Jharna Ray

Gene therapy restores vision in a canine model of childhood blindness

The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65−/− dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.

Molecular pathogenesis of Wilson disease: haplotype analysis, detection of prevalent mutations and genotype–phenotype correlation in Indian patients

Wilson disease (WD) is an autosomal recessive disorder caused by defects in the copper-transporting P-type ATPase gene (ATP7B) resulting in the accumulation of copper in the liver and the brain. We identified prevalent mutations in the ATP7B of Indian WD patients and attempted to correlate those with the disease phenotype. Patients from 62 unrelated families and their first-degree relatives comprising 200 individuals were enrolled in this study. Three dinucleotide repeat markers flanking WD locus and a few intragenic SNPs were used to determine the genotypes and construct haplotypes of the patients. Seven recurring haplotypes accounting for 58% of the total mutant chromosomes were identified, and four underlying defects in the ATP7B representing 37% of WD chromosomes were detected. In addition, five other rare mutations were characterized. Thus a total of nine mutations including five novel changes were identified in the ATP7B of WD patients. Interestingly, homozygotes for different mutations that would be expected to produce similar defective proteins showed significant disparity in terms of organ involvement and severity of the disease. We also observed WD patients with neurological symptoms with little or no manifestation of hepatic pathogenesis. In one WD family, the proband and a sib had remarkably different phenotypes despite sharing the same pair of mutant chromosomes. These findings suggest a potential role for yet unidentified modifying loci for the observed phenotypic heterogeneity among the WD patients.

A Line 1 insertion in the Factor IX gene segregates with mild hemophilia B in dogs.

We undertook the biochemical and molecular characterization of hemophilia in a large pedigree of German wirehaired pointers. Males affected with hemophilia B had approximately 5% normal Factor IX coagulant activity and a proportional reduction of Factor IX protein concentration, indicative of a mild hemophilia B phenotype. Using Southern blot analyses and PCR amplification of genomic DNA, we discovered a large, 1.5-kb insertion in intron 5 of the Factor IX gene of an affected male. The insert consists of a 5′ truncated canine Line-1 followed by an approximately 200-bp 3′ poly (A) tract, flanked by a 15-bp direct repeat. The insert can be traced through at least five generations and segregates with the hemophilia B phenotype in this breed. This is the first description of a Factor IX mutation associated with mild hemophilia B in a non-human species and provides evidence for a recent Line-1 insertion event in the canine genome.

Clinical characterization and evaluation of DYT1 gene in Indian primary dystonia patients

Objectives –  Dystonia is a common movement disorder. The purpose of this study is to examine the relative distribution of the primary dystonia subtypes and identify mutation (s) in the DYT1 gene in Indian patients.

Cloning and characterization of opticin cDNA: evaluation as a candidate for canine oculo-skeletal dysplasia.

Opticin, a novel member of the leucine-rich repeat (LRR) family, has been reported to bind to collagen fibrils. Many members of the LRR family of extracellular matrix proteins have been reported to bind to fibrillar collagen and regulate the diameter of collagen fibrils and lateral fusion between fibrils. Collagen fibrils are important for the maintenance of the vitreous body in the eye and growth plate cartilage of joints. Oculo-skeletal dysplasia (OSD) is a heterogeneous group of heritable genetic disorders affecting humans and a few breeds of dogs. Labrador retrievers and Samoyeds affected with non-allelic forms of OSD exhibit vitreous dysplasia and dwarfism, and could serve as an animal model for the disorder. To test the opticin gene as a candidate for OSD, canine opticin cDNA has been cloned and characterized. The predicted 327 amino acid sequence is 77% homologous to human opticin, and maintains characteristic structural domains including seven LRR domains, two cysteine clusters and potential O-linked glycosylation sites. It shows highest protein sequence identity to epiphycan (37%) and osteoglycin (31%) and belongs to the Class III family of LRR extracellular matrix proteins. In addition to ocular tissues and cartilage, opticin mRNA and protein have been identified in ligament, skin, muscle, and testes. No alteration of opticin expression at the protein level was observed in OSD affected dogs relative to normal controls. Based on linkage analysis using a newly identified intragenic single nucleotide polymorphism opticin has been excluded from having any causal association with the OSD loci in both Samoyeds and Labrador retrievers.

Evaluation of PINK1 variants in Indian Parkinson's disease patients.

Mutations in PINK1 have been identified in familial and sporadic cases of early onset Parkinsons disease (PD). To determine the contribution of PINK1 variants in Indian PD patients, the gene was screened in 250 patients and 205 ethnically matched controls by polymerase chain reaction, single-stranded conformation polymorphism and DNA sequencing. Two potentially pathogenic variants (Arg246Gln & Arg276Gln) were detected in the heterozygous state in 5 patients; none of the patients carried homozygous or compound heterozygous mutations. In addition, 13 other variants were identified, including a known polymorphism (Ala340Thr), a few synonymous or intronic changes, none of which are likely to be pathogenic. Unlike the Chinese population, the Ala340Thr variant did not show any association with PD in Indian population. Six single nucleotide polymorphisms (SNPs) selected from dbSNP were genotyped in 531 normal, healthy individuals representing different ethnic groups of India. Most of the SNP markers were observed to be highly heterozygous among Indians, which could be used for segregation analysis of PINK1 alleles in familial PD cases.

