Deciphering the genetic architecture and ethnographic distribution of IRD in three ethnic populations by whole genome sequence analysis.

Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations.

A missense allele of PEX5 is responsible for the defective import of PTS2 cargo proteins into peroxisomes.

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.

Metabolome profiling of the developing murine lens.

Metabolomics is a study of the entire repertoire of metabolites in a cell at a particular time point. Here, we investigate the mouse lens at multiple embryonic and postnatal time points to establish the metabolome profile during early lens development. The lenses were isolated at six time points including embryonic day 15 (E15) and E18 and postnatal day 0 (P0), P3, P6, and P9. A total of four biological replicates of each time point, each consisting of 25 mg of lens tissue were preserved. Sample preparation was performed by protein precipitation followed by centrifugation to remove proteins and recover metabolites. The resulting extract was subjected to reverse phase/ultra-performance liquid chromatography-tandem mass spectrometry. Metabolome profiling identified a total of 353 metabolites in mouse lens, marked with an abundance of collagen, antioxidant, glycosaminoglycans, lipid, amino acid, and energy-related metabolites. A comparative metabolome analysis identified >200 metabolites exhibiting increased levels (p < 0.05) at latter time points relative to E15. Principal component analysis revealed distinct metabolomic signatures running from E15 to P9 while random forest analysis categorized lipid-, amino acid-, and nucleotide-related metabolites contributing significantly to the separation of the time points. To the best of our knowledge, this is the first report investigating the mouse lens metabolome at multiple embryonic and postnatal time points.

Novel mutations in LTBP2 identified in familial cases of primary congenital glaucoma.

Purpose: Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder caused by developmental defects in the anterior chamber and trabecular meshwork. This disease is an important cause of childhood blindness. In this study, we aim to identify the genetic determinants of PCG in three consanguineous families of Pakistani descent. Methods: Affected members of all three families underwent detailed ophthalmological examination including slit-lamp biomicroscopy. Blood samples were collected from affected and healthy members of all three families, and genomic DNA was extracted. Linkage analysis was performed for the known or reported loci of PCG to localize the disease interval, and logarithm of odds (LOD) scores were calculated. All protein-coding exons of the candidate gene, latent transforming growth factor-beta binding protein 2 (LTBP2), were bidirectionally sequenced to identify the disease-causing mutation. Results: Short tandem repeat (STR) marker-based linkage analysis localized the critical interval to chromosome 14q with a maximum two-point LOD score of 2.86 (PKGL076), 2.8 (PKGL015), and 2.92 (PKGL042). Bidirectional Sanger sequencing of LTBP2 revealed three novel pathogenic variants, i.e., c.3028G>A (p.Asp1010Asn), c.3427delC (p.Gln1143Argfs*35), and c.5270G>A (p.Cys1757Tyr) in PKGL076, PKGL015, and PKGL042, respectively. All three mutations segregated with the disease phenotype in their respective families and were absent in 200 ethnically matched normal chromosomes. Conclusions: We identified three novel mutations, p.D1010N, p.Q1143Rfs*35, and p.C1757Y, in LTBP2 responsible for PCG.

Mutations in FYCO1 identified in families with congenital cataracts.

Purpose: This study was designed to identify the pathogenic variants in three consanguineous families with congenital cataracts segregating as a recessive trait. Methods: Consanguineous families with multiple individuals manifesting congenital cataracts were ascertained. All participating members underwent an ophthalmic examination. A small aliquot of the blood sample was collected from all participating individuals, and genomic DNAs were extracted. Homozygosity-based linkage analysis was performed using short tandem repeat (STR) markers. The haplotypes were constructed with alleles of the STR markers, and the two-point logarithm of odds (LOD) scores were calculated. The candidate gene was sequenced bidirectionally to identify the disease-causing mutations. Results: Linkage analysis localized the disease interval to chromosome 3p in three families. Subsequently, bidirectional Sanger sequencing identified two novel mutations-a single base deletion resulting in a frameshift (c.3196delC; p.His1066IlefsTer10) mutation and a single base substitution resulting in a nonsense (c.4270C>T; p.Arg1424Ter) mutation-and a known missense (c.4127T>C, p.Leu1376Pro) mutation in FYCO1. All three mutations showed complete segregation with the disease phenotype and were absent in 96 ethnically matched control individuals. Conclusions: We report two novel mutations and a previously reported mutation in FYCO1 in three large consanguineous families. Taken together, mutations in FYCO1 contribute nearly 15% to the total genetic load of autosomal recessive congenital cataracts in this cohort.

