Computational protocol: Clinical and biochemical characterization of four patients with mutations in ECHS1

Similar protocols

Protocol publication

[…] Mutations were identified by molecular studies performed at each of the centers. For patients 1 and 2, the genomic DNA was sheared, size selected (~400–600 bp), ligated to sequencing adapters, and PCR amplified following standard library preparation. The library was subsequently enriched for exonic sequences using the SureSelect Human All Exon 51 Mb Kit (Agilent Technologies, Santa Clara, California, USA). The exome enriched samples were sequenced to 100 bp paired-end on a HiSeq2000 (Illumina, San Diego, California, USA). The reads were aligned to the human genome assembly GRCh37 using GSNAP (Genomic Short-read Nucleotide Alignment Program, version 2012-07-20) and variants identified by GATK (Genome Analysis Toolkit, v2.1-8-g5efb575). Only non-synonymous coding variants, coding indels and variants affecting splice sites were retained for further analysis. Common variants present in dbSNP and 1000 Genomes data were filtered out. Parental exome sequencing data was used to analyze variants following various inheritance models including dominant (de novo mutations) and recessive (compound heterozygous, homozygous, and X-linked hemizygous mutations) models. Only mutations in a single gene, ECHS1, were identified using an autosomal recessive inheritance model.For patient 3, exonic sequences were enriched in the DNA sample of the patient using SureSelect Human All Exon 50 Mb Kit (AgilentTechnologies, Santa Clara, California, USA). Sequences were determined by HiSeq2000 (Illumina, San Diego, California, USA) and 100-bp were read paired-end. Reads alignment and variant calling were performed with DNAnexus software (Palo Alto, California, USA) using the default parameters with the human genome assembly hg19 (GRCh37) as a reference. Aligning the patient’s 50.80 million reads to the reference human genome revealed 141,096 variants. After removal of variants of low depth (<×8), deep intronic, and those present in dbSNP132 or in the in-house dbSNP, variants were sorted for autosomal recessive inheritance mode and for predicted pathogenicity by Mutation Taster software. Only a single set of heterozygous mutations was identified, and these were localized in the ECHS1 gene.In patient 4, genomic DNA was extracted from cultured skin fibroblasts using standard methods. The exons and flanking intronic sequences of the ECHS1 gene were sequenced after amplification by PCR from genomic DNA using intronic primers with −21 M13 (5′-TGTAAAACGACGGCCAGT-3′) or M13-Rev (5′-CAGGAAACAGCTATGACC-3′) extensions. PCR fragments were sequenced with −21 M13 and M13-Rev sequence primers using BigDye Terminator cycle sequencing kits (Applied Biosystems, Foster City, CA, USA). Mutations in patients 1–3 were verified by Sanger sequencing. Sequence data were compared to the reference ECHS1 sequence [GenBank:NM_004092] with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. […]

Pipeline specifications

Software tools GSNAP, GATK, DNAnexus
Applications Miscellaneous, WES analysis
Organisms Homo sapiens
Diseases Acidosis, Acidosis, Lactic, Basal Ganglia Diseases, Brain Diseases, Pyruvate Dehydrogenase Complex Deficiency Disease, Heredodegenerative Disorders, Nervous System, Leukoencephalopathies
Chemicals Amino Acids, Pyruvic Acid