Cloning and expression of type II collagen mRNA: evaluation as a candidate for canine oculo-skeletal dysplasia

The disease phenotype of oculo-skeletal dysplasia (OSD) detected in Labrador retrievers and Samoyeds shows a large degree of similarity with human Stickler and Kniest dysplasia. Type II collagen (COL2A1) mRNA, which is defective in a larger number of Stickler and Kniest patients, has been cloned and characterized from normal dog. The amino acid sequence of the canine type II procollagen is predicted to contain 1487 residues, with high degree of homology with its human homologue, and maintains all the characteristic structural domains. In addition to cartilage, expression of COL2A1 has also been detected in canine retina and testes. In testes, the N-propeptide region of COL2A1 displayed differential splicing and expressed both splice variants, IIA (with exon 2) and IIB (without exon 2), suggesting the importance of both forms in testis maturation and maintenance. Despite a severe decrease of type II collagen protein in the vitreous of OSD affected Labrador retrievers, COL2A1 gene has been excluded from having any causal association with the disease locus by linkage analysis. Using an intragenic RFLP marker, COL2A1 gene has also been tested as a candidate gene for the non-allelic form of the other canine OSD identified in Samoyeds, and excluded by linkage analysis. Oculo-skeletal dysplastic Labrador retriever and Samoyed provide two animal models for chondrodysplasia with genetic heterogeneity.

Role of tau kinases (CDK5R1 and GSK3B) in Parkinson's disease: A study from India

Glycogen synthase kinase-3β (GSK3B) and cyclin-dependent kinase 5 (CDK5) are the 2 major protein kinases involved in abnormal phosphorylation of tau. To determine their potential role in the pathogenesis of Parkinsons disease (PD) we analyzed 2 functional single nucleotide polymorphisms (SNPs) of GSK3B (rs334558 and rs6438552) and rs735555 of CDK5 regulatory subunit 1 (CDK5R1) in 373 PD cases and 346 healthy controls of eastern India. The C,C and T,C haplotypes of GSK3B were respectively moderately associated with increased risk and protection for late onset PD (LOPD) (odds ratio [OR], 1.399; 95% confidence interval [CI], 1.069-1.829; p = 0.015, and OR, 0.436; 95% CI, 0.222-0.853; p = 0.016, respectively). Moreover, moderate to significant interaction between different loci were observed for the entire PD cohort or late onset PD only. However, among these interactions, individuals carrying the (C/C) genotype at both loci (rs6438552 and rs735555) had almost twice the risk of developing PD than those without this genotypic combination (OR, 1.871; 95% CI, 1.181-2.964; p = 0.009). Thus, synergistic effect between the 2 major tau kinases, through these SNPs, appears to determine the risk profile for PD.

Microtubule-associated protein tau (MAPT) influences the risk of Parkinson's disease among Indians.

Parkinsons disease (PD) is a neurodegenerative disease of the central nervous system and its prevalence increases with age. Microtubule-associated protein tau (MAPT), a neuronal protein is involved in the pathogenesis of several neurodegenerative diseases including PD. To determine the broader significance of this association with PD, replicative studies in distinct ethnic populations are required. In this study, we investigated MAPT for its potential association with PD using five haplotype-tagging SNPs and the del-In9 polymorphism of MAPT in 301 PD patients and 243 healthy controls from eastern India. Our case-control analysis did not show a significant association with any of the markers and PD. However, a risk haplotype [GAC+G] for PD was identified (OR=1.563; 95% CI=1.045-2.337; p=0.03). In addition, haplotype AAC+A (OR=2.787; 95% CI=1.372-5.655; p=0.004) was strongly associated with early onset PD (age at onset < or =40 years) and AAC+G haplotype showed a weak association (OR=2.233; 95% CI=1.018-4.895; p=0.045) with late onset PD (age at onset >40 years). This observation highlights the significance of rs7521 in modifying the age at onset of PD under a common haplotype background. We also identified AGC+A as a risk haplotype for sporadic cases (OR=2.773, 95% CI=1.198-6.407, p=0.016). This is the first association study from India conducted on MAPT among PD patients and provides valuable information for comparison with other ethnic groups.

DJ-1 Variants in Indian Parkinson’s Disease Patients

Parkinson’s disease (PD) is a common neurodegenerative movement disorder. Among the candidate genes, DJ-1 accounts for about 1% of the cases in different populations. We aim to find the contribution of the gene towards PD among Indians. By screening DJ-1 in 308 PD patients of eastern India and 248 ethnically matched controls, a total of 21 nucleotide variants – including two nonsynonymous changes – were detected. p.Arg98Gln was identified in 6 unrelated patients and 2 controls while p.Val35Ile, a novel change, was found only in 2 unrelated patients. A SNP (rs7517357) was observed to be moderately associated with increased risk of PD (p < 0.05). The deletion allele (g.168–185del) of a known 18 bp del/ins/dup polymorphism was found to be over represented (p < 0.05) among older patients (> 40 years) compared to the controls (> 45 years). Two of the patients, also heterozygotes for PINK1 mutation, had more severe disease phenotypes, consistent with the reported interaction between PINK1 and DJ-1 gene products [19]. Our results demonstrate that up to 3.9% (12/308) of PD patients of eastern India harbor DJ-1 variants that should be explored further for any causal relationship with PD.