Case#: 22-year-old man

DiseaseAssertion: NCCM and mildly reduced LV function

FamilyInfo: Mother and brothers have also been diagnosed with NCCM

ParentalTesting: Mother had cardio magnetic resonance imaging done

CasePresentingHPOs: not reported

CaseHPOFreeText:

CaseNotHPOs: HP:0001681, HP:0001279 Angina pectoris andSyncope

CaseNotHPOFreeText: no arrhythmic events

CasePreviousTesting: electrocardiogram, transthoracic echocardiography, cardiac-magnetic resonance imaging

Genotyping Method: next generation sequencing

FunctionalAnalysis: not reported

Variant: NM_001018005.1:c.20_22del; p.Lys7del

ClinVar:181682

CAID:CA018708

gnomAD:15-63042841-CAAG-C

AdditionalInfo:

Case#: male; age 44

DiseaseAssertion: Hypertrophic, restrictive, and dilated cardiomyopathy

FamilyInfo: The patient’s unaffected 6-year-old daughter was tested for the variant. His brother and parents were healthy

ParentalTesting: Parents refused genetic testing

CasePresentingHPOs: HP:0001639 HP:0001723 HP:0001644

CaseHPOFreeText: Hypertrophic cardiomyopathy; restrictive left-ventricular filling pattern; diastolic dysfunction

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: 25-gene Ion AmpliSeq panel with Ion PGM semiconductor sequencing; Sanger sequencing

FunctionalAnalysis: NR

Variant: TPM1 c.292G>A (p.Glu98Lys; p.E98K; rs730881131); missense variant; heterozygous

ClinVar: 1472862

CAID: CA392720843

gnomAD: NR

Case#: 29 year old female; diagnosed with DCM 8 years before death; died suddenly at home on 22 February 2017; forensic autopsy case

DiseaseAssertion: Dilated cardiomyopathy (DCM) with sudden cardiac death; MYBPC3-related

FamilyInfo: Family from Central-South China with a history of SCD. Father (I:1) reportedly died of a heart attack at 37, not genetically tested. Mother (I:2) and sister (II:2) are unaffected and do not carry the variant. No other affected relatives reported.

ParentalTesting: Not performed, both parents were not genotyped for the variant; the father was deceased before the study. Maternal non-carriage confirmed by Sanger, so the variant is either paternal or de novo; phase not established.

CasePresentingHPOs: HP:0001644; HP:0001645

CaseHPOFreeText: Diagnosed with DCM 8 years before death, with no other abnormalities. Found unresponsive at home at noon and expired; no prodromal symptoms documented. Autopsy: enlarged heart with valve thickening, myocardial fibrosis, and multi-organ congestion; no other fatal injury or disease. HE staining of left ventricle showed no hypertrophy or other anomalies. Toxicological screening for ethanol, benzodiazepines, barbiturates, amphetamine, methamphetamine, ketamine, morphine, MDMA, and THC-acid was negative; serum IgE normal (27 IU/ml); eosinophils 4.7% with no mast-cell degranulation.

CaseNotHPOs: HP:0001639; HP:0001634; HP:0003124

CaseNotHPOFreeText: No cardiomyocyte hypertrophy on histology, distinguishing this from HCM. No coronary, valvular, or congenital structural disease identified. No toxicological cause of death.

CasePreviousTesting: Whole-exome sequencing on postmortem blood, retaining synonymous variants; filtering against gnomAD and CMDB; in silico prediction with MutationTaster, PolyPhen-2, and SIFT; NetGene2-2.42 used to predict splice effects. Five variants in cardiomyopathy-related genes were found; only the MYBPC3 variant was classified likely pathogenic.

Genotyping Method: WES with Sanger confirmation on ABI 3100; family members genotyped by Sanger.

FunctionalAnalysis: Minigene assay pcDNA3.1-MYBPC3 construct spanning exon 1-intron 1-exon 2, transfected into HEK293 cells: wild-type and the c.10G>A negative control both produced a ~1,500 bp full-length band and a ~300 bp spliceosome band; the c.24A>C construct produced no spliceosome band, indicating abnormal splicing with intron 1 retention and frameshift. Immunohistochemistry showed decreased MYBPC3 protein in the proband's left ventricle versus a control heart.

Variant: MYBPC3 NM_000256.3:c.24A>C (p.Pro8Pro); heterozygous synonymous variant at the penultimate base of exon 1; disrupts the splice donor site; novel

ClinVar: 164098

CAID: CA11138

gnomAD: 0.00001541

AdditionalInfo: Classified likely pathogenic with PS3, PM2, PP1, PP3; note the paper's Discussion relabels the prediction code as PP4, an internal inconsistency. The authors describe it as the first reported deleterious synonymous MYBPC3 variant, acting by intron retention and reduced protein. The same splice site is affected by the known HCM variants c.24+1G>A and c.24+3A>C. Four other variants were found and classified likely benign or VUS: LDB3 c.2131T>C (VUS), BBS2 c.422A>G, EVC2 c.2643G>C, and BBS4 c.1548_1549del.

Case1: Caucasian. Sex and exact age not specified for the proband

DiseaseAssertion: Dilated cardiomyopathy (DCM)

FamilyInfo: Family A was notable for severe HF and sudden death in young children. Individuals I-2, III-10 and IV-7 died suddenly at ages 3 years, 13 months, and 5 months respectively, without prior evidence of heart disease. Two siblings, IV-5 and IV-8, presented with DCM and advanced HF at 5 months of age (LVEF 27% and 29%). Individuals III-3 and IV-1 developed end-stage HF refractory to medical therapy as teenagers. III-3 presented at age 13 with refractory HF, underwent mitral and tricuspid valve replacement for functional regurgitation and died weeks later of multisystem organ failure. IV-1 was asymptomatic when diagnosed with DCM at 7 years during family screening, but developed severe HF at age 17 and underwent transplant at 18 years of age. Individuals III-4, III-7 and III-12 developed mild exercise intolerance in their 5th decade.

ParentalTesting: NR

CasePresentingHPOs: HP:0001635, HP:0001644

CaseHPOFreeText: No family members with TPM1 D230N had evidence of cardiac conduction disease, and with one exception, none had evidence of skeletal myopathy

CaseNotHPOs: NR

CaseNotHPOFreeText: No family members with TPM1 D230N had evidence of cardiac conduction disease, and with one exception, none had evidence of skeletal myopathy.

CasePreviousTesting: No prior testing.

Genotyping Method: Genomic DNA was isolated from blood using standard methods in probands with DCM. Direct DNA sequence analysis of all coding regions and intron/exon boundaries was performed in 6 sarcomere genes.

FunctionalAnalysis: Functional analysis performed on recombinant proteins but not patient tissue. In vitro studies demonstrated major inhibitory effects on sarcomere function with reduced Ca2+-sensitivity, maximum activation, and Ca2+ affinity compared to wildtype TPM1.

Variant: NM_000366.5:c.688G>A (p.Asp230Asn)

ClinVar: NR

CAID: UnregisteredVariant

gnomAD: NR

AdditionalInfo: The paper does not explicitly identify which pedigree member is the Family A proband

Case2: Caucasian. Sex and exact age not specified for the proband

DiseaseAssertion: Dilated cardiomyopathy (DCM)

FamilyInfo: Young members of this family also presented with severe HF. Individual III-1 developed failure to thrive at 10 weeks of age and echocardiography revealed severe LV dilation (LVIDD Z-score 15.6) and systolic dysfunction (LVEF 15%). Transplantation was considered, but she had substantial recovery with basic medical therapy. By age 8 years, she was asymptomatic with only mild LV systolic dysfunction (LVIDD Z-score 1.3, LVEF 46%). At age 20, II-6 underwent cardiac transplantation shortly after presenting with refractory HF symptoms and severe LV systolic dysfunction (LVEF < 20%). Individual II-7 died of HF at 12 years, prior to genetic testing. In addition to DCM, he had congenital cataracts and skeletal muscle weakness. I-1 had unexplained syncope at age 56 years. Cardiac studies revealed ventricular tachycardia and DCM (LVEF 25%) which improved with medical management. Three other adults (II-2, II-3, II-4, ages 38, 36 and 33 years, respectively) had asymptomatic LV systolic dysfunction.

ParentalTesting: NR

CasePresentingHPOs: HP:0001635, HP:0001644

CaseHPOFreeText: NR

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: No prior testing.

Genotyping Method: Genomic DNA was isolated from blood using standard methods in probands with DCM. Direct DNA sequence analysis of all coding regions and intron/exon boundaries was performed in 6 sarcomere genes.

FunctionalAnalysis: Functional analysis performed on recombinant proteins but not patient tissue. In vitro studies demonstrated major inhibitory effects on sarcomere function with reduced Ca2+-sensitivity, maximum activation, and Ca2+ affinity compared to wildtype TPM1.

Variant: NM_000366.5:c.688G>A (p.Asp230Asn)

ClinVar: NR

CAID: UnregisteredVariant

gnomAD: NR

AdditionalInfo: The paper does not explicitly identify which pedigree member is the Family B proband

Case#: Patient 1; 89-year-old female; identical twin (Patient 2) with mild HCM

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM) associated with the pathogenic MYBPC3 nonsense variant NM_000256.3.787G>T (p.Gly263Ter). Patient 1 is an 89-year-old female with severe symptomatic obstructive HCM, previously requiring septal ablation. Her maximum reported left ventricular wall thickness was 29 mm by echocardiography in 2015.

FamilyInfo: Patient 1 belongs to a family with hereditary hypertrophic cardiomyopathy associated with the MYBPC3 p.Gly263Ter founder variant. Her monozygotic twin sister (Patient 2) carries the same pathogenic variant but has a substantially milder HCM phenotype, with a maximum left ventricular wall thickness of 13 mm and no reported symptoms at an advanced age. The twin pair belongs to the extended family that also includes Patients 3 and 4, another pair of monozygotic twins carrying the same variant. The pedigree documents additional affected relatives and a history of sudden death, although not all family members have confirmed genetic testing.

ParentalTesting: NR – The family pedigree supports familial occurrence of hypertrophic cardiomyopathy, but the specific parent from whom Patient 1 inherited the MYBPC3 p.Gly263Ter variant cannot be conclusively determined from the available genetic testing data. Her monozygotic twin sister (Patient 2) was confirmed to carry the same variant.

CasePresentingHPOs: HP:0001639 (Hypertrophic cardiomyopathy); HP:0002094 (Dyspnea); HP:0001712 (Left ventricular hypertrophy); HP:0032092 (Left ventricular outflow tract obstruction)

CaseHPOFreeText: Echocardiography documented a maximum left ventricular wall thickness of 29 mm in 2015. She previously experienced significant dyspnea, with symptoms classified as NYHA III/IV, associated with severe left ventricular outflow tract obstruction. She underwent septal ablation, which improved her clinical symptoms. At the time of the study, she remained minimally symptomatic.

CaseNotHPOs: HP:0000822 (Hypertension)

CaseNotHPOFreeText: Explicitly reported that Patient 1 did not have hypertension. No additional negative phenotypic findings were specifically documented for this patient.

CasePreviousTesting: Previous cardiac evaluations included echocardiography demonstrating severe left ventricular hypertrophy, with a maximum wall thickness of 29 mm in 2015, and clinical identification of severe left ventricular outflow tract obstruction. The patient underwent septal ablation for symptomatic obstructive hypertrophic cardiomyopathy. Electrocardiographic findings were also evaluated following the procedure. The article does not provide a detailed history of genetic testing performed before the reported familial MYBPC3 investigation.

Genotyping Method: Genetic testing of index cases was performed using targeted next-generation sequencing (NGS) with Ion Torrent technology. The earlier index cases were evaluated using a nine-gene sarcomeric panel, including MYBPC3. Familial screening for the MYBPC3 NM_000256.3.787G>T (p.Gly263Ter) variant was performed using Sanger sequencing on an ABI3130XL sequencer. Monozygosity of the twin pairs was independently confirmed using the PowerPlex 16 HS System, which analyzes 16 microsatellite markers. Article does not specify whether Patient 1's individual MYBPC3 genotype was initially established through NGS or other targeted Sanger sequencing.

FunctionalAnalysis: The MYBPC3 NM_000256.3.787G>T (p.Gly263Ter) variant is a nonsense mutation that introduces a premature termination codon. The authors classified this founder variant as pathogenic based on its predicted loss-of-function effect, absence from the population reference databases examined, and segregation among affected family members. Computational evidence supported a deleterious effect. However, no patient-specific functional assays measuring MYBPC3 RNA expression, protein abundance, or protein function were reported in this study. The molecular consequences were inferred from the variant type and existing evidence rather than directly demonstrated experimentally in Patient 1.

Variant: MYBPC3 NM_000256.3:c.787G>T p.Gly263Ter; Nonsense; Autosomal dominant; Clinical significance: Pathogenic (1 submission). Review status: 1 out of 4 stars (criteria provided, single submitter).

ClinVar: 1460339

CAID: CA380333920

gnomAD: GRCh38 variant 11-47347891-C-A. African/African American population: AC = 0; AF = 0.0000.

AdditionalInfo: Patient’s monozygotic twin sister (Patient 2) carries the same variant but exhibits a substantially milder clinical phenotype. Patient 1 had a maximum left ventricular wall thickness of 29 mm in 2015, severe left ventricular outflow tract obstruction, and symptomatic dyspnea requiring septal ablation. In contrast, Patient 2 had a maximum wall thickness of 13 mm and was asymptomatic. This illustrates variable expressivity of MYBPC3-associated hypertrophic cardiomyopathy, even among genetically identical individuals. The authors discuss environmental and epigenetic factors as possible contributors to phenotypic variability, although no specific modifying mechanism was experimentally demonstrated in these patients.

Case#: Patient 5; 47-year-old male; identical twin (Patient 6) with less severe hypertrophy

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM)

FamilyInfo: Patient has a monozygotic twin brother (Patient 6), who is also affected by HCM. Both individuals carry the same pathogenic MYBPC3 nonsense variant, NM_000256.3.787G>T (p.Gly263Ter). However, Patient 5 demonstrates more pronounced left ventricular hypertrophy than Patient 6. Cardiac magnetic resonance imaging (CMR) revealed maximum left ventricular wall thicknesses of 28 mm and 19 mm, respectively.

ParentalTesting: NR – Parental genetic testing results were not explicitly reported for Patient 5 or his monozygotic twin brother (Patient 6)

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

CaseHPOFreeText: Patient diagnosed with hypertrophic cardiomyopathy (HCM), characterized by substantial left ventricular hypertrophy. Echocardiography performed in 2022 demonstrated a maximum left ventricular wall thickness of 18 mm, while cardiac magnetic resonance imaging (CMR) showed a maximum thickness of 28 mm.

CaseNotHPOs: HP:0032092 (Left ventricular outflow tract obstruction); HP:0000822 (Hypertension)

CaseNotHPOFreeText: Patient 5 did not exhibit left ventricular outflow tract obstruction (LVOTO), even though having substantial left ventricular hypertrophy (max wall thickness of 28 mm). The study also explicitly reports that none of the six patients, including Patient 5, had hypertension.

CasePreviousTesting: Patient 5 underwent clinical cardiac evaluation, including echocardiography, cardiac magnetic resonance imaging (CMR), and electrocardiography (ECG).

Genotyping Method: Genetic analysis was performed using next-generation sequencing (NGS) with Ion Torrent technology for index patients and targeted Sanger sequencing for familial screening. The study reports that recent index cases associated with Patients 5–6 were evaluated using an expanded 195-gene NGS panel, as described in Supplementary Table S2. The pathogenic MYBPC3 variant NM_000256.3.787G>T (p.Gly263Ter) was identified in both monozygotic twins. However, the article does not explicitly specify whether Patient 5 underwent the full NGS panel or targeted familial testing. Monozygosity was independently confirmed using the PowerPlex 16 HS System.

FunctionalAnalysis: NR – No patient-specific functional assays were reported for Patient 5 (computational evidence supporting a deleterious effect, absence from the ESP, 1000 Genomes, and ExAC population databases, and segregation of the variant among affected family members).

