Search

PurposeThe Pakistan Punjab population has been a rich source for identifying genes causing or contributing to autosomal recessive retinal degenerations (arRD). This study was carried out to delineate the genetic architecture of arRD in the Pakistani population.MethodsThe genetic origin of arRD in a total of 144 families selected only for having consanguineous marriages and multiple members affected with arRD was examined. Of these, causative mutations had been identified in 62 families while only the locus had been identified for an additional 15. The remaining 67 families were subjected to homozygosity exclusion mapping by screening of closely flanking microsatellite markers at 180 known candidate genes/loci followed by sequencing of the candidate gene for pathogenic changes.ResultsOf these 67 families subjected to homozygosity mapping, 38 showed homozygosity for at least one of the 180 regions, and sequencing of the corresponding genes showed homozygous cosegregating mutations in 27 families. Overall, mutations were detected in approximately 61.8 % (89/144) of arRD families tested, with another 10.4% (15/144) being mapped to a locus but without a gene identified.ConclusionsThese results suggest the involvement of unmapped novel genes in the remaining 27.8% (40/144) of families. In addition, this study demonstrates that homozygosity mapping remains a powerful tool for identifying the genetic defect underlying genetically heterogeneous arRD disorders in consanguineous marriages for both research and clinical applications.

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.

PurposeThis study was undertaken to identify causal mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in consanguineous families.MethodsLarge consanguineous families were ascertained from the Punjab province of Pakistan. An ophthalmic examination consisting of a fundus evaluation and electroretinography (ERG) was completed, and small aliquots of blood were collected from all participating individuals. Genomic DNA was extracted from white blood cells, and a genome-wide linkage or a locus-specific exclusion analysis was completed with polymorphic short tandem repeats (STRs). Two-point logarithm of odds (LOD) scores were calculated, and all coding exons and exon-intron boundaries of RP1 were sequenced to identify the causal mutation.ResultsThe ophthalmic examination showed that affected individuals in all families manifest cardinal symptoms of RP. Genome-wide scans localized the disease phenotype to chromosome 8q, a region harboring RP1, a gene previously implicated in the pathogenesis of RP. Sanger sequencing identified a homozygous single base deletion in exon 4: c.3697delT (p.S1233Pfs22*), a single base substitution in intron 3: c.787+1G>A (p.I263Nfs8*), a 2 bp duplication in exon 2: c.551_552dupTA (p.Q185Yfs4*) and an 11,117 bp deletion that removes all three coding exons of RP1. These variations segregated with the disease phenotype within the respective families and were not present in ethnically matched control samples.ConclusionsThese results strongly suggest that these mutations in RP1 are responsible for the retinal phenotype in affected individuals of all four consanguineous families.

To delineate the genetic determinants associated with retinitis pigmentosa (RP), a hereditary retinal disorder, we recruited four large families manifesting cardinal symptoms of RP. We localized these families to regions on the human genome harboring the α and β subunits of phosphodiesterase 6 and identified mutations that were absent in control chromosomes. Our data suggest that mutations in PDE6A and PDE6B are responsible for the retinal phenotype in these families.

PurposeTo identify pathogenic mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in consanguineous familial cases.MethodsSeven large familial cases with multiple individuals diagnosed with retinitis pigmentosa were included in the study. Affected individuals in these families underwent ophthalmic examinations to document the symptoms and confirm the initial diagnosis. Blood samples were collected from all participating members, and genomic DNA was extracted. An exclusion analysis with microsatellite markers spanning the TULP1 locus on chromosome 6p was performed, and two-point logarithm of odds (LOD) scores were calculated. All coding exons along with the exon-intron boundaries of TULP1 were sequenced bidirectionally. We constructed a single nucleotide polymorphism (SNP) haplotype for the four familial cases harboring the K489R allele and estimated the likelihood of a founder effect.ResultsThe ophthalmic examinations of the affected individuals in these familial cases were suggestive of RP. Exclusion analyses confirmed linkage to chromosome 6p harboring TULP1 with positive two-point LOD scores. Subsequent Sanger sequencing identified the single base pair substitution in exon14, c.1466A>G (p.K489R), in four families. Additionally, we identified a two-base deletion in exon 4, c.286_287delGA (p.E96Gfs77*); a homozygous splice site variant in intron 14, c.1495+4A>C; and a novel missense variation in exon 15, c.1561C>T (p.P521S). All mutations segregated with the disease phenotype in the respective families and were absent in ethnically matched control chromosomes. Haplotype analysis suggested (p

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

PurposeThis study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin.MethodsOphthalmic 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.ResultsA 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.Conclusionswe 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.

