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#: female; age 65

DiseaseAssertion: chemotherapy-induced cardiomyopathy with severe left ventricular systolic dysfunction

FamilyInfo: NR

ParentalTesting: NR

CasePresentingHPOs: HP:0001644 HP:0001635 HP:0001640

CaseHPOFreeText: Dilated left ventricle; left ventricular dysfunction; cardiomegaly; bilateral pleural effusion; dyspnea

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: Previous anthracycline-associated cardiomyopathy 7 years earlier

Genotyping Method: TruSight One Expanded 6700-gene sequencing panel on an Illumina NextSeq 550

FunctionalAnalysis: NR

Variant: c.452G>A (p.Arg151Gln; rs730881101); missense variant; heterozygous

ClinVar: 181617

CAID: NR

gnomAD: NR

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.

Case#:reports one patient with this variant

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM)

FamilyInfo: N/A

ParentalTesting:N/A

CasePresentingHPOs: HP:0001639 "Hypertrophic cardiomyopathy"

CaseHPOFreeText: The study cohort consisted of 23 patients diagnosed with HCM.

CaseNotHPOs: N/A

CaseNotHPOFreeText:N/A

CasePreviousTesting:N/A

Genotyping Method: Sanger sequencing

FunctionalAnalysis: none preformed

Variant: MYBPC3 c.1037G>C, p.Arg346Pro

ClinVar: No exact match, closest is MYBPC3 c.1037G>A

CAID: N/A

gnomAD:N/A

AdditionalInfo: variant is suspected to be novel and potentially pathenogenic

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

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)

Case#: 31 year old man of European ancestry. Patient ID NR.

DiseaseAssertion: Nonischemic dilated cardiomyopathy.

FamilyInfo: No reported family history of dilated cardiomyopathy. Both parents had normal heart exams and echocardiograms.

ParentalTesting: Both biological parents tested negative for the TPM1 variant. Sequencing coverage was 500 to 600×. The authors thought the variant likely arose de novo. They could not rule out parental germline mosaicism.

CasePresentingHPOs: HP:0001644, HP:0025169, HP:0001635, HP:0001640, HP:0002202, HP:0002875, HP:0002094, HP:0012764, HP:0034807

CaseHPOFreeText: He first felt short of breath during activity. Later he felt short of breath at rest. He also had a persistent cough and abdominal swelling. Chest CT showed an enlarged heart and fluid around both lungs. The echo showed that both ventricles were enlarged and pumped poorly. His left ventricular ejection fraction was 12%. His heart failure worsened and he needed a heart transplant.

CaseNotHPOs: HP:4000004 (no myocardial late gadolinium enhancement on cardiac MRI).

CaseNotHPOFreeText: The biopsy and exam of the removed heart did not show evidence of active myocarditis.

CasePreviousTesting: NR

Genotyping Method: The Invitae Dilated Cardiomyopathy Panel used next generation sequencing to test 41 genes.

FunctionalAnalysis: Thin filaments with T237S needed more calcium to reach the same level of activation. At low calcium the mutant filaments were less likely to be open and myosin ATPase activity was lower. Omecamtiv mecarbil restored calcium sensitivity in the lab assay. See Figures 3 through 5 and Tables 1 and 2.

Variant: NM_001018005.2(TPM1):c.710C>G (p.Thr237Ser). Heterozygous.

ClinVar: 1067773

CAID: CA392724554

gnomAD: No variant record found in gnomAD v4.1.

AdditionalInfo: PMID 36739943. Figure 1 shows the echo and biopsy results. The clinical report called the variant a VUS. The study authors considered it likely pathogenic after lab testing.

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#: a male in the age range of 36-40

DiseaseAssertion: Dilated cardiomyopathy and generated hiPSC-CMS

FamilyInfo: not reported

ParentalTesting: not reported

CasePresentingHPOs: HP:0002875, HP:0031352 Exertional dyspnea and chest tightness

CaseHPOFreeText: nocturnal dyspnea and reduced exercise tolerance

CaseNotHPOs:

CaseNotHPOFreeText:

CasePreviousTesting: echocardiogram and electrocardiograms

Genotyping Method: peripheral blood was extracted and use to sequence the entire genome

FunctionalAnalysis: hiPSCs were looked at in this case

Variant: NM_000256.3: c.194 C > T (p.Thr65Met)

ClinVar:407324

CAID: CA047700

gnomAD: 11-47372888-G-A

AdditionalInfo:F

Case#: Female with DCM symptoms beginning at age 30

DiseaseAssertion: Dilated cardiomyopathy (DCM) associated with a heterozygous missense variant in MYBPC3, c.3373G>A (p.Val1125Met). The authors propose autosomal dominant inheritance in this DCM case.

FamilyInfo: NR – Detailed family relationships, segregation testing, and individual relatives' phenotypes are not described for the current DCM proband.

ParentalTesting: NR

CasePresentingHPOs: HP:0001644 (Dilated cardiomyopathy); HP:0002094 (Dyspnea); HP:0001962 (Palpitations); HP:0004756 (Ventricular tachycardia)

CaseHPOFreeText: The patient experienced symptoms beginning at age 30, including frequent palpitations and dyspnea. ECG monitoring revealed multiple premature ventricular contractions and nonsustained ventricular tachycardia. Echocardiography demonstrated severe spherical left ventricular enlargement, severe systolic and diastolic dysfunction, LVEF of 10%, and mild pulmonary hypertension. She was classified as NYHA functional class II–III and received medical therapy and an implantable cardioverter-defibrillator.

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR – Article describes patient's clinical evaluation, including ECG monitoring and echocardiography, before genetic analysis.

Genotyping Method: Genomic DNA was extracted from peripheral blood. Targeted next-generation sequencing was performed using a custom NimbleGen panel covering the coding regions and exon–intron boundaries of 23 genes associated with DCM. Sequencing was conducted on an Illumina HiSeq 2000 platform. Reads were aligned to the hg19 reference genome using BWA, and SAMtools was used for variant analysis. MYBPC3 variant confirmed using Sanger sequencing.

