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#: 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#: Single case; 37-year-old male construction worker; sudden unexplained death (SUD); found dead in bed ~1 hr after lunch, August 2005; molecular autopsy via WES in 2019 from autopsy tissue

DiseaseAssertion: Sudden unexplained death (SUD); no structural cardiac cause at autopsy; MYBPC3/TTN/ACADVL/RYR2-related per WES reevaluation

FamilyInfo: No family history or segregation data reported. The paper is a single molecular autopsy; no relatives were genotyped or phenotyped. No pedigree assessment performed.

ParentalTesting: Not performed, no family members tested.

CasePresentingHPOs: HP:0001698 (Sudden cardiac death); HP:0004756 (Ventricular tachycardia), the latter only as a speculative arrhythmic mechanism, not a documented clinical event

CaseHPOFreeText: Found dead in bed ~1 hr after lunch in August 2005. External examination showed no traumatic injury; toxicological blood screen negative. Clinical history unremarkable. Heart weighed 350 g (reference 331±57 g in men) with regular morphology and volume. Microscopy showed small foci of interstitial fibrosis, deemed not significant. No other organ showed significant abnormality. Case dismissed as SUD, reevaluated in 2019.

CaseNotHPOs: HP:0001639; HP:0001644; HP:0004308; HP:0001279

CaseNotHPOFreeText: No structural cardiac disease at autopsy, no hypertrophy, no dilation, normal heart morphology and volume. No evidence of any cardiomyopathy phenotype. No documented arrhythmia or syncope in life.

CasePreviousTesting: Autopsy (2005) with toxicology, histology. WES (2019) on DNA from FFPE left ventricular tissue; 562-gene cardiac panel (234 SCD/heart-disease genes + 328 heart-expressed genes); filtering against gnomAD (rare = MAF <0.1%); in silico prediction with DANN, MutationTaster, FATHMM, MutationAssessor, PolyPhen-2, SIFT, PROVEAN; ACMG 2015 classification; WebGestalt enrichment analysis.

Genotyping Method: WES on Illumina 500 (TruSeq library prep, xGen exome capture), SeqMule pipeline, ENLIS Genome Research annotation; variants confirmed by Sanger sequencing. Aligned to GRCh37.

FunctionalAnalysis: None performed by the authors, no functional assays in this paper. (ACADVL p.V383A residual activity ~20% is cited from a 2007 external study, not performed here.)

Variant: Four heterozygous missense variants, one per gene

ClinVar:926037

CAID: CA221696968

gnomAD: N/A

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#: 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.

Case1: 11 year old female

DiseaseAssertion: hypertrophic cardiomyopathy, restrictive cardiomyopathy

FamilyInfo: twin sister carrier identical mutation, both sisters expired, no family history of cardiomyopathy

ParentalTesting: sanger sequencing

CasePresentingHPOs:HP:0001629, HP:0001640, HP:0034314,HP:0002098, HP:0001723

CaseHPOFreeText:bilateral atrial dilation with mid ventricular hypertrophy, accelerated cardiac sound, liver palpable 2cm below costal margin, cold extremities, cardiac condition was New York Heart Association class (NYHA) III.

CaseNotHPOs: NR

CaseNotHPOFreeText: no gallop rhythm, no murmur, no blood test abnormalities, no metabolic disorders

CasePreviousTesting: cardiac catheterization, cardiac magnetic resonance, echocardiography, Chest Xray, electrocardiography,

Genotyping Method: sanger sequencing

FunctionalAnalysis: NR

Variant: TNNI3 c.574 C>T p.Arg192Cys

ClinVar: 165510

CAID: CA021951

gnomAD:NR

Case 2: 15 year old female

DiseaseAssertion: restrictive cardiomyopathy, hypertrophic cardiomyopathy

FamilyInfo: twin sister carrier identical mutation, both sisters expired, no family history of cardiomyopathy

ParentalTesting: Sanger Sequencing

CasePresentingHPOs:HP:0002875, HP:0001723

CaseHPOFreeText: Abnormal ECG, LV hypertrophy, enlargement of both atria, high LVEF levels, accelerated cardiac sound, liver was palpable 3 cm, extremities were warm, and cardiac condition was NYHA II.

