Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.
Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound mixed hearing loss locus, DFNB88 on chromosome 2p12-p11.2. Exome enrichment followed by massive parallel sequencing revealed a c.794T>C transition mutation in ELMOD3 that segregated with DFNB88-associated hearing loss in a large Pakistani family. This transition mutation is predicted to substitute a highly invariant leucine residue with serine (p.Leu265Ser) in the engulfment and cell motility (ELMO) domain of the protein. No biological activity has been described previously for the ELMOD3 protein. We investigated the biochemical properties and ELMOD3 expression to gain mechanistic insights into the function of ELMOD3 in the inner ear. In rodent inner ears, ELMOD3 immunoreactivity was observed in the cochlear and vestibular hair cells and supporting cells. However, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Ex vivo, ELMOD3 is associated with actin-based structures, and this link is impaired by the DFNB88 mutation. ELMOD3 exhibited GAP activity against Arl2, a small GTPase, providing a potential functional link between Arf family signaling and stereocilia actin-based cytoskeletal architecture. Our study provides new insights into the molecules that are necessary for the development and/or function of inner ear sensory cells.
Human hereditary deafness at the DFNB29 autosomal locus on chromosome 21q22.1 is caused by recessive mutations of CLDN14, encoding claudin 14. This tight junction protein is tetra-membrane 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 pre-lingual, bi-lateral, 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) while 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 approximately 67% of the mutant alleles of CLDN14 in our cohort. Combined with 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 (e.g. drug and noise exposure) and/or genetic modifiers.
CLDN14; claudin 14; DFNB29; mild hearing loss; profound deafness; Pakistan
Mesenchymal stem cells (MSCs) have the potential for treatment of diabetic cardiomyopathy; however, the repair capability of MSCs declines with age and disease. MSCs from diabetic animals exhibit impaired survival, proliferation, and differentiation and therefore require a strategy to improve their function. The aim of the study was to develop a preconditioning strategy to augment the ability of MSCs from diabetes patients to repair the diabetic heart.
Diabetes was induced in C57BL/6 mice (6 to 8 weeks) with streptozotocin injections (55 mg/kg) for 5 consecutive days. MSCs isolated from diabetic animals were preconditioned with medium from cardiomyocytes exposed to oxidative stress and high glucose (HG/H-CCM).
Gene expression of VEGF, ANG-1, GATA-4, NKx2.5 MEF2c, PCNA, and eNOS was upregulated after preconditioning with HG/H-CCM, as evidenced by reverse transcriptase/polymerase chain reaction (RT-PCR). Concurrently, increased AKT phosphorylation, proliferation, angiogenic ability, and reduced levels of apoptosis were observed in HG/H-CCM-preconditioned diabetic MSCs compared with nontreated controls. HG/H-CCM-preconditioned diabetic-mouse-derived MSCs (dmMSCs) were transplanted in diabetic animals and demonstrated increased homing concomitant with augmented heart function. Gene expression of angiogenic and cardiac markers was significantly upregulated in conjunction with paracrine factors (IGF-1, HGF, SDF-1, FGF-2) and, in addition, reduced fibrosis, apoptosis, and increased angiogenesis was observed in diabetic hearts 4 weeks after transplantation of preconditioned dmMSCs compared with hearts with nontreated diabetic MSCs.
Preconditioning with HG/H-CCM enhances survival, proliferation, and the angiogenic ability of dmMSCs, augmenting their ability to improve function in a diabetic heart.
Mesenchymal stem cells; Diabetic heart; Preconditioning; Oxidative stress
Diabetes mellitus is affecting more than 300 million people worldwide. Current treatment strategies cannot prevent secondary complications. Stem cells due to their regenerative power have long been the attractive target for the cell-based therapies. Mesenchymal stem cells (MSCs) possess the ability to differentiate into several cell types and to escape immune recognition in vitro. MSCs can be differentiated into insulin-producing cells (IPCs) and could be an exciting therapy for diabetes but problems like poor engraftment and survivability need to be confronted. It was hypothesized that stromal cell derived factor- 1alpha (SDF-1alpha) will enhance therapeutic potential of stem cell derived IPCs by increasing their survival and proliferation rate.