FunctionalAnalysis: In silico analyses were performed to investigate the potential functional consequences of MYBPC3 p.Val1125Met. SIFT, PolyPhen-2, and MutationTaster predicted potentially damaging effects, while CADD produced a score of 33. Multiple-sequence alignment demonstrated that Val1125 is highly conserved across species. Protein modeling using I-TASSER and Phyre2 predicted structural differences involving the region surrounding residue 1125, located within the C9 fibronectin type III domain of cardiac myosin-binding protein C (cMyBP-C). The authors proposed that the substitution could affect protein structure and function. No direct experimental functional validation of this variant was reported.

Variant: MYBPC3 NM_000256.3:c. 3373G>A (p.Val1125Met); rsID: rs121909378; missense; heterozygous

ClinVar: 8604

CAID: CA014038

gnomAD: MYBPC3 c.3373G>A (p.Val1125Met), GRCh38: 11-47332931-C-T. In gnomAD v4, the reported allele frequencies are 0.00001920 (exomes), 0.00003943 (genomes), and 0.00002111 (total). The highest ancestry-specific allele frequency identified was in the Ashkenazi Jewish population (AC = 5; AF = 0.0001695).

AdditionalInfo: Authors propose variant dosage, interactions with other variants, and genetic modifiers may contribute to differences in disease phenotype. However, the proposed autosomal dominant association with DCM was not supported by documented segregation testing in the current proband's family, and the functional analyses were computational rather than experimental.

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

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

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

ParentalTesting: NR

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

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

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

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

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

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

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

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

ClinVar: 693982

CAID: CA221697326

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

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

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#: 38 year old woman

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM)

FamilyInfo: Father and one brother had HCM, no sudden deaths reported in family

ParentalTesting: Father carried the same heterozygous variant

CasePresentingHPOs: HP:0001962, HP:0031352 “palpitation and chest distress”

CaseHPOFreeText: myocardial fibrosis

CaseNotHPOs: N/A

CaseNotHPOFreeText: Unremarkable

CasePreviousTesting: N/A

Genotyping Method:PCR

FunctionalAnalysis: N/A

Variant: TNNI3 heterozygous c.235C>T (p.Arg79Cys)

ClinVar: Variation ID: 43367

CAID:N/A

gnomAD:N/A

AdditionalInfo: authors describe the variant as novel

Case#:27-year-old woman

DiseaseAssertion: Familial restrictive cardiomyopathy (RCM)

FamilyInfo: Strong family history of cardiac disease and sudden cardiac death

ParentalTesting:N/A

CasePresentingHPOs: “ Fatigue, dyspnoea , palpitations, atrial fibrillation, pulmonary hypertension, and fluid congestion.”

CaseHPOFreeText: age 27, she presented with progressive fatigue and limited exercise capacity, 13-year symptom-free interval, she developed rapid clinical deterioration with dyspnoea and palpitations

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: focused screening of MYH7, TNNT2, and MYBPC3 was unremarkable,

Genotyping Method: NGS

FunctionalAnalysis:N/A

Variant: TNNI3, NP_000354.4: p.Lys193Glu; NC_000019.9: g.55663258T>C. Heterozygous reported as novel

ClinVar: N/A

CAID:N/A

gnomAD: N/A

AdditionalInfo: patient improved with restored sinus rhythm and low-dose diuretic treatment and was symptom-free at the time of reporting. later had an ischaemic stroke

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#:7 ¾ year old girl

DiseaseAssertion: Hypertrophic cardiomyopathy (HCM), with ventricular tachycardia and Stokes–Adams syndrome.

FamilyInfo: Parents not consanguineous, no family history of cardiovascular disease or sudden cardiac death.

ParentalTesting: Parents not consanguineous, no symptoms

CasePresentingHPOs: HP:0001639, HP:0001695, HP:0001663, HP:0001250, HP:0001678, HP:0001712 “Hypertrophic cardiomyopathy, cardiac arrest, ventricular tachycardia, seizures, atrioventricular block, left ventricular hypertrophy:”

CaseHPOFreeText: Diagnosed with cardiomyopathy in utero. Frequent cardiac arrests from 3-7, multiple seizures a year, cardiac mri showed heart enlargement, hypertrophy, after icd implant no cardiac arrests after 1 year.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: diagnosed with cardiomyopathy in utero, no other testing stated.

Genotyping Method: Whole exon sequencing

FunctionalAnalysis: protein modeling only, revealed to be “probably damaging” score 1.0

Variant: MYH7 NM_000257.3:c.2723T>C (p.Leu908Pro; p.L908P)

ClinVar: no id but is listed under https://www.ncbi.nlm.nih.gov/clinvar/RCV000158849.1/?cmd=HTOn&utm_source=chatgpt.com

CAID: N/A

gnomAD:N/A

AdditionalInfo: patient underwent ICD implantation and had no further arrests after a years time.

Case#: CMP-83-03; sex NR; pediatric age, exact age NR

CasePresentingHPOs: HP:0001644

CaseHPOFreeText: Biopsy proven myocarditis with dilated cardiomyopathy

Genotyping Method: Targeted next-generation sequencing

Variant: TNNT2 c.460C>T (p.Arg154Trp); heterozygous; missense single-nucleotide variant

ClinVar: 132943

CAID: CA004554

gnomAD: 0.000032

Case#: Male, 47

DiseaseAssertion: hypertrophic cardiomyopathy

FamilyInfo: NR

ParentalTesting: NR

CasePresentingHPOs: HP:0025169, HP:0034307, HP:0001712,

CaseHPOFreeText: Severe alterations in inter ventricular septum thickness and left ventricular posterior wall thickness