CaseNotHPOs:NR

CaseNotHPOFreeText: no gallop rhythm, no murmur, no blood test abnormalities, no metabolic disorders

CasePreviousTesting: cardiac catheterization, cardiac auscultation, myocardial disarray with myocyte hypertrophy

Genotyping Method: Sanger sequencing

FunctionalAnalysis: NR

Variant: TNNI3 c.574 C>T p.Arg192Cys

ClinVar: 165510

CAID: CA021951

gnomAD:NR

Case#:7-year-old boy

DiseaseAssertion: restrictive cardiomyopathy

FamilyInfo: his parents were in generally good health and other family members denied a history of syncope, sudden death or cardiomyopathy

ParentalTesting: sanger sequencing

CasePresentingHPOs:HP:0007185, HP:0001695, HP:0005943, HP:0002516, HP:0001663

CaseHPOFreeText:electrocardiogram (ECG) revealed Ptf-V 1 < –0.04 mm⋅s and biphasic P waves, indicating left atrial enlargement and the chest X-ray revealed an enlarged heart shadow. The echocardiogram showed biatrial enlargement, especially in the left atrium (LA: 32 mm, RA: 30 mm), decreased diastolic function (E/A = 2.6; E Peak deceleration time 130 ms; Constant volume diastolic time 49 ms), moderate mitral valve regurgitation and left ventricular ejection fraction of 51.8%.

CaseNotHPOs:HP:0000969

CaseNotHPOFreeText:Except for bilateral enlargement, cardiac magnetic resonance (CMR) indicated no abnormalities

CasePreviousTesting: brain MRI, video electrocephalogram, electrocardiogram

Genotyping Method: sanger sequencing

FunctionalAnalysis:

Variant: TNNI3 c.611(exon8)G>A,p.R204H

ClinVar:177679

CAID:CA022060

gnomAD:chr19-55151856-C-T

Case#: Female, 20, non-Hispanic white

DiseaseAssertion: Peripartum cardiomyopathy

FamilyInfo: NR

ParentalTesting: NR

CasePresentingHPOs: HP:0034807, HP:0001640

CaseHPOFreeText: NA

CaseNotHPOs: NA

CaseNotHPOFreeText: NA

CasePreviousTesting: NR

Genotyping Method: Sanger sequencing

FunctionalAnalysis: demonstrated decreased calcium sensitivity, which indicated that this mutation was likely to be disease causing

Variant: NM_001276345.2:c.505C>G

ClinVar: 2953222

CAID: CA344204620

gnomAD: NR

AdditionalInfo: PPCM was diagnosed one week postpartum.

Case1: 2-year-old female

DiseaseAssertion: restrictive cardiomyopathy, dilated cardiomyopathy

FamilyInfo:The maternal family history was negative for any form of cardiomyopathy or sudden death. Paternal family history was unknown, the mother (1-II:2) and maternal half-brother (1-III:2) were both heterozygous for the TNNI3 p.Arg136∗ variant, mother (1-II:2, age 20) and half-brother (1-III:2, age 21 months) demonstrated normal heart function on echocardiography. ParentalTesting: Clinical exome sequencing done on mother of proband

CasePresentingHPOs: HP:0032025, HP:0002140, HP:0005180, HP:0001653, HP:0005117

CaseHPOFreeText: left-sided facial and extremity weakness, enlarged cardiac silhouette with interstitial opacities, dilated left and right atria, unusual morphology of the chordae tendineae of both atrioventricular valves was noted, leading to a tethering phenomenon

CaseNotHPOs: HP:0001712,

CaseNotHPOFreeText: normal systolic function

CasePreviousTesting: Chest Xray, Echocardiogram, Head CT

Genotyping Method: clinical exome sequencing

FunctionalAnalysis: Immunostaining on heart tissue from patient, Quantitative analysis of heart tissue, Transmission electron microscopy with immersion fixed heart samples

Variant:homozygous TNNI3 nonsense variant, c.406C>T (p.Arg136∗)

ClinVar:488853

CAID:CA407440677

gnomAD:chr19-55154173-G-A v4

Case#: A 2-year-old girl with dilated cardiomyopathy (DCM) and left ventricular noncompaction, who received a heart transplant at 26 months.

DiseaseAssertion: DCM with left ventricular noncompaction

FamilyInfo: Family history revealed that three of the father’s (A.II.2) sisters (A.II.4-A.II.6) died in infancy due to cardiac failure.