Variant: MYBPC3 NM_000256.3:c.787G>T p.Gly263Ter; Premature termination codon; Nonsense; Autosomal dominant; Clinical significance: Pathogenic (1 submission). Review status: 1 out of 4 stars (criteria provided, single submitter).

ClinVar: 1460339

CAID: CA380333920

gnomAD: gnomAD: GRCh38 variant 11-47347891-C-A. African/African American population: AC = 0; AF = 0.0000.

AdditionalInfo: Patient’s monozygotic twin brother (Patient 6) carries the same pathogenic variant but exhibits less pronounced left ventricular hypertrophy. The authors suggest that additional environmental or epigenetic modifiers may influence disease severity, although no specific modifying mechanism was experimentally established.

Case1: 63-year-old male

DiseaseAssertion: Atrial flutter(54), VT & ICD(57); CVA(58) & DCM(58); HF(59)

FamilyInfo: There are 14 affected family members (Fig 1): six living (three sisters III-1, III-4, III-6 and three nieces IV-5, IV-15, IV-16) and eight deceased individuals (I-3, II-8, II-13, III-8, III-10, IV-1, IV-4, IV-6).

ParentalTesting:

CasePresentingHPOs:

CaseHPOFreeText: atrial flutter during evaluation for non-cardiac surgery. Subsequent evaluations led to the diagnosis of SSS. At 54 years old, echocardiogram showed mild systolic dysfunction and left ventricle dilation (EF 45%, LVEDD 58 mm). Myocardial perfusion study showed exercise intolerance but no signs of ischemia. At presentation, he felt well noting mild exercise intolerance for the past year with no palpitations or chest pain; he took no medications. At age 49, he had normal systolic function and LV dilation by echocardiogram (EF 53%, LVEDD 61 mm) and a normal myocardial perfusion study.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: Sanger sequencing

FunctionalAnalysis: NR

Variant: NM_170707.4(LMNA):c.357-2A>G

ClinVar: 652066

CAID: CA342814936

gnomAD: NR

AdditionalInfo:

Case#: 43-year-old man family b

DiseaseAssertion: Familial dilated cardiomyopathy (FDCM)

FamilyInfo: Sister II:1 had previously been diagnosed with DCM and carried both reported variants. Brother II:2 died of sudden cardiac death.

ParentalTesting: father was negative for either varient

CasePresentingHPOs: HP:0031352, HP:0002094, HP:0002321 HP:0001644 “Dyspnea; chest tightness; dizziness; dilated cardiomyopathy

CaseHPOFreeText: Severe DCM symptoms

CaseNotHPOs: not reported

CaseNotHPOFreeText: Father I:1 and daughter III:1 were reported to have no clinical symptoms

CasePreviousTesting: Previously diagnosed with FDCM

Genotyping Method: NGS. PCR, Sanger

FunctionalAnalysis: Putative pathogenic mutations were analysed computationally using the PolyPhen-2, SIFT, and MutationTaster

Variant: MYH7 NM_000257:c.3134G>A; p.Arg1045His; heterozygous missense

ClinVar: 651054

CAID: N/A

gnomAD: 14-23422291-C-T

AdditionalInfo: two variants may have a “dose effect” and associate their combination with the proband’s severe phenotype

Case#: male, 43 yrs,

DiseaseAssertion: FDCM

FamilyInfo: Family B; sister also proband; brother died of SCD; daughter and father asymptomatic

CasePresentingHPOs: HP:0012250 (ST segment depression)

CaseHPOFreeText: severe cardiac enlargement, increased LVEDD (77.8 mm), decreased LVESD (69.8 mm), decreased LVEF (21% < 50%), heart rate 78 beats/min, widened QRS complex (120 ms > 110 ms)

CaseNotHPOs:

CaseNotHPOFreeText:

CasePreviousTesting: N/A

CaseGenotypingMethod: Sanger sequencing, Genome Analyzer IIx, ECG,

Variant: NM_000257.4(MYH7):c.3134G>A (p.Arg1045His)

ClinVar: 651054 https://www.ncbi.nlm.nih.gov/clinvar/variation/651054/

CAID: N/A

gnomAD: absent

Case#: female

DiseaseAssertion: FDCM

FamilyInfo: Family B; brother also proband; brother died of SCD, niece and father asymptomatic

CasePresentingHPOs: N/A

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

CaseGenotypingMethod: Sanger sequencing, Genome Analyzer IIx, ECG,

Variant: NM_000257.4(MYH7):c.3134G>A (p.Arg1045His)

ClinVar: 651054 https://www.ncbi.nlm.nih.gov/clinvar/variation/651054/

CAID: N/A

gnomAD: absent

Case1: 5 week old female

DiseaseAssertion: Feeding problems and cyanosis. X-thorax showed a grossly enlarged heart

FamilyInfo: Family history of both parents was positive for HCM and sudden death

ParentalTesting: Initial echocardiography revealed no abnormalities in the 32-year-old mother, who was heterozygous for c.2373dup, nor in the 31-year-old father, heterozygous for c.2827C>T. ECG in the father showed mild repolarization abnormalities (ST elevation of 0.5 mm in V1, followed by a negative T in V1–V4). Re-evaluation after 7 years revealed moderate septal hypertrophy (HCM) with an interventricular septum of 14 mm in the mother, and interventricular septal measurements at the upper limit of the normal range (12 mm) in the father.

CasePresentingHPOs: HP:0001640, HP:0001639,

CaseHPOFreeText: Severe cardiomegaly and dilatation with a total weight of 115  g (normal weight at this age, 30  g). Right ventricular thickening was noted, especially of the posterior wall. The anterior wall of the left ventricle was severely thickened and showed abnormal trabeculation and multiple intertrabecular recesses as seen in noncompaction cardiomyopathy. Secundum ASD was confirmed. No other congenital malformations were found. Microscopic examination of cardiac tissue (Figure 1a) showed myofibrillar disarray in both the ventricular septum and the left ventricular wall. Hypertrophic myocytes with a diameter of 20–30 μm (normal 12  μm) and multiple vacuoles on electron microscopy suggested glycogenosis

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: NR

FunctionalAnalysis: Microscopic postmortem examination of heart muscle from patient 1. Hypertrophic myocytes with myofibrillar disarray typical of HCM due to sarcomeric protein variations were present, albeit without a significant amount of interstitial fibrosis (hematoxylin and eosin staining, magnification × 100). (b) Electron micrograph showing a large, irregular vacuole.

Variant: NC_000011.10:g.47337730dup, NM_000256.3(MYBPC3):c.2827C>T (p.Arg943Ter)

ClinVar: 37039, 42619

CAID: CA013001, CA012139

gnomAD: 11-47356671-G-A, 11-47359280-A-AC

AdditionalInfo: NR

Case4: 6 week old female

DiseaseAssertion: dyspnea, hypotension, feeding difficulties, and failure to thrive

FamilyInfo: Her mother was diagnosed with postpartum HCM with diastolic dysfunction, confirmed by cardiac MRI, at age 26. Echocardiography of the father was normal; ECG showed a right bundle branch block. A paternal uncle of patient 4 had died aged 3 months of unknown cause

ParentalTesting: Both parents were heterozygous for c.2827C>T p.(Arg943*) nonsense mutation in MYBPC3

CasePresentingHPOs: HP:0001639

CaseHPOFreeText: Severe HCM with features of noncompaction, and severe diastolic and systolic left ventricular dysfunction (IVSd 11 mm, Z+10; LVPWd 10 mm, Z+10, FS 13% (LVID 17/20) and left ventricular mass 255 g/m2). A secundum ASD with a left–right shunt was also observed.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: NR

FunctionalAnalysis: NR

Variant: NM_000256.3(MYBPC3):c.2827C>T (p.Arg943Ter)

ClinVar: 37039

CAID: CA013001

gnomAD: 11-47356671-G-A

AdditionalInfo: NR

Case3: 7 week old female

DiseaseAssertion:

FamilyInfo: A cousin of the father presented with cardiac arrest due to HCM. He had a heterozygous pathogenic c.2373dup p.(Trp792fs) mutation in MYBPC3. We subsequently confirmed homozygosity for the c.2373dup variant in DNA isolated from stored fibroblasts from the deceased proband. Both parents of the proband were heterozygous for this truncating variant (Figure 2b). Cardiac re-evaluation showed only slightly reduced ECG voltages in the father (age 38) and borderline hypertrophy (septal thickness 11 mm) in the mother (age 41).

ParentalTesting:

CasePresentingHPOs: HP:0001640, HP:0002119

CaseHPOFreeText: Cardiomegaly was seen on chest X-ray, and echocardiography showed HCM with dilated ventricles. M-Mode measurements were unavailable. Noncompaction was not a clear feature, but could not be excluded. There was a small foramen ovale and mitral valve insufficiency

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: NR

FunctionalAnalysis: DNA isolated from stored fibroblasts from the deceased proband.

Variant: NC_000011.10:g.47337730dup

ClinVar: 42619

CAID: CA012139

gnomAD: 11-47359280-A-AC

AdditionalInfo: NR

Case 1: One proband; (53-year-old woman)

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM) with fasciculoventricular bypass tract (FVBT)

FamilyInfo: Autosomal dominant family with HCM and pre-excitation. Mother has an ICD for nonischemic cardiomyopathy and progressed from HCM to DCM; sister (49) carries the variant with HCM and NSVT; younger daughter (25) carries the variant with pre-excitation but no HCM. Maternal aunt died suddenly at 42; maternal uncle had a sudden cardiac arrest at 40 with subsequent ICD.

CasePresentingHPOs: HP:0001962; HP:0001639; HP:0004756; HP:0001677; HP:0001671; HP:0004308

CaseHPOFreeText: Palpitations prompted workup. ECG showed pre-excitation; Holter documented NSVT; echo showed HCM without significant outflow obstruction; EP study demonstrated an FVBT with inducible AF; CMR showed midventricular septal thickness of 22 mm with patchy enhancement involving 25% of myocardial mass.

CaseNotHPOs: HP:0001635; HP:0001279; HP:0001645

CaseNotHPOFreeText: No heart failure reported; no syncope; alive at publication.

CasePreviousTesting: ECG (pre-excitation), Holter (NSVT), echo (HCM without significant outflow obstruction), EP study (FVBT with inducible AF), CMR (midventricular septum 22 mm, patchy enhancement 25% of mass).

GenotypingMethod: HCMNext panel (Ambry Genetics); heterozygous c.655G>C (p.Val219Leu) in MYBPC3, reported pathogenic.

FunctionalAnalysis: None performed.

Variant: MYBPC3 NM_000256.3:c.655G>C (p.Val219Leu); heterozygous missense

ClinVar: 42784

CAID: CA015670

gnomAD: 0.00002

AdditionalInfo:N/A

Case1: Pedigree mother; affected carrier; HCM progressing to DCM; clinical case

DiseaseAssertion: Hypertrophic cardiomyopathy progressing to dilated cardiomyopathy, with pre-excitation; MYBPC3-related

FamilyInfo: Carries the familial MYBPC3 variant. ICD in place for nonischemic cardiomyopathy despite optimal medical therapy; never received a shock. Mother of the proband and the sister.

ParentalTesting: Not applicable, this is the transmitting generation.

CasePresentingHPOs: HP:0001639; HP:0001644; HP:0004308

CaseHPOFreeText: Serial echocardiography over 11 years documented progression from hypertrophic to dilated phenotype. ECG showed pre-excitation. ICD implanted; no appropriate or inappropriate therapy to date.

CaseNotHPOs: HP:0001279 ; HP:0001645

CaseNotHPOFreeText: No syncope reported; alive at publication.

CasePreviousTesting: Serial echocardiography; ECG; ICD interrogation.

Genotyping Method: Family screening; carrier status reported.

FunctionalAnalysis: None performed.

Variant: MYBPC3 NM_000256.3:c.655G>C (p.Val219Leu); heterozygous missense

ClinVar: 42784

CAID: CA015670

gnomAD: 0.00002

AdditionalInfo:N/A

Case#: Proband and family; distal arthrogryposis phenotype; clinical case

DiseaseAssertion: Distal arthrogryposis (DA); MYBPC1-related

FamilyInfo: Two MYBPC1 variants segregate in the family. The paper provides the pedigree and segregation data.

ParentalTesting: Not stated

CasePresentingHPOs: HP:0002804

CaseHPOFreeText: Distal arthrogryposis phenotype in affected family members, per the paper's clinical description.

CaseNotHPOs: Not stated

CasePreviousTesting: Clinical evaluation, genetic testing for MYBPC1.

Genotyping Method: Genetic testing confirmed MYBPC1 variants; specific method in the paper.

FunctionalAnalysis: Confirm whether a functional assay was performed.

Variant: MYBPC1, two variants, specific nucleotide changes need confirmation from the paper

ClinVar:N/A

CAID:N/A

gnomAD:N/A

AdditionalInfo: N/A

Case2: Sex not stated, age 8 months at diagnosis, ethnicity not stated

DiseaseAssertion: Restrictive cardiomyopathy (RCM)

FamilyInfo: Patients 2 and 3, monozygotic twins, were diagnosed with RCM at the age of 8 and 13 months, respectively. No familial history of cardiac disorders was reported in the R170W case, parental testing confirmed that the R170W variant occurred de novo (however, no parental proof was performed)

ParentalTesting: Parental testing confirmed that the R170W variant occurred de novo (however, no parental proof was performed). Parents do not carry the variant

CasePresentingHPOs: HP:0001723, HP:0002875, HP:0005110

CaseHPOFreeText: Echocardiographic examination revealed a restrictive filling pattern and enlarged atria. Both children died at the age of 12 and 18 months, respectively, due to congestive heart failure and atrial fibrillation

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: Total genomic DNA was extracted from peripheral blood using FlexiGene DNA Kit (Qiagen) according to the manufacturer’s protocol. The TNNI3 gene was screened for mutations using dye terminator sequencing. In both cases additional target sequencing including a panel of 108 cardiomyopathy- and channelopathy-associated genes was performed using Illumina MiSeq as previously described

FunctionalAnalysis: Functional analysis performed on recombinant proteins. Both cTnI-R170G/W strongly enhanced the Ca2+-sensitivity of skinned fibres, as is typical for RCM-mutations. Both mutants strongly enhanced the affinity of troponin (cTn) to tropomyosin compared to wildtype cTn. The stability of reconstituted thin filaments was reduced as revealed by electron microscopy.

Variant: NM_000363.5:c.508C>T (p.Arg170Trp)

ClinVar: 179285

CAID: CA021778

gnomAD: chr19-55154071-G-A

AdditionalInfo: Monozygotic twin

Case1: Male, age 3 at diagnosis, ethnicity not stated

DiseaseAssertion: Restrictive cardiomyopathy (RCM)

FamilyInfo: Familial screening in the R170G proband documented a hypertrophic cardiomyopathy with restrictive filling pattern in patient’s father, but the family refused additional genetic testing. No other family history of cardiac disorders stated.

ParentalTesting: The family refused additional genetic testing

CasePresentingHPOs: HP:0001723, HP:0001650, HP:0001654

CaseHPOFreeText: Patient 1 showed an impaired left ventricular function and pulmonary hypertension at three years of age. Heart biopsy showed mild cardiomyocyte hypertrophy and interstitial fibrosis. He was diagnosed with RCM and died one year later of ventricular fibrillation.

CaseNotHPOs: NR

CaseNotHPOFreeText: Target sequencing including a panel of 108 cardiomyopathy- and channelopathy-associated genes revealed no additional pathogenic or likely pathogenic variants

CasePreviousTesting: Additional target sequencing including a panel of 108 cardiomyopathy- and channelopathy-associated genes was performed using Illumina MiSeq as previously described.

Genotyping Method: Total genomic DNA was extracted from peripheral blood using FlexiGene DNA Kit (Qiagen) according to the manufacturer’s protocol. The TNNI3 gene was screened for mutations using dye terminator sequencing. In both cases additional target sequencing including a panel of 108 cardiomyopathy- and channelopathy-associated genes was performed using Illumina MiSeq as previously described

FunctionalAnalysis: Functional analysis performed on recombinant proteins. Both cTnI-R170G/W strongly enhanced the Ca2+-sensitivity of skinned fibres, as is typical for RCM-mutations. Both mutants strongly enhanced the affinity of troponin (cTn) to tropomyosin compared to wildtype cTn. The stability of reconstituted thin filaments was reduced as revealed by electron microscopy.

