Development of a high-throughput microarray-based resequencing system for neurological disorders and its application to molecular genetics of amyotrophic lateral sclerosis.

BACKGROUND Comprehensive resequencing of the causative and disease-related genes of neurodegenerative diseases is expected to enable (1) comprehensive mutational analysis of familial cases, (2) identification of sporadic cases with de novo or low-penetrant mutations, (3) identification of rare variants conferring disease susceptibility, and ultimately (4) better understanding of the molecular basis of these diseases. OBJECTIVE To develop a microarray-based high-throughput resequencing system for the causative and disease-related genes of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. DESIGN Validation of the system was conducted in terms of the signal-to-noise ratio, accuracy, and throughput. Comprehensive gene analysis was applied for patients with ALS. Subjects Ten patients with familial ALS, 35 patients with sporadic ALS, and 238 controls. RESULTS The system detected point mutations with 100% accuracy and completed the resequencing of 270 kilobase pairs in 3 working days with greater than 99.9% accuracy of base calls, or the determination of base(s) at each position. Analysis of patients with familial ALS revealed 2 SOD1 mutations. Analysis of the 35 patients with sporadic ALS revealed a previously known SOD1 mutation, S134N, a novel putative pathogenic DCTN1 mutation, R997W, and 9 novel variants including 4 nonsynonymous heterozygous variants consisting of 2 in ALS2, 1 in ANG, and 1 in VEGF that were not found in the controls. CONCLUSION The DNA microarray-based resequencing system is a powerful tool for high-throughput comprehensive analysis of causative and disease-related genes. It can be used to detect mutations in familial and sporadic cases and to identify numerous novel variants potentially associated with genetic risks.