ParentalTesting: The father and paternal grandmother tested positive for the same TNNT2 variant

CasePresentingHPOs: HP:0001644, HP:0030682, HP:0001635

CaseHPOFreeText: The heart failure signs began at 2 months and got worse. Then got a heart transplant at 26 months.

CaseNotHPOs: not reported

CaseNotHPOFreeText: nothing found

CasePreviousTesting: a genetic panel test, including the genes LDB3, LMNA, MYH7, MYBPC3, and TNNT2, was performed.

Genotyping Method: Variants were confirmed in first-degree family members by Sanger sequencing.

FunctionalAnalysis: not reported

Variant: c.316G>A (p.Glu106Lys)

ClinVar: 224775

CAID: CA088557

gnomAD: not reported

AdditionalInfo: not reported

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#: 33 year-old female; first cardiologic evaluation at age 24 (LV wall thickness 13 mm); re-evaluated at 33 after recurrent palpitations

DiseaseAssertion: Noonan syndrome (PTPN11) with sarcomeric hypertrophic cardiomyopathy (MYBPC3); non-obstructive HCM, mild phenotype

FamilyInfo: Father carries the MYBPC3 c.1790G>A variant and has HCM; the PTPN11 variant was absent in both parents, consistent with de novo. Both parents and the proband's brothers underwent full cardiologic evaluation, no cardiovascular disease in the other family members. Parents declined genetic testing of the underage siblings. Grandparents (I-1, I-2) and II-2, II-3 have unknown phenotypes (pedigree Fig. 2).

ParentalTesting: Both parents genotyped for the two proband variants (Table 1). Father heterozygous for MYBPC3 c.1790G>A; neither parent carries PTPN11 c.1510A>G, supporting de novo status. Phase between the two variants therefore established as in trans (different genes).

CasePresentingHPOs: HP:0001639; HP:0001962; HP:0004322; HP:0000271; HP:0001647; HP:0000508

CaseHPOFreeText: Regular psychophysical development. First echo at 24 showed mild increase in LV wall thickness (13 mm). At 33, presented with several episodes of palpitations; exam showed mild facial dysmorphisms (wide mouth, sloping forehead, prominent nasal bridge, prominent nose, low-set ears), short stature, and a systolic heart murmur. Echo: asymmetric LV hypertrophy, maximum wall thickness 16 mm at the anterior interventricular septum, mild mitral regurgitation, non-obstructive. No other cardiovascular signs.

CaseNotHPOs: HP:0001635; HP:0001650; HP:0001642; HP:0001250

CaseNotHPOFreeText: No heart failure, no LV outflow obstruction, no pulmonary stenosis, no other congenital heart defect. No intellectual disability; psychophysical development was regular. No other signs of cardiovascular disease on examination.

CasePreviousTesting: NGS panel of 325 cardiomyopathy-associated genes (Supplementary Table S1); MAF threshold 5%; variants validated by Sanger sequencing; cascade testing restricted to the two proband variants.

Genotyping Method: NGS targeted panel (Maxwell 16 DNA extraction, Agilent Alyssa analysis, Illumina Variant Interpreter, in-house pipeline) with Sanger validation; parental Sanger genotyping for the same two variants.

FunctionalAnalysis: None performed by the authors. They cite prior HeLa cell work showing the MYBPC3 c.1790G>A change causes exon 18 inclusion in the transcript and an almost undetectable 248 bp product.

Variant 1: PTPN11 NM_002834.3:c.1510A>G (p.Met504Val); heterozygous missense; de novo; classified pathogenic, explains Noonan syndrome

ClinVar: Not stated in the paper; verify the current record before submitting.

CAID: N/A

gnomAD: Not reported in the paper; I have not checked gnomAD for this variant.

Variant 2: MYBPC3 NM_000256.3:c.1790G>A (p.Arg597Gln); heterozygous missense; exon 18, last nucleotide of the exon, affecting a highly conserved residue and causing a splicing variation; inherited from the father

ClinVar: Not stated in the paper; authors note it is classified pathogenic/likely pathogenic for HCM in several genetic databases.