Novel culture conditions were developed to differentiate bone marrow derived mesenchymal stem cells (BMSCs) into IPCs by using endocrine differentiation inducers and growth factors via a three stage protocol. In order to enhance their therapeutic potential, we preconditioned IPCs with SDF-1alpha.
Our results showed that SDF-1alpha increases survival and proliferation of IPCs and protects them from glucotoxicity under high glucose conditions in vitro. SDF-1alpha also enhances the glucose responsive insulin secretion in IPCs in vitro. SDF-1alpha preconditioning reverses hyperglycemia and increase serum insulin in drug induced diabetic rats.
The differentiation of BMSCs into IPCs and enhancement of their therapeutic potential by SDF-1alpha preconditioning may contribute to cell based therapies for diabetes.
Diabetes mellitus; Mesenchymal stem cells; Differentiation; Preconditioning; SDF-1α
We ascertained two large Pakistani consanguineous families (PKDF231 and PKDF608) segregating profound hearing loss, vestibular dysfunction, and retinitis pigmentosa, the defining features of Usher syndrome type 1 (USH1). To date seven USH1 loci have been reported. Here, we map a novel locus, USH1K, on chromosome 10p11.21-q21.1. In family PKDF231 we performed a genome-wide linkage screen and found a region of homozygosity shared among the affected individual at chromosome 10p11.21-q21.1. Meiotic recombination events in family PKDF231 define a critical interval of 11.74 cM (20.20 Mb) bounded by markers D10S1780 (63.83 cM) and D10S546 (75.57 cM). Affected individuals of family PKDF608 were also homozygous for chromosome 10p11-21-q21.1 linked STR markers. Of the 85 genes within the linkage interval, PCDH15, GJD4, FZD4, RET, and LRRC18 were sequenced in both families, but no potential pathogenic mutation was identified. The USH1K locus overlaps the non-syndromic deafness locus DFNB33 raising the possibility that the two disorders may be caused by allelic mutations.
deafness; DFNB33; retinitis pigmentosa; Usher syndrome; USH1K; vestibular dysfunction; 10p11.21-q21.1
Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy for liver fibrosis. Issues concerning poor MSC survival and engraftment in the fibrotic liver still persist and warrant development of a strategy to increase MSC potency for liver repair. The present study was designed to examine a synergistic role for Interleukin-6 (IL-6) and MSCs therapy in the recovery of carbon tetrachloride (CCl4) induced injured hepatocytes in vitro and in vivo.
Injury was induced through 3 mM and 5 mM CCl4 treatment of cultured hepatocytes while fibrotic mouse model was established by injecting 0.5 ml/kg CCl4 followed by treatment with IL-6 and MSCs. Effect of MSCs and IL-6 treatment on injured hepatocytes was determined by lactate dehydrogenase release, RT-PCR for (Bax, Bcl-xl, Caspase3, Cytokeratin 8, NFκB, TNF-α) and annexin V apoptotic detection. Analysis of MSC and IL-6 treatment on liver fibrosis was measured by histopathology, PAS, TUNEL and Sirius red staining, RT-PCR, and liver function tests for Bilirubin and Alkaline Phosphatase (ALP).
A significant reduction in LDH release and apoptosis was observed in hepatocytes treated with a combination of MSCs and IL-6 concomitant with upregulation of anti-apoptotic gene Bcl-xl expression and down regulation of bax, caspase3, NFκB and TNF-α. Adoptive transfer of MSCs in fibrotic liver pretreated with IL-6 resulted increased MSCs homing and reduced fibrosis and apoptosis. Hepatic functional assessment demonstrated reduced serum levels of Bilirubin and ALP.
Pretreatment of fibrotic liver with IL-6 improves hepatic microenvironment and primes it for MSC transplantation leading to enhanced reduction of liver injury after fibrosis. Synergistic effect of IL-6 and MSCs seems a favored therapeutic option in attenuation of liver apoptosis and fibrosis accompanied by improved liver function.
