logarithm of odds (LOD) scores were calculated. Protein coding exons of CRYAB were sequenced, bi-directionally. Evolutionary conservation was investigated by aligning CRYAB orthologues, and the expression of Cryab in embryonic and postnatal mice lens was investigated with TaqMan probe. The clinical and ophthalmological examinations suggested that all affected individuals had nuclear cataracts. Genome-wide linkage analysis suggested a potential region on chromosome 11q23 harboring CRYAB. DNA sequencing identified a missense variation: c.34C>T (p.R12C) in CRYAB that segregated with the disease phenotype in the family. Subsequent interrogation of our entire cohort of familial cases identified a second familial case localized to chromosome 11q23 harboring a c.31C>T (p.R11C) mutation. In silico analyses suggested that the mutations identified in familial cases, p.R11C and p.R12C will not be tolerated by the three-dimensional structure of CRYAB. Real-time PCR analysis identified the expression of Cryab in mouse lens as early as embryonic day 15 (E15) that increased significantly until postnatal day 6 (P6) with steady level of expression thereafter. Here, we report two novel missense mutations, p.R11C and p.R12C, in CRYAB associated with autosomal recessive congenital nuclear cataracts.

Pathogenic mutations of MARVELD2, encoding tricellulin, a tricelluar tight junction protein, cause autosomal recessive non-syndromic hearing loss (DFNB49) in families of Pakistan and Czech Roma origin. In fact, they are a significant cause of prelingual hearing loss in the Czech Roma, second only to GJB2 variants. Previously, we reported that mice homozygous for p.Arg497* variant of Marveld2 had a broad phenotypic spectrum, where defects were observed in the inner ear, heart, mandibular salivary gland, thyroid gland and olfactory epithelium. The current study describes the types and frequencies of MARVELD2 alleles and clinically reexamines members of DFNB49 families. We found that MARVELD2 variants are responsible for about 1.5 % (95 % CI 0.8–2.6) of non-syndromic hearing loss in our cohort of 800 Pakistani families. The c.1331+2T>C allele is recurrent. In addition, we identified a novel large deletion in a single family, which appears to have resulted from non-allelic homologous recombination between two similar Alu short interspersed elements. Finally, we observed no other clinical manifestations co-segregating with hearing loss in DFNB49 human families, and hypothesize that the additional abnormalities in the Marveld2 mutant mouse indicates a critical non-redundant function for tricellulin in other organ systems.