Prevalence of primary mutations in Leber hereditary optic neuropathy: A five-year report from a tertiary eye care center in India.

PURPOSE: Genetic testing for primary mutations m.3460G>A, m.11778G>A, and m.14484T>C in ND1, ND4, and ND6 genes of mitochondrial DNA is the recommended assay for Leber hereditary optic neuropathy (LHON; OMIM 535000). This report discusses the outcome of molecular genetic screening for these three primary mutations in suspected LHON cases in India. METHODS: Two hundred and seventy-eight unrelated presumed LHON patients who were seen at the neuro-ophthalmology clinic of a tertiary eye care center from 2014-2018 were analyzed. They were genotyped for the three common variants by polymerase chain reaction-based direct sequencing, and their plasmy status was also determined by restriction enzyme digestion. RESULTS: Eighty two of 278 patients were positive for one of the 3 common mutations with m.11778G>A in ND4 gene more frequently distributed (N=72) in homoplasmic state (N=59/82). The mean onset age of visual loss was 21.1years (SD, 9.8 years; range, 5-58 years) in patients harboring the primary mutation. The most common clinical presentation was bilateral sequential painless vision loss with central and cecocentral scotomas in the visual field due to optic disc atrophy. CONCLUSIONS: The study subjects are a sample of a much larger number of suspected LHON cases tested for primary mutations in India. (N= 278) and 29.4% (82/278) of patients harbour one of the 3 common mutations. Screening the entire mitochondrial genome and the other nuclear genes encoding mitochondrial protein, would probably aid in identifying the other less common mtDNA mutations causing LHON in Indian population.

Ophthatome™: an integrated knowledgebase of ophthalmic diseases for translating vision research into the clinic.

BACKGROUND: Medical big data analytics has revolutionized the human healthcare system by introducing processes that facilitate rationale clinical decision making, predictive or prognostic modelling of the disease progression and management, disease surveillance, overall impact on public health and research. Although, the electronic medical records (EMR) system is the digital storehouse of rich medical data of a large patient cohort collected over many years, the data lack sufficient structure to be of clinical value for applying deep learning methods and advanced analytics to improve disease management at an individual patient level or for the discipline in general. Ophthatome™ captures data contained in retrospective electronic medical records between September 2012 and January 2018 to facilitate translational vision research through a knowledgebase of ophthalmic diseases. METHODS: The electronic medical records data from Narayana Nethralaya ophthalmic hospital recorded in the MS-SQL database was mapped and programmatically transferred to MySQL. The captured data was manually curated to preserve data integrity and accuracy. The data was stored in MySQL database management system for ease of visualization, advanced search functions and other knowledgebase applications. RESULTS: Ophthatome™ is a comprehensive and accurate knowledgebase of ophthalmic diseases containing curated clinical, treatment and imaging data of 581,466 ophthalmic subjects from the Indian population, recorded between September 2012 and January 2018. Ophthatome™ provides filters and Boolean searches with operators and modifiers that allow selection of specific cohorts covering 524 distinct ophthalmic disease types and 1800 disease sub-types across 35 different anatomical regions of the eye. The availability of longitudinal data for about 300,000 subjects provides additional opportunity to perform clinical research on disease progression and management including drug responses and management outcomes. The knowledgebase captures ophthalmic diseases in a genetically diverse population providing opportunity to study genetic and environmental factors contributing to or influencing ophthalmic diseases. CONCLUSION: Ophthatome™ will accelerate clinical, genomic, pharmacogenomic and advanced translational research in ophthalmology and vision sciences.

Family-Based Genome-Wide Association Study of South Indian Pedigrees Supports WNT7B as a Central Corneal Thickness Locus.