Mesenchymal stem cells; Liver fibrosis; Hepatocytes; Interleukin-6
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.
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.
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.
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.
MSCs; SNAP; Pre-conditioning; Renal ischemia; Cytoprotective factors
To identify pathogenic mutations responsible for autosomal recessive retinitis pigmentosa in 5 consanguineous Pakistani families.
Affected individuals in the families underwent a detailed ophthalmological examination that consisted of fundus photography and electroretinography. Blood samples were collected from all participating family members, and genomic DNA was extracted. A genome-wide linkage scan was performed, followed by exclusion analyses among our cohort of nuclear consanguineous families with microsatellite markers spanning the TULP1 locus on chromosome 6p. Two-point logarithm of odds scores were calculated, and all coding exons of TULP1 were sequenced bidirectionally.
The results of ophthalmological examinations among affected individuals in these 5 families were suggestive of retinitis pigmentosa. The genome-wide linkage scan localized the disease interval to chromosome 6p, harboring TULP1 in 1 of 5 families, and sequential analyses identified a single base pair substitution in TULP1 that results in threonine to alanine substitution (p.T380A). Subsequently, we investigated our entire cohort of families with autosomal recessive retinitis pigmentosa and identified 4 additional families with linkage to chromosome 6p, all of them harboring a single base pair substitution in TULP1 that results in lysine to arginine substitution (p.K489R). Results of single-nucleotide polymorphism haplotype analyses were suggestive of a common founder in these 4 families.
Pathogenic mutations in TULP1 are responsible for the autosomal recessive retinitis pigmentosa phenotype in these consanguineous Pakistani families, with a single ancestral mutation in TULP1 causing the disease phenotype in 4 of 5 families.
Clinical and molecular characterization of pathogenic mutations in TULP1 will increase our understanding of retinitis pigmentosa at a molecular level.
To identify disease-causing mutations in two consanguineous Pakistani families with fundus albipunctatus.
Affected individuals in both families underwent a thorough clinical examination including funduscopy and electroretinography. Blood samples were collected from all participating members and genomic DNA was extracted. Exclusion analysis was completed with microsatellite short tandem repeat markers that span all reported loci for fundus albipunctatus. Two-point logarithm of odds (LOD) scores were calculated, and coding exons and exon–intron boundaries of RLBP1 were sequenced bi-directionally.
The ophthalmic examination of affected patients in both families was consistent with fundus albipunctatus. The alleles of markers on chromosome 15q flanking RLBP1 segregated with the disease phenotype in both families and linkage was further confirmed by two-point LOD scores. Bi-directional sequencing of RLBP1 identified a nonsense mutation (R156X) and a missense mutation (G116R) that segregated with the disease phenotype in their respective families.
These results strongly suggest that mutations in RLBP1 are responsible for fundus albipunctatus in the affected individuals of these consanguineous Pakistani families.
Congenital stationary night blindness is characterized by impaired night vision, decreased visual acuity, nystagmus, myopia, and strabismus. A genome-wide linkage scan was completed that localized the critical interval to the short arm of chromosome 3 and sequencing identified a novel missense mutation in GNAT1.
Congenital stationary night blindness is a nonprogressive retinal disorder manifesting as impaired night vision and is generally associated with other ocular symptoms, such as nystagmus, myopia, and strabismus. This study was conducted to further investigate the genetic basis of CSNB in a consanguineous Pakistani family.
A consanguineous family with multiple individuals manifesting cardinal symptoms of congenital stationary night blindness was ascertained. All family members underwent detailed ophthalmic examination, including fundus photographic examination and electroretinography. Blood samples were collected and genomic DNA was extracted. Exclusion and genome-wide linkage analyses were completed and two-point LOD scores were calculated. Bidirectional sequencing of GNAT1 was completed, and quantitative expression of Gnat1 transcript levels were investigated in ocular tissues at different postnatal intervals.
