Pathogenic/Likely Pathogenic (P/LP)

We further subset variants in this group into three categories: (1) Tier1A_TwoPlus: ClinVar TwoPlus variants were considered high confidence known pathogenic variants and automatically classified as P/LP. Novel single nucleotide variants that result in known amino acid codon change that has a ClinVar TwoPlus status were also categorized as P/LP. (2)Tier1A_Conflicting: Variants in ClinVar with conflicting interpretations but ≥4P/LP submissions were considered P/LP whereas those with 1-3P/LP submissions were manually curated according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines⁶⁴, taking into consideration allele frequency, in silico scores and reports in literature (Human Gene Mutation Database (HGMD)⁶⁵ and PubMed). Known variants that occurred in cis such as GAA c.752C > T;c.761 C > T were counted as one event. (3) Tier1B: LOF variants (frameshift insertions/deletions, nonsense, essential splice site variants) that were either absent in ClinVar or that were in ClinVar but did not meet our preceding criteria, were manually curated according to the ACMG/AMP PVS1 criterion using the high-throughput, automated application AutoPVS1 (v1.1)⁶⁶. Variants fulfilling the following criteria were considered P/LP: (a) LOF consequence in MANE transcript; for genes without MANE transcript, ClinVar and the National Center for Biotechnology Information (NCBI) were referenced to determine the LOF variant affected a clinically relevant transcript, and (b) AutoPVS1 indicated PVS1 strength of “Very Strong”, or (c) there are ≥2 ClinVar TwoPlus P/LP variants located downstream of the variant. Truncating variants in TTN were separately assessed using CardioClassifier⁶⁷ (v.0.2.0) for P/LP classification.