Purpose: To identify genetic risk factors contributing to central corneal thickness (CCT) in individuals from South India, a population with a high prevalence of ocular disorders. Methods: One hundred ninety-five individuals from 15 large South Indian pedigrees were genotyped using the Omni2.5 bead array. Family-based association for CCT was conducted using the score test in MERLIN. Results: Genome-wide association study (GWAS) identified strongest association for single nucleotide polymorphisms (SNPs) in the first intron of WNT7B and CCT (top SNP rs9330813; ß = -0.57, 95% confidence interval [CI]: -0.78 to -0.36; P = 1.7 × 10-7). We further investigated rs9330813 in a Latino cohort and four independent European cohorts. A meta-analysis of these data sets demonstrated statistically significant association between rs9330813 and CCT (ß = -3.94, 95% CI: -5.23 to -2.66; P = 1.7 × 10-9). WNT7B SNPs located in the same genomic region that includes rs9330813 have previously been associated with CCT in Latinos but with other ocular quantitative traits related to myopia (corneal curvature and axial length) in a Japanese population (rs10453441 and rs200329677). To evaluate the specificity of the observed WNT7B association with CCT in the South Indian families, we completed an ocular phenome-wide association study (PheWAS) for the top WNT7B SNPs using 45 ocular traits measured in these same families including corneal curvature and axial length. The ocular PheWAS results indicate that in the South Indian families WNT7B SNPs are primarily associated with CCT. Conclusions: The results indicate robust evidence for association between WNT7B SNPs and CCT in South Indian pedigrees, and suggest that WNT7B SNPs can have population-specific effects on ocular quantitative traits.

Analysis of candidate genes ZEB1 and LOXHD1 in late-onset Fuchs' endothelial corneal dystrophy in an Indian cohort.

BACKGROUND: Fuchs' endothelial corneal dystrophy (FECD) is a complex degenerative disease of the corneal endothelium with genetic predisposition. Pathogenic rare variants have been identified in SLC4A11, LOXHD1, ZEB1, and AGBL1. Association of single nucleotide polymorphisms (SNPs) and CTG trinucleotide repeat expansions in the intron of TCF4 gene to FECD has been studied across multiple ethnicities. Recently, genome-wide association studies have also identified KANK4, LAMC1, and ATP1B1 as novel loci for FECD. Here, we report the contribution of ZEB1 and LOXHD1 genes in our sporadic late-onset FECD cohort. MATERIALS AND METHODS: In the experimental study, coding regions of ZEB1 and LOXHD1 were screened by Sanger DNA sequencing in 52 late-onset and 5 early-onset FECD cases of Indian origin, recruited at a tertiary eye care center. Further, bioinformatics analysis was done. RESULTS: One reported missense mutation, c.2522A>C; p.(Q841P), and one variant of uncertain significance (VUS), c.619A>G; p.(S207G), were identified in the ZEB1 gene. One VUS, c.6413G>Ap.(R2138Q), was observed in LOXHD1. A 3D structural bioinformatic analysis of the missense variant in LOXHD1 predicted the variant to affect the structure-function relationship of the protein. DISCUSSION: While mutations in ZEB1 contributed to 2% of the late-onset FECD cases, the exact role of the two VUS identified in ZEB1 and LOXHD1 in FECD pathogenesis needs to be studied.

Retinal findings in a patient of French ancestry with CABP4-related retinal disease.