The results of ophthalmic examinations were suggestive of early-onset stationary night blindness with no extraocular anomalies. The genome-wide scan localized the critical interval to chromosome 3, region p22.1-p14.3, with maximum two-point LOD scores of 3.09 at θ = 0, flanked by markers D3S3522 and D3S1289. Subsequently, a missense mutation in GNAT1, p.D129G, was identified, which segregated within the family, consistent with an autosomal recessive mode of inheritance, and was not present in 192 ethnically matched control chromosomes. Expression analysis suggested that Gnat1 is expressed at approximately postnatal day (P)7 and is predominantly expressed in the retina.
These data suggest that a homozygous missense mutation in GNAT1 is associated with autosomal recessive stationary night blindness.
To investigate the genetic basis and molecular characteristics of the isolated form of ectopia lentis.
We ascertained a consanguineous Pakistani family with multiple individuals with ectopia lentis. All affected as well as unaffected members with isolated ectopia lentis underwent detailed ophthalmologic and medical examination. Blood samples were collected and DNA was extracted. A genome-wide scan was completed with 382 polymorphic microsatellite markers, and logarithm of odds (LOD) scores were calculated.
Maximum 2-point LOD scores of 5.68 and 2.88 at θ=0 were obtained for markers D8S285 and D8S260, respectively, during the genome-wide scan. Additional microsatellite markers refined the disease locus to a 16.96-cM (14.07-Mb) interval flanked by D8S1737 proximally and D8S1117 distally.
We report on a new locus for nonsyndromic autosomal recessive ectopia lentis on chromosome 8q11.23-q13.2 in a consanguineous Pakistani family.
Identification of genetic loci and genes involved in ectopia lentis will enhance our understanding of the disease at a molecular level, leading to better genetic counseling and family screening and possible future development of better treatment.
Retinitis pigmentosa (RP) is one of the most common ophthalmic disorders affecting one in approximately 5000 people worldwide. A nuclear family was recruited from the Punjab province of Pakistan to study the genetic basis of autosomal recessive RP.
All affected individuals underwent a thorough ophthalmic examination and the disease was characterised based upon results for fundus photographs and electroretinogram recordings. Genomic DNA was extracted from peripheral leucocytes. Exclusion studies were performed with short tandem repeat (STR) markers flanking reported autosomal recessive RP loci. Haplotypes were constructed and results were statistically evaluated.
The results of exclusion analyses suggested that family PKRP173 was linked to chromosome 2q harbouring mer tyrosine kinase protooncogene (MERTK), a gene previously associated with autosomal recessive RP. Additional STR markers refined the critical interval and placed it in a 13.4 cM (17 Mb) region flanked by D2S293 proximally and D2S347 distally. Significant logarithm of odds (LOD) scores of 3.2, 3.25 and 3.18 at θ=0 were obtained with markers D2S1896, D2S2269 and D2S160. Sequencing of the coding exons of MERTK identified a mutation, c.718G→T in exon 4, which results in a premature termination of p.E240X that segregates with the disease phenotype in the family.
Our results strongly suggest that the nonsense mutation in MERTK, leading to premature termination of the protein, is responsible for RP phenotype in the affected individuals of the Pakistani family.
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.
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.
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.
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.
Liver fibrosis; Mesenchymal stem cells (MSCs); Hepatic stellate cells (HSCs); Nitric oxide
There are 68 sex-linked syndromes that include hearing loss as one feature and five sex-linked nonsyndromic deafness loci listed in the OMIM database. The possibility of additional such sex-linked loci was explored by ascertaining three unrelated Pakistani families (PKDF536, PKDF1132, PKDF740) segregating X-linked recessive deafness. Sequence analysis of POU3F4 (DFN3) in affected members of families PKDF536 and PKDF1132 revealed two novel nonsense mutations, p.Q136X and p.W114X, respectively. Family PKDF740 is segregating congenital blindness, mild to profound progressive hearing loss that is characteristic of Norrie disease (MIM#310600). Sequence analysis of NDP among affected members of this family revealed a novel single nucleotide deletion c.49delG causing a frameshift and premature truncation (p.V17fsX1) of the encoded protein. These mutations were not found in 150 normal DNA samples. Identification of pathogenic alleles causing X-linked recessive deafness will improve molecular diagnosis, genetic counseling, and molecular epidemiology of hearing loss among Pakistanis.
