Reasoning
PS3 is met at Supporting strength. The variant-specific study of seven MYBPC3 c.2373dupG carriers used cardiac left-ventricular septal biopsies and demonstrated abnormal transcript processing, markedly reduced mutant mRNA, absence of detectable truncated cMyBP-C, approximately 33% reduction of full-length cMyBP-C, and reduced maximal cardiomyocyte force with increased calcium sensitivity. These findings are biologically relevant and directly support a damaging loss-of-function effect. The VCEP states that individual in vitro cardiomyopathy assays are unlikely to support PS3 above Supporting because no validated gold-standard assay reliably predicts the clinical phenotype; the evidence does not satisfy the VCEP's separate Strong splicing-assay requirements, including a clearly documented reproducible splice product comparable to wild type, control comparison, and Sanger confirmation.
VCEP rule
_**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**
While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.
As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level.
Evidence considered
- PMID:19273718, Methods/Results: cardiac left-ventricular septal biopsies from seven MYBPC3 c.2373dupG carriers with hypertrophic obstructive cardiomyopathy were compared with nonfailing donor myocardium using allele-specific RT-PCR, immunoblotting, protein analysis, and force-pCa measurements.
- PMID:19273718, variant-specific findings: c.2373dupG was reported to induce a cryptic donor splice site with skipping of 40 nucleotides; mutant mRNA was approximately 23% of total cMyBP-C mRNA, truncated cMyBP-C was not detected by immunoblotting, full-length cMyBP-C was reduced by approximately 33%, and carrier cardiomyocytes had reduced maximal active force and increased calcium sensitivity.
- cspec, PS3 Supporting rule: the VCEP limits individual in vitro cardiomyopathy studies to Supporting because there are no validated gold-standard assays that reliably predict the clinical phenotype.
Still missing
- The available extraction does not document all VCEP requirements for Strong in vitro splicing evidence, including control comparison and Sanger confirmation of the abnormal splice product.
Sources