INTRODUCTION: CABP4-related retinal dysfunction is a cone-rod synaptic transmission disorder with electronegative electroretinogram (ERG) waveform. It is a rare retinal dysfunction that can be classified into the incomplete form of congenital stationary night blindness. Absent foveal reflex and overall foveal thinning were previously reported, but in most cases the fundus appearance was described as nearly normal. We report here peculiar macular changes in a patient of French ancestry harbouring CABP4 mutations. METHODS: Complete ocular examination and full-field ERG were performed at the initial presentation and follow-up. Multimodal fundus imagining, including spectral-domain optical coherence tomography, colour, infrared reflectance and short-wavelength autofluorescence photographs, was performed during follow-up visits. RESULTS: A 7-month-old infant was addressed to our department for visual unresponsiveness and nystagmus. ERG had an electronegative waveform, even for light-adapted stimuli, thus supporting the diagnosis of photoreceptor-bipolar cell transmission disorder. Genetic investigations discovered a compound heterozygous mutation in CABP4: c.646C > T, p.Arg216*/c.673C > T, p.Arg225*. Multimodal fundus imaging, performed at follow-up visits, showed fine radial folds at the vitreomacular interface and dark foveal dots in both eyes. Optic coherence tomography revealed a focal foveal ellipsoid zone gap. DISCUSSION: Initial presentation was misleading with Leber congenital amaurosis. The electronegative ERG waveform reoriented the genetic investigations and thus establishing a correct diagnosis. To the best of our knowledge, the peculiar fundus changes observed in our patient were never reported before. We hypothesized that a foveal ellipsoid zone interruption discovered in our patient could reflect mostly a cone dysfunction. It was unclear whether the fine radial folds in both maculae were linked with high hyperopia or were an intrinsic feature of the retinal disease. CONCLUSION: CABP4-related retinal disease is a cone-rod system disorder with possible foveal abnormalities.

Association of polymorphisms in the intron of TCF4 gene to late-onset Fuchs endothelial corneal dystrophy: An Indian cohort study.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is a progressive degenerative disease of the corneal endothelium. It is genetically heterogeneous and follows either an autosomal dominant or sporadic pattern of inheritance. Here, we have explored the association of four previously reported intronic single nucleotide polymorphisms and intronic CTG repeat expansions in TCF4 gene to FECD in an Indian cohort. METHODS: The cohort consisting of 52 sporadic late-onset cases, 5 early-onset cases, and 148 controls was taken for the study. rs2286812 and rs613872 were genotyped by allele specific polymerase chain reaction (ASPCR) and PCR-based restriction digestion, respectively; rs17595731 and rs9954153 were genotyped by Taqman assay using real-time PCR. The quantitative assessment of the CTG repeat region was performed by PCR/Sanger DNA sequencing. The repeats were assessed qualitatively by short tandem repeat and triplet repeat primed PCR assays. The statistical analysis was performed using two-tailed Fisher's exact probability test. RESULTS: SNPsrs613872 (G/T) for the 'G' allele (P value: 4.57 × 10-5) and rs17595731 (C/T) for the 'C' allele (P value: 1.87 × 10-5), respectively, showed a significant association to sporadic late-onset FECD. CTG repeat expansions were found to be associated with FECD with a P value = 2.4 × 10-3. CONCLUSION: rs613872, rs17595731, and CTG repeat expansions in intronic region of TCF4 are associated with increased risk of sporadic late-onset FECD in the Indian cohort studied.

Leber's Hereditary Optic Neuropathy-Specific Mutation m.11778G>A Exists on Diverse Mitochondrial Haplogroups in India.

Purpose: Leber's hereditary optic neuropathy (LHON; OMIM 535000) is one of the most common maternally inherited mitochondrial disorders. Three mitochondrial DNA point mutations-m.3460G>A (MT-ND1), m.11778G>A (MT-ND4), and m.14484T>C (MT-ND6)-account for the majority of reported LHON cases. Only approximately 50% of males and approximately 10% of females carrying these mutations develop optic neuropathy and blindness. Additional factors, such as mtDNA/nuclear genetic background and environmental modifiers, are likely to contribute toward the observed incomplete penetrance and gender bias. We aimed to investigate whether mtDNA haplogroup influences LHON clinical expression in Indian patients harboring the m.11778G>A mutation. Methods: Detailed clinical assessment and complete mitochondrial genome sequencing was undertaken in 64 LHON families harboring the m.11778G>A mutation. Mitochondrial haplogroup was assigned based on evolutionarily conserved mtDNA variations. Results: A total of 543 individuals (295 male, 248 female) from 64 unrelated families harboring the m.11778G>A mutation were recruited to the study. The overall disease penetrance was 27.07% (146 of 543) and higher in males (37.9%; 112 of 295) than females (13.7%; 34 of 248). The mtDNA haplogroup analysis revealed that all affected probands belonged to different mtDNA haplogroups. No association between the m.11778G>A mutation and the background mtDNA haplogroup was detected. Conclusions: The first detailed study of Indian LHON patients confirm that the m.11778G>A-related LHON in India coexists with multiple different mtDNA haplogroups, unlike the preferential association of west Eurasian haplogroup J and the reported increased clinical penetrance with the J2 subhaplogroup. However, we observed variable penetrance of LHON in different Indian mtDNA haplogroup backgrounds, indicating their possible influence on clinical expression. These data suggest that a similar heterogeneity, resulting from the mtDNA haplogroup, might also exist in other mitochondrial diseases among Indian populations.