DFN3; Norrie disease; POU3F4; NDP; hearing loss; gushers
Hepatitis C virus (HCV) is a major cause of chronic liver diseases including steatosis, cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. The development of in-vitro models such as HCV infection system, HCV sub-genomic replicon, HCV producing pseudoparticles (HCVpp) and infectious HCV virion provide an important tool to develop new antiviral drugs of different targets against HCV. These models also play an important role to study virus lifecycle such as virus entry, endocytosis, replication, release and HCV induced pathogenesis. This review summarizes the most important in-vitro models currently used to study future HCV research as well as drug design.
Recessive mutations of fibroblast growth factor 3 (FGF3) can cause LAMM syndrome (OMIM 610706), characterized by fully penetrant complete labyrinthine aplasia, microtia and microdontia.
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.
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.
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.
This study was designed to identify pathogenic mutations causing autosomal recessive retinitis pigmentosa (RP) in consanguineous Pakistani families.
Two consanguineous families affected with autosomal recessive RP were identified from the Punjab Province of Pakistan. All affected individuals underwent a thorough ophthalmologic examination. Blood samples were collected, and genomic DNAs were extracted. Exclusion analysis was completed, and two-point LOD scores were calculated. Bidirectional sequencing of the β subunit of phosphodiesterase 6 (PDE6β) was completed.
During exclusion analyses both families localized to chromosome 4p, harboring PDE6β, a gene previously associated with autosomal recessive RP. Sequencing of PDE6β identified missense mutations: c.1655G>A (p.R552Q) and c.1160C>T (p.P387L) in families PKRP161 and PKRP183, respectively. Bioinformatic analyses suggested that both mutations are deleterious for the native three-dimensional structure of the PDE6β protein.
These results strongly suggest that mutations in PDE6β are responsible for the disease phenotype in the consanguineous Pakistani families.
To identify the disease locus for autosomal recessive congenital cataract in a consanguineous Pakistani family.
All affected individuals underwent detailed ophthalmologic and medical examination. Blood samples were collected and DNA was extracted. A genome-wide scan was performed with polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and logarithm of odds (LOD) scores were calculated.
The clinical records and ophthalmological examinations suggested that all affected individuals have nuclear cataracts. Maximum LOD scores of 5.01, 4.38, and 4.17 at θ=0 were obtained with markers D7630, D7S657, and D7S515, respectively. Fine mapping refined the critical interval and suggested that markers in a 27.78 cM (27.96 Mb) interval are flanked by markers D7S660 and D7S799, which co-segregate with the disease phenotype in family PKCC108.
We have identified a new locus for autosomal recessive congenital cataract, localized to chromosome 7q21.11-q31.1 in a consanguineous Pakistani family.
To investigate the genetic basis of autosomal recessive congenital cataracts in a consanguineous Pakistani family.
All affected individuals underwent a detailed ophthalmological and clinical examination. Blood samples were collected and genomic DNAs were extracted. A genome-wide scan was performed with polymorphic microsatellite markers. Logarithm of odds (LOD) scores were calculated, and Eph-receptor type-A2 (EPHA2), residing in the critical interval, was sequenced bidirectionally.
The clinical and ophthalmological examinations suggested that all affected individuals have nuclear cataracts. Genome-wide linkage analyses localized the critical interval to a 20.78 cM (15.08 Mb) interval on chromosome 1p, with a maximum two-point LOD score of 5.21 at θ=0. Sequencing of EPHA2 residing in the critical interval identified a missense mutation: c.2353G>A, which results in an alanine to threonine substitution (p.A785T).
