*2.3. Genetic Testing*

Genetic screening was carried out with DNA samples from the 38 LVNC recruited patients. All of them were NGS sequenced for a gene panel including *MYBPC3*, *MYH7*, *TNNI3*, *TNNT2*, *TPM1*, *TNNC*, *MYL1*, *MYL2*, *ACTC1*, *FLNC*, *MIB1*, *TAZ*, *LDB3*, *DTNA*, *HCN4*, *RYR2*, *LMNA*, *NKX2-5*, *MYH6*, *PRDM16*, *ACTN2*, *DMD*, *DNAJC19*, *FHL1*, *PLN*, and *TTN* genes by Ion Torrent semiconductor chip technology in a Ion GeneStudio S5 Sequencer (Thermo Fisher Scientific, Waltham, MA, USA), according to previously described protocols [45,46]. Overall coverage of the gene panel was >95% (Supplementary Table S1). Variant Caller v5 software was used to variant identification (Thermo Fisher Scientific, Waltham, MA, USA). Ion Reporter (Thermo Fisher Scientific, Waltham, MA, USA) and HD Genome One (DREAMgenics S.L., Oviedo, Spain) software were used for variant annotation, including population, functional, disease-related, and in silico predictive algorithms databases.

Data acquisition and analysis was performed in compliance with protocols evaluated by the Ethical Local Committee of the Hospital Universitario Central de Asturias (No. 2020.224). Written informed consent was obtained from all 38 participants, prior to genetic study.

Interpretation of all gene variants with an allele frequency <0.01 was based the American College of Medical Genetics and Genomics (ACMG-AMP) 2015 Standards and Guidelines [37,47,48]. All genetic variants identified in this cohort were reviewed by two biologists and two cardiologists trained in cardiogenetics. Results provided will be divided in 3 groups: (1) pathogenic (P) or likely pathogenic (LP) variants carriers; (2) negative genetic result (benign or likely benign variants); (3) carriers of variants of uncertain significance (VUS). If a P or LP variant was identified direct Sanger sequencing was performed for family screening.