CAID: N/A

gnomAD: N/A

PMID: 32824488

Gene: MYBPC3

AdditionalInfo: Authors describe this as the first reported young adult with HCM and mild NS carrying variants in both PTPN11 and MYBPC3. Note the terminology problem: the abstract calls them "compound heterozygous," but the variants are in different genes on different chromosomes, so this is digenic (double heterozygosity), not compound heterozygosity. The authors argue the mild phenotype contrasts with the usual expectation that multiple sarcomeric hits worsen the disease, and that red flags justified the broad panel.

Case1: 59-year-old male

DiseaseAssertion: frequent premature ventricular contractions

FamilyInfo:Significant for dilated cardiomyopathy with multiple sudden cardiac deaths. His sister’s initial genetic testing panel was negative for pathogenic variants at that time

ParentalTesting:NR

CasePresentingHPOs: HP:0006682, HP:0025169, HP:0004751, HP:5210340

CaseHPOFreeText: Transthoracic echocardiogram (TTE) demonstrated a left ventricular (LV) ejection fraction (EF) of 20%-25% and an LV diastolic diameter of 6.6 cm. revealed bulky mediastinal adenopathy. A transbronchial biopsy of lung parenchyma showed noncaseating granulomas, and ultimately the patient was found to have pulmonary sarcoidosis.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting:NR

Genotyping Method: NR

FunctionalAnalysis: NR

Variant:NM_001458.5(FLNC):c.4737+5G>A

ClinVar: 934656

CAID: CA1139660256

gnomAD: NR

AdditionalInfo: NR

Case#: Family #6; 36-year-old male

DiseaseAssertion: Arrhythmogenic right ventricular cardiomyopathy (ARVC); diagnosed according to the 2010 revised Task Force Criteria

FamilyInfo: Family #6. No informative segregation evidence specifically linking the MYL3 variant to ARVC was reported. The authors noted that family screening was incomplete or normal in other families, but did not specify which circumstance applied to Family #6.

ParentalTesting: NR

CasePresentingHPOs: HP:0006682 (PVCs)

CaseHPOFreeText: Symptomatic presentation; major right ventricular structural abnormality; minor ECG repolarization criterion; 1,181 ventricular ectopic beats on Holter monitoring; sustained ventricular arrhythmia (specific rhythm not reported).

CaseNotHPOs: HP:0001279 (Syncope); HP:0005162 (Abnormal left ventricular function)

CaseNotHPOFreeText: No cardiac syncope or left ventricular dysfunction reported. Absence of cardiac syncope does not necessarily exclude every form of syncope.

CasePreviousTesting: No pathogenic or likely pathogenic variants identified in established ARVC-associated genes (PKP2, DSG2, DSP, DSC2, JUP, TMEM43, PLN, SCN5A). Subsequent screening included nine sarcomere genes: ACTC1, MYBPC3, MYH7, MYL2, MYL3, TNNC1, TNNI3, TNNT2, and TPM1. The study used broad genetic testing, including whole-exome sequencing in a subset of probands; whether Family #6 underwent whole-exome sequencing was not specified.

Genotyping Method: Sarcomere-gene sequencing and deletion/duplication analysis through clinical cardiomyopathy panels, research-based next-generation sequencing panels, or whole-exome sequencing. The exact initial platform for Family #6 was not specified. Identified variants were confirmed by PCR amplification and Sanger sequencing.

FunctionalAnalysis: NR

Variant: MYL3 NM_000258.2:c.461G>A (p.Arg154His); missense variant; exon 4

ClinVar: 14062

CAID: CA013861

gnomAD: MYL3 c.461G>A (p.Arg154His), GRCh38: 3-46859495-C-T. gnomAD v4: Exome AF = 0.00003557; Genome AF = 0.00001971; Total AF = 0.00003408. European (non-Finnish): AC = 52, AF = 0.00004407. https://gnomad.broadinstitute.org/variant/3-46859495-C-T?dataset=gnomad_r4

AdditionalInfo: Murray et al. (2018), Tables 1 and 2, Family #6. The MYL3 variant was classified as likely pathogenic (LP) according to ACMG criteria, with a PolyPhen-2 score of 0.861. The proband met criteria for definite ARVC, with no reported left ventricular dysfunction. Although the variant was rare and computationally predicted to be damaging, the authors emphasized that evidence demonstrating a causal relationship between sarcomere variants and ARVC remained insufficient. No direct functional analysis of this variant was performed in the study.

