Case#: 12 yr, female,

DiseaseAssertion: HCM

FamilyInfo: Father and younger brother also diagnosed, same mutation, brother asymptomatic,

CasePresentingHPOs: Syncope, Left bundle branch block, third degree atrioventricular block, amaurosis fugax, vertigo, ventricular septal hypertrophy, mitral regurgitation (HP:0001279, HP:0011713, HP:0001709, HP:0100576, HP:0002321, HP:0005144, HP:0001653)

CaseHPOFreeText: bilateral atrial enlargement to 44mm for left and 40 mm for right, interventricular septum thickened to 23mm, posterior left ventricle thickened to 10 mm, left ventricular ejection fraction of 51.8%, elevated plasma level B-type Natriuretic peptic 1165 pg/ml

CaseNotHPOs: heart murmur (HP:0030148)

CaseNotHPOFreeText: normal liver and spleen size, no edema, pericardial abnormality, no obstruction of left ventricular outflow tract, normal pasma amino acids and urine organic acids

CasePreviousTesting: whole exome sequencing

CaseGenotypingMethod: heart rate of 102/min; respiratory rate of 25/min; blood pressure of 127/74 mmHg; ECG; 175 QRS complex; Holter monitor; MRI; Etiologic investigations; Bioinformatic analysis based on Polymorphism Phenotyping v2, Sorting Intolerant From Tolerant;

Variant: NM_000257.4(MYH7):c.2155C>T (p.Arg719Trp)

ClinVar: 14104 https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

CAID: N/A

gnomAD: 0.00006481 https://gnomad.broadinstitute.org/variant/14-23895180-G-A

Table 1 and 4

Case A13: Male, 10yo, proband (III-2)

DiseaseAssertion: malignant HCM

FamilyInfo: positive family history (fig 1), grandfather died at 42yo from heart disease, father died at 40yo due to sudden cardiac death, mother (A11, II-1) had no clinical symptoms, sister (A12, III-1) diagnosed at 21yo with severe HCM

CasePresentingHPOs: HP:0100749, HP:0001279 (chest pain, syncope)

CaseHPOFreeText: interventricular septum thickness = 21.0mm, left ventricular posterior wall thickness = 12.0mm, left atrium = 40.0mm, left ventricular ejection fraction = 48%, septal asymmetry, decreased left ventricular systolic and diastolic function

CasePreviousTesting: 12-lead ECG, echocardiography, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1), additional testing (LMNA, PRKAG2, SCN5A, MYH6)

CaseGenotypingMethod: genomic DNA collected from peripheral whole blood samples, PCR, high-throughput sequencing using PGM Sequencing 400 kit, variants verified via Sanger Sequencing

Variant: NM_000257.3(MYH7):c.2155C>T (p.Arg719Trp)

ClinVar: 14104, https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: 0.00003185, https://gnomad.broadinstitute.org/variant/14-23895180-G-A

MultipleGeneVariants:

(1) PRKAG2 Variant:

Variant: NM_016203.3(PRKAG2):c.298G>A (p.Gly100Ser)

ClinVar: 36697, https://www.ncbi.nlm.nih.gov/clinvar/variation/36697/

gnomAD: 0.006770, https://gnomad.broadinstitute.org/variant/7-151478406-C-T

Table 1 and 4

Case A12: Female, 21yo

DiseaseAssertion: HCM

FamilyInfo: positive family history

CaseHPOFreeText: interventricular septum thickness = 25.9mm, left ventricular end diastolic diameter = 32.0mm, left ventricular end systolic diameter = 18.4mm, left ventricular posterior wall thickness = 9.4mm, left atrium = 26.9mm, left ventricular ejection fraction = 75%

CasePreviousTesting: 12-lead ECG, echocardiography, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1), additional testing (LMNA, PRKAG2, SCN5A, MYH6)

CaseGenotypingMethod: genomic DNA collected from peripheral whole blood samples, PCR, high-throughput sequencing using PGM Sequencing 400 kit, variants verified via Sanger Sequencing

Variant: NM_000257.3(MYH7):c.2155C>T (p.Arg719Trp)

ClinVar: 14104, https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: 0.00003185, https://gnomad.broadinstitute.org/variant/14-23895180-G-A

CaseCM1856: Male, 15 years diagnosis, Japan, Asian

CasePresentingHPOs: HP:0001639 (Hypertrophic cardiomyopathy)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001939 (Abnormality of metabolism/homeostasis)

NotHPOsFreeText: Neuromuscular disease, Malformation syndromes

CaseAddInfo: Positive family history of cardiomyopathy or sudden death, Proband found in Supplementary Table 2, Mother with an ECG abnormality and 2 brothers with HCM tested positive for this variant, Father with no phenotypes tested negative for this variant

CasePreviousTesting: 8 patients with HCM and one with RCM were analyzed by NGS for the eight sarcomere-encoding genes and MYP.

