Test results of: SAMPLE REPORT

Admera Health, LLC • CLIA ID 31D2038676 126 Corporate Boulevard • South Plainfield, NJ 07080 1-908-222-0533 • [email protected] • http://www.admerahealth.com

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PATIENT INFORMATION SAMPLE REFERRING PHYSICIAN

Patient Id Sample number Name

Sex Male Source Institution

DOB Date received

Reference Date of report Aug 10, 2016

Test results of: SAMPLE REPORT

Reason for the study: Hypertrophic cardiomyopathy

Test(s) requested: Hypertrophic cardiomyopathy - Full panel (90 genes)

RESULT: POSITIVE

A previously described mutation in the MYH7 gene has been identified. This mutation has been extensively described in

association with the development of an overlapping phenotype of hypertrophic cardiomyopathy, dilated

cardiomyopathy, and left ventricular non-compaction that could be different stages of the same disease. Prognosis

seems to be adverse in terms of sudden death occurrence or development of severe ventricular dysfunction, even in

young individuals.

Gene Variant Result Pathogenicity Population frequency

Number of references

MYH7 NP_000248.2:p.Gly716Arg NM_000257.3:c.2146G>A NC_000014.8:g.23895189C>T

Heterozygosis Pathogenic or disease-causing (+++)

Mutation (not found in controls)

48

Clinical interpretation

The presence of a genetic variant does not exclude the existence of additional genetic alterations in other genes potentially

involved in the development of the disease.

Technical aspects of the study

This sample has been studied by a massive parallel sequencing method using a library that included 90 genes related to

hypertrophic cardiomyopathy. Both sensitivity and specificity are above 99% for SNVs and small INDELs (≤ 20 bp).

Signatures

James Dermody, PhD Laboratory Director

ABMG Certified, Clinical Molecular Genetics Admera Health LLC

Doc. Lorenzo Monserrat Iglesias Cardiologist and Scientific Director

Health in Code


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DETAILED RESULTS

Gene: MYH7 (Encoding the protein: Myosin-7)

NP_000248.2:p.Gly716Arg/NC_000014.8:g.23895189C>T

Heterozygous carrier: mutation occurs in only one copy of the gene.

Mutation nomenclature: Nucleotide code: NM_000257.3:c.2146G>A, NC_000014.8:g.23895189C>T. Amino acid code:

NP_000248.2:p.Gly716Arg. dbSNP ID: rs121913638. Alternative names at the DNA level: c2146G>A. Alternative names at

the protein level: Gly716Arg, NP_000248.2:p.G716R. Located in: exon 19.

Pathogenicity: pathogenic or disease-causing (+++).

Population frequency: mutation (not found in controls).

Number of articles/communications that cite it: 48. Number of described families: 23. Number of families with additional

unpublished information: 4.

Major phenotypes Carriers (families)

Non-carriers

Without genetic study

Total

Hypertrophic cardiomyopathy 31 (22) 0 9 40

Hypertrophic cardiomyopathy + Dilated cardiomyopathy 3 (1) 0 0 3

Sudden death 2 (1) 0 13 15

Restrictive cardiomyopathy 2 (1) 0 0 2

No phenotypic study 1 (1) 1 12 14

Hypertrophic cardiomyopathy + Left ventricular non-compaction/hypertrabeculation

1 (1) 0 0 1

Dilated cardiomyopathy 1 (1) 0 1 2

Left ventricular non-compaction/hypertrabeculation 1 (1) 0 0 1

Not affected or healthy 0 (0) 23 7 30

Clinical information

Gly716Arg in MYH7 is a clearly pathogenic mutation which has not been reported in public databases of genotyping in the

general population. It has been described in families from different ethnic backgrounds and is considered one of the

"malignant" mutations in this gene.

Cosegregation with disease was demonstrated in the reported families with complete penetrance (in adults), and the

variant was associated with the development of an overlapping phenotype (also within members of the same family)

consisting of hypertrophic cardiomyopathy, left ventricular non-compaction, and dilated cardiomyopathy that could

represent different stages of the same disease. Some cases also developed restrictive cardiomyopathy. In the majority of

cases, the diagnosis was made below the age of 25 (mean age of diagnosis was 22 years old), including young females.

Moreover, seven of the patients in which the age of diagnosis was available were diagnosed during their childhood and

presented a severe phenotype (see below). Myocardial hypertrophy was asymmetrically distributed (septal), with an

average maximal wall thickness of 22±5 mm (range 15-33 mm). One of the cases who developed a hypertrophy greater

than 30 mm presented a second mutation in the MYH7 gene. Some families deserve a special comment, such as a Korean


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family with a reported high incidence of sudden death, even in individuals younger than 16 years old. In those relatives

who survived, the disease progressed to its end-stage (developed symptomatic dilated cardiomyopathy [DCM], requiring

heart transplantation even below 25 years of age in some cases). The development of left ventricular dysfunction/dilatation

was relatively prevalent. In fact, DCM was the initial diagnosis in some cases. A restrictive behavior was also observed. It

was described in two out of six carriers of a family from the United States, and it was also observed in a proband referred

to our center who developed severe biatrial enlargement and mild impaired ventricular function. Finally, non-compaction

cardiomyopathy was observed in a family referred to our center. The proband and his sister presented severe biventricular

dysfunction, both of them requiring a heart transplant. The proband's daughter, a carrier of the mutation, presented a

phenotype also compatible with non-compaction at 7 years old, which in subsequent echocardiographic evaluation was

manifested as obstructive hypertrophic cardiomyopathy, indicating that both phenotypes could be different stages of the

same disease.

This mutation is located in a crucial functional domain (converter domain) in which more than 50 pathogenic mutations

have been described. In general terms, these mutations showed a worse prognosis than other missense pathogenic

mutations in MYH7. Moreover, there is a specific sub-region of the converter domain (helix motif)—where Gly716Arg is

located—which clusters mutations with the worst prognosis of the converter region (see below: “Information about other

described mutations affecting the same region” and survival curves in Figure 1). These mutations share several features

with Gly716Arg, such as high penetrance, early expression of the disease, high prevalence of sudden death (in some cases),

and progression to ventricular dysfunction. They are also associated with overlapping phenotypes including hypertrophic

cardiomyopathy, left ventricular non-compaction/dysfunction, and restrictive cardiomyopathy. Some well-described

mutations affecting surrounding amino acids (Arg719Trp, Arg719Gln, and Asp717Gly) present practically the same bad

prognosis as Gly716Arg. Survival curves indicate that half of the carriers had presented a serious adverse event (sudden

death, heart failure death, cardiac transplantation, ICD implantation) by the age of 40 years. These events occurred in early

stages of life in several carriers, but their incidence notably increased in the fourth decade of life in carriers of Gly716Arg.