Case1: 6 year old, female

DiseaseAssertion: Restrictive cardiomyopathy

FamilyInfo:Both parents were heterozygous carriers, supporting an autosomal recessive inheritance pattern, younger brother showed no abnormalities or signs of the same illness

ParentalTesting: Sanger Sequencing

CasePresentingHPOs:HP:0002094, HP:0012378

CaseHPOFreeText: well below normal weight and height range for patients age, preserved systolic function and diastolic dysfunction, revealed preserved left ventricular systolic function with an ejection fraction, impaired diastolic function, enlarged atrium, wall thickening, impaired ventricular filling

CaseNotHPOs: NR

CaseNotHPOFreeText: comprehensive initial workup, including blood tests and urinalysis, revealed no additional abnormalities

CasePreviousTesting: Echocardiogram, blood tests, urinalysis

Genotyping Method: Whole-exome sequencing

FunctionalAnalysis: NR

Variant: TNNI3 c.406 C > T:p.R136

ClinVar: 488853

gnomAD: not found

Case#: 2 year old, male, from Yunnan Province, China

DiseaseAssertion: Restrictive cardiomyopathy

FamilyInfo:no family history of sudden cardiac death or cardiomyopathy

ParentalTesting: Sanger Sequencing

CasePresentingHPOs:HP:0034031, HP:0001635, HP:0002240,

CaseHPOFreeText:a little puffiness of the eyelids, jugular veins were full but not inflamed, atypical heart beat, edema of the lower limbs, bi-atrial enlargement, left ventricular ejection fraction of 64.6%, normal septal and ventricular wall thickness, trace pericardial effusion atrial and ventricular septum had uneven signals in the localized areas on perfusion imaging under resting state, and gadolinium-delayed enhancement showed a suspicious stripe enhancement of the septum, (Figures 1-3)

CaseNotHPOs:OMIM:145500, HP:0000819, HP:0001698,

CaseNotHPOFreeText: no history of connective tissue disease or radiation therapy, no endocardial thickening

CasePreviousTesting: electrocardiogram, cardiac magnetic resonance,

Genotyping Method: Whole-exome sequencing, sanger sequencing

FunctionalAnalysis: NR

Variant:heterozygous (c.574C > T, p.Arg192Cys) TNNI3

ClinVar: 165510

gnomAD: not found

Case1:eight-year-old female

DiseaseAssertion: Restrictive cardiomyopathy (RCM)

FamilyInfo: The patient's parents denied any positive family history of cardiac attacks, cardiovascular issues, hypertension, or coronary artery diseases. There was also no reported history of diabetes or obesity among family members. No inherited diseases, including cardiomyopathies or metabolic disorders, were identified in the family history

ParentalTesting: NR

CasePresentingHPOs:HP:0012378, HP:0001695, HP:0100539, HP:0031352, HP:0034030

CaseHPOFreeText:bilateral pedal edema, shortness of breath after engaging in physical activity, typically lasting for 15 min, enlarged bilateral atrium, segmental thickening of the interventricular septum, segmentally diffuse reduction and lack of coordination in ventricular wall pulse amplitude, mild mitral regurgitation, and diminished left ventricular systolic and diastolic function identified by increased parameters of early diastolic velocity (E wave) and low late filling velocity (A wave) (Figure 1A) abnormal bilateral atrial signals, premature atrial beats, junctional ectopic beats, first-degree atrioventricular block, complete right bundle branch block, prolonged QT interval, and abnormal Q wave (Figure 1B).

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: echocardiography, genetic screening, imaging assessments, blood gas analysis, blood cell counts, carbon dioxide retention

Genotyping Method: whole-exome sequencing (WES)

FunctionalAnalysis: computational analysis

Variant:TNNI3 c.509G>A p.R170Q

ClinVar:165516

gnomAD: not found

Case1: Male, Infant, ethnicity not stated

DiseaseAssertion: congenital fiber-type disproportion (CFTD) and fatal infantile cardiomyopathy

FamilyInfo: The father was a healthy 20-yr old with no particular medical concerns, and there was no family history of neuromuscular or genetic disorders on either side. Both parents confirmed as heterozygous carriers of the MYL2 c.188del variant by Sanger sequencing