PurposeThis study was undertaken to investigate the causal mutations responsible for autosomal recessive congenital stationary night blindness (CSNB) in consanguineous Pakistani families.MethodsTwo consanguineous families with multiple individuals manifesting symptoms of stationary night blindness were recruited. Affected individuals underwent a detailed ophthalmological examination, including fundus examination and electroretinography. Blood samples were collected and genomic DNA was extracted. Exclusion analyses were completed by genotyping closely spaced microsatellite markers, and two-point logarithm of odds (LOD) scores were calculated. All coding exons, along with the exon-intron boundaries of GRM6, were sequenced bidirectionally.ResultsAccording to the medical history available to us, affected individuals in both families had experienced night blindness from the early years of their lives. Fundus photographs of affected individuals in both the families appeared normal, with no signs of attenuated arteries or bone spicule pigmentation. The scotopic electroretinogram (ERG) response were absent in all of the affected individuals, while the photopic measurements show reduced b-waves. During exclusion analyses, both families localized to a region on chromosome 5q that harbors GRM6, a gene previously associated with autosomal recessive CSNB. Bidirectional sequencing of GRM6 identified homozygous single base pair changes, specifically c.1336C>T (p.R446X) and c.2267G>A (p.G756D) in families PKRP170 and PKRP172, respectively.ConclusionsWe identified a novel nonsense and a previously reported missense mutation in GRM6 that were responsible for autosomal recessive CSNB in patients of Pakistani decent.

A genome-wide scan of individuals affected with autosomal-recessive congenital stationary night blindness (CSNB) is undertaken to identify the causative gene for the disorder. Results suggest that a 2 bp deletion mutation in the SLC24A1 gene located at chromosome 15q is associated with the cause of CSNB.

A splice-site mutation in an unknown exon of BBS8 (MIM 608132), one of the genes involved in pleiotropic Bardet-Biedi syndrome (BBS) which is to cause nonsyndromic recessive RP in a consanguineous Pakistani family is reported. The exon which represented the major BBS8 mRNA species in the mammalian photoreceptor indicated that the encoded 10 amino acids play a pivotal role in the function of BBS8 in retina.

A study uses targeted capture and next-generation sequencing to identify the gene which causes nonsyndromic deafness DFNB79. Results reveal that a nonsense mutation in the gene C9orf75, also called TPRN, which encodes the protein taperin, is responsible for the nonsyndromic deafness DFNB79.

Tricellulin is concentrated in the tight-type tight junctions (TJs) of mammalian inner-ear epithelia, including the tricellular junctions of the reticular lamina of the organ of Corti. It is also demonstrated that there are multiple alternatively spliced isoforms of TRIC in various tissues and that mutations of TRIC associated with hearing loss remove most of a conserved region in the cytosolic domain that binds to the cytosolic scaffolding protein ZO-1.

Abstract Background Ischemia is the major cause of acute kidney injury (AKI), associated with high mortality and morbidity. Mesenchymal stem cells (MSCs) have multilineage differentiation potential and can be a potent therapeutic option for the cure of AKI. Methods MSCs were cultured in four groups SNAP (S-nitroso N-acetyl penicillamine), SNAP + Methylene Blue (MB), MB and a control for in vitro analysis. Cultured MSCs were pre-conditioned with either SNAP (100 μM) or MB (1 μM) or both for 6 hours. Renal ischemia was induced in four groups (as in in vitro study) of rats by clamping the left renal padicle for 45 minutes and then different pre-conditioned stem cells were transplanted. Results We report that pre-conditioning of MSCs with SNAP enhances their proliferation, survival and engraftment in ischemic kidney. Rat MSCs pre-conditioned with SNAP decreased cell apoptosis and increased proliferation and cytoprotective genes’ expression in vitro. Our in vivo data showed enhanced survival and engraftment, proliferation, reduction in fibrosis, significant improvement in renal function and higher expression of pro-survival and pro-angiogenic factors in ischemic renal tissue in SNAP pre-conditioned group of animals. Cytoprotective effects of SNAP pre-conditioning were abrogated by MB, an inhibitor of nitric oxide synthase (NOS) and guanylate cyclase. Conclusion The results of these studies demonstrate that SNAP pre-conditioning might be useful to enhance therapeutic potential of MSCs in attenuating renal ischemia reperfusion injury.