Variant: NM_000363.5:c.508C>G (p.Arg170Gly)

ClinVar: 165517

CAID: CA021769

gnomAD: NR

AdditionalInfo: NR

Case#: II.3, 30-year-old man from an Indian family.

DiseaseAssertion: Familial hypertrophic cardiomyopathy.

FamilyInfo: Figure 1A. His father (I.1), brother (II.1), and sister (II.2) had HCM and carried TPM1 p.Gln68Arg. His mother (I.2) had a normal echocardiogram and tested negative for the variant. The authors report autosomal dominant segregation.

ParentalTesting: Father positive for TPM1 p.Gln68Arg. Mother negative.

CasePresentingHPOs: HP:0001639, HP:0001712, HP:0005144, HP:0031656, HP:0001653, HP:0010872, HP:4000004

CaseHPOFreeText: Echo showed asymmetric septal hypertrophy and trivial mitral regurgitation. The interventricular septum measured 2.1 cm and the left ventricular posterior wall measured 1.2 cm. ECG showed a left ventricular hypertrophy strain pattern with T-wave inversions in leads II, III, aVF, and V2–V6. Cardiac MRI showed patchy STIR hyperintensity and late gadolinium enhancement in the thickened septum.

CaseNotHPOs: HP:0000822 (no history of hypertension), HP:0032092 (no left ventricular outflow tract obstruction at rest)

CaseNotHPOFreeText: Asymptomatic at presentation. Both atria and the right ventricle were normal in size and function. Left ventricular ejection fraction was 63%.

CasePreviousTesting: NR (prior genetic testing details not available in the article).

Genotyping Method: Custom targeted amplicon sequencing panel of nine sarcomeric genes on the Ion Torrent PGM. Sanger sequencing confirmed the TPM1 variant.

FunctionalAnalysis: NR (functional assay results not available in the article).

Variant: NM_001018005.2(TPM1).203A>G (p.Gln68Arg), heterozygous.

ClinVar: 1490086

CAID: NR (CAID not listed in the article).

gnomAD: No variant record found in gnomAD v4.1 for 15-63044115-A-G.

AdditionalInfo: PMID 38223010. Figure 1A identifies the proband and shows the pedigree. Figures 1B–D show cardiac imaging. Figure 1E shows Sanger sequencing. Figure 1F shows conservation of the affected residue. The authors also report computational predictions of a damaging effect.

Case1: Male, 24 years old at diagnosis, ethnicity not stated

DiseaseAssertion: Dilated cardiomyopathy (DCM)

FamilyInfo: Three affected family members carry the D94A mutation, mother (I.2, diagnosed age 21 with peripartum cardiomyopathy), sister (II.1, diagnosed age 29), and proband (II.2, diagnosed age 24). Sanger sequencing confirmed the MYL2 variant was present in all three affected family members (I.2, II.1, and II.2).

ParentalTesting: Mother confirmed as heterozygous carrier of D94A mutation with DCM phenotype. Father not a carrier, unaffected

CasePresentingHPOs: HP:0001644, HP:0006699

CaseHPOFreeText: The proband (II.2) and his sister (II.1) were diagnosed at ages 24 and 29 after preventive cardiovascular screening revealed mild global left ventricular systolic dysfunction. Medical therapy was initiated upon evidence of decreased ejection fraction. Patients II.1 and II.2 (proband) displayed abnormal ECG findings and systolic dysfunction as manifested by reduced EF. However, the LV and septal wall thickness were within the normal range.

CaseNotHPOs: NR

CaseNotHPOFreeText: Septal wall thickness measurements were within normal range in all affected family members. LV wall thickness within normal range.

CasePreviousTesting: No prior testing.

Genotyping Method: Twenty two variants, meeting our established criteria for exome sequencing analysis, were identified in the three affected family members of the pedigree. Exome sequencing performed; variant confirmed by Sanger sequencing. Sanger sequencing confirmed the MYL2 variant was present in all three affected family members (I.2, II.1, and II.2)

FunctionalAnalysis: Functional analysis performed on recombinant proteins, not directly on patient samples. Far-UV CD spectroscopy was used to analyze the effect of the D94A mutation on the secondary structure of the RLC. The mutation was observed to induce a reduction in the α-helical content of the RLC and imposed intra-molecular rearrangements. The mutation was seen to impair the binding of RLC to the MHC (myosin heavy chain), and its incorporation into the RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared to ATPase of WT

Variant: NP_000423.2:p.Asp94Ala

ClinVar: 4710041

CAID: CA386698152

gnomAD: NR

AdditionalInfo: Three affected family members carry the variant (mother I.2, sister II.1, proband II.2)

Case1: Female, 85 at diagnosis, ethnicity not stated

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM)

FamilyInfo: An index patient’s brother (II.6) was reported with moderate HCM and another brother (II.7) with sudden cardiac death at the age of 40 years. The clinical screening led to the diagnosis of HCM in two of the index patient’s descendants, a daughter and a son. Daughter (III.4, age 59) with septal hypertrophy of 16 mm confirmed by cardiac MRI, son (III.8, age 47) with septal hypertrophy of 15 mm. Daughter (III.6, age 50) is an unaffected carrier. Five other relatives (III.1, III.5, III.6, III.7, IV.1) were healthy with normal cardiac evaluation. Pedigree shown in Figure 1.

ParentalTesting: Not preformed.

CasePresentingHPOs: HP:0001639, HP:0001279

CaseHPOFreeText: Admitted to the emergency unit with chest pain after a pre-syncopal episode at the age of 85. Electrocardiogram showed sinus rhythm, negative T waves in V1-V3 and signs of ventricular hypertrophy. Transthoracic echocardiography detected asymmetric septal hypertrophy of 25 mm with preserved ejection fraction, moderate diastolic dysfunction and mild left atrial enlargement. Twenty-four-hour Holter monitoring documented episodes of non-sustained ventricular tachycardia defined as ≥ 3 beats at a rate of ≥ 120 bpm

CaseNotHPOs: NR

CaseNotHPOFreeText: Coronary artery disease was ruled out

CasePreviousTesting: NR

Genotyping Method: The index patient’s genomic DNA was subjected to massively parallel sequencing to identify the genetic origin of the disease using a 251-gene cardiomyopathy diagnostic panel. Targeted massively parallel sequencing of the index patient’s genomic DNA was performed with an Agilent SureSelect custom library covering 251 genes. For the MYBPC3 gene: all exons including the complete sequence of introns. Sequencing performed on NovaSeq 6000 (Illumina), bioinformatics pipeline using BWA alignment and GATK variant calling, reference genome GRCh37/hg19. Sanger sequencing confirmed both variants

FunctionalAnalysis: Minigene and patient blood RNA splicing assays performed. Both the minigene and patients’ RNA splicing assays revealed that the MYBPC3 c.3331−26T>G variant promoted complete intron 30 retention in the mRNA, and also partial retention of the second half of intron 30. Normal MYBPC3 mRNA was not detected in the MUT lanes. The corresponding normal MYBPC3 fusion protein was not detected with either of the two antibodies in cells transfected with the MUT plasmid.

Variant: NM_000256.3:c.3331−26T>G

ClinVar: 1320039

CAID: CA2573146361

gnomAD: chr11-47332999-A-C

AdditionalInfo: This paper also identified two additional unrelated HCM probands carrying c.3331−26T>G in a large cohort of 9,611 HCM probands

Case#: 80-year-old male (I-1) diagnosed with HCM at approximately 50–55 years of age; familial case with his 3 adult offspring (II-2, II-3, and II-4)

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM) associated with a heterozygous frameshift variant in MYBPC3.

FamilyInfo: The proband (I-1) is an elderly male with familial hypertrophic cardiomyopathy. Three adult offspring (II-2, II-3, and II-4) were evaluated and diagnosed with HCM based on interventricular septal hypertrophy. All four individuals carried the heterozygous MYBPC3 c.1042_1043insCGGCA variant. Individual II-2 experienced chest discomfort and palpitations, whereas II-3 and II-4 were asymptomatic at evaluation. These findings demonstrate segregation of the variant with HCM in the examined family members and are consistent with autosomal dominant inheritance.

ParentalTesting: NR – Genetic testing of the proband's parents was not reported but the variant was identified in his three adult offspring, supporting familial segregation.

CasePresentingHPOs: HP:0001639 (Hypertrophic cardiomyopathy); HP:0002094 (Dyspnea); HP:0001635 (Congestive heart failure)

CaseHPOFreeText: The proband experienced chest tightness, dyspnea, and recurrent heart failure. Echocardiography demonstrated marked interventricular septal hypertrophy (39 mm during diastole and 41 mm during systole), a left atrial diameter of 44 mm, and a left ventricular ejection fraction of 60%. Additional findings included premature atrial beats, intraventricular conduction block, and NYHA functional class II. A history of hypertension was reported.

CaseNotHPOs: HP:0001279 (Syncope); HP:0001681 (Angina pectoris); HP:0001695 (Cardiac arrest)

CaseNotHPOFreeText: The proband had no reported history of syncope, angina pectoris, or cardiac arrest. No surgical myectomy was reported.

CasePreviousTesting: NR – Previous genetic testing was not reported but the proband underwent clinical cardiovascular evaluation, including electrocardiography, echocardiography, and BNP measurement.

Genotyping Method: Whole-genome sequencing (WGS) was performed using peripheral blood DNA on an Illumina NovaSeq 6000 platform with 150-bp paired-end reads and approximately 30× sequencing depth. Reads were aligned to the hs37d5 reference genome using BWA, and variants were annotated using ANNOVAR.

FunctionalAnalysis: Cardiac tissue from the proband was analyzed using RT-qPCR and Western blotting. MYBPC3 mRNA expression and cardiac myosin-binding protein C (cMyBP-C) abundance were significantly reduced (P < 0.05), supporting a haploinsufficiency mechanism. These findings do not independently establish the functional effect of the specific variant, and the Western blot analysis lacked biological replicates.

Variant: MYBPC3 NM_000256.3.1038_1042dup (p.Met348ThrfsTer4). Five-base-pair duplication resulting in a frameshift and premature termination codon. Reported in Zhang et al. (2025) as c.1042_1043insCGGCA.

ClinVar: No exact match identified for MYBPC3 c.1042_1043insCGGCA using the reported HGVS notation. ClinVar Variation ID not established.

CAID: CA273429

gnomAD: No exact matching entry identified in gnomAD v4 for MYBPC3 NM_000256.3:c.1038_1042dup (CA273429). Population allele frequency could not be established.

AdditionalInfo: The variant was identified in the proband and three adult offspring with interventricular septal thickening. The study proposed MYBPC3 haploinsufficiency as a disease mechanism based on reduced transcript and protein abundance. The available familial and functional evidence supports an association with HCM, although the specific variant's pathogenicity requires interpretation alongside the limitations of the study.

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 2; male; 74 years old at first observation; 27 mm LV wall thickness; family history of HCM

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

FamilyInfo: Reported family history of hypertrophic cardiomyopathy. Echocardiographic evaluation demonstrated a maximum left ventricular wall thickness of 27 mm and left ventricular outflow tract (LVOT) obstruction, with a gradient of 50 mmHg. The patient was classified as NYHA functional class II.

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:0032092 (Left ventricular outflow tract obstruction); HP:0001712 (Left ventricular hypertrophy)

CaseHPOFreeText: Echocardiography demonstrated a maximum left ventricular wall thickness of 27 mm and left ventricular outflow tract (LVOT) obstruction with a measured gradient of 50 mmHg. The patient was classified as New York Heart Association (NYHA) functional class II, indicating mild limitation of physical activity.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: The patient previously underwent targeted next-generation sequencing using the Illumina TruSight Cardio panel covering 174 genes associated with hereditary cardiac diseases. The initial analysis produced a negative molecular result. Subsequent reanalysis of the existing sequencing data identified the MYBPC3 intronic variant c.1224-80G>A.

Genotyping Method: Genomic DNA was extracted from peripheral blood and sequenced using the Illumina TruSight Cardio panel on a MiniSeq platform. Initial bioinformatics analysis was performed using Illumina BaseSpace software. Raw sequencing data were subsequently reanalyzed using an intronic-variant hotspot approach, with variant visualization in Integrative Genomics Viewer (IGV). The identified MYBPC3 variant was validated by Sanger sequencing.

FunctionalAnalysis: The MYBPC3 intronic variant c.1224-80G>A creates a novel 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 and is associated with approximately 50% reduction in MYBPC3 transcript abundance. Previous studies demonstrated the splice-altering effect through functional analyses, while SpliceAI predicted a strong effect on splicing (Δscore = 0.94). The current study identified the variant through reanalysis of existing NGS data and confirmed its presence using Sanger sequencing; it did not perform new patient-specific RNA functional experiments.

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: The variant was detected by expanding the analysis to include previously characterized intronic splice-altering regions and was subsequently confirmed by Sanger sequencing. Functional evidence for altered splicing was obtained from previously published research rather than new patient-specific RNA experiments.

Case#: Family B, Case IV.2; 8-year-old male; diagnosed at age 6

DiseaseAssertion: Dilated cardiomyopathy (DCM)

FamilyInfo: Positive family history of sudden cardiac death (SCD). The proband's brother (IV.1) died at age 2 of SCD and renal failure. Both parents (III.1 and III.2) were asymptomatic heterozygous carriers of MYL3 c.106G>T. Two unaffected siblings were tested: IV.3 was wild-type and IV.4 was heterozygous (Fig. 1b; Supplementary Fig. S2b).

ParentalTesting: Both parents (III.1 and III.2) were confirmed heterozygous carriers of MYL3 c.106G>T by PCR and bidirectional Sanger sequencing, supporting autosomal recessive inheritance (Supplementary Genetic Findings; Fig. S2b).

CasePresentingHPOs: HP:0001644 (Dilated cardiomyopathy); HP:0000085 (Horseshoe kidney); HP:0005162 (Reduced left ventricular ejection fraction).

CaseHPOFreeText: DCM diagnosed at age 6; heart transplantation performed shortly after diagnosis. Clinical evaluation revealed horseshoe kidney, severe left ventricular dysfunction (LVEF 15–20%), moderate right ventricular dysfunction (TAPSE 9 mm), chamber cardiomegaly on coronary CT angiography, and rapid ventricular tachycardia on ECG (Main text, Family B; Supplementary Results, p. 6).

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR (no prior diagnostic genetic testing reported before the study's exome sequencing)

Genotyping Method: Exome sequencing identified the homozygous MYL3 c.106G>T (p.Glu36Ter) variant in the proband (IV.2). PCR and bidirectional Sanger sequencing validated the variant and confirmed segregation in the parents and two unaffected siblings (Supplementary Methods; Supplementary Genetic Findings; Fig. S2b).

FunctionalAnalysis: Zebrafish cmlc1 knockdown and rescue experiments were performed using human MYL3 RNA containing c.106G>T (p.Glu36Ter). Unlike wild-type MYL3 RNA, the mutant RNA failed to rescue the cardiac phenotype, with persistent cardiac edema, ventricular dysfunction, and atrial dilation (Fig. 2; Supplementary Methods; Supplementary Movie S7).

Variant: MYL3 NM_000258.2:c.106G>T (p. Glu36Ter); homozygous nonsense variant

ClinVar: NR

CAID: CA352499542

gnomAD: NR

AdditionalInfo: The authors classified the homozygous MYL3 c.106G>T (p.Glu36Ter) nonsense variant as pathogenic. The variant introduces a premature termination codon at amino acid 36. The proband was born to asymptomatic, consanguineous (first-cousin) parents of Bakhtiyari ancestry. Family segregation and zebrafish functional studies provide supporting evidence (Supplementary Results; Supplementary Genetic Findings; Supplementary Table S2).

Case#: Family C, Case VI.1; female; diagnosed at age 2.5 years; died at age 8

DiseaseAssertion: Unclassified cardiomyopathy

FamilyInfo: Family C has a positive family history of childhood-onset cardiomyopathy and sudden cardiac death. The affected individual (VI.1) was diagnosed with cardiomyopathy at age 2.5 years and died at age 8. Three maternal relatives (V.5, V.6, and V.8) are depicted as affected and deceased in the pedigree (Fig. 1C). Both parents (V.1 and V.2) were clinically unaffected heterozygous carriers of the MYL3 c.482-1G>A splice-acceptor variant. The genotype of VI.1 could not be confirmed because DNA was unavailable.

