Computational protocol: Behr’s Syndrome is Typically Associated with Disturbed Mitochondrial Translation and Mutations in the C12orf65 Gene

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Protocol publication

[…] Whole exome sequencing was performed in genomic DNA of patients 1, 2 and 3 and direct sequencing of C12orf65 was performed in patient 4 based on the clinical phenotype. DNA was 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: dbSNP135, 1000 genomes (February 2012 data release), the National Heart, Lung and Blood Institute (NHLBI, NIH, Bethesda, MD) Exome Sequencing Project (ESP) 6500 exomes, and 343 unrelated in-house controls. 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 []. Candidate genes were prioritized if previously associated with a disease phenotype []. Putative pathogenic variants were confirmed by Sanger sequencing using custom-designed primers ( on an ABI 3130XL (Life Technologies, CA), allowing segregation analyses where possible. The following primers were used for genomic DNA analysis of C12orf65 (NM 152269): exon 2: forward: 5′ GCATAATCTTGAGGGCAGATG-3′, reverse: 5′-GGCCCAAGCCAGAAAAATA-3′, exon 3: forward: 5′-GCGAACAGGTTGAATTTAATGA-3′, reverse: 5′-CACTATAATAATGCTGGTGATGGA-3′.Direct Sanger sequencing of the cDNA of C12orf65 was performed in patient 4 with the following primers: forward: 5′- GCAACCAACAAAACCAGCAA-3′ and reverse: 5′- CAGGACTGTTTTCACCATTGTAG-3′. […]

Pipeline specifications

Software tools VarScan, Dindel, ANNOVAR
Databases dbSNP
Application WES analysis
Organisms Homo sapiens
Diseases Nervous System Diseases, Nystagmus, Pathologic, Optic Atrophy, Peripheral Nervous System Diseases, Mitochondrial Diseases
Chemicals Oxygen