Homozygosity mapping guided next generation sequencing to identify the causative genetic variation in inherited retinal degenerative diseases.

Inherited retinal degeneration (IRD) are a group of genetically heterogeneous disease of which retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are the most common and severe type. In our study we had taken three unrelated South Indian consanguineous IRD families. Homozygosity mapping was done using Affymetrix 250K Nsp1 GeneChip in each of LCA, Cone-Rod dystrophy (CRD) and autosomal recessive RP (arRP) families followed by targeted re-sequencing by next generation sequencing (NGS) on Illumina MiSeq. Known candidate genes ranging from 1-8 in numbers within the homozygous blocks were identified by homozygosity mapping and targeted NGS revealed the causative mutations; RDH12 c.832A>C, ABCA4 c.1462G>T, CDHR1c.1384_1392delCTCCTGGACinsG, in the LCA, CRD and arRP families, respectively. The identified mutations were validated by Sanger sequencing, segregation in the families and their absence in 200 control chromosomes. Homozygosity mapping guided targeted NGS, especially when more numbers of known candidate genes within the homozygous blocks are observed is a comprehensive method for mutation identification. Molecular data from a larger retinal degenerative disease cohort would reveal the spectrum and prevalence of mutations and genes in Indian population. Molecular diagnosis also aids in genetic counseling, offering carrier and prenatal testing to family members.

Homozygosity Mapping in Leber Congenital Amaurosis and Autosomal Recessive Retinitis Pigmentosa in South Indian Families.

Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) are retinal degenerative diseases which cause severe retinal dystrophy affecting the photoreceptors. LCA is predominantly inherited as an autosomal recessive trait and contributes to 5% of all retinal dystrophies; whereas RP is inherited by all the Mendelian pattern of inheritance and both are leading causes of visual impairment in children and young adults. Homozygosity mapping is an efficient strategy for mapping both known and novel disease loci in recessive conditions, especially in a consanguineous mating, exploiting the fact that the regions adjacent to the disease locus will also be homozygous by descent in such inbred children. Here we have studied eleven consanguineous LCA and one autosomal recessive RP (arRP) south Indian families to know the prevalence of mutations in known genes and also to know the involvement of novel loci, if any. Complete ophthalmic examination was done for all the affected individuals including electroretinogram, fundus photograph, fundus autofluorescence, and optical coherence tomography. Homozygosity mapping using Affymetrix 250K HMA GeneChip on eleven LCA families followed by screening of candidate gene(s) in the homozygous block identified mutations in ten families; AIPL1 - 3 families, RPE65- 2 families, GUCY2D, CRB1, RDH12, IQCB1 and SPATA7 in one family each, respectively. Six of the ten (60%) mutations identified are novel. Homozygosity mapping using Affymetrix 10K HMA GeneChip on the arRP family identified a novel nonsense mutation in MERTK. The mutations segregated within the family and was absent in 200 control chromosomes screened. In one of the eleven LCA families, the causative gene/mutation was not identified but many homozygous blocks were noted indicating that a possible novel locus/gene might be involved. The genotype and phenotype features, especially the fundus changes for AIPL1, RPE65, CRB1, RDH12 genes were as reported earlier.