Events

Major events Carriers Non-carriers Without genetic study Total

Sudden death 6 0 13 19

Cardiac transplantation 5 0 0 5

Heart failure death 4 0 1 5

Cerebrovascular accident (without death) 1 0 0 1

Appropriate ICD discharge 1 0 0 1

Almost all families in which several relatives were informed presented more than one member who suffered a serious

event that occurred below the age of 45 in the majority of the reported cases. A high proportion of them were present

during childhood. Sudden death occurred in more than half of those families, affecting a proportion by 12% to 25% of all

members. Ventricular dysfunction, which leads to heart failure or cardiac transplant, was also a frequent event occurring

in the rest of families evaluated in detail (families in which no sudden death cases were reported). Some families presented

both events within their members. Several other probands have been reported to not present events; however, the lack of

family details or familial antecedent descriptions questions whether some bias could exist.

In summary, this mutation seems to be associated with a high prevalence of serious adverse events in terms of an elevated

risk of sudden death or complications such as progression to an end-stage of the disease (heart failure).


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Survival curves

Figure 1. Kaplan-Meier survival curves: Comparative survival estimate (probability) for carriers of Gly716Arg (blue) versus

of mutations located in the specific helix region (orange), the whole converter region (green), and the whole MYH7 gene

(red) over time (years). The analysis included all mutation carriers and all affected relatives with unknown genotype.

Functional study / Animal model

This mutation affects a highly conserved residue (Gly716) and causes a change from a non-polar amino acid to a basic polar

positive one. As a result, there are moderate changes in the physicochemical properties such as mass, polarity, charge, and

hydrophobicity. Fujita et al. performed a functional study of this mutation in the eukaryotic cell model Dictyostelium

discoideum and observed that the actin-myosin interaction was affected. They showed a decreased Vmax of actin-activated

ATPase activity with a weakened affinity for actin, which was among the lowest affinity values observed compared to other

mutations evaluated. Since Gly716Arg is located in the subdomain that amplifies the movements of myosin motor domain

(converter domain), it is possible to infer that mutations similarly located in this subdomain might reduce the affinity to

actin filaments by affecting the swing motion.

Other mutations located in the converter domain were also functionally evaluated. Although their behavior differed from

that observed for Gly716Arg, these mutations remarkably affected the actomyosin cross-bridges, thereby pointing out the

relevance of the converter domain of the myosin head for the vital functions of the motor molecule. Most of the mutations

affecting this important functional domain were associated with a malignant phenotype.


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Mutation effect predictors (These predictors have limited clinical utility and should be used only as supporting evidence)

Predictor Prediction Score Version

SIFT Damaging 0.03 Computed from ENSEMBL 55 (September, 2014)

Polyphen-2 (HumVar) Probably damaging 0.983 v2.2.2 (September, 2014)

Polyphen-2 (HumDiv) Probably damaging 0.999 v2.2.2 (September, 2014)

MutationTaster Disease-causing 1 date of accession: Jul 13, 2016

DANN 0.999306 July, 2015

SIFT: it ranges from 0 to 1. Scores <0.05 are called "Damaging" and all others are called "Tolerated". Polyphen: it ranges from 0 (benign) to 1 (damaging). MutationTaster: it

ranges from 0 to 1. Value close to 1 indicates a high 'security' of the prediction. DANN: the higher score the more potential pathogenicity. Values between 0 and 1.

Conclusions

The Gly716Arg mutation in MYH7 is associated with the development of an overlapping phenotype of hypertrophic

cardiomyopathy, dilated cardiomyopathy, and left ventricular non-compaction that could be different stages of the same

disease. Penetrance is complete (considering young adults), and clinical manifestations have an early onset. Prognosis

seems to be adverse in terms of sudden death occurrence or development of severe ventricular dysfunction, even in

young individuals.


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APPENDIX 1: SUPPLEMENTAL INFORMATION

Detailed description of the families (mutation: p.Gly716Arg, gene: MYH7)

Family 155 [reference: 1]

Country of origin of the family (or of the publication): Italy. Ethnicity: Western Caucasian.

Mutation carriers with available information: 3 (hypertrophic cardiomyopathy: 3). Mutation non-carriers with available

information: 4 (not affected or healthy: 4). Relatives without genetic study: 2 (not affected or healthy: 1, without

phenotypic study: 1).

Adverse events reported in the family ─ no sudden death has been reported; death of unknown cause: 1 (without genetic

study: 1); transplant: 1 (with the mutation: 1); (see tables).

Family 197 [references: 4, 20]

Country of origin of the family (or of the publication): Korea, Republic of. Ethnicity: Oriental.

Mutation carriers with available information: 10 (hypertrophic cardiomyopathy + dilated cardiomyopathy: 3, hypertrophic

cardiomyopathy: 3, possible left ventricular non-compaction/hypertrabeculation + hypertrophic cardiomyopathy: 1,

sudden deaths of unknown cause: 2, without phenotypic study: 1). Mutation non-carriers with available information: 10

(not affected or healthy: 10). Relatives without genetic study: 10 (sudden deaths of unknown cause: 5, not affected or

healthy: 5).

Adverse events reported in the family ─ sudden death: 8 (with the mutation: 3, without genetic study: 5); heart failure

death: 3 (with the mutation: 3); transplant: 1 (with the mutation: 1); (see tables).

Particularly highly penetrant and with poor prognosis, 13 out of 32 members affected. Four sudden death in patients

younger than 16 years of age (at 9 playing, at 12 in primary school, at 13 running, at 16 immediately after running to catch

a bus). Other SDs: at 40 with no details, at 42 with no details, at 32 after a night of drinking alcohol. Most sudden deaths

apparently had no prior symptoms; the family members were not aware of having any cardiac condition (only one of the

children had a diagnosis, a 9-year-old girl). There is also one heart failure death (age 47), and alive patient (niece of 38

years) had systolic impairment (EF 40%) with enlarged ventricles (LVED 50 mm). None of the five patients with echo details

had obstruction (apparently, reported no SAM in all of them). Most patients (including those with impaired systolic

function) had minor symptoms (NYHA I-II); no syncope recorded in any of the cases. 2/5 had chest pain. Palpitations not

recorded. ECG features: most popular Q wave and ST changes, no conduction or bundle branch blocks. There is no mention

on ECG rhythm, arrhythmias, Holter, exercise, medications, or some other echo parameters such as diastolic dysfunction.

The major phenotypic characteristics of the MYH7 Gly716Arg mutation were HCM with frequent deterioration to DCM or

SCD at a young age. All 15 patients with the mutation exhibited clinical phenotype or clinical events such as SCD or HF with

deterioration to DCM.

- Patient 922: He was diagnosed with HCM with normal LVEF at age 23. Biopsy with hypertrophy, disarray, and fibrosis. He

was a heavy alcohol drinker. At age 38, LVEDD was 60 mm and LVEF was 39%. He continued to drink alcohol despite

aggravation of symptoms. Clinical events of AF, LV thrombus, and renal infarction developed during follow-up. At age 45,

LVEDD was further enlarged to 71 mm and LVEF was reduced to 20%. He died of HF while awaiting cardiac transplantation

at age 46.