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: Sanger sequencing

FunctionalAnalysis: NA

Variant: NM_001276345.2:c.423G>A

ClinVar: NR

CAID: CA422532259

gnomAD: NR

AdditionalInfo: IVST, average of 19.2 mm and LVPWT; average of 11.5 mm

Case#: Jenni a 7 year-old female

DiseaseAssertion: Dilated cardiomyopathy with LVNC

FamilyInfo: MYH7 maternally inherited and MYBPC3 paternally inherited

ParentalTesting: Mother has PPCM

CasePresentingHPOs: HP:0004755

heart failure and supraventicular tachycardia

CaseHPOFreeText: Intermittent Wolff-Parkinson- White

CaseNotHPOs:

CaseNotHPOFreeText:

CasePreviousTesting: Echocardiogram and MRI

Genotyping Method: Segregation analysis, targeted next generation sequencing

FunctionalAnalysis: electrophysiology, transplant evaluation, CT angiography

Variant: NM_000256.3(MYBPC3):c.2672G>A (p.Arg891Gln)

ClinVar:177884

CAID: CA012826

gnomAD: 11-47357493-C-T

AdditionalInfo:

Case#: AD-FDC1, IV:14, male, age 45

DiseaseAssertion: Familial dilated cardiomyopathy

FamilyInfo: multigenerational DCM family

CasePresentingHPOs: HP:0001644 HP:4000141 HP:0012664

Genotyping Method: Whole-exome sequencing using Agilent SureSelect 50 Mb exome; HiSeq 2000 sequencing followed by Sanger sequencing confirmation

Variant: TNNT2 c.517C>T (p.Arg173Trp); missense single-nucleotide variant; heterozygous

ClinVar: 228409

CAID: CA10576372

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

Case#: Male, 65

DiseaseAssertion: Idiopathic dilated cardiomyopathy

FamilyInfo:None of his five siblings were affected

ParentalTesting: NR

CasePresentingHPOs: HP:0005110

CaseHPOFreeText: Slow progression to severe heart failure

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

CasePreviousTesting: NR

Genotyping Method: Bidirectional dna sequencing

FunctionalAnalysis: NR

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

ClinVar: 4881578

CAID: CA344201883

gnomAD: NA

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

Case#: 15

DiseaseAssertion: Dilated cardiomyopathy

FamilyInfo: NR

ParentalTesting: NR

CasePresentingHPOs: NA

CaseHPOFreeText: decreased cardiac contractility

CaseNotHPOs: NA

CaseNotHPOFreeText: NA

CasePreviousTesting: NR

Genotyping Method: Sanger sequencing

FunctionalAnalysis: NR

Variant: NM_001276345.2:c.577C>T

ClinVar: 834548

CAID: CA088264

gnomAD: NR

AdditionalInfo: NA

Patient7: Male

CasePresentingHPOs: HP:0002194

HPOsFreeText: ERT dosage = 20 mg/kg/2 w, CRIM positive

CaseNotHPOs: HP:0004887

NotHPOsFreeText: no reversal of HCM

CaseAddInfo: Symptom onset and diagnosis at 3mo, ERT at 4mo, ERT lasted for 24mo, died at 28mo

CasePMIDs: 22644586, 14695532

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.655G>A (p.Gly219Arg)

ClinVar: 189065, https://www.ncbi.nlm.nih.gov/clinvar/variation/189065/

gnomAD: 1.666e-05, http://exac.broadinstitute.org/variant/17-78079656-G-A

Patient11: Female

CasePresentingHPOs: HP:0004887, HP:0002100, HP:0006597, HP:0200136, HP:0011471

HPOsFreeText: ERT dosage = 40mg/kg/w, HCM reversal, CRIM positive, able to run, diaphragmatic paralysis required plication at 3yo

CaseNotHPOs: HP:0009046

NotHPOsFreeText:

CaseAddInfo: Symptom onset at 2mo, diagnosis at 4mo, ERT at 5mo, ERT lasted for 67mo, ventilation required at 62mo, currently 6yo

CasePMIDs: 14695532, 14972326

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.1064T>C (p.Leu355Pro)

ClinVar: 284093, https://www.ncbi.nlm.nih.gov/clinvar/variation/284093/

gnomAD: 8.304e-06, http://exac.broadinstitute.org/variant/17-78082197-T-C

CaseOPTPD-5: Female, Taiwan, Birth diagnosis

HPOsFreeText: GAA (MS/MS) enzyme activity at 1.27 umol/hr/L

GenotypingMethod: GAA Gene Sequencing and PCR Amplification

Variant1: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant2: NM_000152.4:c.1222A>G (p.Met408Val)

ClinVar: 371235 https://www.ncbi.nlm.nih.gov/clinvar/variation/371235/

gnomAD: European (Non-Finnish) 1.790e-5, Overall 8.058e-6 http://gnomad.broadinstitute.org/variant/17-78082523-A-G

CaseAddInfo: One Pompe Mutation allele and Two Pseudodeficency Mutation alleles, Nomenclature indicates patient is homozygous for Variant 1 and Variant 2 is in cis with one copy

CaseLP2-12: Male, Taiwan, Birth diagnosis

HPOsFreeText: GAA (MS/MS) enzyme activity at 0.43 umol/hr/L, Serum CK at 29 U/L

GenotypingMethod: GAA Gene Sequencing and PCR Amplification

Variant1: NM_000152.4:c.1935C>A (p.Asp645Glu)

ClinVar: 4029 https://www.ncbi.nlm.nih.gov/clinvar/variation/4029/

gnomAD: East Asian 1.729e-3, Overall 1.240e-4 http://gnomad.broadinstitute.org/variant/17-78086721-C-A

Variant2: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant3: NM_000152.4:c.2482-5T>C

ClinVar: 558320 https://www.ncbi.nlm.nih.gov/clinvar/variation/558320/

gnomAD: Not in gnomAD

CaseAddInfo: Late Onset Pompe Disease, Nomenclature indicates Variants 1 and 2 are in cis one one allele and Variants 2 and 3 are in cis on the other