Abstract Background Liver fibrosis is a major health problem worldwide and poses a serious obstacle for cell based therapies. Mesenchymal stem cells (MSCs) are multipotent and important candidate cells for future clinical applications however success of MSC therapy depends upon their homing and survival in recipient organs. This study was designed to improve the repair potential of MSCs by transplanting them in sodium nitroprusside (SNP) pretreated mice with CCl4 induced liver fibrosis. Methods SNP 100 mM, a nitric oxide (NO) donor, was administered twice a week for 4 weeks to CCl4-injured mice. MSCs were isolated from C57BL/6 wild type mice and transplanted in the left lateral lobe of the liver in experimental animals. After 4 weeks, animals were sacrificed and liver improvement was analyzed. Analysis of fibrosis by qRT-PCR and sirius red staining, homing, bilirubin and alkaline phosphatase (ALP) serum levels between different treatment groups were compared to control. Results Liver histology demonstrated enhanced MSCs homing in SNP-MSCs group compared to MSCs group. The gene expression of fibrotic markers; αSMA, collagen 1α1, TIMP, NFκB and iNOS was down regulated while cytokeratin 18, albumin and eNOS was up-regulated in SNP-MSCs group. Combine treatment sequentially reduced fibrosis in SNP-MSCs treated liver compared to the other treatment groups. These results were also comparable with reduced serum levels of bilirubin and ALP observed in SNP-MSCs treated group. Conclusion This study demonstrated that NO effectively augments MSC ability to repair liver fibrosis induced by CCl4 in mice and therefore is a better treatment regimen to reduce liver fibrosis.

Abstract Background Recessive mutations of fibroblast growth factor 3 (FGF3) can cause LAMM syndrome (OMIM 610706), characterized by fully penetrant complete labyrinthine aplasia, microtia and microdontia. Methods We performed a prospective molecular genetic and clinical study of families segregating hearing loss linked to FGF3 mutations. Ten affected individuals from three large Pakistani families segregating FGF3 mutations were imaged with CT, MRI, or both to detect inner ear abnormalities. We also modeled the three dimensional structure of FGF3 to better understand the structural consequences of the three missense mutations. Results Two families segregated reported mutations (p.R104X and p.R95W) and one family segregated a novel mutation (p.R132GfsX26) of FGF3. All individuals homozygous for p.R104X or p.R132GfsX26 had fully penetrant features of LAMM syndrome. However, recessive p.R95W mutations were associated with nearly normal looking auricles and variable inner ear structural phenotypes, similar to that reported for a Somali family also segregating p.R95W. This suggests that the mild phenotype is not entirely due to genetic background. Molecular modeling result suggests a less drastic effect of p.R95W on FGF3 function compared with known missense mutations detected in fully penetrant LAMM syndrome. Since we detected significant intrafamilial variability of the inner ear structural phenotype in the family segregating p.R95W, we also sequenced FGF10 as a likely candidate for a modifier. However, we did not find any sequence variation, pointing out that a larger sample size will be needed to map and identify a modifier. We also observed a mild to moderate bilateral conductive hearing loss in three carriers of p.R95W, suggesting either a semi-dominant effect of this mutant allele of FGF3, otitis media, or a consequence of genetic background in these three family members. Conclusions We noted a less prominent dental and external ear phenotype in association with the homozygous p.R95W. Therefore, we conclude that the manifestations of recessive FGF3 mutations range from fully penetrant LAMM syndrome to deafness with residual inner ear structures and, by extension, with minimal syndromic features, an observation with implications for cochlear implantation candidacy.