- Patient 924: Normal echo at 7 years old and abnormal at 9 years of age.

- Patient 931: Alcohol was a possible precipitating factor at the time of SCD.

- Patient 923: Deterioration to DCM could be aggravated by pregnancy and emotional stress.


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Country of origin of the family (or of the publication): Canada.

Mutation carriers with available information: 2 (hypertrophic cardiomyopathy: 2).

No adverse events have been reported.

Two carriers diagnosed at 25 and 75 years of age. One of them had a max LV wall thickness of 23, LVEDD of 51, and LA

diameter of 48 (probably the 75-year-old one).

Family 324 [Unpublished]

Country of origin of the family (or of the publication): Spain. Ethnicity: Western Caucasian.

Mutation carriers with available information: 3 (left ventricular non-compaction/hypertrabeculation: 1, hypertrophic

cardiomyopathy: 2). Mutation non-carriers with available information: 1 (not affected or healthy: 1). Relatives without

genetic study: 2 (possible dilated cardiomyopathy: 1, not affected or healthy: 1).

Adverse events reported in the family ─ no sudden death has been reported; heart failure death: 1 (without genetic study:

1); transplant: 2 (with the mutation: 2); (see tables).



Mutation carriers with available information: 1 (dilated cardiomyopathy: 1). Mutation non-carriers with available

information: 1 (possible arrhythmogenic cardiomyopathy: 1). Relatives without genetic study: 8 (sudden deaths of

unknown cause: 1, without phenotypic study: 7).

Adverse events reported in the family ─ sudden death: 1 (without genetic study: 1); non-cardiovascular death: 1 (with the

mutation: 1); death of unknown cause: 3 (without genetic study: 3); transplant: 1 (with the mutation: 1); (see tables).


Country of origin of the family (or of the publication): France.




Country of origin of the family (or of the publication): France.




Country of origin of the family (or of the publication): United States. Ethnicity: Western Caucasian.

Mutation carriers with available information: 1 (hypertrophic cardiomyopathy: 1). Relatives without genetic study: 10

(hypertrophic cardiomyopathy: 3, sudden deaths of unknown cause: 3, without phenotypic study: 4).

Adverse events reported in the family ─ sudden death: 3 (without genetic study: 3); (see tables).


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Country of origin of the family (or of the publication): United States.




Country of origin of the family (or of the publication): India. Ethnicity: Hindu.

Mutation carriers with available information: 1 (hypertrophic cardiomyopathy: 1). Mutation non-carriers with available

information: 3 (not affected or healthy: 3).

Adverse events reported in the family ─ sudden death: 1 (with the mutation: 1); (see tables).


Country of origin of the family (or of the publication): Italy. Ethnicity: Western Caucasian.


Adverse events reported in the family ─ sudden death: 1 (with the mutation: 1); appropriate icd discharge: 1 (with the

mutation: 1); (see tables).


Country of origin of the family (or of the publication): United Kingdom.




Country of origin of the family (or of the publication): United Kingdom.




Country of origin of the family (or of the publication): United States.

Mutation carriers with available information: 6 (hypertrophic cardiomyopathy: 4, restrictive cardiomyopathy: 2). Mutation

non-carriers with available information: 5 (not affected or healthy: 5). Relatives without genetic study: 10 (hypertrophic

cardiomyopathy: 1, possible hypertrophic cardiomyopathy: 5, sudden deaths of unknown cause: 4).


Country of origin of the family (or of the publication): France. Ethnicity: Western Caucasian.




Country of origin of the family (or of the publication): United States. Ethnicity: Spanish American.



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Adverse events reported in the family ─ no sudden death has been reported; heart failure death: 1 (with the mutation: 1);

stroke without death: 1 (with the mutation: 1); (see tables).

- Patient 51559: He had a syncopal episode at age 35 for which he received an ICD. At age 35, he developed paroxysmal

atrial fibrillation. He subsequently developed dilated cardiomyopathy with progressive congestive heart failure. He had an

ablation for atrial flutter. Because of advanced heart failure, he was considered a candidate for cardiac transplantation.

The patient underwent autologous peripheral blood cell harvest followed by direct myocardial injection of autologous

precursor cells as a means of augmenting heart function. After intra-myocardial injection, he developed ventricular

fibrillation that required defibrillation. He developed acute renal failure for which he received hemodialysis. Due to overall

poor conditioning exacerbated by his post-arrest course, he was not a candidate for heart transplantation. His left

ventricular function remained severely reduced and showed no improvement. He developed an ischemic right lower

extremity due to an embolism for which he was treated with a percutaneous intervention and anticoagulated with

bivalirudin. Shortly after he had a hemorrhagic stroke. Support was withdrawn and he expired. The heart was 660 g (normal,

350 g) at autopsy, with four-chamber dilation and biventricular hypertrophy (left ventricle, 1.5 cm; right ventricle, 0.7 cm).

Histologically, marked diffuse interstitial fibrosis was present and there was myofiber disarray. There were multiple small

areas of necrosis.


Country of origin of the family (or of the publication): Greece. Ethnicity: Western Caucasian.




Country of origin of the family (or of the publication): Brazil.












Country of origin of the family (or of the publication): Italy.




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Country of origin of the family (or of the publication): Italy.


Adverse events reported in the family ─ sudden death: 1 (with the mutation: 1); (see tables).

Other mutations in the family: carriers with p.Gln1654His (gene MYH7): 1.





Total number of controls reported: 860; controls in the reference 1: 2; controls in the reference 10: 106; controls in the

reference 11: 100; controls in the reference 15: 100; controls in the reference 18: 200; controls in other references: 352.


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Table 1: Genetic and clinical characteristics of mutant carriers who suffered adverse events (mutation: p.Gly716Arg, gene: MYH7)

Patient Id

Family Id

Mutation Other mut.

Clinical diagnosis Sex Age at DX

Age last FU

NYHA Atr. fib.

FHSD LVH (MLVWT)

VT-VF-EV

Previous syncope

Abn. BP resp.

Obstr. (grad.)