CaseOPOPD-52: Male, Taiwan, Birth diagnosis

HPOsFreeText: GAA (MS/MS) enzyme activity at 1.05 umol/hr/L

GenotypingMethod: GAA Gene Sequencing and PCR Amplification

Variant1: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant2: NM_000152.4:c.1843G>A (p.Gly615Arg)

ClinVar: 188786 https://www.ncbi.nlm.nih.gov/clinvar/variation/188786/

gnomAD: East Asian 2.223e-4, Overall 1.640e-5 http://gnomad.broadinstitute.org/variant/17-78086465-G-A

CaseAddInfo: One Pompe Mutation allele and One Pseudodeficency Mutation allele, 1 other patient has the same genotype

CaseOPOPD-45: Female, Taiwan, Birth diagnosis

HPOsFreeText: GAA (MS/MS) enzyme activity at 1.46 umol/hr/L

GenotypingMethod: GAA Gene Sequencing and PCR Amplification

Variant1: NM_000152.4:c.1411_1414delGAGA (p.Glu471Profs)

ClinVar: 188874 https://www.ncbi.nlm.nih.gov/clinvar/variation/188874/

gnomAD: East Asian 2.513e-4, Overall 1.781e-5 http://gnomad.broadinstitute.org/variant/17-78083827-CGAGA-C

Variant2: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

CaseAddInfo: One Pompe Mutation allele and One Pseudodeficency Mutation allele, 1 other has the same genotype

CaseOPOPD-4: Female, Taiwan, Birth diagnosis

HPOsFreeText: GAA (MS/MS) enzyme activity at 1.16 umol/hr/L

GenotypingMethod: GAA Gene Sequencing and PCR Amplification

Variant1: NM_000152.4:c.460_465del (p.Arg154_Thr155del)

ClinVar: 551306 https://www.ncbi.nlm.nih.gov/clinvar/variation/551306/

gnomAD: Not in gnomAD

Variant2: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

CaseAddInfo: One Pompe Mutation and One Pseudodeficency Mutation

Case59

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.1441T>C (p.Trp481Arg)

ClinVar: 189007 https://www.ncbi.nlm.nih.gov/clinvar/variation/189007/

gnomAD: African 8.031e-5, Overall 2.126e-5 http://gnomad.broadinstitute.org/variant/17-78084529-T-C

Variant2: NM_000152.4:c.-32-13T>G

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

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

CaseAddInfo: Adult Late Onset Pompe Disease

Case90

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.925G>A (p.Gly309Arg)

ClinVar: 188797 https://www.ncbi.nlm.nih.gov/clinvar/variation/188797/

gnomAD: Ashkenazi Jewish 1.010e-4, Overall 2.860e-5 http://gnomad.broadinstitute.org/variant/17-78081665-G-A

Variant2: NM_000152.4:c.-32-13T>G

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

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

CaseAddInfo: Adult Late Onset Pompe Disease, 1 other patient has the same genotype

Case69

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.2238G>A (p.Trp746*)

ClinVar: 370904 https://www.ncbi.nlm.nih.gov/clinvar/variation/370904/

gnomAD: African 4.011e-5, Overall 1.063e-5 http://gnomad.broadinstitute.org/variant/17-78090815-G-A

Variant2: NM_000152.4:c.-32-13T>G

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

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

CaseAddInfo: Adult Late Onset Pompe Disease, 1 other patient has the same genotype

Case35

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.2238G>A (p.Trp746*)

ClinVar: 370904 https://www.ncbi.nlm.nih.gov/clinvar/variation/370904/

gnomAD: African 4.011e-5, Overall 1.063e-5 http://gnomad.broadinstitute.org/variant/17-78090815-G-A

Variant2: NM_000152.4:c.1843G>A (p.Gly615Arg)

ClinVar: 188786 https://www.ncbi.nlm.nih.gov/clinvar/variation/188786/

gnomAD: East Asian 2.223e-4, Overall 1.640e-5 http://gnomad.broadinstitute.org/variant/17-78086465-G-A

CaseAddInfo: Infantile Onset Pompe Disease

Case38

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.2481+102_2646+31del (p.Gly828_Asn882del) (exon 18 del)

ClinVar: 4031 https://www.ncbi.nlm.nih.gov/clinvar/variation/4031/

gnomAD: Not in gnomAD

Variant2: NM_000152.4:c.1912G>T (p.Gly638Trp)

CAID: 280954 https://www.ncbi.nlm.nih.gov/clinvar/variation/280954/

gnomAD: African 6.752e-5, Overall 1.239e-5 http://gnomad.broadinstitute.org/variant/17-78086698-G-T

CaseAddInfo: Infantile Onset Pompe Disease

Case24

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.2560C>T (p.Arg854*)

ClinVar: 4034 https://www.ncbi.nlm.nih.gov/clinvar/variation/4034/

gnomAD: African 1.891e-3, Overall 2.090e-4 http://gnomad.broadinstitute.org/variant/17-78092070-C-T

Variant2: NM_000152.4:c.2459_2461delCTG (p.Ala820del)

ClinVar: 456404 https://www.ncbi.nlm.nih.gov/clinvar/variation/456404/

gnomAD: European (Non-Finnish) 6.492e-5, Overall 3.190e-5 http://gnomad.broadinstitute.org/variant/17-78091524-GGCT-G

CaseAddInfo: Infantile Onset Pompe Disease

Patient30: Female, 1 year onset

CasePresentingHPOs: HP:0001324, HP:0001263 (Muscle weakness, Developmental delay)

HPOsFreeText: GAA activity 3% from leukocyte

CaseNotHPOs: HP:0002098, HP:0002240, HP:0001640 (Respiratory distress, Hepatomegaly, Cardiomegaly)

NotHPOsFreeText: No wheelchair needed, Respiratory Assistance not needed

CaseAddInfo: LOPD

Patient19: Male, 21 years report, 14 year onset

CasePresentingHPOs: HP:0001324, HP:0002098 (Muscle weakness, Respiratory distress)

