Computational protocol: Nucleotide substitutions in CD101, the human homolog of a diabetes susceptibility gene in non‐obese diabetic mouse, in patients with type 1 diabetes

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Protocol publication

[…] The present study was approved by the institutional review board committee at the National Center for Child Health and Development, and was carried out after obtaining written informed consent. We carried out molecular analyses of a Japanese family (family A) consisting of two patients with type 1 diabetes and three unaffected relatives. The male proband (case 1) and his mother (case 2) developed diabetes at the ages 2.6 and 18 years, respectively (Table ). At disease onset, case 1 was positive for the insulin autoantibody, whereas case 2 was positive for the islet cell surface antibody. Human leukocyte antigen (HLA) typing showed known risk alleles of the Japanese population, DRB1*04:05 and DQB1*04:01, in cases 1 and 2 and two unaffected family members, and DQB1*03:02 in case 1 and his unaffected father (Table ). The unaffected grandmother of case 1 (the mother of case 2) carried two risk alleles, DRB1*04:05 and DQB1*04:01, together with a protective DQB1*03:01 allele. Cases 1 and 2 showed no additional clinical features. No family history of other autoimmune diseases was recorded in this family.We carried out whole‐exome sequencing using genomic DNA samples obtained from cases 1 and 2 and three unaffected family members (the father and two older siblings of the proband). DNA libraries were constructed using a SureSelect Kit (51 Mb version 4; Agilent Technologies, Santa Clara, CA, USA), and sequenced using a Hiseq 1000 sequencer (Illumina, San Diego, CA, USA). Nucleotide alterations were called by Avadis NGS 1.3.1 (DNA Chip Research, Yokohama, Japan) or SAMtools 0.1.17 software (https://sourceforge.net/projects/samtools/files/samtools/). We searched for nucleotide alterations shared by cases 1 and 2, but absent from the three unaffected relatives. We focused on exonic mutations that alter protein sequences and intronic substitutions located within a 5‐bp region from an exon–intron boundary. Known polymorphisms with an allele frequency of more than 1.0% in the general population (NCBI Browser, http://www.ncbi.nlm.nih.gov/), and mutations whose functional outcomes were predicted as ‘benign’ by in silico analysis using PolyPhen‐2 (http://genetics.bwh.harvard.edu/pph2/) were excluded from further analysis. We referred to the OMIM database (https://www.ncbi.nlm.nih.gov/omim) to examine whether the genes identified in the present study were associated with any human disorders. We also searched the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) for previous reports on these genes. Nucleotide alterations in CD101 (NM_004258) and GAD2 (NM_000818.2) were confirmed by Sanger sequencing. Primer sequences are available on request. In addition, we analyzed the parental origin of a CD101 mutation identified in case 2. Copy‐number alterations in case 1 were analyzed by array‐based comparative genomic hybridization (SurePrint G3 Human Microarray, 2 × 400 k format; Agilent Technologies). We referred to the Database of Genomic Variants (http://projects.tcag.ca/variation/) to exclude known benign variants. […]

Pipeline specifications

Software tools Strand NGS, SAMtools, PolyPhen
Databases DGV OMIM
Application WES analysis
Organisms Homo sapiens, Mus musculus