Biosynthetic and functional defects in newly identified SLC4A11 mutants and absence of COL8A2 mutations in Fuchs endothelial corneal dystrophy.

Late-onset Fuchs endothelial corneal dystrophy (FECD) shows genetic heterogeneity. Identification of SLC4A11 as a candidate gene for congenital hereditary endothelial dystrophy with similar corneal endothelial defects as FECD and reduced mRNA expression of SLC4A11 in the endothelium of FECD cases suggested that this gene may also be involved in pathogenesis of FECD. Mutations in SLC4A11 give rise to SLC4A11 protein marked by retention in the endoplasmic reticulum as a result of mis-folding. We screened 45 sporadic late-onset, 4 early-onset FECD patients and an early-onset autosomal dominant FECD family. We identified three previously unreported missense mutations: c.719G>C (p.W240S), c.1519G>A (p.V507I) and c.1304C>T (p.T434I) in unrelated individuals. These SLC4A11 mutants, expressed in HEK293 cells, had defects in either their cell surface expression or functional activity (rate of osmotically driven water flux). SLC4A11 mutations contribute to 11% (5/45) of sporadic late-onset FECD in the cohort studied. COL8A2, which causes some cases of early-onset FECD, was also screened in this cohort. No mutations were identified in COL8A2, in neither the late-onset cohort nor the early-onset family, suggesting genetic heterogeneity in this FECD family.

Molecular profiling of complete congenital stationary night blindness: a pilot study on an Indian cohort.

PURPOSE: Congenital stationary night blindness (CSNB) is a non-progressive retinal disorder that shows genetic and clinical heterogeneity. CSNB is inherited as an autosomal recessive, autosomal dominant, or X-linked recessive trait and shows a good genotype-phenotype correlation. Clinically, CSNB is classified as the Riggs type and the Schubert-Bornschein type. The latter form is further sub-classified into complete and incomplete forms based on specific waveforms on the electroretinogram (ERG). There are no molecular genetic data for CSNB in the Indian population. Therefore, we present for the first time molecular profiling of eight families with complete CSNB (cCSNB). METHODS: The index patients and their other affected family members were comprehensively evaluated for the phenotype, including complete ophthalmic evaluation, ERG, fundus autofluorescence, optical coherence tomography, and color vision test. The known gene defects for cCSNB, LRIT3, TRPM1, GRM6, GPR179, and NYX, were screened by PCR direct sequencing. Bioinformatic analyses were performed using SIFT and PolyPhen for the identified missense mutations. RESULTS: All eight affected index patients and affected family members were identified as having cCSNB based on their ERG waveforms. Mutations in the TRPM1 gene were identified in six index patients. The two remaining index patients each carried a GPR179 and GRM6 mutation. Seven of the patients revealed homozygous mutations, while one patient showed a compound heterozygous mutation. Six of the eight mutations identified are novel. CONCLUSIONS: This is the first report on molecular profiling of candidate genes in CSNB in an Indian cohort. As shown for other cohorts, TRPM1 seems to be a major gene defect in patients with cCSNB in India.

Leber's congenital amaurosis as the retinal degenerative phenotype in thiamine responsive megaloblastic anemia: a case report.