HPOsFreeText: GAA activity 9.3% from leukocyte, Respiratory Assistance needed

CaseNotHPOs: HP:0001640, HP:0002240 (Cardiomegaly, Hepatomegaly)

NotHPOsFreeText: Wheelchair not needed

CaseAddInfo: LOPD

Patient14: Male, 59 years report, 40 year onset

CasePresentingHPOs: HP:0001324, HP:0002098 (Muscle weakness, Respiratory distress)

HPOsFreeText: GAA activity 6.9% from dried blood spot, Respiratory Assistance needed

CaseNotHPOs: HP:0001640, HP:0002240 (Cardiomegaly, Hepatomegaly)

NotHPOsFreeText: Wheelchair not needed

CaseAddInfo: LOPD

Patient20: Male, 22 years report, 12 year onset

CasePresentingHPOs: HP:0001324, HP:0002098 (Muscle weakness, Respiratory distress)

HPOsFreeText: GAA activity 11.6% from leukocyte, Respiratory Assistance needed

CaseNotHPOs: HP:0001640, HP:0002240 (Cardiomegaly, Hepatomegaly)

NotHPOsFreeText: Wheelchair not needed

CaseAddInfo: LOPD

Case#: 1, age 36, age 30 (report)

DiseaseAssertion: Pompe Disease

FamilyInfo: Family 1, no further information

CasePresenting HPOs:HP:0002878, HP:0012378, HP:0003546, HP:0001324 (Respiratory failure, fatigue, exercise intolerance, muscle weakness)

CaseHPOFreeText: myopathy with vacuoles, biopsy with vacuolar myopathy with PAS +

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: single gene sequencing/analysis, 48.47 NaG/AaGIA, 89.61% inhibition of GAA; NaG/AaGIA above 40 and %INH above 87 are in the affected range

Variants:

Variant 1:NM_000152.5(GAA):c.1941C>G (p.Cys647Trp)

ClinVar: 550327

Variant 2: Not reported

ParentalGenotypes: N/A

PreviouslyPublished: N/A

Patient1: 7yo

CasePresentingHPOs: HP:0001638

HPOsFreeText: rhGAA dosage = 20QW, independent walker

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 17days old at ERT start

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.1935C>A (p.Asp645Glu)

ClinVar: 4029, https://www.ncbi.nlm.nih.gov/clinvar/variation/4029/

gnomAD: 0.0001721, http://exac.broadinstitute.org/variant/17-78086721-C-A

Variant2: NM_000152.4(GAA):c.784G>A (p.Glu262Lys)

ClinVar: 188806, https://www.ncbi.nlm.nih.gov/clinvar/variation/188806/

gnomAD: 1.66e-05, http://exac.broadinstitute.org/variant/17-78081447-G-A

Patient2: Female, 37yo

CasePresentingHPOs: HP:0003077, HP:0001513

HPOsFreeText: LOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 10yr disease duration, ERT lasted for 33mo

CasePMIDs: PMID: 25614309

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.1655T>C (p.Leu552Pro)

ClinVar: 279811, https://www.ncbi.nlm.nih.gov/clinvar/variation/279811/

gnomAD: n/a

Variant2: NM_000152.4(GAA):c.1478C>T (p.Pro493Leu)

ClinVar: 379593, https://www.ncbi.nlm.nih.gov/clinvar/variation/379593/

gnomAD: 0.00001416, http://gnomad.broadinstitute.org/variant/17-78084566-C-T

Case7: Female, 3 years onset, 3 years diagnosis, 27 years report, France

CasePresentingHPOs: HP:0002540, HP:0002878, HP:0004887 (Inability to walk, Respiratory failure, Respiratory failure requiring assisted ventilation)

HPOsFreeText: Lost ambulation at 11 years, Started invasive ventilation at 11 years, Started ERT given every other week at a dose of 20 mg/kg at 18 years for 114 months, Peak Anti-rhGAA antibodies at 1:102,400, FVC at 0.22/6 (L / % expected), MIP at 10 (cm H2O), MEP at 10 (cm H2O), Time off ventilation stayed constant at 0 min, Total Motor Function Measure from 23 to 19, 6MWT stayed constant at 0 meters

CaseNotHPOs: HP:0030148 (Heart murmur)

NotHPOsFreeText: No adverse reaction to ERT was recorded

CaseAddInfo: Late Onset Pompe Disease

Patient25: Male, Southern China

CasePresentingHPOs:

HPOsFreeText: IOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: c.971dupC (p.Ser325GlufsX5) no ClinVar or CAID

Variant2: NM_000152.4(GAA):c.1802C>T (p.Ser601Leu)

ClinVar: 194154, https://www.ncbi.nlm.nih.gov/clinvar/variation/194154/

gnomAD: n/a

Patient10: Male, Southern China

CasePresentingHPOs:

HPOsFreeText: IOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.2105G>A (p.Arg702His)

ClinVar: 426278, https://www.ncbi.nlm.nih.gov/clinvar/variation/426278/

gnomAD: 6.695e-05, http://exac.broadinstitute.org/variant/17-78087081-G-A

Variant2: NM_000152.4(GAA):c.2297A>C (p.Tyr766Ser)

ClinVar: 420102, https://www.ncbi.nlm.nih.gov/clinvar/variation/420102/

gnomAD: 8.35e-06, http://exac.broadinstitute.org/variant/17-78090874-A-C

Case: Female, 59yo, Japanese, non-consanguineous family

CasePresentingHPOs: HP:0002515, HP:0001290, HP:0009055, HP:0002015, HP:0002098, HP:0002315, HP:0007340, HP:0004944, HP:0002138, HP:0001297, HP:0000822, HP:0001288, HP:0002788, HP:0003236, HP:0002119, HP:0002415, HP:0012444, HP:0001922, HP:0012529

