Whole Exome Sequencing
Genomic DNA was extracted from peripheral blood samples according to standard methods. WES was performed using the Whole Exome Solution kit by Sophia Genetics, SA, following the manufacturer’s recommendations. The resulting libraries were subjected to paired-end sequencing on an Illumina NextSeq 500 platform. Library preparation and sequencing were outsourced (Genotypos-Science Labs, Athens). Raw data were converted to FastQ files for computational analysis which included read alignment to the human genome reference sequence (GRCh37/hg19), duplicate marking, base quality score recalibration, indel realignment and variant calling, using the SOPHiA DDM® platform (Sophia Genetics, SA). Variant annotation with ANNOVAR algorithm (K. Wang et al., 2010) and variant filtration process were performed using VarAFT v2.16 (http://varaft.eu) application (Desvignes et al., 2018).
Variant filtering involved a six-step phenotypic-driven strategy as presented in Figure 2 and summarized as follows: (1) Variant filtering by phenotype and/or in silico gene list; (2) Variant filtering by population frequency (Minor Allele Frequency, MAF ≤1%); (3) Variant filtering by variant type and/or ClinVar pathogenicity and/or in-silico pathogenic prediction tools; (4) Variant filtering by genotype; (5) Variant filtering by American College of Medical Genetics and Genomics (ACMG) classification; (6) Clinical/phenotypic (re)evaluation of retained variant(s).
The accepted WES data quality control parameters included a mean depth of coverage >50x, with >98% regions at 25x. Compared to the human genome reference hg19, ~30.000 variants were identified per exome including only exonic and flanking (+/- 10bp) regions. Subsequent to variant filtration using criteria outlined in Fig. 1, distinct variants were selected and set for further phenotype/genotype assessment and evaluation by a multidisciplinary team including the referring clinician