Comparative transcriptome analysis of hESC- and iPSC-derived corneal endothelial cells.

The corneal endothelium (CE), a monolayer of hexagonal cells constitutes the innermost layer of the cornea that is critical in maintaining clarity by mediating hydration through barrier and pump functions. Corneal endothelial cells (CECs) have limited proliferative potential and therefore generation of CECs has been undertaken by many groups. We previously reported generation of CECs from peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs). In here, we extend our analysis through next-generation seqeuncing based transcriptome profiling of H9 human embryonic stem cell (hESC)- and human PBMC-originated, iPSC-derived CECs. The differentiating CECs on day 20 (D20) exhibited a tightly packed hexagonal/polygonal shape expressing zona occludens-1 (ZO-1) and N-cadherin at the cell boundaries. Next-generation RNA sequencing of hESC- and iPSC-derived CECs detected expression (≥0.659 RPKM) of 13,546 and 13,536 genes, respectively. Comparative transcriptome analysis of hESC- and iPSC-derived CECs revealed 13,208 (>96%) genes common in both transcriptomes. Among the 13,208 genes common in these transcriptomes, 12,580 (>95%) exhibited a quantitatively similar expression. To the best of our knowledge, this is the first report presenting comparative transcriptome analysis of hESC- and iPSC-derived CECs.

Non-coding RNA profiling of the developing murine lens.

Non-coding RNAs (ncRNAs) are emerging as an important player in the regulation of genome integrity and gene expression, and they have been implicated in the pathogenesis of many diseases. The aim of the present study is to identify the repertoire of ncRNAs expressed in the developing mouse lens. We previously reported the mouse lens transcriptome, including mRNA and microRNA (miRNA) profiling at two embryonic (E15 and E18) and four postnatal (P0, P3, P6, and P9) time points. We analyzed the data from small RNA-Seq and mRNA-Seq libraries to investigate the ncRNA profile. Our analysis revealed expression of 12 different classes of ncRNA in the murine lens at six developmental time points. Annotation of small RNA data showed expression of 1,756 antisense ncRNA (asncRNA) in the mouse lens transcriptome. Likewise, we identified 82 P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA), 345 transfer RNA (tRNA), 12 small nuclear RNA (snRNA), 167 small nucleolar RNA (snoRNA), 19 small Cajal body-specific RNA (scaRNA), six ribosomal RNA (rRNA), 18 tRNA-like structures, one MALAT1-associated small cytoplasmic RNA (mascRNA), one Vault RNA (vtRNA), and one Y RNA expressed in the developing mouse lens. In parallel, bioinformatic investigation of mRNA-Seq data identified expression of 1,952 long intergenic ncRNA (lincRNA) in the developing mouse lens. In conclusion, we report a comprehensive ncRNA profile in the murine lens at six developmental time points. To the best of our knowledge, this is first report investigating different classes of ncRNAs in the developing mouse lens and will be monumental in elucidating processes essential for the development of the ocular lens and the maintenance of its transparency.

Splice-site mutations identified in PDE6A responsible for retinitis pigmentosa in consanguineous Pakistani families.