HPOsFreeText: 3mm aneurysm in left middle cerebral artery, underwent cerebral arterial clipping, muscle atrophy more significant in proximal muscles of lower limbs, hypotonia more significant in right thigh, CPK = 327mg/dL, alanine aminotransferase = 44mg/dL, aspartate aminotransferase = 42mg/dL, lactic dehydrogenase = 266mg/dL, low-density lipoprotein = 162mg/dL, flattened diaphragm, left atrial ventricular enlargement with mass = 156g, left ventricular index = 106g/m^2, extensor muscle atrophy, diffuse fibrosis in thighs, multiple infractions, bilateral tortuous irregular cerebral arteries, GAA activity = 1.3 pmol hour^-1 punch^-1, vacuolated platelets, glycogen accumulation, large lysosomes filled with membrane bound deposition in fibroblasts

CaseNotHPOs: HP:0003691, HP:0012195, HP:0030850, HP:0010535, HP:0000365, HP:0000505, HP:0002111

NotHPOsFreeText: regurgitation

CaseAddInfo: Hypertension developed at 40yo and was treated with antihypertensive drugs. Cerebral stroke at 57yo. Father died at 82yo due to cardiac aneurism and arterial thrombosis. Mother had left ventricular hypertrophy and died at 62yo due to colon carcinoma. Sister had psychiatric illness. Her 2 sons were healthy.

CasePreviousTesting: CT scan, chest x-ray, echocardiogram, MRI, DBS, electron microscopy of blood cells, skin biopsy

CaseGenotypingMethod: PCR and direct DNA sequencing of GAA gene, cDNA-sequencing analysis

Variant: NM_000152.4(GAA):c.546G>T (p.Thr182=), homozygous

ClinVar: 370637, https://www.ncbi.nlm.nih.gov/clinvar/variation/370637/

gnomAD: n/a

PatientF3P1: Female, 3 months diagnosis, Multiracial (maternal European and Native American and paternal Czechoslovakian, German, and Australian)

CasePresentingHPOs: HP:0004755, HP:0001290 (Supraventricular tachycardia, Generalized hypotonia)

CaseAddInfo: Proband

Patient7: Male, 3.8mo at ERT start, 3.3yo at study end

CasePresentingHPOs:

HPOsFreeText: treated with 40mg/kg/wk, patient had 10 infusion associated reactions

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NC_000017.11:g.(?80118193)(80118357_?)del (c.2481 + 102_2646 + 31del)

ClinVar: 456368, https://www.ncbi.nlm.nih.gov/clinvar/variation/456368/

gnomAD: n/a

Variant2: NM_000152.4(GAA):c.525delT (p.Glu176Argfs)

ClinVar: 4033, https://www.ncbi.nlm.nih.gov/clinvar/variation/4033/

gnomAD: 0.00009959, http://gnomad.broadinstitute.org/variant/17-78078909-CT-C

Patient4: 19yo

CasePresentingHPOs:

HPOsFreeText: hyperCKemia, GAA = 24%, BMI = 26.1, normal CTA and CT

CaseNotHPOs:

NotHPOsFreeText: No cardiovascular risk

CaseAddInfo: 12yr disease duration

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.1771C>T (p.Arg591Trp)

ClinVar: 551782, https://www.ncbi.nlm.nih.gov/clinvar/variation/551782/

gnomAD: 8.662e-06, http://exac.broadinstitute.org/variant/17-78086393-C-T

CasePD/Pathogenic-9: 3 weeks diagnosis

HPOsFreeText: GAA activity 8.4 (nmol · h−1 · mg−1), Mean percentage of normal 11.7%

GenotypingMethod: Newborn Screening Panels

Variant1: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant2: NM_000152.4:c.2065G>A (p.Glu689Lys)

ClinVar: 4030 https://www.ncbi.nlm.nih.gov/clinvar/variation/4030/

gnomAD: East Asian 0.2355, Overall 0.05524 http://gnomad.broadinstitute.org/variant/17-78087041-G-A

Variant3: NM_000152.4:c.2662G>T (p.Glu888Ter)

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

gnomAD: East Asian 2.719e-4, Overall 1.998e-5 http://gnomad.broadinstitute.org/variant/17-78092467-G-T

CaseAddInfo: Patient has one pseudodeficency allele and one pathogenic allele, Nomenclature claims Variant 1 and 2 are in cis

CaseLOPD-16: 2.5 weeks diagnosis

HPOsFreeText: GAA activity 2.5 (nmol · h−1 · mg−1), Mean percentage of normal 3.6%

GenotypingMethod: Newborn Screening Panels

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 http://www.ncbi.nlm.nih.gov/clinvar/variation/4027

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.2065G>A (p.Glu689Lys)

ClinVar: 4030 https://www.ncbi.nlm.nih.gov/clinvar/variation/4030/

gnomAD: East Asian 0.2355, Overall 0.05524 http://gnomad.broadinstitute.org/variant/17-78087041-G-A

Multiple interpretations of p.W746C possible, below is considered the most likely

Variant3: NM_000152.4:c.2238G>C (p.Trp746Cys)

ClinVar: 265160 https://www.ncbi.nlm.nih.gov/clinvar/variation/265160/

gnomAD: European (Non-Finnish) 5.673e-4, Overall 3.083e-4 http://gnomad.broadinstitute.org/variant/17-78090815-G-C

CaseAddInfo: Late Onset Pompe Disease, Nomenclature claims Variant 2 and 3 are in cis

Case10HPOsFreeText: 20 mg/kg GAA treatment, GAA activity at 0.2 pmol/punch/hr, GAA activity at 33% lower limit of normal

CaseAddInfo: Late Onset Pompe Disease

CaseGenotypingMethod: Method not described, only that the diagnosis was "confirmed … based on 2 GAA gene mutations"

Variant1: NM_000152.4:c.-32-13T>G

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

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 https://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.1548G>A (p.Trp516Ter)

ClinVar: 189025 https://www.ncbi.nlm.nih.gov/clinvar/variation/189025/

gnomAD: European (Non-Finnish) 1.552e-5, Overall 7.079e-6 https://gnomad.broadinstitute.org/variant/17-78084636-G-A