Syst. dysf. (EF)

Event (Age at event)

618 155 + No Hypertrophic cardiomyopathy Male ? 43 + (III) ? ? +(25) ? - ? - + Cardiac transplantation (42)

922 197 + No Hypertrophic cardiomyopathy + Dilated cardiomyopathy

Male 23 46 + (II) ? + +(23) ? - ? - +(16) Heart failure death (46)

923 197 + No Hypertrophic cardiomyopathy + Dilated cardiomyopathy

Female 18 50 + (IV) ? + +(21) ? - ? - +(13) Cardiac transplantation (45)

926 197 + No Hypertrophic cardiomyopathy Male 5 11 - (I) ? + +(25) ? - ? ? - Sudden death (11)

927 197 +(O) No Sudden death of unknown cause

Male ? 35 ? ? ? ? ? ? ? ? ? Sudden death (35)

928 197 +(O) No Sudden death of unknown cause

Female ? 42 - (I) ? + ? ? ? ? ? ? Sudden death (42)

929 197 +(O) No Hypertrophic cardiomyopathy + Dilated cardiomyopathy

Female ? 47 + (IV) ? + +(22) ? ? ? - +(33) Heart failure death (47)

930 197 +(O) No ? Male ? 25 + (?) ? + ? ? ? ? ? ? Heart failure death (25)

2079 324 + No Left ventricular non-compaction/hypertrabeculation

Male 20 42 + (II) + (P)

- +(15) ? - ? - +(34) Cardiac transplantation (42)

2080 324 + No Hypertrophic cardiomyopathy Female 25 43 + (III) + (P)

- +(22) [2] - - - +(44) Cardiac transplantation (43)

3244 477 + No Dilated cardiomyopathy Male 49 74 + (III) ? + +(15) [2] - ? - +(39) Non-cardiovascular death (74) Cardiac transplantation (53)

14489 2670 + No Hypertrophic cardiomyopathy Male 20 21 ? ? ? +(31) ? ? ? ? ? Sudden death (21)


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Patient Id

Family Id

Mutation Other mut.


Age last FU

NYHA Atr. fib.

FHSD LVH (MLVWT)

VT-VF-EV

Previous syncope

Abn. BP resp.

Obstr. (grad.)

Syst. dysf. (EF)


21179 4487 + No Hypertrophic cardiomyopathy Male 7 ? - (I) ? - +(24) ? + - ? ? Sudden death Appropriate ICD discharge

51559 18867 + No Hypertrophic cardiomyopathy Male 9 41 + (?) + (P)

+ + [5] + ? ? +(16) Heart failure death (41) Stroke without death (41)

119003 53026 + Yes Hypertrophic cardiomyopathy Female 10 ? - (I) ? - +(33) ? ? ? - -(66) Sudden death (17)

Mutation: + = yes, +(O) = obligate carrier, +(H) = homozygous carrier; NYHA: New York Heart Association functional class; Atr. fib.: atrial fibrillation ([C] chronic, [P] paroxysmal); FHSD: family history of sudden death; LVH (MLVWT): left ventricular hypertrophy

(maximal ventricular wall thickness in mm); VT-VF-EV: [1] non-sustained ventricular tachycardia, [2] sustained monomorphic ventricular tachycardia, [3] sustained polymorphic ventricular tachycardia, [4] torsade de pointes, [5] ventricular fibrillation, [6] frequent

premature ventricular beats; Abn. BP resp.: abnormal blood pressure response on exercise; Obstr. (grad.): gradient in mmHg; Syst. dysf. (EF): systolic dysfunction (ejection fraction). Non-sustained ventricular tachycardia, previous syncope, family history of

premature sudden death, severe hypertrophy (maximum wall thickness >30 mm) and abnormal blood pressure response on exercise are considered risk factors of sudden death. Shaded rows represent index cases.

Patient119003: mutation p.Gln1654His (gene MYH7).


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Table 2: Genetic and clinical characteristics of mutant carriers without documented adverse events (mutation: p.Gly716Arg, gene: MYH7)

Patient Id

Family Id

Mutation Other mut.


Age last FU

NYHA Atr. fib.

FHSD LVH (MLVWT)

VT-VF-EV

Previous syncope

Abn. BP resp.

Obstr. (grad.)

Syst. dysf. (EF)

Comment

625 155 + No Hypertrophic cardiomyopathy Male ? 22 + (II) ? ? +(17) ? + ? - +

626 155 + No Hypertrophic cardiomyopathy Male ? 16 - (I) ? ? +(20) ? - ? - ?

924 197 + No Hypertrophic cardiomyopathy Female 10 20 - (I) ? + +(15) ? - ? - -

925 197 + No Left ventricular non-compaction/hypertrabeculation? + Hypertrophic cardiomyopathy

Male 9 24 - (I) ? + +(23) ? - ? - -

1204 220 + No Hypertrophic cardiomyopathy ? 25 25 ? ? ? ? ? ? ? ? ?

1205 220 + No Hypertrophic cardiomyopathy ? 75 75 ? ? ? +(23) ? ? ? ? ?

2083 324 + No Hypertrophic cardiomyopathy Female 7 18 - (I) - - +(24) ? + + +(75) -(91)

6988 939 + No Hypertrophic cardiomyopathy ? ? ? ? ? ? + ? ? ? ? ?

6989 940 + No Hypertrophic cardiomyopathy ? ? ? ? ? ? + ? ? ? ? ?

7159 1045 + No Hypertrophic cardiomyopathy Male 24 32 ? ? + +(26) ? - ? +(80) -

12308 2001 + No Hypertrophic cardiomyopathy ? ? <15 ? ? ? + ? ? ? ? ?



35305 9948 + No Hypertrophic cardiomyopathy ? ? ? ? ? + + ? ? ? ? ?




35309 9948 + No Restrictive cardiomyopathy Female ? ? ? ? + ? ? ? ? ? -


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Patient Id

Family Id

Mutation Other mut.


Age last FU

NYHA Atr. fib.

FHSD LVH (MLVWT)

VT-VF-EV

Previous syncope

Abn. BP resp.

Obstr. (grad.)

Syst. dysf. (EF)

Comment

35310 9948 + No Restrictive cardiomyopathy Female ? ? ? ? + ? ? ? ? ? -

36975 10707 + No Hypertrophic cardiomyopathy ? ? ? ? ? ? ? ? ? ? ? ?

38080 197 + No Hypertrophic cardiomyopathy Male 32 43 - (I) ? + +(21) ? ? ? - -

58662 22224 + No Hypertrophic cardiomyopathy Male ? ? ? ? ? + ? ? ? ? ?




118270 52743 + No Hypertrophic cardiomyopathy Female 28 33 - (I) ? ? +(28) ? ? ? - +(68)

122253 54513 + No Hypertrophic cardiomyopathy Female ? ? ? ? ? ? ? ? ? ? ?






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Table 3: Clinical characteristics of relatives without genetic diagnosis who had adverse cardiovascular events potentially related to the family disease (mutation:

p.Gly716Arg, gene: MYH7)

Patient Id

Family Id

Other mut.


Age last FU

NYHA Atr. fib.

FHSD LVH (MLVWT)

VT-VF-EV

Previous syncope

Abn. BP resp.

Obstr. (grad.)

Syst. dysf. (EF)


931 197 No Sudden death of unknown cause

Male ? 32 - (I) ? + ? ? ? ? ? ? Sudden death (32)


Male ? 12 - (I) ? + ? ? ? ? ? ? Sudden death (12)


Female ? 13 - (I) ? + ? ? ? ? ? ? Sudden death (13)


Female ? 45 ? ? ? ? ? ? ? ? ? Sudden death (45)



2081 324 No Dilated cardiomyopathy?

Male ? ? ? ? ? ? ? ? ? ? ? Heart failure death (47)

3247 477 No ? Male ? 55 ? ? ? ? ? ? ? ? ? Death of unknown cause (55)




Female ? 46 ? ? ? ? ? ? ? ? ? Sudden death (46)


Female ? 16 ? ? + ? ? ? ? ? ? Sudden death (16)


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Patient Id

Family Id

Other mut.