ParentalTesting: PCR and bidirectional Sanger sequencing confirmed that both parents (V.1 and V.2) were heterozygous carriers of MYL3 c.482-1G>A. Direct testing of the affected daughter (VI.1) was not possible because DNA was unavailable. Her homozygous genotype was therefore suspected but not confirmed (Fig. 1C; Supplementary Genetic Findings).

CasePresentingHPOs: HP:0001638 (Cardiomyopathy); HP:0001645 (Sudden cardiac death).

CaseHPOFreeText: Female diagnosed with unclassified cardiomyopathy at age 2.5 years; died of sudden cardiac death at age 8 (Family C, Fig. 1C).

CaseNotHPOs: NR (no patient-specific negative HPO findings confirmed for VI.1).

CaseNotHPOFreeText: The authors reported no evidence of muscle weakness by neurological examination or history among affected individuals from the three families; however, it is unclear whether this assessment specifically included VI.1.

CasePreviousTesting: NR (no prior genetic testing reported for VI.1).

Genotyping Method: Genetic analysis of the affected individual's mother (V.2) identified MYL3 c.482-1G>A. PCR and bidirectional Sanger sequencing validated the variant and confirmed both parents (V.1 and V.2) as heterozygous carriers. VI.1 was not directly genotyped because DNA was unavailable (Supplementary Genetic Findings; Fig. 1C and S2C).

FunctionalAnalysis: A minigene splicing assay in human HEK293FT cells demonstrated that MYL3 c.482-1G>A causes abnormal RNA splicing. The predominant outcome (~75%) was skipping of the 78-bp exon 5, producing an in-frame deletion (p.Gly161_Glu186del). A less frequent outcome (~25%) was deletion of the first nucleotide of exon 5, causing a frameshift (p.Gly161Valfs*4). Both outcomes are predicted to disrupt EF-hand domains. The authors note that the minigene assay may not fully reflect in vivo splicing frequencies (Supplementary Results, pp. 7–8; Fig. 2E–I).

Variant: MYL3 c.482-1G>A (rs727503300)

ClinVar: 164486

CAID: CA013932

gnomAD: NR MYL3 c.482-1G>A was not found in the gnomAD v4 (GRCh38) region results at chr3:46,860,813. Allele frequency not reported for this variant in the searched dataset.

AdditionalInfo: The affected individual (VI.1) died at age 8, and DNA was unavailable for direct genetic testing. Both clinically unaffected parents (V.1 and V.2) were confirmed heterozygous carriers of MYL3 c.482-1G>A, suggesting that VI.1 may have inherited the variant from both parents; however, her genotype remains unconfirmed. A minigene assay demonstrated that the variant disrupts RNA splicing, predominantly causing exon 5 skipping, with a less frequent frameshift-producing splicing event. These functional findings support a damaging effect of the variant but do not establish VI.1's genotype (Fig. 1C; Fig. 2E–I; Supplementary Genetic Findings and Minigene Assay Results).

Case#: Family A, Case III.7; 38-year-old female; diagnosed at age 26

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM)

FamilyInfo: Positive family history of HCM and SCD. Mother (II.4), brother (III.8), uncle (II.7), and cousin (III.11) died of SCD at ages 55, 26, 57, and 24, respectively. The proband's three children (IV.3, IV.4, IV.5) were homozygous for the MYL3 variant but had normal echocardiograms at ages 18, 13, and 6.

ParentalTesting: Supplementary Table S2 reports parental testing supportive of PM3 (variants in trans). However, DNA was unavailable for the proband's deceased mother (II.4), and individual parental genotypes were not specified in the supplementary genetic findings.

CasePresentingHPOs: HP:0001639 (Hypertrophic cardiomyopathy); HP:0002094 (Dyspnea); HP:0001653 (Mitral regurgitation); HP:0001712 (Left ventricular hypertrophy)

CaseHPOFreeText: HCM diagnosed at age 26 following dyspnea. At age 38, echocardiography showed reverse-curvature HCM with asymmetric left ventricular hypertrophy involving anterior, anterolateral, and lateral segments; maximum wall thickness 22 mm; severe mitral regurgitation; LVEF 55%; E/e' ratio 16.4; resting LVOT gradient 9 and post-maneuver gradient 30. Holter monitoring showed no arrhythmias (Supplementary Results, pp. 5–6; Table S1; Fig. S1).

CaseNotHPOs: HP:0001324 (Muscle weakness)

CaseNotHPOFreeText: Neurological evaluation revealed normal muscle tone, strength 5/5 in upper and lower limbs, no distal or proximal muscle atrophy or fasciculations, and normal creatine phosphokinase levels.

CasePreviousTesting: Exome sequencing was performed; no rare, likely pathogenic variants in other cardiomyopathy-associated genes identified after filtering.

Genotyping Method: Exome sequencing identified the MYL3 variant; bidirectional Sanger sequencing validated the variant. Familial genotyping supported recessive inheritance (Fig. 1a).

FunctionalAnalysis: Zebrafish cmlc1 knockdown and rescue experiments were performed using human MYL3 RNA carrying c.170C>A (p.Ala57Asp). Mutant RNA showed partial phenotypic rescue but no statistically significant improvement in ventricular contractility compared with knockdown alone (Fig. 2a-d).

Variant: MYL3 NM_000258.2:c.170C>A (p.Ala57Asp); homozygous missense variant; rs139794067

ClinVar: 43121

CAID: CA013575

gnomAD: Allele frequency 0.000163 (0 homozygotes), as reported in Supplementary Table S2.

AdditionalInfo: Family A, Case III.7. Clinical characteristics and genetic findings described in the Results section (Fig. 1a and 1d-e). Functional experiments described in Fig. 2. The authors classified c.170C>A as a variant of uncertain significance (VUS).

Case#: Patient 1, individual II.1 (Fig. 2A); 22-year-old Salvadoran female

DiseaseAssertion: Restrictive cardiomyopathy (RCM) with advanced heart failure

FamilyInfo: Patient denied a family history of cardiomyopathy, although supporting records were unavailable. Her mother (I.2) was clinically unaffected at age 45 and heterozygous for both MYL3 p.Glu143Lys and MYL2 p.Gly57Glu. Other family members were unavailable for evaluation (Fig. 2A; Table I).

ParentalTesting: Mother (I.2) was confirmed heterozygous for MYL3 c.427G>A (p.Glu143Lys) and MYL2 c.170G>A (p.Gly57Glu). Paternal testing was not reported.

CasePresentingHPOs: HP:0012764 (Orthopnea); HP:0004749 (Atrial flutter); HP:0031295 (Left atrial enlargement); HP:0030718 (right atrial enlargement)

CaseHPOFreeText: Worsening fatigue and exertional dyspnea over 12 months, paroxysmal nocturnal dyspnea, palpitations, NYHA Class III–IV heart failure, and ascites suggestive of congestive hepatopathy. Echocardiography showed severe biatrial enlargement and advanced left ventricular diastolic dysfunction with preserved systolic function. Additional findings included myocardial fibrosis and myofiber disarray on biopsy.

CaseNotHPOs: NR

CaseNotHPOFreeText: At initial presentation, patient denied dizziness, syncope, and lower-extremity edema. No S4 heart sound was detected. Recurrent syncope developed later.

CasePreviousTesting: Targeted sequencing of eight sarcomere genes (ACTC, MYBPC3, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPM1). Genome-wide analysis was not reported. An additional heterozygous MYL2 variant, c.170G>A (p.Gly57Glu), was identified (ClinVar Variation ID: 43458; CAID: CA009910).

Genotyping Method: Sequencing of coding exons and intron–exon boundaries of MYL3 and seven additional sarcomere genes, performed by a commercial CLIA-certified laboratory (Correlagen Diagnostics, Inc.). The specific sequencing platform was not reported.

FunctionalAnalysis: NR

Variant: MYL3 NM_000258.2:c.427G>A (p.Glu143Lys); homozygous

ClinVar: 14063

CAID: CA013774

gnomAD: MYL3 c.427G>A (p.Glu143Lys). Highest reported ancestry-specific allele frequency: 0.0002333 (0.02333%) in the Admixed American population (AC = 14). Overall AF (exomes): 0.000008209; AF (genomes): 0.00001314; AF (total): 0.000008674. https://gnomad.broadinstitute.org/variant/3-46859529-C-T?dataset=gnomad_r4.

AdditionalInfo: Patient developed recurrent syncope and received an implantable cardioverter-defibrillator. She underwent orthotopic heart transplantation six months after RCM diagnosis. Authors considered the homozygous MYL3 and heterozygous MYL2 variants likely contributors to disease, although their individual contributions were not established. Clinical and family findings are reported in Patient 1, Figure 2A, and Table I.

Case#: Female, 6, non-Hispanic white

DiseaseAssertion: Dilated Cardiomyopathy

FamilyInfo: Father and Paternal uncle diagnosed with DCM at age 34.

ParentalTesting: Her father underwent cardiac screening based on family history several years later at age 34

CasePresentingHPOs: HP:0001640

CaseHPOFreeText: NA

CaseNotHPOs: NA

CaseNotHPOFreeText: Acute viral illness and gastroenteritis

CasePreviousTesting: NR

Genotyping Method: Sanger Sequencing

FunctionalAnalysis: NR

Variant: NM_001276345.2:c.149A>G

ClinVar: 2005620

CAID: CA344207232

gnomAD: 1:201337304 T / C

AdditionalInfo: NA

Family 6 - Patients 6a and 6b

FamilyPresentingHPOs: HP:0007340, HP:0003701, HP:0000467, HP:0003327, HP:0200136, HP:0003691, HP:0003306, HP:0002650, HP:0040081, HP:0002111, HP:0100284

HPOsFreeText: myopathic muscle unit action potentials, 6a muscle pathology with vacuolar myopathy with glycogen excess

FamilyNotHPOs: HP:0010628

Ethnicity: Iranian

ProbandLabel: 6a, male, 12yo at onset, 25yo at diagnosis

FamilyAddInfo: 6b (male) dx at 33yo, 6a MRC sum score = 78, 6b MRC sum score = 79, both patients required use of NIV

FamilyPreviousTesting: Respiratory Evaluation: 6a FVC = 19%, 6b FVC = 38%, 6a CK = 820 U/L, 6b CK = 503 U/L, muscle pathology (examined only in 6a), DBS enzyme activity (with inhibition) for 6a = 0 μmol/L/h, for 6b = 0.21 μmol/L/h, NCS, EMG

FamilyGenotypingMethod: GAA analyzed with Sanger Sequencing for entire coding region and conserved exon-intron splice junctions, PCR performed for deletion screening

Variant: NM_000152.4(GAA):c.1979G>A (p.Arg660His)

ClinVar: 189172, https://www.ncbi.nlm.nih.gov/clinvar/variation/189172/

gnomAD: 3.279e-05, http://exac.broadinstitute.org/variant/17-78086765-G-A

Family 7 - Patient 7

FamilyPresentingHPOs: HP:0007340, HP:0003701, HP:0003306, HP:0002111, HP:0040081

HPOsFreeText: muscle pathology with vacuolar myopathy with glycogen excess, myopathic muscle unit action potentials

FamilyNotHPOs: HP:0010628, HP:0002015, HP:0003691, HP:0002650

Ethnicity: Iranian

ProbandLabel:

FamilyAddInfo: male, 20yo at onset, 23yo at diagnosis, MRC sum score = 96, patient required use of NIV

FamilyPreviousTesting: Respiratory Evaluation: FVC = 20%, CK = 2,550 U/L, muscle pathology examined, DBS enzyme activity (with inhibition) = 0.1 μmol/L/h, NCS, EMG

FamilyGenotypingMethod: GAA analyzed with Sanger Sequencing for entire coding region and conserved exon-intron splice junctions, PCR performed for deletion screening

VariantAllele1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

VariantAllele2: NM_000152.4(GAA):c.2662G>T (p.Glu888Ter)

ClinVar: 578595, https://www.ncbi.nlm.nih.gov/clinvar/variation/578595/

gnomAD: 1.65e-05, http://exac.broadinstitute.org/variant/17-78092467-G-T

Family 1 - Patient 1

FamilyPresentingHPOs: HP:0002355, HP:0003701, HP:0040081, HP:0100284

HPOsFreeText: myopathic muscle unit action potentials

FamilyNotHPOs HP:0010628, HP:0002015, HP:0003691, HP:0003306, HP:0002650

NotHPOsFreeText:

Ethnicity: Iranian

ProbandLabel: Patient 1, male

FamilyAddInfo: 2yo at onset, 4yo at diagnosis, MRC sum score not available, consanguineous family

FamilyPreviousTesting: Respiratory evaluation: FVC = 90%, CK = 2,960 U/L,DBS enzyme activity (with inhibition) = 0.3 μmol/L/h, no muscle pathology performed, cardiac evaluations, NCS, EMG

FamilyGenotypingMethod: GAA analyzed with Sanger Sequencing for entire coding region and conserved exon-intron splice junctions, PCR performed for deletion screening

Variant: NM_000152.4(GAA):c.854C>G (p.Pro285Arg)

ClinVar: 225114, https://www.ncbi.nlm.nih.gov/clinvar/variation/225114/

gnomAD: n/a

Family 8 - Patients 8a and 8b

FamilyPresentingHPOs: HP:0007340, HP:0003701, HP:0000467, HP:0003327, HP:0010628, HP:0002015, HP:0003691, HP:0002650, HP:0040081

HPOsFreeText: 8b muscle pathology with mild lysosomal vacuolar myopathy with slight glycogen excess, no metachromatic debris, myopathic muscle unit action potentials

FamilyNotHPOs: HP:0003306

Ethnicity: Iranian

ProbandLabel: 8a, female, 27yo at onset, 32yo at diagnosis

FamilyAddInfo: 8b (male) dx at 40yo, 8a MRC sum score = 82, 8b MRC sum score = 60, 8b is wheelchair bound, both patients required use of NIV

FamilyPreviousTesting: Respiratory Evaluation: 8a FVC = 102%, 8b FVC = 17%, 8a CK = 268 U/L, 8b CK = 199 U/L, muscle pathology (examined only in 8b), DBS enzyme activity (with inhibition) for 8a = 0.11 μmol/L/h, for 8b = 0.15 μmol/L/h, NCS, EMG

FamilyGenotypingMethod: GAA analyzed with Sanger Sequencing for entire coding region and conserved exon-intron splice junctions, PCR performed for deletion screening

VariantAllele1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

VariantAllele2: NM_000152.4(GAA):c.2078dupA (p.Ala694Glyfs)

ClinVar: 180143, https://www.ncbi.nlm.nih.gov/clinvar/variation/180143/

gnomAD: n/a

Family 3 - Patient 3

FamilyPresentingHPOs: HP:0002355, HP:0003701, HP:0000467, HP:0003327, HP:0002111, HP:0040081, HP:0100284

HPOsFreeText: Vacuolar myopathy, myopathic muscle unit action potentials

FamilyNotHPOs: HP:0010628, HP:0002015, HP:0003691, HP:0003306, HP:0002650

NotHPOsFreeText:

Ethnicity: Iranian

ProbandLabel:

FamilyAddInfo: female, 2yo at onset, 26yo at diagnosis, MRC sum score = 76, NIV used beginning at 22yo

FamilyPreviousTesting: Respiratory Evaluation: FVC = 31%, CK = 1,551 U/L, Vacuolar myopathy present in muscle pathology exam, DBS enzyme activity (with inhibition) = 5.1 μmol/L/h, NCS, EMG

FamilyGenotypingMethod: GAA analyzed with Sanger Sequencing for entire coding region and conserved exon-intron splice junctions, PCR performed for deletion screening

VariantAllele1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

VariantAllele2: NM_000152.4(GAA):c.1004G>A (p.Gly335Glu)

ClinVar: 180142, https://www.ncbi.nlm.nih.gov/clinvar/variation/180142/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

Family 9 - Patient 9

FamilyPresentingHPOs: HP:0007340, HP:0003701, HP:0000467, HP:0003327, HP:0010628, HP:0003306, HP:0002111, HP:0040081

HPOsFreeText: muscle pathology with slight vacuolar myopathy with mild glycogen excess

FamilyNotHPOs: HP:0002015, HP:0003691, HP:0002650

Ethnicity: Iranian

ProbandLabel:

FamilyAddInfo: onset at 28yo, diagnosed at 35yo, MRC sum score = 81

FamilyPreviousTesting: Respiratory Evaluation: FVC = 71%, CK = 748 U/L, muscle pathology examined, DBS enzyme activity (with inhibition) = 3.5 μmol/L/h

FamilyGenotypingMethod: GAA analyzed with Sanger Sequencing for entire coding region and conserved exon-intron splice junctions, PCR performed for deletion screening

VariantAllele1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

VariantAllele2: NM_000152.4(GAA):c.1799G>A (p.Arg600His)

ClinVar: 370130, https://www.ncbi.nlm.nih.gov/clinvar/variation/370130/

gnomAD: 8.675e-06, http://exac.broadinstitute.org/variant/17-78086421-G-A

Case: Patient A, male, 14 years old at WIL-29 study entry; ethnicity not reported.

