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.

Mutations in GRM6 identified in consanguineous Pakistani families with congenital stationary night blindness.

PURPOSE: This study was undertaken to investigate the causal mutations responsible for autosomal recessive congenital stationary night blindness (CSNB) in consanguineous Pakistani families. METHODS: Two 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. RESULTS: According 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. CONCLUSIONS: We identified a novel nonsense and a previously reported missense mutation in GRM6 that were responsible for autosomal recessive CSNB in patients of Pakistani decent.

Incidence of Rhegmatogenous Retinal Detachment (RRD) in a Tertiary Care Center of Pakistan.

Background Regardless of the advancements in ophthalmology, rhegmatogenous retinal detachment (RRD) remains a substantial issue for physicians. The present study assessed the incidence of RRD among our population. Methodology A cross-sectional study was performed at the Layton Rehmatullah Benevolent Trust (LRBT) between June 2020 and May 2021. All the patients of RRD, irrespective of gender, within the age bracket of 20 years or more and diagnosed by a consultant ophthalmologist were included in the research study. Patients with serous retinal or tractional detachment and RRD with vitreous leakage were excluded from the study. A slit lamp and dilated fundus examination was performed preoperatively to assess the type of retinal detachment and associated factors as mentioned above. All data were collected on predesigned pro forma. Results About 25,000 individuals were presented to the outpatient department during the study period. Out of these, 100 patients were diagnosed with RRD. The incidence rate of the RRD in our center was 0.4%. There were a majority of the males. The mean age of patients did not vary significantly with respect to gender (p < 0.797). The most common type of RD was the total RD with a frequency of 53 cases followed by inferior RD with 19 cases. The majority of those with total RRD were males, i.e., 37%; however, the difference was statistically insignificant (p = 0.476). The study revealed that most of the RRD was diagnosed in patients < 45 years of age; however, the difference was not statistically significant (p < 0.227). Conclusion The present study highlighted the incidence of RRD and explored the sociodemographic and other clinical features in the Pakistani population. However, it is possible that the RRD condition is still under-diagnosed in our hospital settings. Further exploration is warranted to study comprehensively the risk factors associated with RRD.

Mapping of a novel locus associated with autosomal recessive congenital cataract to chromosome 8p.

PURPOSE: To identify the disease locus for autosomal recessive congenital cataracts in a consanguineous Pakistani family. METHODS: 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. RESULTS: 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. CONCLUSIONS: Here, we report a new locus for autosomal recessive congenital cataract mapped to chromosome 8p in a consanguineous Pakistani family.

Mapping of a new locus associated with autosomal recessive congenital cataract to chromosome 3q.

PURPOSE: To localize the disease interval for autosomal recessive congenital cataracts in a consanguineous Pakistani family. METHODS: 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. RESULTS: 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. CONCLUSIONS: Here, we report a new locus for autosomal recessive congenital cataract localized to chromosome 3q in a consanguineous Pakistani family.

Autosomal recessive congenital cataract in consanguineous Pakistani families is associated with mutations in GALK1.

PURPOSE: To identify the pathogenic mutations responsible for autosomal recessive congenital cataracts in consanguineous Pakistani families. METHODS: 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. RESULTS: 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 theta=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. CONCLUSIONS: Here, we report pathogenic mutations in GALK1 that are responsible for autosomal recessive congenital cataracts in consanguineous Pakistani families.

Molecular Genetic Analysis of Pakistani Families With Autosomal Recessive Congenital Cataracts by Homozygosity Screening.

Purpose: To identify the genetic origins of autosomal recessive congenital cataracts (arCC) in the Pakistani population. Methods: Based on the hypothesis that most arCC patients in consanguineous families in the Punjab areas of Pakistan should be homozygous for causative mutations, affected individuals were screened for homozygosity of nearby highly informative microsatellite markers and then screened for pathogenic mutations by DNA sequencing. A total of 83 unmapped consanguineous families were screened for mutations in 33 known candidate genes. Results: Patients in 32 arCC families were homozygous for markers near at least 1 of the 33 known CC genes. Sequencing the included genes revealed homozygous cosegregating sequence changes in 10 families, 2 of which had the same variation. These included five missense, one nonsense, two frame shift, and one splice site mutations, eight of which were novel, in EPHA2, FOXE3, FYCO1, TDRD7, MIP, GALK1, and CRYBA4. Conclusions: The above results confirm the usefulness of homozygosity mapping for identifying genetic defects underlying autosomal recessive disorders in consanguineous families. In our ongoing study of arCC in Pakistan, including 83 arCC families that underwent homozygosity mapping, 3 mapped using genome-wide linkage analysis in unpublished data, and 30 previously reported families, mutations were detected in approximately 37.1% (43/116) of all families studied, suggesting that additional genes might be responsible in the remaining families. The most commonly mutated gene was FYCO1 (14%), followed by CRYBB3 (5.2%), GALK1 (3.5%), and EPHA2 (2.6%). This provides the first comprehensive description of the genetic architecture of arCC in the Pakistani population.

Autosomal recessive retinitis pigmentosa is associated with mutations in RP1 in three consanguineous Pakistani families.

PURPOSE: To localize and identify the gene and mutations causing autosomal recessive retinitis pigmentosa in three consanguineous Pakistani families. METHODS: Blood samples were collected and DNA was extracted. A genome-wide scan was performed by using 382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and lod scores were calculated. RESULTS: A genome-wide scan of 25 families gave an hlod = 4.53 with D8S260. Retinitis pigmentosa in all three families mapped to a 14.21-cM (21.19-Mb) region on chromosome 8 at q11, flanked by D8S532 and D8S260. This region harbors RP1, which is known to cause autosomal dominant retinitis pigmentosa. Sequencing of the coding exons of RP1 showed mutations in all three families: two single-base deletions, c.4703delA and c.5400delA, resulting in a frame shift, and a 4-bp insertion, c.1606insTGAA, all causing premature termination of the protein. All affected individuals in these families are homozygous for the mutations. Parents and siblings heterozygous for the mutant allele did not show any signs or symptoms of RP. CONCLUSIONS: These results provide strong evidence that mutations in RP1 can result in recessive as well as dominant retinitis pigmentosa. The findings suggest that truncation of RP1 before the BIF motif or within the terminal portion results in a simple loss of RP1 function, producing a recessive inheritance pattern. In contrast, disruption of RP1 within or immediately after the BIF domain may result in a protein with a deleterious effect and hence a dominant inheritance pattern.

Association of pathogenic mutations in TULP1 with retinitis pigmentosa in consanguineous Pakistani families.

OBJECTIVE: To identify pathogenic mutations responsible for autosomal recessive retinitis pigmentosa in 5 consanguineous Pakistani families. METHODS: 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. RESULTS: 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. CONCLUSION: 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 RELEVANCE: Clinical and molecular characterization of pathogenic mutations in TULP1 will increase our understanding of retinitis pigmentosa at a molecular level.

Ectopia lentis in a consanguineous pakistani family and a novel locus on chromosome 8q.

OBJECTIVE: To investigate the genetic basis and molecular characteristics of the isolated form of ectopia lentis. METHODS: 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. RESULTS: Maximum 2-point LOD scores of 5.68 and 2.88 at theta = 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. CONCLUSIONS: We report on a new locus for nonsyndromic autosomal recessive ectopia lentis on chromosome 8q11.23-q13.2 in a consanguineous Pakistani family. Clinical Relevance 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.