Molecular Diagnosis of Wilson Disease Using Prevalent Mutations and Informative Single-Nucleotide Polymorphism Markers

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2007-09, Vol.53 (9), p.1601-1608
• Main Authors: Gupta, Arnab, Maulik, Mahua, Nasipuri, Poonam, Chattopadhyay, Ishita, Das, Shyamal K, Gangopadhyay, Prasanta K, The Indian Genome Variation Consortium, Ray, Kunal
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - genetics; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cation Transport Proteins - genetics; Chromosomes; Copper-transporting ATPases; Diagnostic tests; Families & family life; Fundamental and applied biological sciences. Psychology; Genetic counseling; Genetic disorders; Genetic Markers; Genetics; Genotype; Hepatolenticular Degeneration - diagnosis; Hepatolenticular Degeneration - ethnology; Hepatolenticular Degeneration - genetics; Heterozygote; Humans; India - epidemiology; Industrial research; Investigative techniques, diagnostic techniques (general aspects); Linkage Disequilibrium; Medical sciences; Molecular biology; Molecular Diagnostic Techniques; Mutation; Oligonucleotide Array Sequence Analysis; Patients; Pedigree; Polymerase Chain Reaction; Polymorphism; Polymorphism, Single Nucleotide; Population; Population Groups; Siblings; World population
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2007.086066

Wilson disease (WD) is an autosomal recessive disorder caused by defects in the ATPase, Cu(2+) transporting, beta-polypeptide gene (ATP7B) resulting in accumulation of copper in liver and brain. WD can be thwarted if detected at a presymptomatic stage, but occasional recombination during carrier detection with dinucleotide repeat markers flanking the WD locus may lead to faulty diagnosis. We examined the use of intragenic single-nucleotide polymorphism (SNP) markers to avoid this limitation. We prepared genomic DNA from the peripheral blood of Indian WD patients. By use of PCR, we amplified the exons and flanking regions of the WD gene and then performed sequencing to identify the nucleotide variants. We genotyped the SNPs in 1871 individuals by use of the Sequenom mass array system. We made linkage disequilibrium plots using Haploview software. We identified 1 mutation accounting for 11% (19 of 174) of WD chromosomes among patients in addition to 4 prevalent mutations characterized previously. Among 24 innocuous allelic variants identified, we selected 3 SNPs found to have high heterozygosity (>0.40) for the detection of mutant WD chromosomes. On analyzing these SNPs in 28 test individuals, who were sibs to 17 unrelated WD patients, we obtained unequivocal genotyping in 25 cases (approximately 89%). The remaining 3 cases were genotyped by dinucleotide repeat marker (D13S133). Sets of SNP markers are highly heterozygous across most world populations and could be used in combination with analysis of prevalent mutations as a comprehensive strategy for determining presymptomatic and carrier sibs of WD patients.