Conventional approaches can repair minor skin injuries; however, severe burn injuries require innovative approaches for efficient and better wound repair. Recent studies indicate that stem cell‐based regenerative therapies can restore severe damaged skin both structurally and functionally. The current study aims to evaluate the wound healing potential of skin substitute derived from human neonatal stem cells (hNSCs) using a severe burn injury rat model. Amniotic epithelial cells (AECs) and mesenchymal stem cells (MSCs) were isolated from placenta (a source of neonatal stem cells) by explant culture method. After characterization, AECs and umbilical cord‐MSCs were differentiated into keratinocyte and fibroblasts, respectively. Morphological changes, and expression of corresponding keratinocyte and fibroblast specific markers were used to verify differentiation into respective lineage. A skin substitute was developed by mixing hNSCs‐derived skin cells (hNSCs‐SCs) in plasma for transplantation in a rat model of severe burn injury. Results indicated that placenta‐derived AECs and MSCs were efficiently differentiated into skin cells, that is, keratinocytes and fibroblasts, respectively, as indicated by morphological changes, immunostaining, and polymerase chain reaction analysis. Further, transplantation of hNSCs‐SCs seeded in plasma significantly improved basic skin architecture, re‐epithelization rate, and wound healing concurrent with reduced apoptosis. In conclusion, neonatal stem cell‐derived skin substitute efficiently improved severe burn wounds in a rat model of burn injury. Unique properties of placenta‐derived stem cells make them superlative candidates for the development of "off‐the‐shelf" artificial skin substitutes for future use. [ABSTRACT FROM AUTHOR]

Consanguineous Pakistani pedigrees segregating deafness have contributed decisively to the discovery of 31 of the 68 genes associated with nonsyndromic autosomal recessive hearing loss (HL) worldwide. In this study, we utilized genome‐wide genotyping, Sanger and exome sequencing to identify 163 DNA variants in 41 previously reported HL genes segregating in 321 Pakistani families. Of these, 70 (42.9%) variants identified in 29 genes are novel. As expected from genetic studies of disorders segregating in consanguineous families, the majority of affected individuals (94.4%) are homozygous for HL‐associated variants, with the other variants being compound heterozygotes. The five most common HL genes in the Pakistani population are SLC26A4, MYO7A, GJB2, CIB2 and HGF, respectively. Our study provides a profile of the genetic etiology of HL in Pakistani families, which will allow for the development of more efficient genetic diagnostic tools, aid in accurate genetic counseling, and guide application of future gene‐based therapies. These findings are also valuable in interpreting pathogenicity of variants that are potentially associated with HL in individuals of all ancestries. The Pakistani population, and its infrastructure for studying human genetics, will continue to be valuable to gene discovery for HL and other inherited disorders. In this study, we utilized genome‐wide genotyping, Sanger and exome sequencing to identify 163 DNA variants in 41 previously reported hearing loss (HL) genes segregating in 321 Pakistani families. SLC26A4, MYO7A, GJB2, CIB2 and HGF were identified as the five most common genes in our cohort. Our study provides a profile of the genetic etiology of HL in Pakistani families, which will allow for the development of more efficient genetic diagnostic tools, aid in accurate genetic counseling and guide application of future gene‐based therapies. [ABSTRACT FROM AUTHOR]

Aim: The aim of the present study is to use human amniotic membrane (HAM) for in vitro chondrogenesis of placenta-derived mesenchymal stem cells (MSCs) and umbilical cord-derived MSCs. Materials & methods: MSCs from the placenta and umbilical cord were isolated, characterized by immunophenotyping and after analyzing their rate of proliferation, cytotoxicity and viability, chondrogenesis was performed on plastic adherent surface and on HAM. Results: Successfully isolated and characterized placenta-derived MSCs and umbilical cord-derived MSCs revealed positive expression of MSCs markers CD90, CD73, CD105 and CD49d, while they were negative for CD45. Both types of cells in the presence of chondrogenic induction medium on plastic adherent surface and HAM showed aggregates of proteoglycan and strong expression of COL2A1 (collagen 2) and ACAN1 (aggrecan). Conclusion: HAM supported proliferation as well as chondrogenesis of MSCs and provide novelty of HAM utilization as an efficient natural delivery matrix for stem cell transplantation. [ABSTRACT FROM AUTHOR]