DiseaseAssertion: VWD3. The patient was initially suspected to have VWD2A based on the family history but was reclassified as VWD3 after next-generation sequencing.

FamilyInfo: Patient A and Patient B are brothers from a consanguineous family. Both parents were diagnosed with VWD. Figure 2 shows both parents as heterozygous and Patient A and Patient B as homozygous for the familial VWF variant. The pedigree also includes another brother affected with VWD whose genetic testing was underway and a sister whose VWD diagnosis and genetic testing were pending.

ParentalGenotype: Both parents were heterozygous for the familial VWF variant, as shown in Figure 2.

CasePresentingHPOs: HP:0000421, HP:0030129, HP:0012147 (Epistaxis, Impaired ristocetin cofactor assay activity, Reduced quantity of VWF)

CaseHPOFreeText: Recurrent spontaneous epistaxis. Historical VWF:RCo 1% and VWF:Ag 10%. VWF:RCo 1 IU/dL at WIL-31 screening. At the month 0 pharmacokinetic visit, pre-injection FVIII 12 IU/dL and VWF:RCo <5 IU/dL; after 60 IU/kg wilate, virtually no rise in VWF activity (maximum VWF:RCo 9 IU/dL, in vivo recovery 0.07 kg/dL). VWF inhibitors were confirmed present at WIL-31 study entry, and the patient remained inhibitor-positive at the 12-month visit.

CaseNotHPOs: NA

CaseNotHPOFreeText: No anaphylactic reactions or severe treatment-related adverse events were reported.

CasePreviousTesting: Diagnosed with VWD shortly after birth and initially suspected to have VWD2A based on family history. Historical VWF activity and antigen testing, and subsequent pharmacokinetic and VWF inhibitor testing, were performed. No earlier molecular genetic testing was reported.

GenotypingMethod: Next-generation sequencing performed in Bonn, Germany. Additional technical details regarding the sequencing assay or panel were not provided.

Gene: VWF

Variant: c.4094T>C p.Leu1365Pro, homozygous

HGVS: NM_000552.5:c.4094T>C (p.Leu1365Pro)

ClinVar: 2438545

CAID: NA

gnomAD: Absent (not reported in gnomAD; 0/251040 control chromosomes)

Case: Patient B, male, 11 years old at WIL-29 study entry; ethnicity not reported.

DiseaseAssertion: VWD3. The patient was initially suspected to have VWD2A based on the family history but was reclassified as VWD3 after next-generation sequencing.

FamilyInfo: Patient B and Patient A are brothers from a consanguineous family. Both parents were diagnosed with VWD. Figure 2 shows both parents as heterozygous and Patient A and Patient B as homozygous for the familial VWF variant. The pedigree also includes another brother affected with VWD whose genetic testing was underway and a sister whose VWD diagnosis and genetic testing were pending.

ParentalGenotype: Both parents were heterozygous for the familial VWF variant, as shown in Figure 2.

CasePresentingHPOs: HP:0000421, HP:0030129 (Epistaxis, Impaired ristocetin cofactor assay activity)

CaseHPOFreeText: Recurrent spontaneous epistaxis. Historical VWF:RCo 1%; historical VWF:Ag not measured. VWF:RCo 1 IU/dL at WIL-31 screening. At the month 0 pharmacokinetic visit, pre-injection FVIII 16 IU/dL and VWF:RCo <5 IU/dL; after 60 IU/kg wilate, maximum VWF:RCo 65 IU/dL and in vivo recovery 1.02 kg/dL. VWF inhibitors were confirmed present at WIL-31 study entry; no VWF inhibitor was detectable at the 12-month visit.

CaseNotHPOs: NA

CaseNotHPOFreeText: No anaphylactic reactions or severe treatment-related adverse events were reported.

CasePreviousTesting: Diagnosed with VWD shortly after birth and initially suspected to have VWD2A based on family history. Historical VWF activity testing, and subsequent pharmacokinetic and VWF inhibitor testing, were performed. Historical VWF antigen was not measured. No earlier molecular genetic testing was reported.

GenotypingMethod: Next-generation sequencing performed in Bonn, Germany. Additional technical details regarding the sequencing assay or panel were not provided.

Gene: VWF

Variant: c.4094T>C p.Leu1365Pro, homozygous

HGVS: NM_000552.5:c.4094T>C (p.Leu1365Pro)

ClinVar: 2438545

CAID: NA

gnomAD: Absent (not reported in gnomAD; 0/251040 control chromosomes)

Supplementary Data: Table 2

Case B: proband, Female

DiseaseAssertion: Dilated Cardiomyopathy

CasePresentingHPOs: HP:0001644 (dilated cardiomyopathy)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, partial testing (MYH7, LMNA)

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

Supplementary Data: Table 2

Case A: proband, female

DiseaseAssertion: Dilated Cardiomyopathy

CasePresentingHPOs: HP:0001644 (dilated cardiomyopathy)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, partial testing (MYH7, LMNA)

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

Supplementary Data: Table 2

Case H: proband, Female

DiseaseAssertion: left ventricular noncompaction

CasePresentingHPOs: HP:0030682 (left ventricular noncompaction)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, partial testing (MYH7)

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

Supplementary Data: Table 2

Case F: proband, Female

DiseaseAssertion: Left ventricular noncompaction

CasePresentingHPOs: HP:0030682 (left ventricular noncompaction)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1), additional testing listed in supplemental table 2

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

Supplementary Data: Table 2

Case J: proband, Male

DiseaseAssertion: Dilated Cardiomyopathy

CasePresentingHPOs: HP:0001644 (dilated cardiomyopathy)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1), additional testing listed in supplemental table 2

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

Supplementary Data: Table 2

Case D: proband, female

DiseaseAssertion: Hypertrophic cardiomyopathy

CasePresentingHPOs: HP:0001639 (hypertrophic cardiomyopathy)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1), additional testing listed in supplemental table 2

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

MultipleGeneVariants:

(1) DSC2 Variant:

Variant: NM_004949.4(DSC2):c.2194T>G (p.Leu732Val)

ClinVar: 36005, https://www.ncbi.nlm.nih.gov/clinvar/variation/36005/

gnomAD: 0.001181, https://gnomad.broadinstitute.org/variant/18-28650748-A-C

(2) DSG2 Variant:

Variant: NM_001943.4(DSG2):c.1174G>A (p.Val392Ile)

ClinVar: 36009, https://www.ncbi.nlm.nih.gov/clinvar/variation/36009/

gnomAD: 0.002137, https://gnomad.broadinstitute.org/variant/18-29111109-G-A

(3) DSP Variant:

Variant: NM_001008844.2(DSP):c.1778A>G (p.Asn593Ser)

ClinVar: 44867, https://www.ncbi.nlm.nih.gov/clinvar/variation/44867/

gnomAD: 0.0005695, https://gnomad.broadinstitute.org/variant/6-7571692-A-G

Supplementary Data: Table 2

Case C: proband, Male

DiseaseAssertion: Dilated Cardiomyopathy

CasePresentingHPOs: HP:0001644 (dilated cardiomyopathy)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, partial testing (MYH7, CSRP3, DES, LMNA, MYBPC3, TNNT2, TNNI3)

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

Supplementary Data: Table 2

Case E: proband, male

DiseaseAssertion: Dilated Cardiomyopathy

CasePresentingHPOs: HP:0001644 (dilated cardiomyopathy)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1), additional testing listed in supplemental table 2

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

MultipleGeneVariants:

(1) PRDM16 Variant:

Variant: NM_022114.3(PRDM16):c.2666C>T (p.Pro889Leu)

ClinVar: 487607, https://www.ncbi.nlm.nih.gov/clinvar/variation/487607/

gnomAD: 0.0001771, https://gnomad.broadinstitute.org/variant/1-3331186-C-T

Supplementary Data: Table 2

Case G: proband, Male

DiseaseAssertion: left ventricular noncompaction

CasePresentingHPOs: HP:0030682 (left ventricular noncompaction)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, partial testing (MYH7)

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

Supplementary Data: Table 2

Case I: proband, Male

DiseaseAssertion: Dilated Cardiomyopathy

CasePresentingHPOs: HP:0001644 (dilated cardiomyopathy)

CasePreviousTesting: 12-lead electrocardiogram (ECG), echocardiography, exercise test and 24-hour (or 48-hour) Holter monitoring, cardiac MRI, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1), additional testing listed in supplemental table 2

CaseGenotypingMethod: genomic DNA isolated from peripheral blood, initially analyzed via Sanger Sequencing then by next-generation sequencing using targeted gene panels, variant pathogenicity assed using in silico prediction tools, Haplotype analysis was performed using 16 microsatellite markers around MYH7 (DNA from the 14 probands was analysed, and DNA samples of 6 family members carrying the mutation were used to verify the phase and reconstruct the haplotype Supplementary Data: Table 1)

Variant: NM_000257.2(MYH7):c.5754C>R (p.Asn1918Lys)

ClinVar: 66082, https://www.ncbi.nlm.nih.gov/clinvar/variation/66082/

gnomAD: n/a

Case: Patient 18, female, 18yo, congenital onset of symptoms

CasePresentingHPOs: HP:0003327, HP:0009053, HP:0002650, HP:0011713, HP:0003458 (axial muscle weakness, distal lower limb muscle weakness, scoliosis, left bundle branch block, EMG: myopathic abnormalities)

HPOsFreeText: respiratory involvement, NIV required from 7yo, normal CK levels

FamilyInfo: no family history

DiseaseEntity: no assertion

InheritancePattern: no assertion

CasePreviousTesting: see below

CaseGenotypingMethod: 9 cases underwent targeted Sanger sequencing of MYH7, 12 cases underwent Motorplex analysis (NGS of 93 myopathy-related genes - PMID:25214167), all MYH7 variants confirmed via Sanger sequencing, segregation analysis performed on available family members, which cases received which sequencing technique is not specified

Variant: NM_000257.4(MYH7):c.5655G>A (p.Ala1885=)

ClinVar: 378215, https://www.ncbi.nlm.nih.gov/clinvar/variation/378215/

gnomAD: absent

Case: Patient 19, female, 33yo, symptom onset in childhood

CasePresentingHPOs: HP:0009027, HP:0008959, HP:0009053, HP:0008997, HP:0008994, HP:0002650, HP:0003458 (foot drop, Distal upper limb muscle weakness, Distal lower limb muscle weakness, Proximal muscle weakness in upper limbs, Proximal muscle weakness in lower limbs, scoliosis, EMG: myopathic abnormalities)

HPOsFreeText: respiratory involvement - restrictive from 25yo, normal CK levels, minicores present on muscle biopsy

CaseNotHPOs: no cardiac involvement

FamilyInfo: family history

DiseaseEntity: no assertion

InheritancePattern: no assertion

CasePreviousTesting: see below

CaseGenotypingMethod: 9 cases underwent targeted Sanger sequencing of MYH7, 12 cases underwent Motorplex analysis (NGS of 93 myopathy-related genes - PMID:25214167), all MYH7 variants confirmed via Sanger sequencing, segregation analysis performed on available family members, which cases received which sequencing technique is not specified

Variant: NM_000257.4(MYH7):c.4844_4846AGA[2] (p.Lys1617del)

ClinVar: 190401, https://www.ncbi.nlm.nih.gov/clinvar/variation/190401/

gnomAD: absent

Case: 6, femaly, 16yo, congenital onset of symptoms

CasePresentingHPOs: HP:0003327, HP:0008959, HP:0009053, HP:0002650 (axial muscle weakness, distal upper limb muscle weakness, distal lower limb muscle weakness, scoliosis)

HPOsFreeText: normal CK

CaseNotHPOs:

NotHPOsFreeText: no respiratory or cardiac involvement

FamilyInfo: family history, pedigree in Figure 1

DiseaseEntity: no assertion

InheritancePattern: no assertion

CasePreviousTesting: see below

CaseGenotypingMethod: 9 cases underwent targeted Sanger sequencing of MYH7, 12 cases underwent Motorplex analysis (NGS of 93 myopathy-related genes - PMID:25214167), all MYH7 variants confirmed via Sanger sequencing, segregation analysis performed on available family members, which cases received which sequencing technique is not specified

Variant: NM_000257.4(MYH7):c.4807G>C (p.Ala1603Pro)

ClinVar: 190407, https://www.ncbi.nlm.nih.gov/clinvar/variation/190407/

gnomAD: absent

Case: Patient 17, male, 36yo, symptom onset in childhood

CasePresentingHPOs: HP:0003327, HP:0008959, HP:0009053 (axial muscle weakness, Distal upper limb muscle weakness, Distal lower limb muscle weakness)

HPOsFreeText: normal CK levels

CaseNotHPOs: HP:0002650 (scoliosis)

NotHPOsFreeText: no respiratory or cardiac involvement

FamilyInfo: no family history

DiseaseEntity: no assertion

InheritancePattern: no assertion

CasePreviousTesting: see below

CaseGenotypingMethod: 9 cases underwent targeted Sanger sequencing of MYH7, 12 cases underwent Motorplex analysis (NGS of 93 myopathy-related genes - PMID:25214167), all MYH7 variants confirmed via Sanger sequencing, segregation analysis performed on available family members, which cases received which sequencing technique is not specified

Variant: NM_000257.4(MYH7):c.5779A>T (p.Ile1927Phe)

ClinVar: 487639, https://www.ncbi.nlm.nih.gov/clinvar/variation/487639/

gnomAD: 0.00004598, https://gnomad.broadinstitute.org/variant/14-23882979-T-A?dataset=gnomad_r2_1

Case: Patient 4, male, 22yo, symptom onset in childhood

CasePresentingHPOs: HP:0009027, HP:0003327, HP:0008959, HP:0009053, HP:0001701, HP:0030682 (foot drop, axial muscle weakness, distal upper limb muscle weakness, distal lower limb muscle weakness, pericarditis, left ventricular noncompaction)

CaseNotHPOs: HP:0003306 (rigid spine)

HPOsFreeText: restrictive respiratory involvement from age 16, CK = 711U/l, minicores present on muscle biopsy, hypertrophy of gastrocnemii

FamilyInfo: no family history

DiseaseEntity: no assertion

InheritancePattern: no assertion

CasePreviousTesting: see below; variants in SEPN1 and LMNA rules out

CaseGenotypingMethod: 9 cases underwent targeted Sanger sequencing of MYH7, 12 cases underwent Motorplex analysis (NGS of 93 myopathy-related genes - PMID:25214167), all MYH7 variants confirmed via Sanger sequencing, segregation analysis performed on available family members, which cases received which sequencing technique is not specified

Variant: NM_000257.4(MYH7):c.4844_4846AGA[2] (p.Lys1617del)

ClinVar: 190401, https://www.ncbi.nlm.nih.gov/clinvar/variation/190401/

gnomAD: absent

Case: Patient 13, female, 59yo, symptom onset in adulthood

CasePresentingHPOs: HP:0003701, HP:0003306, HP:0003458 (proximal muscle weakness, rigid spine, EMG: myopathic abnormalities)