BACKGROUND: Thiamine responsive megaloblastic anemia syndrome (TRMA), an autosomal recessive disorder is caused by mutations in the SLC19A2 gene which encodes for thiamine transporter 1 (THTR1) protein. TRMA presents with a triad of clinical features that includes diabetes mellitus, megaloblastic anemia and sensorineural hearing loss. Apart from the triad, reported ophthalmic features include cone rod dystrophy, optic atropy and retinitis pigmentosa. MATERIALS AND METHODS: A female child presented with Leber's congenital amaurosis at 10 months of age, later diagnosed with hearing impairment at 1 year, diabetes mellitus and megaloblastic anemia at 3 and a half years of age and hence as a case of thiamine responsive megaloblastic anemia. Six exons of the candidate gene SLC19A2 were screened by PCR and direct sequencing. SIFT and PolyPhen analysis was done to predict the probable effect of the mutation. RESULTS: Sequence analysis of the SLC19A2 coding region revealed a novel missense mutation in exon 2; c.314 G > A (p.G105E). Segregation analysis revealed parents heterozygous for the mutation and unaffected sib homozygous for wild type. SIFT and PolyPhen analyses predicted the mutation to be "damaging" (score-0.02) and "probably damaging" (score-0.994), respectively. CONCLUSIONS: SLC19A2, the high-affinity thiamine transporter, is the only gene known to be associated with TRMA. Here we describe for the first time Leber's congenital amaurosis as the retinal phenotype and also report a novel point mutation in the SLC19A2 gene that co-segregated with the disease in a TRMA patient.

Haplogroup heterogeneity of LHON patients carrying the m.14484T>C mutation in India.

PURPOSE: To investigate the clinical and mitochondrial DNA (mtDNA) haplogroup background of Indian Leber hereditary optic neuropathy (LHON) patients carrying the m.14484T>C mutation. METHODS: Detailed clinical investigation and complete mtDNA sequencing analysis was carried out for eight Indian LHON families with the m.14484T>C mutation. Haplogroup was constructed based on the evolutionarily important mtDNA variants. RESULTS: In the present study, we characterized eight unrelated probands selected from 187 LHON cases. The overall penetrance of the disease was estimated to be 19.75% (16/81) in eight pedigrees with the m.14484T>C mutation and showed substantially higher sex bias (male: female = 13:3). The mtDNA haplogrouping revealed that they belong to diverse haplogroups; i.e., F1c1, M31a, U2a, M*, I1, M6, M3a1, and R30a. Interestingly, we did not find an association of the m.14484T>C mutation with any specific haplogroup within the Indian population. We also did not find any secondary mutation(s) in these pedigrees, which might affect the clinical expression of LHON. CONCLUSIONS: Contrary to earlier reports showing preferential association of the m.14484T>C mutation with western Eurasian haplogroup J and increased clinical penetrance when present in J1 subhaplogroup background, the present study shows that m.14484T>C arose independently in a different mtDNA haplogroup and ethnic background in India, which may influence the clinical expression of the disease.

Molecular genetics analysis of hereditary breast and ovarian cancer patients in India.

BACKGROUND: Hereditary cancers account for 5-10% of cancers. In this study BRCA1, BRCA2 and CHEK2*(1100delC) were analyzed for mutations in 91 HBOC/HBC/HOC families and early onset breast and early onset ovarian cancer cases. METHODS: PCR-DHPLC was used for mutation screening followed by DNA sequencing for identification and confirmation of mutations. Kaplan-Meier survival probabilities were computed for five-year survival data on Breast and Ovarian cancer cases separately, and differences were tested using the Log-rank test. RESULTS: Fifteen (16%) pathogenic mutations (12 in BRCA1 and 3 in BRCA2), of which six were novel BRCA1 mutations were identified. None of the cases showed CHEK2*1100delC mutation. Many reported polymorphisms in the exonic and intronic regions of BRCA1 and BRCA2 were also seen. The mutation status and the polymorphisms were analyzed for association with the clinico-pathological features like age, stage, grade, histology, disease status, survival (overall and disease free) and with prognostic molecular markers (ER, PR, c-erbB2 and p53). CONCLUSION: The stage of the disease at diagnosis was the only statistically significant (p < 0.0035) prognostic parameter. The mutation frequency and the polymorphisms were similar to reports on other ethnic populations. The lack of association between the clinico-pathological variables, mutation status and the disease status is likely to be due to the small numbers.

