Whole exome sequencing:
DNA of the fetus and parents (Figure 1A IV:4) was subjected to whole exome sequencing (WES) according to the manufacturer’s protocols. DNA was fragmented using the Covaris M220 Focused-ultrasonicator and subsequently prepped with the TruSeq DNA Sample Preparation kit (Illumina). The SeqCap EZ Human Exome Library v3.0 kit was used for enrichment (NimbleGen, Roche) and finally, sequencing was carried out on a HiSeq 2000 (Illumina). Analysis of WES data was performed using VariantDB (Vandeweyer, Van Laer, Loeys, Van den Bulcke, & Kooy, 2014). Sanger sequencing was performed to confirm the presence of the variant in the fetus and subsequently to perform segregation analysis in the other family members. A PCR reaction using GOTaq polymerase (Promega) was followed by sequencing using the ABI BigDye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems) and the ABI 3130 Genetic Analyzer (Applied Biosystems).
DNA of members of family B (II:1, II:2 and III:1-4) was subjected to a custom-made panel comprising 100 cardiac/aortic conditions-related candidate genes (SOPHiA Genetics, Switzerland) according to the manufacturer’s protocols. The sequencing was carried out on a MiSeq (Illumina). Variant prioritization was performed by Sophia DDM software (Sophia Genetics, Switzerland). The evaluation was carried out by bioinformatics tools integrated in Varsome Clinical software (Saphetor SA, Switzerland). Presence of the detected variant was validated by Sanger DNA sequencing and family segregation was performed.