ParentalTesting: Both parents were confirmed as heterozygous carriers of MYL2 c.188del p.(Asn63Metfs7) by Sanger sequencing. Father confirmed as heterozygous carrier of NEB c.25435C>T p.(Gln8479)

CasePresentingHPOs: HP:0001290, HP:0001252, HP:0001640, HP:0001638, HP:0001639

CaseHPOFreeText: Newborn exam revealed normal first and second heart sound with gallop rhythm and palpable liver at the right costal margin, as well as generalized hypotonia and diminished reflexes. Echocardiogram was performed at 11 d of age and revealed patent foramen ovale with left to right shunt and right atrial enlargement. At age 5 mo, the proband was admitted to hospital in decompensated heart failure and intubated on inotropic support. Echocardiography revealed left ventricular dilatation, mild to moderate mitral valve regurgitation, and left atrial enlargement, with left ventricular function moderately to severely decreased with shortening fraction of 12% and ejection fraction of 23%. A vastus lateralis biopsy performed at 5 mo of age because of persistent muscle weakness and hypotonia revealed variability in fiber size with checkerboard pattern of 1:1 ratio of type 1 and type 2 fibers exhibiting hypotrophy of type 1 fiber size in comparison to the type 2 fibers. high lactate level (7.8 mmol/L)

CaseNotHPOs: NR

CaseNotHPOFreeText: Gomori trichrome–stained light micrographs and electron microscopy (EM) showed no evidence of ragged red fiber or nemaline rods, and oxidative stains revealed no evidence of cores, targetoid, or target fibers. Normal serum creatine kinase level. Normal birth weight, length, and head circumference. There was no histopathologic evidence of Pompe disease and GAA enzyme-level testing was normal. Genetic testing for POLG and Noonan syndrome genes normal

CasePreviousTesting: Genetic testing was performed for DNA polymerase γ, catalytic subunit (POLG) and Noonan syndrome genes (PTPN11, SOS1, KRAS, RAF1, BRAF, MAP2K1, MAP2K2, and HRAS), all of which were normal. There was no histopathologic evidence of Pompe disease and GAA enzyme-level testing was normal. Muscular dystrophy was considered unlikely given the histopathologic findings and normal serum creatine kinase level.

Genotyping Method: Next-generation sequencing was performed by the Laboratory for Molecular Medicine (LMM) of Partners Healthcare Personalized Medicine using the Agilent SureSelect Design process. The gene panel utilized an all exon approach for 43 genes of interest for neuromuscular and cardiac conditions by targeting coding exons with bait out to coding sequence (CDS) ±65 bases. The complete coding regions of all genes were sequenced to average depth of 316 reads

FunctionalAnalysis: No functional analysis of the variant was performed

Variant: NM_000432.3:c.188del

ClinVar: NR

CAID: UnregisteredVariant

gnomAD: NR

AdditionalInfo: This case also has a heterozygous pathogenic NEB variant c.25435C>T p.(Gln8479*) inherited from the father, classified as pathogenic but considered unlikely to be a significant contributor to disease as no second NEB allele was identified.

Case1: 8 year old female

DiseaseAssertion: Heart failure

FamilyInfo: “ no family history of sudden cardiac death or cardiomyopathy” Pedigree in Figure 5.

ParentalTesting: Variant not found in parents

CasePresentingHPOs:HP:0010741, HP:0001723

CaseHPOFreeText: “biatrial enlargement of ST-segment depression in the inferior and lateral precordial leads” “Echocardiography revealed the enlargement of both atria…slight pericardial effusion, and a LV ejection fraction of 55%”

CaseNotHPOFreeText: “LV global longitudinal strain measured by speckle-tracking echocardiography was normal” “Late gadolinium enhancement was negative.” “no evidence of pulmonary embolism, anomalous pulmonary venous drainage, or pulmonary arteriovenous fistulas” “The following laboratory findings were normal: thyroid function, liver chemistry tests, rheumatic disease antibody levels, troponin I, creatinine, and blood urea nitrogen levels, white blood cell count, urine analysis, D-dimer levels, hepatitis panel screening, and serum free light chain concentrations; HIV antibody levels were negative.”

Genotyping Method: Next generation sequencing

Variant: c.574C > T p.R192C)

ClinVar:CA021951