Age last FU

NYHA Atr. fib.

FHSD LVH (MLVWT)

VT-VF-EV

Previous syncope

Abn. BP resp.

Obstr. (grad.)

Syst. dysf. (EF)



Female ? 42 ? ? + ? ? ? ? ? ? Sudden death (42)


? ? ? ? ? ? ? ? ? ? ? ? Sudden death


? ? ? ? ? ? ? ? ? ? ? ? Sudden death


? ? ? ? ? ? ? ? ? ? ? ? Sudden death


? ? ? ? ? ? ? ? ? ? ? ? Sudden death






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Statistics and summary of the mutant carrier characteristics with available data (mutation:

p.Gly716Arg, gene: MYH7)

Variable Cases with data (n) Positive cases Average (SD) / Range

Demographic variables

Sex 27

Male 16 (59.3%)

Female 11 (40.7%)

Age at diagnosis 18 22.0 (±17.3) / 5-75

Age at time of study or last follow-up 23 36.0 (±16.6) / 11-75

Environmental factors or triggers

Sport 9 0

Symptoms and risk factors

Dyspnea 20 10

NYHA I 10 (50%)

NYHA II 3 (15%)

NYHA III - IV 5 (25%)

NYHA Unknown 2 (10%)

Previous syncope 15 4

Chest pain 13 3 (23.1%)

Ventricular arrhythmias 5 3 (60%)

Abnormal blood pressure response 3 1 (33.3%)

Fibrofatty infiltration 1 1 (100%)

Morphology

LV hypertrophy 31 19 (61.3%)

Asymmetrical septal 13

Maximum LV wall thickness (mm) 20 22.6 (±4.9) / 15-33

LV mass (gr) 4 304 (±79.8) / 205-400

LV dilatation 15 7 (46.7%)

LV end-diastolic diameter (mm) 11 53.1 (±17.3) / 34-90

LV systolic dysfunction 19 10 (52.6%)

Ejection fraction (%) 10 42 (±25.8) / 13-91

LV diastolic dysfunction 6 4 (66.7%)

Abnormal relaxation 1

Restrictive filling 3

Left atrial dilatation 15 10 (66.7%)


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Left atrium diameter (mm) 10 51.4 (±8.2) / 36-60

LV obstruction 16 2 (12.5%)

Subaortic gradient (mmHg) 2 77.5 (±3.5) / 75-80

Electrocardiogram

Abnormal ECG 16 16 (100%)

Previous atrial fibrillation 4 3

Rhythm 23 9 (39.1%)

Sinus 5

Atrial fibrillation flutter 4

Abnormal voltage or repolarization 12 10 (83.3%)

Abnormal repolarization 1

Low voltage 2

High voltage 7

Negative T waves 2

Conduction system disease 7 4 (57.1%)

Abnormal Q wave 11 5 (45.5%)

Treatment

Treatment 9 9 (100%)

Medical 8

Surgery 1

Implantable cardioverter defibrillator 4


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Selected references (mutation: p.Gly716Arg, gene: MYH7)

1. Anan R, Greve G, Thierfelder L, Watkins H, McKenna WJ, Solomon S, et al. Prognostic implications of novel beta cardiac

myosin heavy chain gene mutations that cause familial hypertrophic cardiomyopathy. J Clin Invest 1994. 93(1):280-285.

http://www.ncbi.nlm.nih.gov/pubmed/8282798

2. Rayment I, Holden HM, Sellers JR, Fananapazir L, Epstein ND. Structural interpretation of the mutations in the beta-

cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy. Proc Natl Acad Sci U S A

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2015 Jul 1;101(13):1047-53.








http://lm.nih.gov/pubmed/25132132




Page 23

Information about other described mutations affecting the same region (helix-

converter region)

This section describes available data about the following 6 variants: p.Gly716Ala, p.Asp717Gly, p.Arg719Gln, p.Arg719Pro,

p.Arg719Trp, p.Arg721Lys.

Number of families described associated with the selected variants: 80. Carriers with available information: 147 (affected

or possibly affected: 144, not affected or healthy: 3). Non-carriers with available information: 70 (hypertrophic

cardiomyopathy: 1, not affected or healthy: 63, without phenotypic study: 6). Relatives without genetic study: 121

(hypertrophic cardiomyopathy: 39, possible hypertrophic cardiomyopathy: 6, sudden deaths of unknown cause: 11, not

affected or healthy: 27, without phenotypic study: 38).

Major phenotypes Carriers (families)

Non-carriers

Without genetic study

Total

Hypertrophic cardiomyopathy 139 (76) 1 45 185

Not affected or healthy 3 (3) 63 27 93

Restrictive cardiomyopathy 3 (2) 0 0 3

Left ventricular non-compaction/hypertrabeculation 1 (1) 0 0 1

Hypertrophic cardiomyopathy + Left ventricular non-compaction/hypertrabeculation

1 (1) 0 0 1

No phenotypic study 0 (0) 6 38 44

Sudden death 0 (0) 0 11 11


Page 24

Variant Phenotypes (patients) Families Affected carriers

Healthy carriers

Events (patients) References

MYH7 p.Gly716Ala

Hypertrophic cardiomyopathy(1) 1 1 0 2

MYH7 p.Asp717Gly

0 0 0 1

MYH7 p.Arg719Gln

Hypertrophic cardiomyopathy(46) Restrictive cardiomyopathy(1)

41 47 1 Sudden death (26) Cardiac transplantation (6) Heart failure death (3)

76

MYH7 p.Arg719Pro

Hypertrophic cardiomyopathy(2) 2 2 0 6

MYH7 p.Arg719Trp

Hypertrophic cardiomyopathy(91) Left ventricular non-compaction/hypertrabeculation(2)

35 92 2 Sudden death (33) Heart failure death (5) Cardiac transplantation (4) Appropriate ICD discharge (1) Stroke-related death (1)

105

MYH7 p.Arg721Lys

Restrictive cardiomyopathy(2) 1 2 0 Heart failure death (1) 4


Page 25

Statistics and summary about other described mutations affecting the same region


Demographic variables

Sex 74

Male 32 (43.2%)

Female 42 (56.8%)

Age at diagnosis 46 20.9 (±16.2) / 1-68

Age at time of study or last follow-up 52 33.0 (±17.8) / 1-70

Environmental factors or triggers

Sport 3 1 (33.3%)

Non-competitive 1

Symptoms and risk factors

Dyspnea 40 24

NYHA I 16 (40%)

NYHA II 8 (20%)

NYHA III - IV 13 (32.5%)

NYHA Unknown 3 (7.5%)

Previous syncope 35 13

Chest pain 28 10 (35.7%)

RV failure signs 1 0

Ventricular arrhythmias 21 6 (28.6%)

Abnormal blood pressure response 15 5 (33.3%)