Background Usher syndrome (USH) is a neurosensory disorder characterised by deafness, variable vestibular areflexia and vision loss. The aim of the study was to identify the genetic defect in a Pakistani family (PKDF1051) segregating USH. Methods G enome-wide linkage analysis was performed by using an Illumina linkage array followed by Sanger and exome sequencing. Heterologous cells and mouse organ of Corti explant-based transfection assays were used for functional evaluations. Detailed clinical evaluations were performed to characterise the USH phenotype. Results T hrough homozygosity mapping, we genetically linked the USH phenotype segregating in family PKDF1051 to markers on chromosome 1p36.32-p36.22. The locus was designated USH1M. Using a combination of Sanger sequencing and exome sequencing, we identified a novel homozygous 18 base pair inframe deletion in ESPN. Variants of ESPN, encoding the actinbundling protein espin, have been previously associated with deafness and vestibular areflexia in humans with no apparent visual deficits. Our functional studies in heterologous cells and in mouse organ of Corti explant cultures revealed that the six deleted residues in affected individuals of family PKDF1051 are essential for the actin bundling function of espin demonstrated by ultracentrifugation actin binding and bundling assays. Funduscopic examination of the affected individuals of family PKDF1051 revealed irregular retinal contour, temporal flecks and disc pallor in both eyes. ERG revealed diminished rod photoreceptor function among affected individuals. Conclusion Our study uncovers an additional USH gene, assigns the USH1 phenotype to a variant of ESPN and provides a 12th molecular component to the USH proteome. [ABSTRACT FROM AUTHOR]

Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, DFNB100 on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in PPIP5K2 that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells and spiral ganglion neurons. Mice homozygous for a targeted deletion of the Ppip5k2 phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear. [ABSTRACT FROM AUTHOR]

Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl4injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl4which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl4-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson’s trichome staining was observed. [ABSTRACT FROM PUBLISHER]

The defective insulin production is associated with severely reduced islet cell mass leading to diabetes. Growth factors preconditioned stem cells have arisen as an effective therapy to treat many diseases including diabetes. The current study was designed to assess the effect of pretreatment of ASCs derived IPCs with combination of stromal cell derived factor 1 alpha (SDF1α) and basic fibroblast growth factor (bFGF) in improving glucose tolerance in streptozotocin induced diabetic rats. The results showed maximally significant reduction in hyperglycaemia and fibrosis, while up-regulation of survival and pancreas-specific genes, insulin levels and homing of transplanted cells in SDF-1α + bFGF IPCs transplanted rats as compared with other groups. Moreover, increased expression of insulin, glucagon and Glut-2 in pancreas of the SDF-1α + bFGF IPCs transplanted group indicated more regeneration of pancreas. Hence, the use of IPCs preconditioned with SDF-1α + bFGF would be more effective for treating diabetes. [ABSTRACT FROM PUBLISHER]

Purpose: The aim of this study is to identify the molecular basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous pedigree. Methods: All participating individuals underwent a detailed ophthalmic examination. Each patient’s medical history, particularly of cataracts and other ocular abnormalities, was compiled from available medical records and interviews with family elders. Blood samples were donated by all participating family members and used to extract genomic DNA. Genetic analysis was performed to rule out linkage to known arCC loci and genes. Whole-exome sequencing libraries were prepared and paired-end sequenced. A large deletion was found that segregated with arCC in the family, and chromosome walking was conducted to estimate the proximal and distal boundaries of the deletion mutation. Results: Exclusion and linkage analysis suggested linkage to a region of chromosome 6p24 harboring GCNT2 (glucosaminyl (N-acetyl) transferase 2) with a two-point logarithm of odds score of 5.78. PCR amplifications of the coding exons of GCNT2 failed in individuals with arCC, and whole-exome data analysis revealed a large deletion on chromosome 6p in the region harboring GCNT2. Chromosomal walking using multiple primer pairs delineated the extent of the deletion to approximately 190 kb. Interestingly, a failure to amplify a junctional fragment of the deletion break strongly suggests an insertion in addition to the large deletion. Conclusion: Here, we report a novel insertion/deletion mutation at the GCNT2 locus that is responsible for congenital cataracts in a large consanguineous family. [ABSTRACT FROM AUTHOR]

Purpose: To identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC) in a consanguineous family. Methods: All family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2), was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model. Results: Ophthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD) score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D) in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19) compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15) increased after birth to a level that was sustained through the postnatal time points. Conclusion: A novel missense mutation in LIM2 is responsible for autosomal recessive congenital cataracts. [ABSTRACT FROM AUTHOR]

ABSTRACT Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal-recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness-causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long-term maintenance of stereocilia, mechanosensory sound-transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A. [ABSTRACT FROM AUTHOR]