Computational protocol: ANO10 mutations cause ataxia and coenzyme Q10 deficiency

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Protocol publication

[…] Genetic analysis of mitochondrial DNA (mtDNA) for deletions, depletion, and point mutations in muscle DNA as well as direct sequencing of POLG,PDSS1, PDSS2, COQ2, COQ9, CABC1/ADCK3 and APTX in blood DNA were normal in all patients []. In patient 1, whole exome sequencing was performed in genomic DNA, isolated from lymphocytes (DNeasy®, Qiagen, Valencia, CA), fragmented and enriched by Illumina TruSeq™ 62 Mb exome capture, and sequenced (Illumina HiSeq 2000, 100 bp paired-end reads). The in-house bioinformatics pipeline included alignment to the human reference genome (UCSC hg19), reformatting, and variant detection (Varscan v2.2, Dindel v1.01), as described previously []. On-target variant filtering excluded those with minor allele frequency greater >0.01 in several databases. Rare homozygous and compound heterozygous variants were defined, and protein altering and/or putative ‘disease causing’ mutations, along with their functional annotation, were identified using ANNOVAR []. Putative pathogenic variants were confirmed by Sanger sequencing, using custom-designed primers ( on an ABI 3130XL (Life Technologies, CA, USA), allowing segregation analyses (Fig. a). The primers used for genomic DNA (NG_028216.1) and cDNA (NM_018075.3) analysis of ANO10 are listed in the Supplementary Materials (Supplemental Tables 1 and 2).Fig. 1To measure ANO10 mRNA expression level, skin fibroblasts from the patients were grown and total RNA was extracted using the Pure Link™ RNA Mini Kit (Ambion, Life Technologies). RNA concentration was measured using a Nanodrop. Subsequently, 100 ng of RNA was converted into cDNA using SuperScript VILO cDNA Synthesis Kit (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed using TaqMan Assays for ANO10 and GAPDH (Applied Biosystems). […]

Pipeline specifications

Software tools VarScan, Dindel, ANNOVAR
Application WES analysis
Organisms Homo sapiens
Diseases Cataract, Retinal Degeneration, Spinocerebellar Degenerations, Machado-Joseph Disease, Genetic Diseases, Inborn
Chemicals Calcium