GB virus C

We developed a standardized clinical sequencing nomenclature (CSN) for DNA sequence variant annotation. The aims of CSN are a) to provide a fixed, standardized system in which every variant has a single notation, b) to be identical for all mutation detection methods, c) to use a logical terminology understandable to non-experts, and d) to provide a nomenclature that allows easy visual discrimination between the major classes of variant in clinical genomics. The CSN follows the principles of the HGVS nomenclature, with some minor amendments to ensure compatibility and integration with historical clinical data, whilst also allowing high-throughput automated output from NGS platforms. The CSN is fully detailed in Additional file 1.

The cDNA numbering system was compliant with the Human Genome Variation Society recommendations ver. 15.11 (http://varnomen.hgvs.org (accessed on 25 December 2022)). Amino acid numbering was based upon the start methionine, as codon +1. The reference sequence was NG_011403.2 for genomic positioning and NM_000132.4 for cDNA numbering. As reference databases for pathogenic variants, we used the Factor VIII Variant Database (f8-db.eahad.org (accessed on 25 December 2022)), Human Gene Mutation Database (www.hgmd.cf.ac.uk (accessed on 25 December 2022)), and CHAMP (https://www.cdc.gov/ncbddd/hemophilia/champs.html (accessed on 25 December 2022)).

Genetic testing was performed with whole-exome sequencing (WES) using the Illumina platform [28 (link)]. Genomic DNA was extracted from the whole blood of proband using standard methods. Enrichment was performed with Illumina DNA prep with Enrichment (San Diego, CA, USA) to capture all coding regions and exon-intron junctions (±50 bps) followed by Illumina NextSeq500 (San Diego, CA, USA). We obtained >99% of targeted regions covered more than 30 times and 99X mean depth. The raw data were then processed according to the Genome Analysis Toolkit (GATK 1.6) and were analysed using the software BaseSpace Variant Interpreter Annotation Engine 3.15.0.0 (Illumina). Variants were annotated according to the Human Genome Variation Society guidelines (HGVS), mapped to the human genome build GRCh37/UCSC hg19, and classified according to the criteria of the American College of Medical Genetics and Genomics [29 (link)]. Pathogenicity assessment for all rare genetic variants was performed according to ACMG2015 guidelines. To identify variants that were pathogenic, likely pathogenic or VUS, we looked up the variants’ minor allele frequency (MAF) in the Exome Aggregation Consortium (ExAC). We used 0.01 as an initial filtering criterion to limit the number of variants considered. In addition, further analysis was performed to identify variants associated with a given phenotype.
Exome analysis produced a large number of variants (12,154) of approximately 11,460 single nucleotide variations and approximately 666 indels. Variant annotation (i.e., exonic: intronic, and untranslated regions; exonic: synonymous, nonsynonymous, stop gain/loss, frameshift, allele frequency; and so on) and prioritization were performed with open-source software (Variant Interpreter, Illumina, San Diego, CA, USA).
Different approaches were used to minimize the number of potentially deleterious gene defects: (1) filtering based on a quality score of greater than 30; (2) excluding variants with a minor allele frequency of greater than 0.01 by comparison with the single nucleotide polymorphism database (http://www.ncbi.mln.nih.gov/snp (accessed on 18 November 2022)), Exome Aggregation Consortium (http://exac.broadinstitute.org/ (accessed on 18 November 2022)), Exome Variant Server (http://evs.gs.washington.edu/EVS (accessed on 18 November 2022), 1000 Genomes Projects (http://browser.1000genomes.org (accessed on 18 November 2022)), and published studies; (3) removing variants outside coding regions or synonymous coding variants; (4) selecting variants that segregate according to the presumed pattern of inheritance; and (5) querying disease databases, such as ClinVar (http://www.ncbi.nlm.nih.gov/clinvar (accessed on 18 November 2022)), OMIM, (http://www.omim.org (accessed on 18 November 2022)), and the Human Gene Mutation Database locus-specific database (http://www.hgvs.org/ (accessed on 18 November 2022)) to further prioritize the candidate gene variants. After the functional annotation step, which was undertaken based on effect on protein function and a priori knowledge of phenotype, approximately 116 variants were retained.
Virtual subpanels from these broad sequencing assays have been generated within the BaseSpace Variant Interpreter using a gene list associated to the disease (Supplemental data). After this step, the number of variants were reduced to only 8. Of them, 7 resulted benign or likely benign according to ClinVar annotation, and only the DLG1 p.R519H remained to be further investigated.