CaseAddInfo: Patient is compound heterozygous for this variant

Case3: Female, 18 years onset, 19 years diagnosis, 26 years report

CasePresentingHPOs: HP:0003325, HP:0003327, HP:0003236, HP:0002747 (Limb-girdle muscle weakness, Axial muscle weakness, Increased serum CK, Respiratory muscle weakness)

CaseAddInfo: Late-Onset Pompe Disease

Case6: Female, 22 years onset, 23 years diagnosis, 37 years report

CasePresentingHPOs: HP:0003325, HP:0003327, HP:0003236, HP:0002747 (Limb-girdle muscle weakness, Axial muscle weakness, Increased serum CK, Respiratory muscle weakness)

CaseAddInfo: Late-Onset Pompe Disease

Patient25: Female

CasePresentingHPOs: HP:0004887

HPOsFreeText: CRIM positive, Anti-rh GGA antibodies maximum titer = ≤ 1:400, tracheostomy, able to sit independently

CaseNotHPOs: HP:0025156, HP:0002546, HP:0000365

NotHPOsFreeText: infusion associated reaction

CaseAddInfo: variants in trans, symptom onset at 2yo, diagnosis at 3yo, ERT at 4yo

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.784G>A (p.Glu262Lys)

ClinVar: 188806, https://www.ncbi.nlm.nih.gov/clinvar/variation/188806/

gnomAD: 0.00001417, http://gnomad.broadinstitute.org/variant/17-78081447-G-A

Patient7: Male, 4.5mo at diagnosis and ERT

CasePresentingHPOs:

HPOsFreeText: classic IOPD, moderate response to ERT, Pompe muscle pathology score = 3.5, Glycogen = 31.4 Vv(PAS + /mf)%, larger empty vacuoles = 38.1 Vv (LEV/mf)%, Autophagic vacuoles = 0,06 %/mf

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: homozygous, biopsy taken from  musculus vastus lateralis

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.5(GAA):c.2078dup(p.Ala694Glyfs)

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

gnomAD: n/a

CaseVI,2: Adult onset, 61 years diagnosis

CasePresentingHPOs: HP:0003701, HP:0002795, HP:0003388, HP:0003236, HP:0003198 (Proximal muscle weakness, Abnormal respiration, Easy fatiguability, Increased serum CK, Myopathy)

HPOsFreeText: GAA activity from dried blood spot: 0.22%, Forced Vital Capacity Seated: 2040 (46%), 6-Minute Walking Test: 418 min, Plasma CK 2.5-5x Normal

CaseAddInfo: Late-Onset Pompe Disease

Patient1: Italian

CasePresentingHPOs:

HPOsFreeText: non-classic IOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: homozygous

CasePMIDs:

CasePreviousTesting:

CaseGenotypingMethod: method not described

Variant: NM_000152.4(GAA):c.1655T>C (p.Leu552Pro)

ClinVar: 278911, https://www.ncbi.nlm.nih.gov/clinvar/variation/279811/

gnomAD: 0.00003188, https://gnomad.broadinstitute.org/variant/17-78085800-T-C

Case7: Female, 37 years onset, 38 years diagnosis, Italy

HPOsFreeText: See Group Phenotypes, ERT started at 40, GAA Activity at ~4.5 pmol/min/mg before ERT and ~5.5 after

GroupGenotypingMethod: PCR Amplification and GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

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

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.2237G>A (p.Trp746Ter)

ClinVar: 280063 https://www.ncbi.nlm.nih.gov/clinvar/variation/280063/

gnomAD: European (Non-Finnish) 8.834e-6, Overall 3.988e-6 http://gnomad.broadinstitute.org/variant/17-78090814-G-A

CaseAddInfo: Late Onset Pompe Disease

Case#: Patient 16, female

CasePresentingHPOs: HP:0003325

CaseAddInfo: onset and diagnosis at 32yo

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: NC_000017.11:g.(?80118193)(80118357_?)del

ClinVar: 456368, https://www.ncbi.nlm.nih.gov/clinvar/variation/456368/

gnomAD: n/a

Patient11: Female, 39yo at diagnosis, 53yo at ERT

CasePresentingHPOs:

HPOsFreeText: anti rh-GAA antibodies titre = 1:400, enzyme (μmol/g/h) = 0.41 (3-20), NIV required only after ERT began, mobility aid required before and after ERT

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 21yr disease duration, variants in trans

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.1548G>A (p.Trp516Ter)

ClinVar: 189025, https://www.ncbi.nlm.nih.gov/clinvar/variation/189025/

gnomAD: n/a

Variant2: NM_000152.4(GAA):c.2799+4A>G

ClinVar: 286228, https://www.ncbi.nlm.nih.gov/clinvar/variation/286228/

gnomAD: 8.319e-06, http://exac.broadinstitute.org/variant/17-78092608-A-G

Patient7: female, Serbian

CasePresentingHPOs: HP:0003677, HP:0003236, HP:0003325

HPOsFreeText: CK < 10x increase

CaseNotHPOs: HP:0002093

CaseAddInfo: onset in middle age

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant 1: NM_000152.4(GAA):c.2269C>T (p.Gln757Ter)

ClinVar: 429727, https://www.ncbi.nlm.nih.gov/clinvar/variation/429727/

gnomAD: n/a

Variant2: 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

Case#: Variant not associated with a proband. Variant listed in supplementary materials in Supplementary Table 4

DiseaseAssertion: Mucopolysaccharidosis type I, MPS I

FamilyInfo: N/A

CasePresentingHPOs:

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

CaseEnzymeAssay: N/A

CaseUrineGAGs: N/A

CaseERT: N/A

CaseBMT: N/A

Variant1: A79V

Variant1ClinVarID: 1458769

Variant1CAID: CA356987

Variant2: N/A

Variant2ClinVarID: N/A

Variant2CAID: N/A

AdditionalVariants: N/A

ParentalGenotype: N/A

PreviouslyPublished: N/A

Case#: Variant not associated with a proband. Variant listed in supplementary materials in Supplementary Table 4