HPOsFreeText: normal CK levels, cores present on muscle biopsy

CaseNotHPOs: no respiratory or cardiac involvement

FamilyInfo: family history

DiseaseEntity: no assertion

InheritancePattern: no assertion

CasePreviousTesting: see below

CaseGenotypingMethod: 9 cases underwent targeted Sanger sequencing of MYH7, 12 cases underwent Motorplex analysis (NGS of 93 myopathy-related genes - PMID:25214167), all MYH7 variants confirmed via Sanger sequencing, segregation analysis performed on available family members, which cases received which sequencing technique is not specified

Variant: NM_000257.4(MYH7):c.4315G>C (p.Ala1439Pro)

ClinVar: 190404, https://www.ncbi.nlm.nih.gov/clinvar/variation/190404/

gnomAD: absent

Case: Patient 8, male, 35yo, symptom onset in childhood

CasePresentingHPOs: HP:0009027, HP:0003327, HP:0002460, HP:0002650, HP:0003458 (foot drop, axial muscle weakness, distal muscle weakness, scoliosis, EMG: myopathic abnormalities)

HPOsFreeText: CK = 720U/l, neurogenic EMG, cores present on muscle biopsy

CaseNotHPOs:

NotHPOsFreeText: no respiratory or cardiac involvement

FamilyInfo: pedigree in Figure 1

DiseaseEntity: no assertion

InheritancePattern: no assertion

CasePreviousTesting: see below

CaseGenotypingMethod: 9 cases underwent targeted Sanger sequencing of MYH7, 12 cases underwent Motorplex analysis (NGS of 93 myopathy-related genes - PMID:25214167), all MYH7 variants confirmed via Sanger sequencing, segregation analysis performed on available family members, which cases received which sequencing technique is not specified

Variant 1: NM_000257.4(MYH7):c.4844_4846AGA[2] (p.Lys1617del)

ClinVar: 190401, https://www.ncbi.nlm.nih.gov/clinvar/variation/190401/

gnomAD: absent

Variant 2: NM_000257.4(MYH7):c.4855G>A(p.Glu1619Lys)

ClinVar: 181268, https://www.ncbi.nlm.nih.gov/clinvar/variation/181268/

gnomAD: absent

Table III

Case 748: Italian, Female, 16yo

DiseaseAssertion: HCM

FamilyInfo: Family history of HCM and sudden cardiac death

CasePresentingHPOs: HP:0001712, HP:0004756, HP:0005110, HP:0001716 (left ventricular hypertrophy, ventricular tachycardia, atrial fibrillation, Wolff-Parkinson-White syndrome)

CaseHPOFreeText: maximum left ventricular wall thickness of ≥15 mm, LVWT = 17mm, automatic implantable cardioverter defibrillator, LV ejection fraction = 60%

CasePreviousTesting: echocardiography, Full testing (MYBPC3, MYH7, TNNT2, TNNI3, TPM1, MYL2, MYL3, ACTC1), additional testing: (GLA, LAMP2, PRKAG2), additional genes added with NGS: (TNNC1, MYH6, MYOM1, MYOZ2, ANKRD1, VCL, CALR3, CAV3)

CaseGenotypingMethod: Genomic DNA extracted from peripheral leukocytes, Sanger sequencing (73 samples), Next-gen sequencing (19 samples)

Variant: NM_000257.3(MYH7):c.5287G>A (p.Ala1763Thr)

ClinVar: 177846, https://www.ncbi.nlm.nih.gov/clinvar/variation/177846/

gnomAD: 0.00007423, https://gnomad.broadinstitute.org/variant/14-23884476-C-T

MultipleGeneVariants:

(1) LAMP2 Variant:

Variant: NM_002294.2(LAMP2):c.928G>A (p.Val310Ile)

ClinVar: 9982, https://www.ncbi.nlm.nih.gov/clinvar/variation/9982/

gnomAD: n/a

Case4: 47 year old male

DiseaseAssertion: Carried the MYBPC3 R326Q mutation

FamilyInfo:His father had onset of symptoms at 77 years of age and is stable on medication. Two asymptomatic young children (M21, III:1 and :2) had no signs of hypertrophy or dilatation on echocardiography and normal ECG.

ParentalTesting: NR

CasePresentingHPOs: HP:0011713, HP:0034307, HP:0012665, HP:0001635

CaseHPOFreeText: Had onset of symptoms at 42 years, LBBB on ECG and required cardiac resynchronization therapy. Has left ventricular end diastolic diameter (in mm and indexed by body surface area (cm/m2) of 81 mm and 3.6 cm/m2. A left ventricular ejection fraction as 20%. ECG as left bundle branch block. Clinical status as DCM/ congestive heart failure. CAG as normal. As well with cardiac resynchronization therapy.

CaseNotHPOs: HP:0011675

CaseNotHPOFreeText: No arrhythmia

CasePreviousTesting:NR

Genotyping Method:Genetically screened, also using GenBank with PCR by amplifying the coding region

FunctionalAnalysis: It results in the replacement of a conserved charged arginine with neutral glutamine at residue 326 in the phosphorylatable MyBPC motif of cMyBPC. This charge change is likely to cause functional impairment.

Variant:NM_000256.3:c.977G>A

ClinVar:42813

CAID:CA016212

gnomAD: gnomAD v2: 11:47367871 C / T gnomAD v3: 11:47346320 C / T gnomAD v4: chr11-47346320-C-T

AdditionalInfo:NR

Case5: 65 year-old male

DiseaseAssertion: carried a known TNNT2 K247R mutation as well as two novel intronic variants in MYH7 (IVS39+7C>A) and MYBPC3 (IVS25+13T>A)

FamilyInfo: None of his five siblings were affected, judged by their clinical presentation, nor was there any history of cardiac disease or sudden death in the family.

ParentalTesting:NR

CasePresentingHPOs:HP:0003677, HP:0012828, HP:0001635, HP:0005110, HP:0034307, HP:0012666,

CaseHPOFreeText: had late-onset at age 47 IDC, with a slow progression to severe heart failure and subsequently HTX. He also suffered from atrial fibrillation in the later stages of his disease. Had left ventricular end diastolic diameter (in mm and indexed by body surface area (cm/m2)) with a value of 68mm and 3.4cm/m2. As well with 30% of left ventricular ejection fraction. His ECG is normal, clinical status is heart transplantation from the age of 56. CAG is normal.

CaseNotHPOs:NR

CaseNotHPOFreeText:NR

CasePreviousTesting:NR

Genotyping Method:Genetically screened, also using GenBank with PCR by amplifying the coding region

FunctionalAnalysis: The variant could thus possibly result in the synthesis of a limited amount of the slightly modified β-myosin. The functional significance of this is not easily established, but it suggests that the variant may be a disease modifier.

Variant:NM_001276345.2:c.869A>G

ClinVar:4881578

CAID:CA344201883

gnomAD:NR

AdditionalInfo: MYH7 (IVS39+7C>A) increased the likelihood of a cryptic donor splice site to 70%, possibly resulting in the synthesis of mRNA coding for a β-myosin with a C terminus of –VGLE instead of –GLNEE. As the variant is located in the last exon, the faulty spliced mRNA is probably not degraded but results in the synthesis of a modified protein.

Case 7: 47 year old male

DiseaseAssertion: novel MYH7 R1832C in the index case

FamilyInfo:His father died at the age of 47 years due to IDC, whereas a sibling of the index case died suddenly within her first year. It was not possible to obtain DNA from the deceased relatives.

ParentalTesting:NR

CasePresentingHPOs:HP:0012828,HP:0012835, HP:0034307, HP:0012666

CaseHPOFreeText: He had an early onset of symptoms, in his third decade as being the age of 33, with severe dilatation of his left ventricle and depressed LVEF, and required HTX 3 years after the onset of symptoms. A left ventricular end diastolic diameter (in mm and indexed by body surface area (cm/m2)); value of 86mm and 3.2cm/m2. Has a left ventricular ejection fraction of 20%. His clinical status is heart transplantation.

CaseNotHPOs:NR

CaseNotHPOFreeText:NR

CasePreviousTesting:NR

Genotyping Method:NR

FunctionalAnalysis:NR

Variant:NM_000257.4:c.5494C>T

ClinVar:161322

CAID:CA016134

gnomAD: gnomAD v2: 14:23884269 G / A gnomAD v3: 14:23415060 G / A gnomAD v4: chr14-23415060-G-A

AdditionalInfo: This mutation is located in the rod portion of MYH7 and replaces a basic arginine residue with a reactive neutral cysteine residue in the g-position of the heptad repeat in the α-helical myosin rod, and thus may destabilize the salt bridges required for myosin–myosin dimerization.

Case8: 56 year old male

DiseaseAssertion: A double heterozygous for the known MYBPC3 R326Q mutation.

FamilyInfo: His father died due to IDC in his fourth decade, whereas his brother developed IDC. DNA was not available from either of these individuals.

ParentalTesting:NR

CasePresentingHPOs:HP:0005110, HP:0034307,HP:0012666, HP:0011675, HP:0005110, HP:0100651

CaseHPOFreeText: He also developed atrial fibrillation, well regulated on medication. Had onset conditions at age 36. He had left ventricular end diastolic diameter (in mm and indexed by body surface area (cm/m2)); as a value of 65mm and 3.2 cm/m2. A LVEF, left ventricular ejection fraction as 15%. Arrhythmia level as atrial fibrillation. Clinical status as heart transplantation. He also has type 1 diabetes mellitus

CaseNotHPOs:NR

CaseNotHPOFreeText:NR

CasePreviousTesting:NR

Genotyping Method:NR

FunctionalAnalysis:NR

Variant:NM_000256.3:c.977G>A

ClinVar:42813

CAID:CA016212

gnomAD:gnomAD v2: 11:47367871 C / T gnomAD v3: 11:47346320 C / T gnomAD v4: chr11-47346320-C-T

AdditionalInfo:NR

Case1: A 68-year-old female

DiseaseAssertion: dilated cardiomyopathy (DCM), with the MYH7 p.Lys637Glu (K637E) variant identified in the affected patient. The study investigated sarcomeric gene variants associated with DCM

FamilyInfo: The proband's father (M1, II:3) died suddenly at 36 years of age, 2 years after being diagnosed with DCM.

ParentalTesting: Yes from father

CasePresentingHPOs: HP:0011664, HP:0011713, HP:0003677, HP:0001635,

CaseHPOFreeText: was diagnosed as having left ventricular non-compaction (LVNC) based on echocardiography. A left bundle branch block on her ECG. Echocardiography revealed LVNC and she has had a slow progression towards heart failure and an increasing dilatation of her left ventricle. Age 48 at onset, left ventricular end diastolic diameter 59 mm, left ventricular ejection fraction 25%, ventricular extrasystoles for arrhythmia.

CaseNotHPOs:NR

CaseNotHPOFreeText:NR

CasePreviousTesting:NR

Genotyping Method: genetically screened, also using GenBank with PCR by amplifying the coding region

FunctionalAnalysis:NR

Variant: NM_000257.4:c.1909A>G

ClinVar: 1329402

CAID:CA389049558

gnomAD: NR

AdditionalInfo: NR

Case 3: A 64 year old female

DiseaseAssertion: The case carried the novel MYH7 K637E mutation

FamilyInfo: Her mother died of a suspected but unconfirmed cardiac cause; thus, this was regarded as a non-FDC case. Two healthy sisters declined participation in the study.

ParentalTesting:NR

CasePresentingHPOs: HP:0003674, HP:0034307, HP:0002372, HP:0011675, HP:0005110

CaseHPOFreeText: Slowly from onset of symptoms at the age of 42 years, and received HTX in her sixth decade. Has left ventricular end diastolic diameter (in mm and indexed by body surface area (cm/m2)), 81mm and then 4.2m2. LVEF as 15%, ECG as normal. Arrhythmia as atrial fibrillation. NYHA as 4. Clinical status as heart transplantation. CAG has a normal level.

CaseNotHPOs:NR

CaseNotHPOFreeText:NR

CasePreviousTesting:NR

Genotyping Method: Genetically screened, also using GenBank with PCR by amplifying the coding region

FunctionalAnalysis: NR

Variant:NM_000257.4:c.1909A>G

ClinVar:1329402

CAID:CA389049558

gnomAD:NR

AdditionalInfo:NR

Case6: 48 year old male

DiseaseAssertion: Carried the MYBPC3 E619K mutation

FamilyInfo:Three unaffected children were examined (IV:1, IV:2 and IV:3), all with normal echocardiography without signs of hypertrophy or dilatation. Both affected individuals as well as a 26-year-old unaffected daughter (M29, IV:3) carried the MYBPC3 E619K mutation

ParentalTesting:NR

CasePresentingHPOs: HP:0034307, HP:0012666, HP:0011713, HP:0001635

CaseHPOFreeText: Onset of symptoms in their third decade; the index case required heart transplantation 3 years after the onset of symptoms. Has onset at the age of 37. Has left ventricular end diastolic diameter (in mm and indexed by body surface area (cm/m2)) with the value of 79 mm and 3.2 cm/m2. They have a 20% of left ventricular ejection fraction. ECG as left bundle branch block. Clinical status as DCM/ congestive heart failure. CAG as normal.

CaseNotHPOs:NR

CaseNotHPOFreeText:NR

CasePreviousTesting:NR

Genotyping Method:Genetically screened, also using GenBank with PCR by amplifying the coding region

FunctionalAnalysis:NR

Variant: NM_000256.3:c.1855G>A

ClinVar:161303

CAID:CA011336

gnomAD: gnomAD v2: 11:47362731 C / T gnomAD v3: 11:47341180 C / T gnomAD v4: chr11-47341180-C-T

AdditionalInfo:NR

CaseWJ: 30 years diagnosis, Australia

DiseaseAssertion: Hypertrophic Cardiomyopathy

FamilyInfo: One affected relative Brother (index case) died suddenly aged 34 y (MYH7 p.Asp778Val only)

CasePresentingHPOs: HP:0001639 (Hypertrophic cardiomyopathy)

HPOsFreeText: Maximum left ventricular hypertrophy at 18 mm

CaseNotHPOs: N/A

NotHPOsFreeText: Sudden cardiac death event

CaseAddInfo: N/A

CasePreviousTesting: See Table 1

CaseGenotypingMethod: DNA was isolated from peripheral blood. Most participants underwent testing from the Illumina Cardiomyopathy Sequencing Panel, which includes 46 cardiomyopathy related genes. For others, whole exome sequencing or Sanger squencing was used. After the results were returned, variants were filtered for pathogenicity and rarity.

Variant1: NM_000257.3:c.2333A>T (p.Asp778Val)

ClinVar: 454354 https://www.ncbi.nlm.nih.gov/clinvar/variation/454354/

gnomAD: Not in gnomAD

Variant2: NM_000257.3:c.5452C>T (p.Arg1818Trp)

ClinVar: 217826 https://www.ncbi.nlm.nih.gov/clinvar/variation/217826/

gnomAD: Latino 5.782e-5, Overall 7.956e-6 https://gnomad.broadinstitute.org/variant/14-23884311-G-A

CaseAJV: 17 years diagnosis, Australia

DiseaseAssertion: Hypertrophic Cardiomyopathy

FamilyInfo: Father (index case) died awaiting cardiac transplant (carried both variants). Two possibly affected relatives.

CasePresentingHPOs: HP:0001639, HP:0006536 (Hypertrophic cardiomyopathy, Obstructive lung disease)

HPOsFreeText: Maximum left ventricular hypertrophy at 17 mm, Sudden cardiac death event at 17 years, Maximal wall thickness at 22mm,

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

CasePreviousTesting: See Table 1

CaseGenotypingMethod: DNA was isolated from peripheral blood. Most participants underwent testing from the Illumina Cardiomyopathy Sequencing Panel, which includes 46 cardiomyopathy related genes. For others, whole exome sequencing or Sanger squencing was used. After the results were returned, variants were filtered for pathogenicity and rarity.

