Development of a High-Throughput Microarray-Based Resequencing System for Neurological Disorders and Its Application to Molecular Genetics of Amyotrophic Lateral Sclerosis

Abstract

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 highthroughput 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.

Optineurin mutations in Japanese amyotrophic lateral sclerosis: Table 1

“…In the previous report by Maruyama et al ,1 three types of mutations were found in 92 fALS patients (3.3%). Similarly, mutational frequencies of SOD1 and FUS in Japanese fALS are 20% and 12.2%, respectively 3 5. The mutational frequency of fALS in Japanese is also similar to that of TARDBP , but three- to fivefold less than that of SOD1 and FUS .…”

“…Thus, taken together, the mutational frequency of OPTN in Japanese sALS is 0.23%. The mutational frequency of SOD1 in Japanese sALS is 1/35 (2.9%) 3. In addition, the mutational frequency of TARDBP in Japanese sALS was reported to be 0.29% 4.…”

Neurovascular changes in prolonged migraine aura in FHM with a novel ATP1A2 gene mutation

“…In three members, we performed a genetic analysis for all exons of the CACNA1A and ATP1A2 genes using a DNA microarray-based high-throughput resequencing system 24. We also analysed the SCN1A gene by direct nucleotide sequence analysis.…”

Mutations for Gaucher Disease Confer High Susceptibility to Parkinson Disease

“…24 The analysis was conducted according to the manufacturer's instructions (Affymetrix Inc, Santa Clara, California). All variants were further confirmed at direct nucleotide sequence analysis using a genetic analyzer (ABI PRISM 3100; Applied Biosystems Inc, Foster City, California).…”