CERKL mutations cause an autosomal recessive cone-rod dystrophy with inner retinopathy.

PURPOSE: To define the phenotype of the retinal degeneration associated with mutations in the CERKL gene. METHODS: Six patients (ages, 26-54 years) from three unrelated families with CERKL mutations were studied clinically and by electroretinography, kinetic, and chromatic static perimetry, autofluorescence (AF) imaging, and optical coherence tomography (OCT). RESULTS: Three siblings were homozygotes for p.R257X mutation; two siblings were compound heterozygotes for p.R257X and a novel p.C362X mutation; and one patient had only p.R257X mutation identified to date. There was a spectrum of severity: from mild visual acuity loss to light perception; from full kinetic fields with relative central scotomas to remnant peripheral islands; from reduced ERGs (some with negative waveforms) to nondetectable signals. Maculopathy showed residual foveal islands or extensive central rod and cone scotomas. With AF imaging, there was evidence of hyperautofluorescence at earlier and hypoautofluorescence at later disease stages. Peripheral function was generally less affected than central function. With OCT there were small foveal islands of outer nuclear layer (ONL) in those with preserved acuity. Eccentric to an annular region with no discernible ONL, there could be ONL in the midperiphery. At early disease stages, ganglion cell layer thickness was less affected than ONL. Later disease stages were accompanied by inner nuclear layer and nerve fiber layer abnormalities. CONCLUSIONS: CERKL mutations are associated with widespread retinal degeneration with prominent maculopathy. The clinical presentation is that of an autosomal recessive cone-rod dystrophy. Photoreceptor loss appears at all stages of disease and inner laminopathy complicates the phenotype at later stages.

A missense mutation in the nuclear localization signal sequence of CERKL (p.R106S) causes autosomal recessive retinal degeneration.

PURPOSE: To investigate the genetic basis of autosomal recessive retinal degeneration in a large consanguineous family from Pakistan. METHODS: Ophthalmic examinations were conducted on family members to establish their diagnosis. Genomic DNA extracted from peripheral blood was used for homozygosity mapping to discover the chromosomal region that harbors the defective gene. Direct sequence analysis and restriction enzyme digestion were used to identify and confirm the defect in the gene. RESULTS: There were three affected siblings in the family, each with limited peripheral vision and impaired visual acuity. We established linkage to a region on chromosome 2 that encompasses the RP26 locus. Upon sequencing the ceramide kinase-like (CERKL) gene, which is mutated in the original RP26 family, we identified a C>A transversion in exon 2 (c.316C>A) that substitutes an arginine residue with a serine (p.R106S) in the conserved nuclear localization signal sequence (KLKRR) of the protein. This mutation segregated with retinal degeneration in the Pakistani family and was not observed in the DNA of 174 ethnically matched unaffected controls. CONCLUSIONS: This is the third reported mutation in CERKL causing retinal degeneration but is the first report to show that a single amino acid change in CERKL, rather than a null mutation, can cause retinal disease. Although the function of CERKL is still unknown, the mutation described herein confirms that the nuclear localization signal sequence is important in the physiologic function of the protein.

Association of non-synonymous single nucleotide polymorphisms in the LOXL1 gene with pseudoexfoliation syndrome in India.