Morphology

LV hypertrophy 100 46 (46%)

Apical 1

Asymmetrical septal 26

Atypical 3

Maximum LV wall thickness (mm) 47 20.7 (±7.3) / 5-33

LV mass (gr) 3 240.7 (±130.5) / 90-320

LV dilatation 23 2 (8.7%)

LV end-diastolic diameter (mm) 14 44.8 (±6.1) / 38-60

LV systolic dysfunction 30 9 (30%)

Ejection fraction (%) 20 56.2 (±18.2) / 15-83

LV diastolic dysfunction 11 10 (90.9%)

Abnormal relaxation 3


Page 26


Pseudonormalization 2

Restrictive filling 4

Left atrial dilatation 23 11 (47.8%)

Left atrium diameter (mm) 18 43.8 (±8.5) / 28-61

LV obstruction 37 10 (27%)

Subaortic gradient (mmHg) 5 70.6 (±22.2) / 40-95

Electrocardiogram

Abnormal ECG 37 32 (86.5%)

Previous atrial fibrillation 9 4

Rhythm 79 24 (30.4%)

Sinus 21

Atrial fibrillation flutter 3

Abnormal voltage or repolarization 24 22 (91.7%)

Abnormal repolarization 4

Long QT 8

High voltage 8

Negative T waves 9

Conduction system disease 19 7 (36.8%)

Abnormal Q wave 19 11 (57.9%)

Corrected QT duration (ms) 8 8 488.3 (±16.8) / 470-520

Electrophysiologic study

Electrophysiologic study done 1

Treatment

Treatment 35 27 (77.1%)

Medical 11

Surgery 7

Pacemaker 2

Implantable cardioverter defibrillator 11


Page 27

Selected references

p.Gly716Ala

1. Kassem HS, Azer RS, Ayad MS, Moharem-Elgamal S, Magdy G, Elguindy A, et al. Early Results of Sarcomeric Gene

Screening from the Egyptian National BA-HCM. J Cardiovasc Transl Res. 2013 Feb;6(1):65-80.


2. Colegrave M, Peckham M. Structural implications of β-cardiac myosin heavy chain mutations in human disease. Anat Rec

(Hoboken). 2014 Sep;297(9):1670-80.


p.Asp717Gly



2015 Jul 1;101(13):1047-53.


p.Arg719Gln

1. Lopes LR, Rahman MS, Elliott PM. A systematic review and meta-analysis of genotype-phenotype associations in patients

with hypertrophic cardiomyopathy caused by sarcomeric protein mutations. Heart. 2013 Dec;99(24):1800-11.


2. Pan S, Caleshu CA, Dunn KE, Foti MJ, Moran MK, Soyinka O, Ashley EA. Cardiac structural and sarcomere genes associated

with cardiomyopathy exhibit marked intolerance of genetic variation. Circ Cardiovasc Genet. 2012 Dec;5(6):602-10.



Mar;45(1):69-99.


4. Bos JM, Will ML, Gersh BJ, Kruisselbrink TM, Ommen SR, Ackerman MJ. Characterization of a Phenotype-Based Genetic

Test Prediction Score for Unrelated Patients with Hypertrophic Cardiomyopathy. Mayo Clin Proc. 2014 Jun;89(6):727-37.











8. Kapplinger JD, Landstrom AP, Bos JM, Salisbury BA, Callis TE, Ackerman MJ. Distinguishing Hypertrophic Cardiomyopathy-

Associated Mutations from Background Genetic Noise. J Cardiovasc Transl Res. 2014 Apr;7(3):347-61.














Page 28

9. Bonne G, Carrier L, Richard P, Hainque B, Schwartz K. Familial hypertrophic cardiomyopathy: from mutations to functional

defects. Circ Res. 1998 ;83(6):580-93.


10. Jelusic M, Gall-Troselj K, Jurak I, Pavelic K, Kniewald H, Putarek NR, et al. Genetic analysis of hypertrophic

cardiomyopathy in 12 Croatian families. Eur J Hum Genet. 2002;10:255-.

http://www.distrofiamuscular.net/resumos11.htm

p.Arg719Pro




2. Xu Q, Dewey S, Nguyen S, Gomes AV. Malignant and benign mutations in familial cardiomyopathies: Insights into

mutations linked to complex cardiovascular phenotypes. J Mol Cell Cardiol. 2010 May;48(5):899-909.




2015 Jul 1;101(13):1047-53.


4. Marsiglia JD, Credidio FL, de Oliveira TG, Reis RF, Antunes Mde O, de Araujo AQ, et al. Screening of MYH7, MYBPC3, and

TNNT2 genes in Brazilian patients with hypertrophic cardiomyopathy. Am Heart J. 2013;166(4):775-82.



(Hoboken). 2014 Sep;297(9):1670-80.


p.Arg719Trp

1. Green, E. M.,Wakimoto, H.,Anderson, R. L.,Evanchik, M. J.,Gorham, J. M.,Harrison, B. C.,Henze, M.,Kawas, R.,Oslob, J.

D.,Rodriguez, H. M.,Song, Y.,Wan, W.,Leinwand, L. A.,Spudich, J. A.,McDowell, R. S.,Seidman, J. G.,Seidman, C. E. A small-

molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice. Science

2016;351(6273):617-21.



Mar;45(1):69-99.












http://www.distrofiamuscular.net/resumos11.htm












Page 29

6. Kapplinger JD, Landstrom AP, Bos JM, Salisbury BA, Callis TE, Ackerman MJ. Distinguishing Hypertrophic Cardiomyopathy-

Associated Mutations from Background Genetic Noise. J Cardiovasc Transl Res. 2014 Apr;7(3):347-61.


7. Emory University. EmVClass: Variant Classification Database. 2013.

http://geneticslab.emory.edu/emvclass/emvclass.php

8. Piano MR. Familial hypertrophic cardiomyopathy. J Cardiovasc Nurs. 1999;13(4):46-58.


9. Marian AJ, Roberts R. Familial hypertrophic cardiomyopathy: a paradigm of the cardiac hypertrophic response to injury.

Ann Med. 1998;30 Suppl 1:24-32.


10. Bonne G, Carrier L, Richard P, Hainque B, Schwartz K. Familial hypertrophic cardiomyopathy: from mutations to

functional defects. Circ Res. 1998 ;83(6):580-93.


p.Arg721Lys

1. Xu Q, Dewey S, Nguyen S, Gomes AV. Malignant and benign mutations in familial cardiomyopathies: Insights into

mutations linked to complex cardiovascular phenotypes. J Mol Cell Cardiol. 2010 May;48(5):899-909.




2015 Jul 1;101(13):1047-53.


3. Rai TS, Ahmad S, Ahluwalia TS, Ahuja M, Bahl A, Saikia UN, et al. Genetic and clinical profile of Indian patients of idiopathic

restrictive cardiomyopathy with and without hypertrophy. Mol Cell Biochem. 2009 Nov;331(1-2):187-92.




