Cryptic Splice-Altering Variants in MYBPC3 Are a Prevalent Cause of Hypertrophic Cardiomyopathy

Pathogenic Intronic Splice-Affecting Variants in MYBPC3 in Three Patients with Hypertrophic Cardiomyopathy

“…In general, our laboratory policy is to consider variants with SpliceAI scores >0.2 as potentially splice-altering, while scores >0.5 are considered as very likely to be splice-altering, which is similar to previously described SpliceAI cutoffs used for variant prioritisation [39,45]. However, some reports investigating MYBPC3 intronic splice variants have used very stringent SpliceAI cut-offs of ≥0.9 to initially prioritise variants [27]. Nonetheless, when these studies expanded their inclusion criteria to include variants with lower SpliceAI scores, an MYBPC3 c.1898-23A>G variant with a SpliceAI score of just 0.04 was identified; assessment of patient RNA using RT-PCR showed the inclusion of intron 19, leading to a premature stop codon and presumed NMD [27].…”

“…However, some reports investigating MYBPC3 intronic splice variants have used very stringent SpliceAI cut-offs of ≥0.9 to initially prioritise variants [27]. Nonetheless, when these studies expanded their inclusion criteria to include variants with lower SpliceAI scores, an MYBPC3 c.1898-23A>G variant with a SpliceAI score of just 0.04 was identified; assessment of patient RNA using RT-PCR showed the inclusion of intron 19, leading to a premature stop codon and presumed NMD [27]. In our study, we further demonstrate that intronic variants beneath commonly applied SpliceAI filters do have a significant impact on splicing, and these data re-emphasise that functional investigation alongside in silico assessments remains an important consideration for diagnostic testing in the context of splicing.…”

“…While genetic testing is now common in patients with HCM, the diagnostic yield is only 40-50%, and the causative genotype is unknown for at least 40% of patients, even for those with family histories of the disease [27,28]. There are many factors which may explain this low genetic testing yield.…”

“…There are many factors which may explain this low genetic testing yield. For example, phenocopying occurs in other syndromic conditions such as Noonan syndrome and storage disorders, including Anderson-Fabry disease, while in other cases complex and as-yet unknown genetic mechanisms may cause HCM [27,[29][30][31][32]. However, conventional genetic testing methods may also fail to identify pathogenic non-coding variants, in particular cryptic splice variants (i.e., intronic variants out-with the canonical splice acceptor and splice donor dinucleotides which alter pre-mRNA splicing) in known HCM genes, which may be a major contributor to the missing heritability in HCM.…”

“…However, conventional genetic testing methods may also fail to identify pathogenic non-coding variants, in particular cryptic splice variants (i.e., intronic variants out-with the canonical splice acceptor and splice donor dinucleotides which alter pre-mRNA splicing) in known HCM genes, which may be a major contributor to the missing heritability in HCM. For example, several recent publications have identified novel intronic cryptic splice variants within MYBPC3 in cohorts of HCM patients, which would not have been detected as pathogenic variants by standard genetic testing [27,[33][34][35][36][37]. These intronic variants were shown to alter MYBPC3 splicing and result in allelic loss-of-function and haploinsufficiency.…”