PURPOSE: This study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin. METHODS: Ophthalmic examinations that included funduscopy and electroretinography (ERG) were performed to confirm the affectation status. Blood samples were collected from all participating individuals, and genomic DNA was extracted. A genome-wide scan was performed, and two-point logarithm of odds (LOD) scores were calculated. Sanger sequencing was performed to identify the causative variants. Subsequently, we performed whole exome sequencing to rule out the possibility of a second causal variant within the linkage interval. Sequence conservation was performed with alignment analyses of PDE6A orthologs, and in silico splicing analysis was completed with Human Splicing Finder version 2.4.1. RESULTS: A large multigenerational consanguineous family diagnosed with early-onset RP was ascertained. An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP. A genome-wide scan was performed, and suggestive two-point LOD scores were observed with markers on chromosome 5q. Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family. Subsequently, we performed a second genome-wide scan that excluded the entire genome except the chromosome 5q region harboring PDE6A. Next-generation whole exome sequencing identified a splice acceptor site mutation in intron 16: c.2028-1G>A, which was completely conserved in PDE6A orthologs and was absent in ethnically matched 350 control chromosomes, the 1000 Genomes database, and the NHLBI Exome Sequencing Project. Subsequently, we investigated our entire cohort of RP familial cases and identified a second family who harbored a splice acceptor site mutation in intron 10: c.1408-2A>G. In silico analysis suggested that these mutations will result in the elimination of wild-type splice acceptor sites that would result in either skipping of the respective exon or the creation of a new cryptic splice acceptor site; both possibilities would result in retinal photoreceptor cells that lack PDE6A wild-type protein. CONCLUSIONS: we report two splice acceptor site variations in PDE6A in consanguineous Pakistani families who manifested cardinal symptoms of RP. Taken together with our previously published work, our data suggest that mutations in PDE6A account for about 2% of the total genetic load of RP in our cohort and possibly in the Pakistani population as well.

Targeted capture and next-generation sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79.

Targeted genome capture combined with next-generation sequencing was used to analyze 2.9 Mb of the DFNB79 interval on chromosome 9q34.3, which includes 108 candidate genes. Genomic DNA from an affected member of a consanguineous family segregating recessive, nonsyndromic hearing loss was used to make a library of fragments covering the DFNB79 linkage interval defined by genetic analyses of four pedigrees. Homozygosity for eight previously unreported variants in transcribed sequences was detected by evaluating a library of 402,554 sequencing reads and was later confirmed by Sanger sequencing. Of these variants, six were determined to be polymorphisms in the Pakistani population, and one was in a noncoding gene that was subsequently excluded genetically from the DFNB79 linkage interval. The remaining variant was a nonsense mutation in a predicted gene, C9orf75, renamed TPRN. Evaluation of the other three DFNB79-linked families identified three additional frameshift mutations, for a total of four truncating alleles of this gene. Although TPRN is expressed in many tissues, immunolocalization of the protein product in the mouse cochlea shows prominent expression in the taper region of hair cell stereocilia. Consequently, we named the protein taperin.

Molecular basis of DFNB73: mutations of BSND can cause nonsyndromic deafness or Bartter syndrome.

BSND encodes barttin, an accessory subunit of renal and inner ear chloride channels. To date, all mutations of BSND have been shown to cause Bartter syndrome type IV, characterized by significant renal abnormalities and deafness. We identified a BSND mutation (p.I12T) in four kindreds segregating nonsyndromic deafness linked to a 4.04-cM interval on chromosome 1p32.3. The functional consequences of p.I12T differ from BSND mutations that cause renal failure and deafness in Bartter syndrome type IV. p.I12T leaves chloride channel function unaffected and only interferes with chaperone function of barttin in intracellular trafficking. This study provides functional data implicating a hypomorphic allele of BSND as a cause of apparent nonsyndromic deafness. We demonstrate that BSND mutations with different functional consequences are the basis for either syndromic or nonsyndromic deafness.

A genomic deletion encompassing CRYBB2-CRYBB2P1 is responsible for autosomal recessive congenital cataracts.

Here we report a consanguineous Pakistani family with multiple affected individuals with autosomal recessive congenital cataract (arCC). Exclusion analysis established linkage to chromosome 22q, and Sanger sequencing coupled with PCR-based chromosome walking identified a large homozygous genomic deletion. Our data suggest that this deletion leads to CRYBB2-CRYBB2P1 fusion, consisting of exons 1-5 of CRYBB2 and exon 6 of CRYBB2P1, the latter of which harbors the c.463 C > T (p.Gln155*) mutation, and is responsible for arCC.

Next-generation whole exome sequencing to delineate the genetic basis of primary congenital glaucoma.

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.

The role of FYCO1-dependent autophagy in lens fiber cell differentiation.

FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in FYCO1 cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show fyco1 homozygous knockout (fyco1-/-) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in fyco1-/- mice lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in FYCO1 (c.2206C>T) KI lens-like organoid structures and in fyco1-/- mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis.Abbreviations: CC: congenital cataracts; DE: differentially expressed; ER: endoplasmic reticulum; FYCO1: FYVE and coiled-coil domain containing 1; hESC: human embryonic stem cell; KI: knock-in; OFZ: organelle-free zone; qRT-PCR: quantitative real-time PCR; PE: phosphatidylethanolamine; RNA-Seq: RNA sequencing; SD: standard deviation; sgRNA: single guide RNA; shRNA: shorthairpin RNA; TEM: transmission electron microscopy; WT: wild type.

Cigarette Smoke Triggers Loss of Corneal Endothelial Cells and Disruption of Descemet's Membrane Proteins in Mice.

Purpose: To investigate changes at a molecular level in the mouse corneal endothelium (CE) exposed to chronic cigarette smoke (CS). Methods: Pregnant mice (gestation days 18-20) were placed in a whole-body exposure smoking chamber, and a few days later pups were born. After 3.5 months of CS exposure, a ConfoScan4 scanning microscope was used to examine the corneal endothelial cells (CECs) of CS-exposed and control (Ct) mice. The CE was peeled under a microscope and maintained as four biological replicates (two male and two female) for CS-exposed and Ct mice; each replicate consisted of 16 CEs. The proteome of the CE was investigated through mass spectrometry. Results: The CE images of CS-exposed and Ct mice revealed a difference in the shape of CECs accompanied by a nearly 10% decrease in CEC density (P < 0.00003) following CS exposure. Proteome profiling identified a total of 524 proteins exhibiting statistically significant changes in CE from CS-exposed mice. Importantly, proteins associated with Descemet's membrane (DM), including COL4α1, COL4α2, COL4α3, COL4α4, COL4α5, COL4α6, COL8α1, COL8α2, and FN1, among others, exhibited diminished protein levels in the CE of CS-exposed mice. Conclusions: Our data confirm that exposure to CS results in reduced CEC density accompanied by diminished levels of multiple collagen and extracellular matrix proteins associated with DM.

Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty.

Here, we evaluate the efficacy of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) to form a functional monolayer of corneal endothelium (CE) in rabbits and monkeys. We injected cryopreserved hESC-derived CECs into the anterior chamber of rabbits and monkeys either immediately after mechanical scraping of the central CE or a few days later when corneal edema developed. All preclinical models developed deturgesced and clear corneas following the injection of cryopreserved hESC-derived CECs and remained comparable to the corneas of the untreated eye. Confocal scanning microscopy confirmed an intact structure of hexagonal/polygonal cells and immunohistochemical analysis illustrated a monolayer expressing barrier and pump function proteins in the regenerated CE. The necropsy examination confirmed no remarkable change in multiple tissues assessed for teratoma formation. In conclusion, our data demonstrate the efficacy of cryopreserved hESC-derived CECs to form a functional CE on the denuded Descemet's membrane.

Examining the effects of cigarette smoke on mouse lens through a multi OMIC approach.

Here, we report a multi OMIC (transcriptome, proteome, and metabolome) approach to investigate molecular changes in lens fiber cells (FC) of mice exposed to cigarette smoke (CS). Pregnant mice were placed in a whole-body smoke chamber and a few days later pups were born, which were exposed to CS for 5 hours/day, 5 days/week for a total of 3½ months. We examined the mice exposed to CS for CS-related cataractogenesis after completion of the CS exposure but no cataracts were observed. Lenses of CS-exposed and age-matched, untreated control mice were extracted and lens FC were subjected to multi OMIC profiling. We identified 348 genes, 130 proteins, and 14 metabolites exhibiting significant (p < 0.05) differential levels in lens FC of mice exposed to CS, corresponding to 3.6%, 4.3%, and 5.0% of the total genes, protein, and metabolites, respectively identified in this study. Our multi OMIC approach confirmed that only a small fraction of the transcriptome, the proteome, and the metabolome was perturbed in the lens FC of mice exposed to CS, which suggests that exposure of CS had a minimal effect on the mouse lens. It is worth noting that while our results confirm that CS exposure does not have a substantial impact on the molecular landscape of the mouse lens FC, we cannot rule out that CS exposure for longer durations and/or in combination with other morbidities or environmental factors would have a more robust effect and/or result in cataractogenesis.