(Hoboken). 2014 Sep;297(9):1670-80.



http://geneticslab.emory.edu/emvclass/emvclass.php










Page 30

APPENDIX 2

AVAILABLE INFORMATION ON OTHER IDENTIFIED VARIANTS

We have identified genetic variants that we consider not to be associated with disease development either because they

have been reported in healthy controls or because they do not affect protein structure nor function.

Region Variants found

Exonic 106

Synonymous 69

Nonsynonymous 37

Intronic 158

Intronic splicing 20

UTR 16

Total 280

Only good quality variants were included (QUAL ≥170)

106

1620

138

Exon

UTR

Intron splicing

Intron non-splicing


Page 31

List of probably non-disease causing exonic genetic variants (excluding synonymous)

Gene Variant Function Exonic function

dbSNP dbSNP freq.

1000G MAF

5000G MAF

HiC freq.

AF1 DP Qual

Qual Freq. alt.

AARS2 NP_065796.1:p.Ile339Val; NM_020745.3:c.1015A>G; NC_000006.11:g.44275011T>C

Exon Nonsynonymous rs324136 88.9 11.34 99.74 Hom. 682 255 100

AGL NP_000633.2:p.Pro1067Ser; NM_000642.2:c.3199C>T; NC_000001.10:g.100358103C>T

Exon Nonsynonymous rs3753494 13.1 11.06 13.95 27.03 Het. 1696 255 49

BSCL2 NP_001124174.2:p.Lys268Arg; NM_001130702.2:c.803A>G; NC_000011.9:g.62458275T>C

Exon Nonsynonymous rs6856 17.47 13.5 18.33 36.03 Hom. 627 255 100

COQ2 NP_056512.5:p.Val66Leu; NM_015697.7:c.196G>T; NC_000004.11:g.84205872C>A

Exon Nonsynonymous rs6818847 67.59 64.98 31.48 91.21 Hom. 501 255 99.8

COX15 NP_004367.2:p.Phe374Leu; NM_004376.5:c.1120T>C; NC_000010.10:g.101473218A>G


FHOD3 NP_001268669.1:p.Ser455Leu; NM_001281740.1:c.1364C>T; NC_000018.9:g.34232610C>T

Exon Nonsynonymous rs2848901 28.39 31.95 42.33 Het. 858 255 47.7

FHOD3 NP_001268669.1:p.Val1326Ile; NM_001281740.1:c.3976G>A; NC_000018.9:g.34324091G>A


GAA NP_000143.2:p.His199Arg; NM_000152.3:c.596A>G; NC_000017.10:g.78079597A>G

Exon Nonsynonymous rs1042393 65.72 60.08 32.75 93.29 Het. 922 170 43.4

GAA NP_000143.2:p.Arg223His; NM_000152.3:c.668G>A; NC_000017.10:g.78079669G>A


GAA NP_000143.2:p.Val780Ile; NM_000152.3:c.2338G>A; NC_000017.10:g.78091405G>A

Exon splicing

Nonsynonymous rs1126690 72.27 71.19 26.68 94.2 Het. 1119 255 53.5

GFM1 NP_079272.4:p.Val215Ile; NM_024996.5:c.643G>A; NC_000003.11:g.158366900G>A


GLB1 NP_000395.2:p.Ser532Gly; NM_000404.2:c.1594A>G; NC_000003.11:g.33055688T>C


GLB1 NP_000395.2:p.Cys521Arg; NM_000404.2:c.1561T>C; NC_000003.11:g.33055721A>G


GLB1 NP_000395.2:p.Pro10Leu; NM_000404.2:c.29C>T; NC_000003.11:g.33138549G>A


JPH2 NP_065166.2:p.Ala396Thr; NM_020433.4:c.1186G>A; NC_000020.10:g.42747247C>T



Page 32


dbSNP dbSNP freq.

1000G MAF

5000G MAF

HiC freq.

AF1 DP Qual

Qual Freq. alt.

MYH6 NP_002462.2:p.Val1101Ala; NM_002471.3:c.3302T>C; NC_000014.8:g.23861811A>G


MYL3 NP_000249.1:p.Val156Leu; NM_000258.2:c.466G>T; NC_000003.11:g.46900980C>A

Exon Nonsynonymous rs199474707 0 Het. 1303 255 48.4

OBSL1 NP_056126.1:p.Arg1767Gln; NM_015311.2:c.5300G>A; NC_000002.11:g.220417266C>T

Exon splicing

Nonsynonymous rs59332477 41.86 42 65.65 Het. 890 255 48.7

OBSL1 NP_056126.1:p.Gln1578Arg; NM_015311.2:c.4733A>G; NC_000002.11:g.220419339T>C

Exon Nonsynonymous rs10932814 97.02 3.86 100 Hom. 998 255 100

OBSL1 NP_056126.1:p.Glu1365Asp; NM_015311.2:c.4095G>C; NC_000002.11:g.220421417C>G


OBSL1 NP_056126.1:p.Arg723Lys; NM_015311.2:c.2168G>A; NC_000002.11:g.220430203C>T

Exon Nonsynonymous rs1039898 37.87 17.18 98.42 Hom. 1479 255 99.9

TTN NP_003310.4:p.Gln24857His; NM_003319.4:c.74571G>C; NC_000002.11:g.179399576C>G


TTN NP_003310.4:p.Ile17160Thr; NM_003319.4:c.51479T>C; NC_000002.11:g.179432185A>G

Exon Nonsynonymous rs12463674 15.35 13 22.59 42.89 Het. 2478 255 46.3

TTN NP_003310.4:p.Ala13351Pro; NM_003319.4:c.40051G>C; NC_000002.11:g.179444768C>G


TTN NP_001254479.2:p.Arg11096His; NM_001267550.2:c.33287G>A; NC_000002.11:g.179545859C>T


TTN NP_001254479.2:p.Gly10622Arg; NM_001267550.2:c.31864G>A; NC_000002.11:g.179554305C>T


TTN NP_003310.4:p.Ile3716Val; NM_003319.4:c.11146A>G; NC_000002.11:g.179605725T>C


TTN NP_596870.2:p.Arg4915His; NM_133379.4:c.14744G>A; NC_000002.11:g.179612383C>T


TTN NP_596870.2:p.Asp3747Gly; NM_133379.4:c.11240A>G; NC_000002.11:g.179615887T>C


TTN NP_596870.2:p.Leu3732Phe; NM_133379.4:c.11196G>C; NC_000002.11:g.179615931C>G


TTN NP_001254479.2:p.Gly3751Asp; NM_001267550.2:c.11252G>A; NC_000002.11:g.179620951C>T

Exon splicing

Nonsynonymous rs7585334 83.2 80.45 9.72 98.93 Hom. 1503 255 100


Page 33


dbSNP dbSNP freq.

1000G MAF

5000G MAF

HiC freq.

AF1 DP Qual

Qual Freq. alt.

