Whole exome sequencing
Genomic DNA from the proband’s blood was isolated with an automatic system (MagnaPure, Roche). A library was prepared using Comprehensive Exome Panel technology (Twist Bioscience), sequenced on the NovaSeq 6000 SystemTM (Illumina) and designed with the Comprehensive Exome Panel technology (Twist Bioscience) to capture over 20,000 genes, achieving more than 99% coverage of genes included in the RefSeq, CCDS, and GENCODE databases, encompassing over 85% of gene-mediated disease-related alterations and flanking splicing regions (5-20 bp). The library size is 41.2 MB. These processes were carried out at Nimgenetics laboratory.
Data alignment and calling with the reference genome (GRCh38/hg38) in the target regions (Twist_Exome_RefSeq_targets_hg38.bed) was performed with DRAGENTM software version 07.021.572.3.6.3. Variant annotation was carried out using custom software developed using NIMGenetics database and free publicly available sources. The analysis focused on identifying variants in exonic regions or splicing regions (at least 5 bp), including missense or nonsense mutations, synonymous mutations, small insertions or deletions (indels) with an allele frequency (VAF) greater than 30% of the reads. Identified variants were cross-referenced with specific databases like ClinVar for known phenotype associations and population frequency databases (dbSNP, gnomAD, 1000 Genome Project, or NHLBI-ESP 6500 exomes) to annotate variants commonly found in the general population (at least 1%). Pathogenicity of variants was estimated using CADD and a combination of prediction systems from the dbNSFP database (SIFT, PolyPhen2, MutationTaster, MutationAssessor, LRT, FATHMM, and MetaSVM) for missense mutations.
The impact of mutations in splicing regions on mRNA processing was evaluated using SpliceSiteFinder and MaxEntScan prediction systems. Nucleotide position conservation was assessed using UCSC score ranges from the PhyloP tool. The association of identified variants with OMIM syndromes was evaluated. The nomenclature and classification of variants follow the guidelines of the Human Genome Variation Society and the American College of Medical Genetics and Genomics. Geneticists and clinicians jointly interpreted the data in a multidisciplinary assessment process before issuing the final report.