Ethical approval and consent
Approval of this study was obtained from the ethics review board at Children’s Medical Center (CMC) hospital and Tarbiat Modares Univeristy (TMU) of Tehran, Iran. The understudy family sought medical counselling at Myelin Disorders Clinic, CMC hospital and then referred to Medical genetics department of TMU for genetic investigation. Proper informed consent was obtained and parents were ensured to be informed of the result of this study.
Exome sequencing and data analysis
DNA was extracted from the index patient and her healthy family members using Roche extraction kit (Product No. 11814770001). After quality assessment of extracted DNA, all coding region of the index patient was baited against exonic regions and flanking exon-intron boundary regions of the genome utilizing Agilent’s Sure Select Human All Exon V6 kit (Agilent, SantaClara, CA, USA). Paired-end sequencing was performed on Illumina’s HiSeq4000 instrument in accordance to the manufacturer’s protocols (Illumina Inc, USA). Obtained raw reads from sequencing machine were initially aligned onto human genome reference (NCBI build37/hg19 version) using BWA 0.7.17 after removing low quality reads. Afterward, reads were mark duplicated by implementing PICARD software 2.2 and variants (SNPs and Indels) were called utilizing Genome Analysis Toolkit 4.1. Annotations were incorporated to the called variants using ANNOVAR (17).
Prioritization of variants was applied based on a custom scheme. Given the consanguinity of parents, heterozygous variants were ruled out. Exclusion of intragenic, UTRs regions, intronic and synonymous variants was done and followed by opting variants with less than 1% minor allele frequency using 1000 Genomes Project, dbSNPv152, Exome Sequencing Project (ESP), Exome Aggregation Consortium (ExAC) database and Exome Variant Server. Then, further evaluations on the influence of the variants on protein function according to pathogenicity evaluator tools were performed (Mutation Taster, SIFT, Provean, Polyphen-2, MutPred2, and M-CAP). Genotype-phenotype correlation of variants and their relation to patient’s phenotype was checked in ClinVar and HGMD. Website addresses for online tools or software for pathogenicity prediction, and protein modeling are provided at the end of this article.
Mitochondrial Genome Sequencing
Targeted amplification of the entire mitochondrial genome was done using two overlapping long range PCRs. The amplicons were run on the Bioanalyzer to assess for any putative deletion within the mitochondrial genome. The amplified products were subsequently segmented and Illumina compatible adapters ligated to generate libraries that were sequenced on an Illumina HiSeq4000 HiSeq4000 platform to an average sequencing depth of 1000x.
Raw sequence data analysis, including base calling, demultiplexing, alignment to the Revised Cambridge Reference Sequence (rCRS) of the Human Mitochondrial DNA (NC_012920) and variant calling were performed using a validated in-house software. Following the base calling and primary filtering of low quality reads, standard Bioinformatics pipeline was implemented to annotate detected variants and to filter out probable artefacts. The pipeline confidently detects heteroplasmy levels down to 15%. All identified variants were evaluated with respect to their pathogenicity and causality.
Sanger sequencing was performed for verification and family segregation on the ﬁnal candidate variants. We also evaluated the presence of these variants in a cohort of ethnicity-matched controls. The bidirectional primers listed in Supplementary Table 1 were utilized for PCR ampliﬁcation of the variant region. Cycling conditions were including the initial denaturation (94°C, 3min) followed by 40 cycles of 94°C for 30s, 64°C for 30s, 72°C for 30s, and a ﬁnal extension at 72°C for 10min. Consequently, PCR products were visualized by electrophoresis using 1.5% agarose gel and PCR products were sequenced utilizing the ABI 3130xl Genetic Analyzer. Sequencing chromatograms were analyzed using the SnapGene v.5.0.5 software.
Protein structure, stability and conservation
The study of the affected residue was conducted by employing the available human CYC1 structure (PDB ID: 5XTE), as reported by Guo et al (2017). The PDB file was investigated for possible polar contact changes upon mutation by PyMOL software (The PyMOL Molecular Graphics System, Version 2.3.2, Schrödinger, LLC). The effect of the identified variant on CYC1 Protein stability was estimated using MUPro (Cheng, Randall, & Baldi, 2005). ConSurf and UCSC databases were recruited to provide an evolutionary conservation profile for cyt c1 protein.
1000 Genome Database (http://www.1000genomes.org/)
Pymol software (https://pymol.org/2/)
ExAC Browser (http://exac.broadinstitute.org)