Case#: Patient 3; male; 60 years old at first observation; 14 mm LV wall thickness; family history of HCM

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM) associated with the heterozygous MYBPC3 intronic splice-altering variant c.1224-80G>A, classified as likely pathogenic by the study authors.

FamilyInfo: Patient was reported to have positive family history of HCM. Echocardiography demonstrated a maximum left ventricular wall thickness of 14 mm without left ventricular outflow tract obstruction. The study did not identify specific affected relatives or provide a family pedigree.

ParentalTesting: NR – Patient had a positive family history of HCM but parental genotypes and variant segregation were not documented.

CasePresentingHPOs: HP:0001639 (Hypertrophic cardiomyopathy); HP:0001712 (Left ventricular hypertrophy)

CaseHPOFreeText: Echocardiography demonstrated a maximum left ventricular wall thickness of 14 mm without left ventricular outflow tract (LVOT) obstruction. NYHA functional classification was not reported.

CaseNotHPOs: HP:0032092 (Left ventricular outflow tract obstruction)

CaseNotHPOFreeText: Echocardiographic evaluation showed no left ventricular outflow tract (LVOT) obstruction.

CasePreviousTesting: Patient 3 previously underwent targeted next-generation sequencing using the Illumina TruSight Cardio panel, which included 174 genes associated with inherited cardiac conditions. The initial genetic analysis was negative. Subsequent reanalysis of existing sequencing data identified the MYBPC3 intronic variant c.1224-80G>A.

Genotyping Method: Genomic DNA obtained from peripheral blood was analyzed using the Illumina TruSight Cardio targeted next-generation sequencing panel on an Illumina MiniSeq platform. Previously generated sequencing data were reexamined using an intronic hotspot analysis approach, with variant visualization in Integrative Genomics Viewer (IGV). The MYBPC3 c.1224-80G>A variant was subsequently confirmed by Sanger sequencing.

FunctionalAnalysis: The MYBPC3 intronic variant c.1224-80G>A creates a cryptic splice acceptor site within intron 13, resulting in retention of 78 intronic nucleotides upstream of exon 14. This produces an in-frame insertion of 26 codons. Previous functional studies demonstrated altered RNA splicing and an approximately 50% reduction in MYBPC3 transcript abundance. SpliceAI predicted a strong splice-altering effect (Δscore = 0.94). The current study did not perform patient-specific RNA functional experiments. Instead, researchers identified the variant through reanalysis of existing next-generation sequencing data and confirmed its presence using Sanger sequencing.

Variant: MYBPC3 NM_000256.3.1224-80G>A.

ClinVar: 693982

CAID: CA221697326

gnomAD: The MYBPC3 variant c.1224-80G>A (GRCh38: 11-47343342-C-T) is present at a low frequency in gnomAD v4; Exome allele frequency: 0.00001637; Genome allele frequency: 0.00005261; Total allele frequency: 0.00002005; The highest ancestry-specific allele frequency reported was in the African/African American population, with an allele count (AC) of 8 and allele frequency (AF) of 0.0001113.

AdditionalInfo: Although Patient 3 carried the same variant as Patient 2, their reported cardiac phenotypes differed. Patient 2 exhibited a maximum left ventricular wall thickness of 27 mm and LVOT obstruction with a gradient of 50 mmHg. These differences are consistent with variable clinical expression of MYBPC3-associated HCM, although the study did not determine their underlying causes.

Case#: Patient 1; female; 56 years old at first observation with diagnosis of HCM; 13 mm LV wall thickness

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM) associated with the heterozygous MYBPC3 intronic splice-altering variant c.1224-80G>A, classified as likely pathogenic by the study authors.

FamilyInfo: The study reports a negative family history of HCM. No family pedigree or detailed clinical information about relatives was provided.

ParentalTesting: NR

CasePresentingHPOs: HP:0001639 (Hypertrophic cardiomyopathy); HP:0001712 (Left ventricular hypertrophy); HP:0031656 (Systolic anterior motion of the mitral valve)

CaseHPOFreeText: Hypertrophic cardiomyopathy and a maximum left ventricular wall thickness of 13 mm. Echocardiography demonstrated systolic anterior motion (SAM) of the mitral valve without left ventricular outflow tract (LVOT) obstruction. The patient had no reported family history of hypertrophic cardiomyopathy. NYHA functional classification was NR.

CaseNotHPOs: HP:0032092 (Left ventricular outflow tract obstruction)

CaseNotHPOFreeText: No left ventricular outflow tract (LVOT) obstruction was reported despite the presence of systolic anterior motion of the mitral valve.

CasePreviousTesting: Patient 1 previously underwent targeted next-generation sequencing using the Illumina TruSight Cardio panel covering 174 genes associated with inherited cardiac conditions. Initial genetic analysis was negative. Subsequent reanalysis of existing sequencing data identified the MYBPC3 intronic variant c.1224-80G>A.

Genotyping Method: Genomic DNA from peripheral blood was analyzed using the Illumina TruSight Cardio targeted next-generation sequencing panel on an Illumina MiniSeq platform. Existing sequencing data were reexamined using an intronic hotspot analysis approach and Integrative Genomics Viewer (IGV). The MYBPC3 c.1224-80G>A variant was subsequently confirmed by Sanger sequencing.

FunctionalAnalysis: The MYBPC3 intronic variant c.1224-80G>A creates a cryptic splice acceptor site within intron 13, resulting in the retention of 78 intronic nucleotides upstream of exon 14. This produces an in-frame insertion of 26 codons in the resulting transcript. Previous functional studies demonstrated abnormal RNA splicing and approximately 50% reduction in MYBPC3 transcript abundance. SpliceAI predicted a strong splice-altering effect (Δscore = 0.94). The current study did not perform patient-specific RNA functional experiments. Instead, the researchers identified the variant through reanalysis of existing next-generation sequencing data and confirmed its presence using Sanger sequencing.

Variant: MYBPC3 NM_000256.3.1224-80G>A.

ClinVar: 693982

CAID: CA221697326

gnomAD: The MYBPC3 variant c.1224-80G>A (GRCh38: 11-47343342-C-T) is present at a low frequency in gnomAD v4; Exome allele frequency: 0.00001637; Genome allele frequency: 0.00005261; Total allele frequency: 0.00002005; The highest ancestry-specific allele frequency reported was in the African/African American population, with an allele count (AC) of 8 and allele frequency (AF) of 0.0001113.

AdditionalInfo: Unlike Patients 2 and 3, Patient 1 had no reported family history of HCM. Echocardiography demonstrated systolic anterior motion of the mitral valve without left ventricular outflow tract obstruction, and maximum left ventricular wall thickness was 13 mm. Although all three patients carried the same splice-altering variant, their reported cardiac phenotypes differed. This observation is consistent with variable clinical expression of MYBPC3-associated HCM, although the study did not establish the underlying causes of these differences.