Variant: NM_000257.3:c.1954A>G (p.Arg652Gly)

ClinVar: 177626 https://www.ncbi.nlm.nih.gov/clinvar/variation/177626/

gnomAD: Not in gnomAD

Multiple Gene Variants:

MYBPC3 Variant

Variant: NM_000256.3:c.2980C>T (p.Leu994Phe)

ClinVar: 180992 https://www.ncbi.nlm.nih.gov/clinvar/variation/180992/

gnomAD: European (Non-Finnish) 1.624e-4, Overall 8.461e-5 https://gnomad.broadinstitute.org/variant/11-47355487-G-A

Case KMP 15: Eastern Slovakia, Female, 82yo

DiseaseAssertion: hypertrophic cardiomyopathy

FamilyInfo: positive family history

CaseHPOFreeText: left ventricular outflow tract obstruction, left ventricular ejection fraction = 60%, New York Heart Association Classification = III, implantable cardioverter defibrillator

CasePreviousTesting: 12-lead ECG, echocardiography, partial testing (MYBPC3, MYH7, NEBL, SCN5A, TNNI3, TNNT2)

CaseGenotypingMethod: genomic DNA collected from peripheral blood leukocytes, Sanger sequencing

Variant: NM_000257.3(MYH7):c.2734A>G (p.Lys912Glu)

ClinVar: 520267, https://www.ncbi.nlm.nih.gov/clinvar/variation/520267/

gnomAD: n/a

Case#: KMP 15, female, 82 yrs, East Slovakia

DiseaseAssertion: HCM

FamilyInfo: yes

CasePresentingHPOs: n/a

CaseHPOFreeText: symptomatic, LVOTO yes; LVEF 60%; NYHA III.; ICD yes; ECG; echocardiography;

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a

CasePreviousTesting: Additional Sanger sequencing of exons 12, 24, 25 of MYBPC3, exon 12 NEBL, exons 12, 20, 21 of SCN5A, exon 7 of TNNI3, and exon 9 of TNNT2 gene

CaseGenotypingMethod: Sanger sequencing;

Variant: NM_000257.4(MYH7):c.2734A>G (p.Lys912Glu)

ClinVar: 520267 https://www.ncbi.nlm.nih.gov/clinvar/variation/520267/

CAID: n/a

gnomAD: absent

Case: Patient UK1-IV:1, female, 31yo, Caucasian, Irish ancestry

CasePresentingHPOs: HP:0040083, HP:0002650, HP:0001508, HP:0011471, HP:0002093, HP:0002877, HP:0001620 (toe-walking, scoliosis, poor weight gain, Feeding problem necessitating gastrostomy tube feeding, respiratory impairment, nocturnal hypoventilation, high-pitched voice)

HPOsFreeText: scoliosis required spinal fusion surgery, required nocturnal BiPAP ventilation in adolescence, reduced muscle bulk

Muscle biopsy showed variability in myofiber size, uniformity of type I myofibers on ATPase, moth-eaten appearance of myofibers

CaseNotHPOs:

FamilyInfo: non-consanguineous family

CasePreviousTesting: 32 Illumina genes

CaseGenotypingMethod: NGS (Illumina gene panel) or 32 genes previously associated with congenital myopathies, Sanger sequencing to confirm recessive inheritance

InheritancePattern: autosomal recessive

DiseaseEntity:

Variant 1: NM_000257.3:c.4699C>T (p.Gln1657Ter), maternal

CAID: CA389037829, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA389037829

gnomAD: n/a

Variant 2: NM_000257.4(MYH7):c.4664A>G (p.Glu1555Gly), paternal

ClinVar: 181261, https://www.ncbi.nlm.nih.gov/clinvar/variation/181261/

gnomAD: n/a

Case IV-5: Male, Iranian, 30yo at diagnosis, 31yo at study, proband

DiseaseAssertion: DCM (initially); paper notes that phenotype is more consisted with hypokinetic non-dilated cardiomyopathy based on guideline from ESC working group on myocardial and pericardial diseases (PMID: 26792875)

FamilyInfo: consanguineous family, pedigree in fig. 1

CasePresentingHPOs: HP:0001712, HP:0002375, HP:000516 (left ventricular hypertrophy, hypokinesia, left ventricular dysfunction)

CaseHPOFreeText: New York Heart Association Class = II, left ventricular ejection fraction = 20-25%, left ventricular internal end-diastolic diameter = 5.7cm, left ventricular internal end-systolic diameter = 5.2cm, interventricular septum = 0.8cm, posterior wall = 0.9cm, normal ECG and normal sinus rhythm, Normal heart valve function

CaseNotHPOFreeText: Ischemic heart disease

CasePreviousTesting: ECG, Full testing (https://hyp.is/2c24ajtuEemXQ-fRmGkjTA/www.sciencedirect.com/science/article/pii/S1769721217300484)

CaseGenotypingMethod: WES, targeted exome sequence capture using SureSelectXT Human All Excon v5 kig (Agilent), co-segregation analysis via Sanger sequencing

Variant: NM_000257.3(MYH7):c.2863G>A (p.Asp955Asn)

ClinVar: 264608, https://www.ncbi.nlm.nih.gov/clinvar/variation/264608/

gnomAD: 0.000003976, https://gnomad.broadinstitute.org/variant/14-23893175-C-T

MultipleGeneVariants:

(1) TNNT2 Variant:

Variant: NM_000364.3(TNNT2):c.247A>C (p.Asn83His)

ClinVar: 427814, https://www.ncbi.nlm.nih.gov/clinvar/variation/427814/

gnomAD: n/a

Case IV-8: Female, Iranian, 36yo

DiseaseAssertion: DCM

FamilyInfo: consanguineous family, pedigree in fig. 1

CasePresentingHPOs: HP:0005162 (left ventricular dysfunction)

CaseHPOFreeText: New York Heart Association Class = I, left ventricular ejection fraction = 60%, left ventricular internal end-diastolic diameter = 5.0cm, left ventricular internal end-systolic diameter = 3.1cm, interventricular septum = 0.7cm, posterior wall = 0.7cm, normal sinus rhythm, Normal heart valve function

CaseNotHPOFreeText: Ischemic heart disease

CasePreviousTesting: ECG

CaseGenotypingMethod: co-segregation analysis via Sanger sequencing

Variant: NM_000257.3(MYH7):c.2863G>A (p.Asp955Asn)

ClinVar: 264608, https://www.ncbi.nlm.nih.gov/clinvar/variation/264608/

gnomAD: 0.000003976, https://gnomad.broadinstitute.org/variant/14-23893175-C-T

Case III-10: Female, Iranian, 60yo

DiseaseAssertion: DCM

FamilyInfo: consanguineous family, pedigree in fig. 1

CasePresentingHPOs: HP:0001653, HP:0011713, HP:0005162, HP:0005185, HP:0002375 (mitral regurgitation, left bundle branch block, left ventricular dysfunction, systolic dysfunction, hypokinesia)

CaseHPOFreeText: New York Heart Association Class = II, left ventricular ejection fraction = 15-20%, left ventricular internal end-diastolic diameter = 5.6cm, left ventricular internal end-systolic diameter = 4.3cm, interventricular septum = 0.7cm, posterior wall = 0.6cm, Normal heart valve function, grade II diastolic dysfunction

CaseNotHPOFreeText: Ischemic heart disease

CasePreviousTesting: ECG

CaseGenotypingMethod: co-segregation analysis via Sanger sequencing

Variant: NM_000257.3(MYH7):c.2863G>A (p.Asp955Asn)

ClinVar: 264608, https://www.ncbi.nlm.nih.gov/clinvar/variation/264608/

gnomAD: 0.000003976, https://gnomad.broadinstitute.org/variant/14-23893175-C-T

MultipleGeneVariants:

(1) TNNT2 Variant:

Variant: NM_000364.3(TNNT2):c.247A>C (p.Asn83His)

ClinVar: 427814, https://www.ncbi.nlm.nih.gov/clinvar/variation/427814/

gnomAD: n/a

Case IV-6: Female, Iranian, 34yo at diagnosis, 37yo at study

DiseaseAssertion: DCM (initially); paper notes that phenotype is more consisted with hypokinetic non-dilated cardiomyopathy based on guideline from ESC working group on myocardial and pericardial diseases (PMID: 26792875)

FamilyInfo: consanguineous family, pedigree in fig. 1

CasePresentingHPOs: HP:0001653, HP:0002375 (mitral regurgitation, hypokinesia)

CaseHPOFreeText: New York Heart Association Class = II, left ventricular ejection fraction = 35%, left ventricular internal end-diastolic diameter = 5.2cm, left ventricular internal end-systolic diameter = 4.2cm, interventricular septum = 0.9cm, posterior wall = 1.0cm, normal sinus rhythm, grade I diastolic function

CaseNotHPOFreeText:

CasePreviousTesting: ECG, Full testing (https://hyp.is/2c24ajtuEemXQ-fRmGkjTA/www.sciencedirect.com/science/article/pii/S1769721217300484)

CaseGenotypingMethod: https://hyp.is/IKjfsDtxEem3Sh8RHHdYSg/www.sciencedirect.com/science/article/pii/S1769721217300484) WES, targeted exome sequence capture using SureSelectXT Human All Excon v5 kig (Agilent), co-segregation analysis via Sanger sequencing

Variant: NM_000257.3(MYH7):c.2863G>A (p.Asp955Asn)

ClinVar: 264608, https://www.ncbi.nlm.nih.gov/clinvar/variation/264608/

gnomAD: 0.000003976, https://gnomad.broadinstitute.org/variant/14-23893175-C-T

MultipleGeneVariants:

(1) TNNT2 Variant:

Variant: NM_000364.3(TNNT2):c.247A>C (p.Asn83His)

ClinVar: 427814, https://www.ncbi.nlm.nih.gov/clinvar/variation/427814/

gnomAD: n/a

Table 5

Case LO1: Italian

DiseaseAssertion: HCM (late-onset)

FamilyInfo: positive family history

CasePreviousTesting: echocardiography, Full testing (MYBPC3, MYH7, TNNT2, TNNI3, TPM1, MYL2, MYL3, ACTC1), additional testing: (GLA, LAMP2, PRKAG2, TNNC1, MYH6, CSPR3, PLN, TCAP, CAV3), electrocardiography

CaseGenotypingMethod: Genomic DNA extracted from periphera whole blood, emulsion PCR, 17 HCM genes sequenced using Personal Genome Machine (PGM) IonTorrent sequencer, variants confirmed via Sanger sequencing

Variant: NM_000257.3(MYH7):c.3133C>T (p.Arg1045Cys)

ClinVar: 177753, https://www.ncbi.nlm.nih.gov/clinvar/variation/177753/

gnomAD: 0.00002828, https://gnomad.broadinstitute.org/variant/14-23891501-G-A

Case#: Case III-2, female, 74, early adulthood onset

DiseaseAssertion: Congenital Fiber Type Disproportion

FamilyInfo: Family A, late-onset weakness in her deceased mother (II-5) and maternal aunt (II-4), and lifelong weakness in her older sister (III-3)

CasePresentingHPOs: lower limb muscle weakness, gait disturbance, hearing loss, psoriasiform dermatitis, osteoarthritis, glaucoma, hypertension, muscle dystrophy, left ventricular diastolic dysfunction, hyperlordosis, genu recurvatum, proximal lower limb amyotrophy, foot dorsiflexor weakness, hip flexor weakness (HP:0007340, HP:0001288, HP:0000365, HP:0003765, HP:0002758, HP:0000501, HP:0000822, HP:0003560, HP:0025168, HP:0003307, HP:0002816, HP:0008956, HP:0009027, HP:0012515)

CaseHPOFreeText: Pes cavus and hammertor deformities, bilataeral steppage in gait,

CaseNotHPOs: N/A

CaseNotHPOFreeText: myotatic reflexes normal, ECG normal, normal NCS, no fibrillation potentials, sharp waves, or myotonic discharges

CasePreviousTesting: Analysis was negative for sequence variants affecting other congenital myopathy genes associated with CFTD pathology including ACTA1, RYR1, SEPN1, TPM2, and TPM3

CaseGenotypingMethod: neurological examination, serum creatine kinase (60 unit/L), nerve conduction studies, concentric needle electromyography (early recruitment of small polyphasic MUAP in diffuse pattern), electrocardiogram, echocardiogram, forced vital capacity (2.13% (76)), muscle biopsy, immunohistochemical analyses,

Variant: NM_000257.4(MYH7):c.484T>G (p.Tyr162Asp)

ClinVar: 525040 https://www.ncbi.nlm.nih.gov/clinvar/variation/525040/

CAID: N/A

gnomAD: absent

Case: Case report, female, 41yo

CasePresentingHPOs: HP:0001270, HP:0002094, HP:0007340, HP:0010535, HP:0002540, HP:0003323, HP:0002015, HP:0100749, HP:0001662, HP:0005952 (motor delay, dyspnea, lower limb weakness, sleep apnea, inability to walk, progressive muscle weakness, dysphagia, chest pain, bradychardia, decreased pulmonary function)

HPOsFreeText: non-smoker, onset of neuromuscular disorders in early childhood, written language delay, difficulty completing exercise, dyspnea and lower limb weakness presenting during patient's first pregnancy (33yo), required mechanical ventilation machine, symptoms of skeletal muscle disorder began after delivery of first child, unable to get up without assistance, unable to climb stairs, required use of cane, walker, then eventually a wheelchair, episodes of paralysis in lower limbs, normal tone/bulk of arm muscles but bilateral weakness in shoulders and upper limbs (MRC scale of 3/5), intact fine finger movements, down-going toes, reduced perception of vibration in lower limbs, needle EMG showed tendency of myotonic potentials, muscle biopsy of left quadricep showed features of end-stage process consistent with chronic myopathy (Fig. 2A-D), forced vital capacity = 27%, forced expiratory volume per second = 29%

CaseNotHPOs: HP:0001513, HP:0040129 (obese, abnormal nerve conduction velocity)

NotHPOsFreeText: pronator drift, sensory deficit, ocular muscle involvement

FamilyInfo: mother died at 66yo due to heart attack associated with non-compaction cardiomyopathy, daughter (11yo) has signs of muscle disorders, congenital muscular torticollis, excessive growing pains, and underdeveloped muscles in half of body

CaseGenotypingMethod: WES, variants confirmed via Sanger Sequencing

Variant: NM_000257.4(MYH7):c.1370T>G (p.Ile457Arg)

ClinVar: 560881, https://www.ncbi.nlm.nih.gov/clinvar/variation/560881/

gnomAD: absent

MultipleGeneVariants:

(1) SNCB Variant

Variant: NM_003085.5(SNCB):c.368C>A (p.Pro123His)

ClinVar: 7026, https://www.ncbi.nlm.nih.gov/clinvar/variation/7026/

gnomAD: 0.0001929, https://gnomad.broadinstitute.org/variant/5-176048219-G-T

(2) GDF6 Variant:

Variant: NM_001001557.4(GDF6):c.746C>A (p.Ala249Glu)

ClinVar: 8371, https://www.ncbi.nlm.nih.gov/clinvar/variation/8371/

gnomAD: 0.001948, https://gnomad.broadinstitute.org/variant/8-97157413-G-T

CaseVIII: Brazil, Caucasian

DiseaseAssertion: Hypertrophic cardiomyopathy

FamilyInfo: Family history of HCM, 1 other relative without phenotypes

CasePresentingHPOs: HP:0001712, HP:0001639, HP:0011103 (Left ventricular hypertrophy, Hypertrophic cardiomyopathy, Abnormality of the left ventricular outflow tract)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Cosegregation confirmed for MYH7

CasePreviousTesting: Only MYH7, MYBPC3, and TNNT2 were tested.

CaseGenotypingMethod: DNA was extracted from peripheral blood and the above genes were subject to PCR amplification and directly sequenced. The resulting sequences were compared with the reference and filtered for pathogenicity.