Generation and proteome profiling of PBMC-originated, iPSC-derived lentoid bodies.

Here, we report proteome profiling of peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived, lens-like organoids termed lentoid bodies at two differentiation time points. A small aliquot of the blood sample was ascertained to collect PBMCs that were reprogrammed to iPSCs. The PBMC-originated, iPSCs were differentiated to lentoid bodies employing the "fried egg" method. Quantitative real-time PCR (qRT-PCR) analysis revealed increased expression levels of lens-associated markers in lentoid bodies while transmission electron microscopy identified closely packed lens epithelial- and differentiating fiber-like cells in lentoid bodies. Total cellular protein was extracted from lentoid bodies at differentiation day 25 and mass spectrometry identified a total of 9,473 proteins. The low counts of crystallin proteins at differentiation day 25 prompted us to re-examine the proteome at differentiation day 35 as we reasoned that 10 additional days of differentiation will increase the crystallin count. However, we did not detect any substantial increase in crystallin protein counts at differentiation day 35. In conclusion, we report generation and proteome profiles of PBMC-originated, iPSC-derived lentoid bodies at multiple differentiation time points.

Whole genome sequencing data of multiple individuals of Pakistani descent.

Here we report whole genome sequencing of four individuals (H3, H4, H5, and H6) from a family of Pakistani descent. Whole genome sequencing yielded 1084.92, 894.73, 1068.62, and 1005.77 million mapped reads corresponding to 162.73, 134.21, 160.29, and 150.86 Gb sequence data and 52.49x, 43.29x, 51.70x, and 48.66x average coverage for H3, H4, H5, and H6, respectively. We identified 3,529,659, 3,478,495, 3,407,895, and 3,426,862 variants in the genomes of H3, H4, H5, and H6, respectively, including 1,668,024 variants common in the four genomes. Further, we identified 42,422, 39,824, 28,599, and 35,206 novel variants in the genomes of H3, H4, H5, and H6, respectively. A major fraction of the variants identified in the four genomes reside within the intergenic regions of the genome. Single nucleotide polymorphism (SNP) genotype based comparative analysis with ethnic populations of 1000 Genomes database linked the ancestry of all four genomes with the South Asian populations, which was further supported by mitochondria based haplogroup analysis. In conclusion, we report whole genome sequencing of four individuals of Pakistani descent.

SLC26A4 mutation spectrum associated with DFNB4 deafness and Pendred's syndrome in Pakistanis.

Pendred's syndrome (PDS) is an autosomal-recessive disorder characterized by sensorineural hearing loss and goiter. PDS is caused by mutations of the SLC26A4 gene encoding pendrin, a transmembrane exchanger of Cl(-), I(-) and HCO(3)(-), which is expressed in the thyroid and inner ear. SLC26A4 mutations can also be associated with non-syndromic deafness, DFNB4. The goal of our study was to define the identities and frequencies of SLC26A4 mutations in 563 large, consanguineous Pakistani families segregating severe-to-profound recessive deafness. Sequence analyses of SLC26A4 in 46 unreported families segregating deafness linked to DFNB4/PDS revealed 16 probable pathogenic variants, 8 of which are novel. The novel variants include three missense substitutions (p.R24L, p.G139V and p.V231M), two splice site mutations (c.304+2T>C and c.1341+3A>C), one frameshift (p.C565MfsX8) and two different genomic deletions affecting exons 1-2 and 11-18. Each of six pathogenic variants (p.V239D, p.Q446R, p.S90L, p.Y556C, p.R24L and p.K715N) was found in more than one family and haplotype analyses suggest that they are founder mutations. Combined with earlier reported data, SLC26A4 mutations were identified in 56 (7.2%; 95% CI: 5.6-9.2%) of 775 families. Therefore, SLC26A4 mutations are the most common known cause of genetic deafness in this population. As p.V239D (30%), p.S90L (18%) and p.Q446R (18%) account for approximately two-third of the mutant alleles of SLC26A4, hierarchical strategies for mutation detection would be feasible and cost-efficient genetic tests for DFNB4 deafness and PDS in Pakistanis.