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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.

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling—a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to ‘nucleotide empty’ or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders. Age-related macular degeneration (AMD) has been connected to deficits in autophagy. Here, the authors demonstrate, in mice and dry-AMD patient samples, that calcium and integrin binding protein 2 (CIB2) regulates Rheb-mTORC1 signaling axis, and subsequently autophagy.

Intellectual disability (ID) is a clinically and genetically heterogeneous disorder, affecting 1–3% of the general population. Although research into the genetic causes of ID has recently gained momentum, identification of pathogenic mutations that cause autosomal recessive ID (ARID) has lagged behind, predominantly due to non-availability of sizeable families. Here we present the results of exome sequencing in 121 large consanguineous Pakistani ID families. In 60 families, we identified homozygous or compound heterozygous DNA variants in a single gene, 30 affecting reported ID genes and 30 affecting novel candidate ID genes. Potential pathogenicity of these alleles was supported by co-segregation with the phenotype, low frequency in control populations and the application of stringent bioinformatics analyses. In another eight families segregation of multiple pathogenic variants was observed, affecting 19 genes that were either known or are novel candidates for ID. Transcriptome profiles of normal human brain tissues showed that the novel candidate ID genes formed a network significantly enriched for transcriptional co-expression ( P< 0.0001) in the frontal cortex during fetal development and in the temporal–parietal and sub-cortex during infancy through adulthood. In addition, proteins encoded by 12 novel ID genes directly interact with previously reported ID proteins in six known pathways essential for cognitive function ( P< 0.0001). These results suggest that disruptions of temporal parietal and sub-cortical neurogenesis during infancy are critical to the pathophysiology of ID. These findings further expand the existing repertoire of genes involved in ARID, and provide new insights into the molecular mechanisms and the transcriptome map of ID.

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.

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.

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.

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.

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.

It is proposed that light neutrinos form a triplet in a global SU(3) symmetry in the mass eigenstate basis. Assuming that the SU(3) symmetry is broken in the direction \\((-a\\lambda_{3}+\\frac{b}{\\sqrt{3}}\\lambda_{8})\\), and after going to the flavor basis, we predict the atmospheric mixing angles sin2θ23=0.5 and sinθ13=0, if νμ–ντ symmetry is assumed. In the flavor basis, the diagonal part of the matrix coefficient of b (the dominant part) is found to transform like \\((\\lambda_{3}+\\frac{1}{\\sqrt{3}}\\lambda_{8})\\). Imposing the same condition on the matrix coefficient of a fixes the solar mixing angle, \\(\\sin^{2}\\theta_{12}= \\frac{1}{3}\\). The implications for neutrinoless double beta decay are discussed.