PURPOSE: In the Icelandic and Swedish populations, pseudoexfoliation syndrome (XFS) and pseudoexfoliation glaucoma (XFG) has been significantly associated with LOXL1 exon 1 polymorphisms - allele G of rs1048661 (R141L) and allele G of rs3825942 (G135D). In this study, we looked at the association of rs1048661 and rs3825942 in a southern Indian population. METHODS: Fifty-two cases with XFS (including XFG) and 97 matched controls that had thorough glaucoma evaluations were included in the study. Exon 1 of the LOXL1 gene with the single nucleotide polymorphisms (SNPs) were amplified and sequenced. For statistical significance, Pearson's Chi(2) test was performed. The HAPLOVIEW program v4.0 was used to determine the Hardy-Weinberg equilibrium and haplotype association. RESULTS: In our study population, there was a significant association of allele G of rs3825942 with XFS (p=0.0001) and genotype GG (p=0.000305) with XFS. CONCLUSIONS: Out of the two non-synonymous SNPs in exon 1 of the LOXL1 gene, rs3825942 has a significant association with XFS cases in the patients of the southern Indian population. To the best of our knowledge, this is the first Asian study replicating the European studies.

Identification of a novel splice-site mutation in the Lebercilin (LCA5) gene causing Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is one of the most common causes of hereditary blindness in infants. To date, mutations in 13 known genes and at two other loci have been implicated in LCA causation. An examination of the known genes highlights several processes which, when defective, cause LCA, including photoreceptor development and maintenance, phototransduction, vitamin A metabolism, and protein trafficking. In addition, it has been known for some time that defects in sensory cilia can cause syndromes involving hereditary blindness. More recently evidence has come to light that non-syndromic LCA can also be a "ciliopathy." METHODS: Here we present a homozygosity mapping analysis in a consanguineous sibship that led to the identification of a mutation in the recently discovered LCA5 gene. Homozygosity mapping was done using Affymetrix 10K Xba I Gene Chip and a 24.5cM region on chromosome 6 (6q12- q16.3) was identified to be significantly homozygous. The LCA5 gene on this region was sequenced and cDNA sequencing also done to characterize the mutation. RESULTS: A c.955G>A missense mutation in the last base of exon 6 causing disruption of the splice donor site was identified in both the affected sibs. Since there is a second consensus splice donor sequence 5 bp into the adjacent intron, this mutation results in a transcript with a 5 bp insertion of intronic sequence, leading to a frameshift and premature truncation. CONCLUSIONS: We report a missense mutation functionally altering the splice donor site and leading to a truncated protein. This is the second report of LCA5 mutations causing LCA. It may also be significant that one affected child died at eleven months of age due to asphyxia during sleep. To date the only phenotype unambiguously associated with mutations in this gene is LCA. However the LCA5 gene is known to be expressed in nasopharynx, trachea and lungs and was originally identified in the proteome of bronchial epithelium ciliary axonemes. The cause of death in this child may therefore imply that LCA5 mutations can in fact cause a wider spectrum of phenotypes including respiratory disease.

Mutation analysis of hMSH2 and hMLH1 in colorectal cancer patients in India.

The aim of this work was to study the mutation profile in hMSH2 and hMLH1 genes in hereditary nonpolyposis colorectal cancer (HNPCC) patients in India. On the basis of the Bethesda criteria, 31 colorectal cancer patients were studied first for microsatellite instability, using the five markers recommended by the Bethesda guidelines. Twelve of 31 tumor samples were found to be MSI-H, 9 of 31 were MSI-L, and the rest were MSS. The 12 patients with MSI-H were analyzed for mutations in hMSH2 and hMLH1 genes using PCR-denaturing high-performance liquid chromatography (dHPLC), followed by sequencing of samples showing abnormal peaks. Of the five mutations detected, three were found to be deleterious mutations (hMSH2-R680X, hMLH1-E671X, and a splice junction mutation IVS16-2A --> G); one had a mutation of probable significance (hMLH1-C680G) and one was of unknown significance (hMSH2-R171K). This study has also shown that most of the early-onset colon (4/7) and early-onset rectal (15/21) cancers are MSS or MSI-L. This is the first study to describe the mutation in hMSH2 and hMLH1 in Indian patients, a low incidence region for colorectal cancer. A two-stage procedure using MSI testing followed by PCR-dHPLC was found to be an efficient method in studying the mutation profile in high-risk patients.