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.