CaseGenotypingMethod: Probands were subject to PCR amplification and direct sequencing of 64 cardiomyopathy-associated genes, including the eight sarcomere-encoding genes. The variants were then filtered for rarity and tested for pathogenicity.

Variant: NM_000257.3:c.2155C>T (p.Arg719Trp)

ClinVar: 14104 https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: European (Non-Finnish) 6.481e-5, Overall 3.185e-5 https://gnomad.broadinstitute.org/variant/14-23895180-G-A

Supplementary Data Table S3

Case: 20

DiseaseEntity: HCM

HPOsFreeText: left ventricular hypertrophy (LVH) ≥15 mm

InheritancePattern: n/a

CasePreviousTesting: MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1 (59 genes total tested)

CaseGenotypingMethod: NGS of 59 genes (Supplemental Table S1), pathogenic and likely pathogenic variants confirmed via Sanger Sequencing

Variant: NM_000257.4(MYH7):c.2155C>T (p.Arg719Trp)

ClinVar: 14104, https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: 0.00003185, https://gnomad.broadinstitute.org/variant/14-23895180-G-A

MultipleGeneVariants:

(1) LDB3 Variant

Variant: NM_007078.3(LDB3):c.295C>T (p.Pro99Ser)

ClinVar: 178059, https://www.ncbi.nlm.nih.gov/clinvar/variation/178059/

gnomAD: 0.0009830, https://gnomad.broadinstitute.org/variant/10-88439888-C-T

(2) RBM20 Variant

Variant: NM_001134363.3(RBM20):c.2662G>A (p.Asp888Asn)

ClinVar: 43995, https://www.ncbi.nlm.nih.gov/clinvar/variation/43995/

gnomAD: 0.004046, https://gnomad.broadinstitute.org/variant/10-112581039-G-A

Case15: Female, 48 years diagnosis, China, Asian

DiseaseAssertion: Hypertrophic Cardiomyopathy

FamilyInfo: One parent died of cardiomyopathy/sudden death, one offspring had a heart murmur and found to have HCM

CasePresentingHPOs: HP:0001639, HP:0001279, HP:0005162, HP:0003546 (Hypertrophic cardiomyopathy, Syncope, Left ventricular failure, Poor exercise tolerance)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Heart transplantation done

CasePreviousTesting: Probands were only genotyped for MYH7 and MYBPC3 in that order, stopping once a positive finding was detected.

CaseGenotypingMethod: Genomic DNA was extracted from peripheral blood. Each gene's coding exons and flanking introns were subject to PCR amplification and sequencing. From there, any variants found were filtered for pathogenicity and low frequencies.

Variant: NM_000257.3:c.2155C>T (p.Arg719Trp)

ClinVar: 14104 https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: European (Non-Finnish) 6.481e-5, Overall 3.185e-5 https://gnomad.broadinstitute.org/variant/14-23895180-G-A

CaseMYH7-5: Japan, Asian

CasePresentingHPOs: HP:0001639, HP:0006685 (Hypertrophic cardiomyopathy, Endomyocardial fibrosis)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo:

CaseGenotypingMethod: Genomic DNA was extracted from whole blood from each proband. They each had 95 genes associated with DCM, HCM, and other inherited cardiovascular diseases sequenced with a panel, including both the exons and spicing regions. Variants were then filtered for rarity and pathogenicity.

Variant: NM_000257.3:c.2155C>T (p.Arg719Trp)

ClinVar: 14104 https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: European (Non-Finnish) 6.481e-5, Overall 3.185e-5 https://gnomad.broadinstitute.org/variant/14-23895180-G-A

CaseIII-15: Male, 47 years report, China, Asian

DiseaseAssertion: Hypertrophic Cardiomyopathy

FamilyInfo: 25 people from 74 family members were positively identified for having HCM, Some of them only carried a single variant while others carried multiple, Many other family members died from sudden cardiac death at various ages

CasePresentingHPOs: HP:0001639, HP:0031352 ( Hypertrophic cardiomyopathy, Chest tightness)

HPOsFreeText: Left atrial diameter at 38 mm, Left ventricular end-diastolic diameter at 52 mm, Interventricular septum thickness maximum at 20 mm, Left ventricular posterior wall thickness maximum at 8 mm

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: All below variants are heterozygous with the wild-type

CaseGenotypingMethod: The above family members, along with IV:14 to serve as a control to compare to, were processed through Whole Exome Sequencing. Any variants found were mapped and filtered for pathogenicity, and any that were detected were confirmed by Sanger sequencing.

