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.