RESUMEN
Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism that is caused by mutations in the ATP7B gene. To date, more than 300 mutations have been described in this gene. Molecular diagnostics of WD utilizes restriction enzyme digestion, multiplex ligation-dependent probe amplification or a direct sequencing of the whole gene. To simplify and speed up the screening of ATP7B mutations, we have developed a genotyping microarray for the simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene based on the arrayed primer extension reaction. The patient's DNA is amplified in four multiplex polymerase chain reactions, fragmented products are annealed to arrayed primers spotted on a chip, which enables DNA polymerase extension reactions with fluorescently labeled dideoxynucleotides. The Wilson microarray was validated by screening 97 previously genetically confirmed WD patients. In total, we detected 43 mutations and 15 polymorphisms that represent a majority of the common mutations occurring in the Czech and Slovak populations. All screened sequence variants were detected with 100% accuracy. The Wilson chip appears to be a rapid, sensitive and cost-effective tool, representing the prototype of a disease chip that facilitates and speeds up the screening of potential WD patients.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Transporte de Catión/genética , Degeneración Hepatolenticular/genética , Análisis por Micromatrices/métodos , Mutación Puntual , ATPasas Transportadoras de Cobre , Análisis Mutacional de ADN , Tamización de Portadores Genéticos/métodos , Genotipo , Degeneración Hepatolenticular/diagnóstico , Heterocigoto , Humanos , Análisis por Micromatrices/instrumentación , MutaciónRESUMEN
Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.