Variant: NM_000257.3:c.4402G>A (p.Glu1468Lys)

ClinVar: 235033 https://www.ncbi.nlm.nih.gov/clinvar/variation/235033/

gnomAD: Not in gnomAD

Multiple Gene Variants

MYBPC3 Variant

Variant: NM_000256.3:c.2686G>A (p.Val896Met)

ClinVar: 42652 https://www.ncbi.nlm.nih.gov/clinvar/variation/42652/

gnomAD: European (Finnish) 0.02794, Overall 0.006194 https://gnomad.broadinstitute.org/variant/11-47357479-C-T

Case#: HCM516, male, 23 years,

DiseaseAssertion: HCM

FamilyInfo: Yes, father and mother has HCM

CasePresentingHPOs: syncope, chest tightness (HP:0001279, HP:0031352)

CaseHPOFreeText: inverted T-waves, ST-segment depression, 28mm MLVWT, 67% LVE F, 0.64 E/A ratio, LVOT-PG 44mm

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

CaseGenotypingMethod: sequencing, physical examination, ECG, 3D modeling, statistical analysis

MultipleGeneVariants:

(1) Variant: NM_000257.4(MYH7):c.2631G>A (p.Met877Ile)

ClinVar: 955476 https://www.ncbi.nlm.nih.gov/clinvar/variation/955476/

CAID: N/A

gnomAD: absent

(2) Variant: NM_000257.4(MYH7):c.3134G>A (p.Arg1045His)

ClinVar: 651054 https://www.ncbi.nlm.nih.gov/clinvar/variation/651054/

CAID: N/A

gnomAD: absent

CaseHCM133: 64 yrs report, Vietnam, Asian

DiseaseAssertion: Hypertrophic Cardiomyopathy

FamilyInfo: Family history of sudden cardiac death

CasePresentingHPOs: HP:0100749, HP:0001712, HP:0031295 (Chest pain, Left ventricular hypertrophy, Left atrial enlargement)

HPOsFreeText: Maximal LV thickness 21 mm

CaseNotHPOs: HP:0005110, HP:0001297, HP:0004756 (Atrial fibrillation, Stroke, Ventricular tachycardia)

NotHPOsFreeText: N/A

CaseAddInfo: N/A

CasePreviousTesting: The gene panel included 10 sarcomeric genes (MYH7, MYBPC3, TNNT2, TNNI3, TPM1, MYL2, MYL3, ACTC1, TNNC1, MYH6), 9 genes encoding Z-disk components and Ca2+ homeostasis factors (LDB3, CSRP3, TCAP, VCL, ACTN2, MYOZ2, NEXN, JPH2, PLN), and 4 genes involved in HCM phenocopies (GLA, LAMP2, PRKAG2, TTR), All probands had left ventricular wall thickness >= 15 mm and no other diseases that can cause secondary hypertrophy

CaseGenotypingMethod: The coding region, flanking sequences, and untranslated regions of each gene were enriched by LC-PCR, amplified, and sequenced on the Illumina MiSeq or MiniSeq platform. Identified variants were then filtered for pathogenicity and confirmed by Sanger sequencing.

Variant: NM_000257.4(MYH7):c.3551A>T (p.Gln1184Leu)

ClinVar: 181226 https://www.ncbi.nlm.nih.gov/clinvar/variation/181226/

gnomAD: 0.0006115, http://gnomad.broadinstitute.org/variant/14-23889229-T-A

Multiple Gene Variants

(1) MYH6 Variant

Variant: NM_002471.3(MYH6):c.1252G>A (p.Val418Met)

ClinVar: 44449 https://www.ncbi.nlm.nih.gov/clinvar/variation/44449/

gnomAD: 0.001954, http://gnomad.broadinstitute.org/variant/14-23870076-C-T

Case#: 1. Patient II-4 43 years

DiseaseAssertion: Hypertrophic cardiomyopathy and left ventricular noncompaction

FamilyInfo: Several family members have heart disease. One relative died from cardiac arrest at age 50.

ParentalTesting: NR

CasePresentingHPOs: NR

CaseHPOFreeText: The patient had thickened heart muscle, abnormal heartbeats, and extra ridges and deep spaces in the heart muscle.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: ECG, heart monitoring, heart ultrasound, and genetic testing

Genotyping Method: Next- generation sequencing and sanger sequencing

FunctionalAnalysis: computer testing suggested the ACTC1 gene change could weaken the protein

Variant: NM_005159.4:c.62C>T

Clinvar: 410264

CAID: CA16614451

gnomAD: NR

AdditionalInfo: His heart pumping ability was 55%. This article reported changes in his heart muscle cells.

Case#: 2 Patient II-5 46 years old

DiseaseAssertion: HCM and LVNC

FamilyInfo: Several family members have heart disease. One relative died from cardiac arrest at age 50.

ParentalTesting: NR

CasePresentingHPOs: NR

CaseHPOFreeText: The patient had thickened and enlarged heart muscle, abnormal heartbeats, and deep spaces in his heart muscle. He als had temporary episodes of reduced blood flow to the brain.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: ECG, heart monitoring, heart ultrasound, heart MRI, heart biopsy and genetic testing

Genotyping Method: NGS and sanger sequencing

FunctionalAnalysis:computer testing suggested the ACTC1 gene change could weaken the protein.

Variant: NM_005159.4:c.62C>T

Clinvar: 410264

CAID: CA16614451

gnomAD: NR

AdditionalInfo: His heart pumping ability was 30%. He received a defibrillator and was considered for a heat transplant. He also had a second gene change in TTN.

Case#: 3 Patient II-1 10 years

DiseaseAssertion: left ventricular noncompaction

FamilyInfo: Several family members have heart disease. One relative died from cardiac arrest at age 50.

ParentalTesting: NR

CasePresentingHPOs: NR

CaseHPOFreeText: The patient had extra ridges and deep spaces in his heart muscle. His ECG also showed changes in his heart's electrical activity.

CaseNotHPOs: NR

CaseNotHPOFreeText: Normal heart muscle thickness and normal heart pumping ability.

CasePreviousTesting: ECG, heart monitoring, heart ultrasound, heart biopsy and genetic testing

Genotyping Method: NGS and sanger sequencing

FunctionalAnalysis:computer testing suggested the ACTC1 gene change could weaken the protein.

Variant: NM_005159.4:c.62C>T

Clinvar: 410264

CAID: CA16614451

gnomAD: NR

AdditionalInfo: His heart pumping ability was 63%.He received medication and remained stable during follow up.

Case#: 4 Patient III-2 14 years

DiseaseAssertion: HCM and LVNC

FamilyInfo: Several family members have heart disease. One relative died from cardiac arrest at age 50.

ParentalTesting: NR

CasePresentingHPOs: NR

CaseHPOFreeText: The patient had thickened heart muscle extra ridges and deep spaces in his heart, and episodes of fast or abnormal hearbeats.

CaseNotHPOs: NR

CaseNotHPOFreeText: Heart MRI did not show sign of heart muscle scarring

CasePreviousTesting: ECG, heart monitoring, heart ultrasound, heart biopsy and genetic testing

Genotyping Method: NGS and sanger sequencing

FunctionalAnalysis:computer testing suggested the ACTC1 gene change could weaken the protein.

Variant: NM_005159.4:c.62C>T

Clinvar: 410264

CAID: CA16614451

gnomAD: NR

AdditionalInfo: His heart pumping ability was 60%.He received medication and remained stable during follow up.

Case2: Full term male

DiseaseAssertion: poor feeding, respiratory distress, and cyanosis

FamilyInfo: Healthy non‐consanguineous parents. Positive for HCM on the mother's side: the maternal grandfather died from HCM at the age of 52 years, and a brother of the maternal grandfather was also affected. The parents and siblings of the patient showed no abnormalities on cardiological examination.

ParentalTesting:

CasePresentingHPOs: HP:0001714, HP:0011623, HP:0010438

CaseHPOFreeText: On cardiac catheterisation, both ventricles were found to be hypertrophic. There was mild shunting at the level of the foramen ovale. The pressures in the right ventricle and pulmonary artery were pathologically high, while the pressures in the left atrium and ventricle were normal (though with high A waves). There was a total left to right shunt of 43% of the pulmonary circulation, partly caused by a low muscular ventricular septal defect.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: NR

FunctionalAnalysis: NR

Variant: NM_001242672.3(TTC34):c.2827C>T (p.Arg943Trp)

ClinVar: 3329883

CAID: NR

gnomAD: 1-2573220-G-A

AdditionalInfo: NR

Case1: gestational age 40 weeks female

DiseaseAssertion: Poor feeding, cyanosis, and difficulty with breathing.

FamilyInfo: Healthy non‐consanguineous parents. Echocardiographic in the mother and sister were normal. Father had mild dilatation of the left atrium with an abnormal flow pattern in the mitral valve, otherwise healthy.

ParentalTesting: c.2373_2374insG mutation found in the mother

CasePresentingHPOs:HP:0025169,

CaseHPOFreeText: hypertrophic non‐obstructive cardiomyopathy with poor left and right ventricular systolic function. The left ventricular end diastolic diameter measured 17.3 mm (99% of normal), end systolic diameter 12.9 mm (116% of normal), and left ventricular posterior wall 3.6 mm (111% of normal). The intraventricular septum measured 11.8 mm (288% of normal). The shortening fraction of the left ventricle was decreased to 25%. Multiple abnormal small communications with a minimal flow between a coronary artery and the right ventricle were observed in the apical portion of the interventricular septum. There were no structural congenital heart defects.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method:

FunctionalAnalysis: NR

Variant: NM_006073.4(TRDN):c.1624+1G>A

ClinVar:2152664

CAID: CA147231745

gnomAD:NR

AdditionalInfo:NR

Case3: 39-year-old man

DiseaseAssertion: seronegative rheumatoid arthritis

FamilyInfo: Father was diagnosed with hypertrophic cardiomyopathy

ParentalTesting: Genetic testing revealed a pathogenic MYBPC3 variant in the patient and his father (c.655G>C [p.Val219Leu])

CasePresentingHPOs: HP:5210340, HP:0006682, HP:0031971

CaseHPOFreeText: Rccentric septal hypertrophy with a maximal wall thickness of 1.7 cm, no evidence of outflow tract obstruction, and an LV EF of 65% to 70%. Rare premature ventricular complexes. Mild mediastinal lymphadenopathy, pulmonary sarcoidosis

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: NR

FunctionalAnalysis: NR

Variant: NM_006252.4(PRKAA2):c.148_151del (p.Gln50fs)

ClinVar:4891957

CAID:NR

gnomAD:NR

AdditionalInfo:NR

Case#: Pedigree I-1; 74-year-old Korean male; admitted for dyspnea (NYHA III), palpitations, and bilateral pitting leg edema over several months

DiseaseAssertion: Idiopathic restrictive cardiomyopathy (RCM) with biatrial enlargement, in a family with MYBPC3-related cardiomyopathy

FamilyInfo: Family history of heart disease with two affected daughters (II-2 and II-4), both heterozygous for the same variant and both diagnosed with HCM. Unaffected granddaughter (III-3) tested negative. Both daughters showed asymmetric septal hypertrophy on TTE (18 mm and 17 mm) with mildly dilated LA; daughter II-2 had grade I diastolic dysfunction and mild exertional chest discomfort. CMR confirmed reverse septal curvature hypertrophy consistent with HCM in both daughters, with no LGE in either.⁠

ParentalTesting: Not performed; the proband is the index case in generation I; his parents were not evaluated. Phase established only through the daughters and granddaughter.

CasePresentingHPOs:HP:0002094; HP:0001962; HP:0001677; HP:0001635; HP:0001639; HP:0001698

CaseHPOFreeText: Exertional dyspnea NYHA class III, palpitations, and bilateral leg pitting edema for several months; dyspnea refractory to heart failure medication, with pulmonary venous congestion. ECG and Holter: atrial fibrillation with rare premature ventricular complexes. TTE: RCM with biatrial enlargement and small pericardial effusion. CMR: normal LV chamber size, borderline diffuse LV hypertrophy, very large atria, extensive transmural LGE in the apical wall and mid-myocardial patchy LGE in the ventricular septum. Coronary angiogram normal; no secondary cause of RCM found.⁠

CaseNotHPOs: HP:0000822; HP:0000819; HP:0001658; HP:0001678

CaseNotHPOFreeText: Coronary angiogram normal, so no obstructive coronary disease. No secondary cause of RCM identified; no inflammatory, infiltrative, or systemic disease found. No AV block; the rhythm disturbance was atrial fibrillation.

CasePreviousTesting: Whole exome sequencing (Agilent SureSelect XT Human All Exon v5, Illumina HiSeq2500, GATK. Sanger sequencing of family members showed only the MYBPC3 variant tracked with disease; the other four (KALRN c.1702C>T, KCNH2 c.442C>T, SKI c.30T>G, TTN c.54293T>C) did not correlate with phenotype.⁠

Genotyping Method: WES in the proband, with annotation via dbNSFP and Ensembl VEP; Sanger sequencing confirmed the MYBPC3 frameshift in the proband (I-1), daughters II-2 and II-4, and its absence in granddaughter III-3 (Fig. 3B).⁠

FunctionalAnalysis: None performed. CMR showed extensive LGE in the apical LV, and the authors propose that advanced myocardial fibrosis in end-stage HCM can provoke myocardial atrophy, explaining the relatively normal LV chamber size with a predominantly restrictive phenotype hypothesis.⁠

Variant: MYBPC3 NM_000256.3:c.3313_3314insGG (p.Ala1105Glyfs*85); heterozygous frameshift insertion; absent from gnomAD and the Korean Reference Genome Database.

ClinVar: 1473634

CAID: Not reported.

Gene:MYBPC3

HGNC: HGNC: 7551

PMID: 33803538

gnomAD: Absent per the authors' check of gnomAD as of 8 November 2020, and absent from KRGDB.

AdditionalInfo: Authors conclude the MYBPC3 frameshift is the pathogenic variant, with the same allele producing RCM in the proband and HCM in both daughters; an example of pleiotropy and variable expressivity within one family. The frameshift has previously been reported in Korean HCM. Poor-prognosis features in RCM cited by the authors: male sex, age over 70, high NYHA class, and LA dimension >60 mm.

Case#: P16, 17 year old male

DiseaseAssertion: left ventricular noncompaction cardiomyopathy

FamilyInfo: His mother, sister, and grandfather on his mothers side also had LVNC

ParentalTesting: NR

CasePresentingHPOs: NR

CaseHPOFreeText: The patient had an abnormal structure in the left side of his heart. His heart ultrasound showed extra muscle ridges and deep spaces in the heart muscle.

CaseNotHPOs: NR

CaseNotHPOFreeText: The study did not report fainting, heart rhythm problems, blood clots, or heart failure for this patient.

CasePreviousTesting: Heart ultrasound, ECG, heart MRI, blood tests, and genetic testing.

Genotyping Method: Next generation sequencing followed by Sanger sequencing to confirm this gene change

FunctionalAnalysis: NR

Variant: NM_005159.5:c.329>T

Clinvar: 2137647

CAID: CA391631478

gnomAD: Exact frequency not provided

AdditionalInfo: The patient's heart pumping ability was 70 %on ultrasound. Researchers classified his ACTC1 variant as a variant of uncertain significance meaning they were not sure whether it causes diseases.

Case1: 40-year-old Chinese male

DiseaseAssertion: exertional dyspnea for one year and substernal chest pain for one week.

FamilyInfo: Father experienced sudden cardiac death (SCD) at the age of 41. Targeted region capture and second-generation high throughput sequencing were performed on the proband’s cousin, cousin sister, and sister. Results demonstrated that all family members carried the MYBPC3 c.1827dupC variant. Pedigree in Fig. 3

ParentalTesting: NR

CasePresentingHPOs: HP:0025169, HP:0025168, HP:0031675, HP:0001714,

CaseHPOFreeText: harsh systolic ejection murmur at the left sternal border (3rd–4th intercostal spaces), accentuated by Valsalva maneuver. Echocardiography revealed the following key findings: severe asymmetric septal hypertrophy, characterized by an interventricular septal thickness (IVsd) at end-diastole of 25–30 mm and a left ventricular posterior wall dimensions (LVPWd) of 21 mm, with a preserved left ventricular ejection fraction (LVEF) of 59%

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: target-region capture sequencing and second-generation high throughput sequencing were performed to analyze all exons and their Flanking regions. Rare and known variants were validated using Sanger sequencing and cascade screening was carried out for all available relatives

Genotyping Method: target-region capture sequencing and second-generation high throughput sequencing, Sanger sequencing and cascade screening.

FunctionalAnalysis:NR

Variant: NM_000256.3:c.1827dupC, NM_004415.4:c.1324T>C

ClinVar:NR

CAID:UnregisteredVariant