Here, we report for the first time a missense mutation in EPHA2 associated with autosomal recessive congenital cataracts.
To identify the pathogenic mutations responsible for autosomal recessive congenital cataracts in consanguineous Pakistani families.
All affected individuals underwent detailed ophthalmologic and medical examination. Blood samples were collected and genomic DNA was extracted. A genome-wide scan was performed with polymorphic microsatellite markers on genomic DNA from affected and unaffected family members and logarithm of odds (LOD) scores were calculated. All coding exons of galactokinase (GALK1) were sequenced to identify pathogenic lesions.
Clinical records and ophthalmological examinations suggested that affected individuals have nuclear cataracts. Linkage analysis localized the critical interval to chromosome 17q with a maximum LOD score of 5.54 at θ=0, with D17S785 in family PKCC030. Sequencing of GALK1, a gene present in the critical interval, identified a single base pair deletion: c.410delG, which results in a frame shift leading to a premature termination of GALK1: p.G137fsX27. Additionally, we identified a missense mutation: c.416T>C, in family PKCC055 that results in substitution of a leucine residue at position 139 with a proline residue: p.L139P, and is predicted to be deleterious to the native GALK1 structure.
Here, we report pathogenic mutations in GALK1 that are responsible for autosomal recessive congenital cataracts in consanguineous Pakistani families.
To localize the disease interval for autosomal recessive congenital cataracts in a consanguineous Pakistani family.
All affected individuals underwent detailed ophthalmologic examination. Blood samples were collected and genomic DNA was extracted. A genome-wide scan was performed with fluorescently-labeled microsatellite markers on genomic DNA from affected and unaffected family members and logarithm of odds (LOD) scores were calculated.
Clinical records and ophthalmological examinations suggested that affected individuals have bilateral congenital cataracts. Genome-wide linkage analysis localized the critical interval to chromosome 3q with a maximum LOD score of 3.87 at θ=0; with marker D3S3609. Haplotype analyses refined the critical interval to a 23.39 cM (18.01 Mb) interval on chromosome 3q, flanked by D3S1614 proximally and D3S1262, distally.
Here, we report a new locus for autosomal recessive congenital cataract localized to chromosome 3q in a consanguineous Pakistani family.
To identify the disease locus for autosomal recessive congenital cataracts in a consanguineous Pakistani family.
All affected individuals underwent a detailed ophthalmologic examination. Blood samples were collected and genomic DNA was extracted. A genome-wide scan was completed with fluorescently-labeled microsatellite markers on genomic DNA from affected and unaffected family members. Logarithms of odds (LOD) scores were calculated under a fully penetrant autosomal recessive model of inheritance.
Ophthalmic examination suggested that affected individuals have bilateral cataracts. Linkage analysis localized the critical interval to chromosome 8p with LOD scores of 3.19, and 3.08 at θ=0, obtained with markers D8S549 and D8S550, respectively. Haplotype analyses refined the critical interval to 37.92 cM (16.28 Mb) region, flanked by markers, D8S277 proximally and D8S1734 distally.
Here, we report a new locus for autosomal recessive congenital cataract mapped to chromosome 8p in a consanguineous Pakistani family.
Usher syndrome (USH) is a hereditary disorder associated with sensorineural hearing impairment, progressive loss of vision attributable to retinitis pigmentosa and variable vestibular function. Three clinical types have been described with type I (USH1) being the most severe. To date six USH1 loci have been reported. We ascertained two large Pakistani consanguineous families segregating profound hearing loss, vestibular dysfunction, and retinitis pigmentosa, the defining features of USH1. In these families we excluded linkage of USH to the 11 known USH loci, and subsequently performed a genome-wide linkage screen. We found a novel USH1 locus designated USH1H that mapped to chromosome 15q22-23 in a 4.92 cM interval. This locus overlaps the non-syndromic deafness locus DFNB48 raising the possibility that the two disorders may be caused by allelic mutations.
deafness; DFNB48; retinitis pigmentosa; Usher syndrome; USH1H; vestibular dysfunction; 15q22-23