Studies on Copper Homeostasis Pathway Genes With Special Reference to Wilson's Disease

Gupta, Arnab (2006) Studies on Copper Homeostasis Pathway Genes With Special Reference to Wilson's Disease. PhD thesis, Jadavpur University.

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                  Supervisors

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                  Ray, Kunal

                  Abstract

                  Wilson’s disease is an autosomal recessive disorder, which manifests as hepatolenticular degeneration that results in the accumulation of copper in the brain, liver, kidney and cornea. The disease is diagnosed on the basis of typical symptoms and conventional biochemical indicators, which include low serum ceruloplasmin concentration, and elevated excretion of urinary copper. The disease has a worldwide frequency of 1/5,000 to 1/30,000 live births and a carrier frequency of 1 in 90, based on lower frequency. The gene, ATP7B, causally associated with the disease codes for a P-type ATPase that has six copper binding regions and other conserved domains consistent with other metal transporter. The studies undertaken for this dissertation were focused on the following areas: (i) identifying the molecular defects in Indian WD patients and correlating them with the disease phenotype, (ii) utilizing the genotypic variations (microsatellites, SNPs and mutations) in the Indian population for diagnosis of presymptomatic individuals and carriers in Wilson’s disease families and (iii) understanding the role of modifier locus (if any) responsible for phenotypic variations commonly observed in WD. Wilson’s disease patients from 82 unrelated families and their first-degree relatives comprising 402 individuals were enrolled in this study. Three dinucleotide repeat markers (D13S316, D13S133 and D13S314) flanking WD locus and a few intragenic Single Nucleotide Polymorphisms were used to determine the genotypes and construct haplotypes of the patients. Seven distinct haplotypes were identified which accounted for approximately 49% of all the mutant chromosomes. We hypothesized that this 49% of the mutations in the test population resulted from probably seven founder defects in the ATP7B, while the rest of the WD chromosomes were likely to harbor independent rare mutations. Five prevalent mutations were detected in a total of 65 chromosomes accounting for ~40% of the all the mutant WD chromosomes in our patient pool. Twelve rare mutations and a total of 24 single nucleotide variants were also detected. We wished to carryout a correlation of identified mutations with the disease phenotype. 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 despite reports on the earlier accumulation of copper in the liver than in the brain. In one patient, an unnatural feature of extensive involvement of white matter coupled with drug resistance WD was noticed. In a WD family, the proband and a sib had remarkably different phenotypes despite sharing the same pair of mutant chromosomes. These findings suggested potential role(s) for yet unidentified modifying loci for the observed phenotypic heterogeneity among the WD patients. Unlike most other genetic diseases, available treatment regimen for WD provides hope to those persons predisposed to the disease due to harboring two mutant copies of ATP7B, if identified at a pre-clinical stage. We assessed available markers and discovered new ones, to determine the presymptomatic individuals in multi-sib WD families, so that necessary measures could be taken to thwart the disease or to alleviate the disease progression. Using the three extragenic microsatellite markers, already described, we identified six presymptomatic individuals in 30 multisib families of our patient pool. To avoid the potential problem of recombination due to large distance of the microsatellites from the ATP7B gene, SNP markers were also utilized to determine the status of sibs in WD affected families. Four SNP with high minor allele frequency in WD chromosomes were evaluated for heterozygosity and Linkage disequilibrium among themselves in the entire Indian population to assess their utility as suitable markers. The efficacy of the SNPs to track mutant chromosomes, was evaluated using 17 multisib families affected with WD. Although a single gene disorder, as mentioned above, Wilson’s disease patients manifest a wide range of phenotypic heterogeneity, suggesting putative involvement of disease modifying loci. In the WD patients we sequenced MURR1, a gene implicated in copper toxicosis in dogs, to understand the cause of underlying phenotypic heterogeneity. Three nucleotide changes were identified in MURR1, of which two represented SNPs, c.492GAT>GAC (Asp164Asp) and c.170+122C>T (IVS1+122C>T), and the third one a putative mutation (c.521ACG>ATG; Thr174Met) represented a novel variant. The patient harboring the mutation Thr174Met showed remarkably high levels of 24-hours urinary copper uncommon in most WD patients and other features consistent with increased apoptosis. Role of MURR1 has been earlier established in caspase-3 mediated apoptosis (Mufti et al, Mol. Cell 21: 775-785). So far, a single mutation c.813TGC>TGA (Cys271Stop) has been detected in the ATP7B of the patient. The remaining exons of ATP7B are being screened to find the second mutation. Once we complete the study of screening ATP7B, we would have a better idea, whether the disease is caused by a digenic mode of inheritance involving two genes of copper homeostasis pathway with one allele of each gene being defective or that MURR1 mutation modifies the WD phenotype caused by defect in both alleles of ATP7B. Currently work is in progress to decipher the underlying molecular event for the unusual pathologies observed in the WD patients. Most of the studies presented in this dissertation have been already published or has been accepted for publication. Interested readers can refer to the following publications: (i) Gupta et al. Hum Genet. 2005; 118: 49-57, (ii) Gupta et al. Ind J Med Res. 2003; 118: 208-214, (iii) Gupta et al. Clin Chem. 2006 (In press), (iv) Aikath et al. Neurology. 2006 (In press).

                  Item Type: Thesis (PhD)
                  URI: http://www.eprints.iicb.res.in/id/eprint/1595
                  Subjects: Molecular & Human Genetics
                  Divisions: Indian Institute of Chemical Biology
                  Depositing User: Mr Santanu Sadhukhan
                  Date Deposited: 14 Aug 2012 11:57
                  Last Modified: 14 Aug 2012 11:57
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