Variant: NM_000257.3:c.2155C>T (p.Arg719Trp)

ClinVar: 14104 https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: European (Non-Finnish) 6.481e-5, Overall 3.185e-5 https://gnomad.broadinstitute.org/variant/14-23895180-G-A

MultipleGeneVariants:

(1) MYOZ2 Mutation

Variant: NM_016599.4:c.505A>C (p.Lys169Gln)

CAID: CA16609625 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_016599.4%3Ac.505A%3EC

gnomAD: Not in gnomAD

(2) DNAJC19 Mutation

Variant: NM_145261.3:c.59G>A (p.Arg20His)

ClinVar: 466328 https://www.ncbi.nlm.nih.gov/clinvar/variation/466328/

gnomAD: South Asian 4.246e-4, Overall 5.304e-5 https://gnomad.broadinstitute.org/variant/3-180705881-C-T

(3) CTNNA3 Mutation

Variant: NM_001127384.2:c.2441T>A (p.Leu814Ter)

CAID: CA377089103 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_013266.3%3Ac.2441T%3EA

gnomAD: Not in gnomAD

Supplementary Data: S2 Table

Case 232: Spanish, 32yo, Female

DiseaseAssertion: Hypertrophic Cardiomyopathy

CasePreviousTesting: Full Testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1), additional testing listed in supplementary data table S1

CaseGenotypingMethod: genomic DNA obtained from blood or saliva, Next-gen sequencing panels of 55 or 78 genes

Variant: NM_000257.3(MYH7):c.2155C>T (p.Arg719Trp)

ClinVar: 14104, https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: 0.00003185, https://gnomad.broadinstitute.org/variant/14-23895180-G-A

MultipleGeneVariants:

(1) TNNT2 Variant:

Variant: NM_000364.3(TNNT2):c.878G>A (p.Arg293His) listed in paper as c.857G>A (p.Arg286His)

ClinVar: 43677, https://www.ncbi.nlm.nih.gov/clinvar/variation/43677/

gnomAD: 0.00006936, https://gnomad.broadinstitute.org/variant/1-201328348-C-T

(2) ACTN2 Variant:

Variant: NM_001103.3(ACTN2):c.1930G>A (p.Ala644Thr)

ClinVar: 43920, https://www.ncbi.nlm.nih.gov/clinvar/variation/43920/

gnomAD: 0.0001629, https://gnomad.broadinstitute.org/variant/1-236917337-G-A

CaseMYH7-28: Australia

DiseaseAssertion: Hypertrophic Cardiomyopathy

FamilyInfo: N/A

CasePresentingHPOs: HP:0001639 (Hypertrophic cardiomyopathy)

HPOsFreeText: N/A

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.2155C>T (p.Arg719Trp)

ClinVar: 14104 https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: European (Non-Finnish) 6.481e-5, Overall 3.185e-5 https://gnomad.broadinstitute.org/variant/14-23895180-G-A

Supplementary Material 4

Case M-14: Japanese

DiseaseAssertion: idiopathic cardiomyopathy (HCM and DCM)

CasePreviousTesting: Clinical evaluation, chest radiography, electrocardiography, echocardiography, cardiac catheterization, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1)

CaseGenotypingMethod: 8 sarcomeric genes amplified from genomic DNA via PCR then screened using bidirectional direct sequencing

Variant: NM_000257.3(MYH7):c.2155C>T (p.Arg719Trp)

ClinVar: 14104, https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: 0.00003185, https://gnomad.broadinstitute.org/variant/14-23895180-G-A

Supplementary Material 4

Case M-15: Japanese

DiseaseAssertion: idiopathic cardiomyopathy (HCM and DCM)

CasePreviousTesting: Clinical evaluation, chest radiography, electrocardiography, echocardiography, cardiac catheterization, full testing (MYBPC3, TNNT2, TNNI3, MYL2, MYL3, TPM1, ACTC1)

CaseGenotypingMethod: 8 sarcomeric genes amplified from genomic DNA via PCR then screened using bidirectional direct sequencing

Variant: NM_000257.3(MYH7):c.2155C>T (p.Arg719Trp)

ClinVar: 14104, https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: 0.00003185, https://gnomad.broadinstitute.org/variant/14-23895180-G-A

Supplementary Data, Table 2

Case: Australian

DiseaseAssertion: HCM

CaseHPOFreeText: maximum left ventricular wall thickness of ≥15 mm

CasePreviousTesting: electrocardiography, echocardiography, Full testing (MYBPC3, MYH7, TNNT2, TNNI3, TPM1, MYL2, MYL3, ACTC1)

CaseGenotypingMethod: DNA isolated from peripheral blood or buccal swabs, PCR, Sanger sequencing, segregation analysis, haplotype analysis of MYBPC3 and MYH7

Variant: NM_000257.3(MYH7):c.2155C>T (p.Arg719Trp)

ClinVar: 14104, https://www.ncbi.nlm.nih.gov/clinvar/variation/14104/

gnomAD: 0.00003185, https://gnomad.broadinstitute.org/variant/14-23895180-G-A