DiseaseAssertion: Mucopolysaccharidosis type I, MPS I

FamilyInfo: N/A

CasePresentingHPOs:

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

CaseEnzymeAssay: N/A

CaseUrineGAGs: N/A

CaseERT: N/A

CaseBMT: N/A

Variant1: H33Q

Variant1ClinVarID: 2134405

Variant1CAID: CA355945479

Variant2: N/A

Variant2ClinVarID: N/A

Variant2CAID: N/A

AdditionalVariants: N/A

ParentalGenotype: N/A

PreviouslyPublished: N/A

Case#: Variant not associated with a proband. Variant listed in supplementary materials in Supplementary Table 4

DiseaseAssertion: Mucopolysaccharidosis type I, MPS I

FamilyInfo: N/A

CasePresentingHPOs:

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

CaseEnzymeAssay: N/A

CaseUrineGAGs: N/A

CaseERT: N/A

CaseBMT: N/A

Variant1: R89W

Variant1ClinVarID: 580286

Variant1CAID: CA2801156

Variant2: N/A

Variant2ClinVarID: N/A

Variant2CAID: N/A

AdditionalVariants: N/A

ParentalGenotype: N/A

PreviouslyPublished: N/A

Case#: Variant not associated with a proband. Variant listed in supplementary materials in Supplementary Table 4

DiseaseAssertion: Mucopolysaccharidosis type I, MPS I

FamilyInfo: N/A

CasePresentingHPOs:

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

CaseEnzymeAssay: N/A

CaseUrineGAGs: N/A

CaseERT: N/A

CaseBMT: N/A

Variant1: A75T

Variant1ClinVarID: 222993

Variant1CAID: CA356994

Variant2: N/A

Variant2ClinVarID: N/A

Variant2CAID: N/A

AdditionalVariants: N/A

ParentalGenotype: N/A

PreviouslyPublished: N/A

Case#: Variant not associated with a proband. Variant listed in supplementary materials in Supplementary Table 4

DiseaseAssertion: Mucopolysaccharidosis type I, MPS I

FamilyInfo: N/A

CasePresentingHPOs:

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

CaseEnzymeAssay: N/A

CaseUrineGAGs: N/A

CaseERT: N/A

CaseBMT: N/A

Variant1: G51D

Variant1ClinVarID: 193061

Variant1CAID: CA274902

Variant2: N/A

Variant2ClinVarID: N/A

Variant2CAID: N/A

AdditionalVariants: N/A

ParentalGenotype: N/A

PreviouslyPublished: N/A

Case#: Variant not associated with a proband. Variant listed in supplementary materials in Supplementary Table 4

DiseaseAssertion: Mucopolysaccharidosis type I, MPS I

FamilyInfo: N/A

CasePresentingHPOs:

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

CaseEnzymeAssay: N/A

CaseUrineGAGs: N/A

CaseERT: N/A

CaseBMT: N/A

Variant1: Y76C

Variant1ClinVarID: 552686

Variant1CAID: CA2801140

Variant2: N/A

Variant2ClinVarID: N/A

Variant2CAID: N/A

AdditionalVariants: N/A

ParentalGenotype: N/A

PreviouslyPublished: N/A

Case#: Patient 38, male, Han origin, 5 years old (age at diagnosis)

DiseaseAssertion: MPS1-H/S

FamilyInfo: N/A

CasePresentingHPOs: Macrocephaly, Joint stiffness, Mongolian macula, Hepatosplenomegaly, Hernia (HP:0000256, HP:0001387, HP:0011369, HP:0001433, HP:0100790)

CaseHPOFreeText: N/A

CaseNotHPOs: Global Developmental Delay, Airway obstruction (HP:0001263, HP:0006536)

CaseNotHPOFreeText: Cardiac disease

CaseEnzymeAssay: IDUA activity 1.5 nmol/h/mg protein. The normal range in Chinese controls was found to be 27.2–52 nmol/h/mg protein.

CaseUrineGAGs: Diagnosis was confirmed biochemically by the demonstration of an increased excretion of urinary dermatan sulfate and heparan sulfate.

CaseERT: N/A

CaseBMT: N/A

Variant1: c.236C>T (p.A79V)

Variant1ClinVarID: 1458769

Variant1CAID: CA356987

Variant2: c.236C>T (p.A79V)

Variant2ClinVarID: 1458769

Variant2CAID: CA356987

AdditionalVariants: N/A

ParentalGenotype: The transmission of sequence variations found in each patient was inferred by sequencing the corresponding exons of the patient's parents - confirmed in trans by parental testing.

PreviouslyPublished N/A

Case#: Patient 22, male, Han origin, 1 year 8 months old (age at diagnosis)

DiseaseAssertion: MPS1-H

FamilyInfo: N/A

CasePresentingHPOs: Global Developmental Delay, Macrocephaly, Joint stiffness, Mongolian macula, Hepatosplenomegaly, Hernia, Airway obstruction (HP:0001263, HP:0000256, HP:0001387, HP:0011369, HP:0001433, HP:0100790, HP:0006536)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOFreeText: Cardiac disease

CaseEnzymeAssay: IDUA activity 1.8 nmol/h/mg protein. The normal range in Chinese controls was found to be 27.2–52 nmol/h/mg protein.

CaseUrineGAGs: Diagnosis was confirmed biochemically by the demonstration of an increased excretion of urinary dermatan sulfate and heparan sulfate.

CaseERT: N/A

CaseBMT: N/A

Variant1: c.236C>T (p.A79V)

Variant1ClinVarID: 1458769

Variant1CAID: CA356987

Variant2: c.236C>T (p.A79V)

Variant2ClinVarID: 1458769

Variant2CAID: CA356987

AdditionalVariants: N/A

ParentalGenotype: The transmission of sequence variations found in each patient was inferred by sequencing the corresponding exons of the patient's parents - confirmed by parental testing.

PreviouslyPublished N/A