TTN NP_001254479.2:p.Ala3576Thr; NM_001267550.2:c.10726G>A; NC_000002.11:g.179621477C>T


TTN NP_003310.4:p.Ser3373Asn; NM_003319.4:c.10118G>A; NC_000002.11:g.179623758C>T


TTN NP_003310.4:p.Val3215Met; NM_003319.4:c.9643G>A; NC_000002.11:g.179629461C>T


TTN NP_003310.4:p.Ser1249Leu; NM_003319.4:c.3746C>T; NC_000002.11:g.179644035G>A


TTN NP_003310.4:p.Lys1155Glu; NM_003319.4:c.3463A>G; NC_000002.11:g.179644855T>C


TTN NP_003310.4:p.Thr765Ile; NM_003319.4:c.2294C>T; NC_000002.11:g.179650408G>A


Function: location of the variant according to RefSeq annotation database: exonic, intronic, splicing, UTR. dbSNP: identification of the Single Nucleotide Polymorphism Database. dbSNP freq.: variant frequency taken from dbSNP (%). 1000G MAF: minor allele

frequency taken from the 1000 Genomes Project (%). 5000G MAF: minor allele frequency taken from the 5000 Genomes Project (%). HiC freq.: variant frequency taken from our HiC database (%). AF1: heterozygous, hemizygous or homozygous. DP Qual: depth of

coverage after filtering low quality bases or low quality alignments. Qual: quality of the variant reported by SAMtools (maximum value is 255 and means that the variant has a high probability of being different from homozygous wild type. Low values indicate that

it has a high probability of being homozygous for wild type and thus having a low probability of being a true variant). Freq. alt.: the frequency of alternative allele in high quality fragments (%). Only good quality variants were included (QUAL ≥170).


Page 34

List of probably non-disease causing intronic genetic variants in splicing zones

Gene Variant dbSNP dbSNP freq. 1000G MAF 5000G MAF HiC freq. AF1 DP Qual Qual Freq. alt.

AARS2 NM_020745.3:c.243+6G>A; NC_000006.11:g.44280812C>T rs167772 47.37 45.33 49.74 80.51 Het. 299 255 55.2

ACADVL NM_000018.3:c.1605+6T>C; NC_000017.10:g.7127718T>C rs17671352 52.88 51.98 43.83 87 Hom. 424 255 100

AGL NM_000642.2:c.2001+8T>C; NC_000001.10:g.100346741T>C rs3736296 54.22 55.85 49.58 75.89 Het. 1200 255 45.2

CALR3 NM_145046.4:c.397+10C>T; NC_000019.9:g.16601168G>A rs3810197 11.68 11.4 12.96 25.21 Het. 1256 170 52.2

CASQ2 NM_001232.3:c.420+6T>C; NC_000001.10:g.116283343A>G rs9428083 67.55 64.36 29.16 96.11 Hom. 1027 255 100

DLD NM_000108.4:c.439-7T>C; NC_000007.13:g.107545799T>C rs10263341 70.61 71.61 35.15 83.81 Het. 1173 255 46.8

GAA NM_000152.3:c.547-4C>G; NC_000017.10:g.78079544C>G rs3816256 44.95 60.28 32.73 93.29 Het. 809 255 41.3

GAA NM_000152.3:c.858+7_858+8insAGCGGGC; NC_000017.10:g.78081528_78081529insAGCGGGC rs3071247 59.29 92.36 Het. 785 255 47.6

GLB1 NM_000404.2:c.1233+8T>C; NC_000003.11:g.33063050A>G rs13093698 18.2 16.35 21.25 36.41 Het. 716 255 44.5

MAP2K1 NM_002755.3:c.1023-8C>T; NC_000015.9:g.66782048C>T rs41306345 8.2 8.93 9.18 18.56 Het. 1589 255 48.5

MTO1 NM_133645.2:c.938+7G>A; NC_000006.11:g.74189574G>A rs7766570 99.96 100 99.95 Hom. 1481 255 99.9

OBSL1 NM_015311.2:c.5309-4C>G; NC_000002.11:g.220416942G>C rs34490902 42.92 43.53 66.08 Het. 1146 255 46.2

RYR2 NM_001035.2:c.3599-9delT; NC_000001.10:g.237753084delT rs371111802 28.51 50.18 Het. 1094 223 50.9

RYR2 NM_001035.2:c.13783-6A>G; NC_000001.10:g.237957161A>G rs790901 71.93 75.76 34.86 86.62 Hom. 1155 255 100

RYR2 NM_001035.2:c.14091-11_14091-10insT; NC_000001.10:g.237965145_237965146insT rs35563566 50 37.8 39.05 Hom. 594 233 82.7

SLC22A5 NM_003060.3:c.652+6A>G; NC_000005.9:g.131719999A>G rs4551059 100 100 99.95 Hom. 1076 255 100

TNNI3 NM_000363.4:c.373-10T>G; NC_000019.9:g.55665584A>C rs7252610 100 100 99.95 Hom. 367 255 99.5

TNNI3 NM_000363.4:c.25-8T>A; NC_000019.9:g.55668509A>T rs3729836 42.6 45.81 31.62 43.51 Het. 768 255 46.3

TNNT2 NM_001001430.2:c.53-11_53-7delCTTCT; NC_000001.10:g.201341181_201341185delAGAAG rs45533739 53.11 52.04 81.66 Hom. 1062 255 100

TTN NM_003319.4:c.4342+6C>T; NC_000002.11:g.179642425G>A rs719201 91.59 91.15 5.35 99.69 Hom. 1499 255 99.9 dbSNP: identification of the Single Nucleotide Polymorphism Database. dbSNP freq.: variant frequency taken from dbSNP (%). 1000G MAF: minor allele frequency taken from the 1000 Genomes Project (%). 5000G MAF: minor allele frequency taken from the 5000

Genomes Project (%). HiC freq.: variant frequency taken from our HiC database (%). AF1: heterozygous, hemizygous or homozygous. DP Qual: depth of coverage after filtering low quality bases or low quality alignments. Qual: quality of the variant reported by

SAMtools (maximum value is 255 and means that the variant has a high probability of being different from homozygous wild type. Low values indicate that it has a high probability of being homozygous for wild type and thus having a low probability of being a

true variant). Freq. alt.: the frequency of alternative allele in high quality fragments (%). Only good quality variants were included (QUAL ≥170


Page 35

APPENDIX 3

Detailed technical aspects

CardioGxOne was performed using a multiple approach based on targeted Next Generation Sequencing (NGS) combined

with the gold standard Sanger technique. Patient specimens (blood, saliva, tissue) were subjected to automated genomic

DNA purification (QIAsymphony SP®, Qiagen), and sample preparation was carried out using the Agilent SureSelectXT

Target Enrichment technology for Illumina paired-end multiplexed sequencing method. Enrichment was performed using

a custom SureSelect library (Agilent) for the coding regions and adjacent intronic areas for the selected genes. After cluster

generation on a cBot (Illumina), captured DNA was sequenced on either Illumina HiSeq 1500, MiSeq, or the NextSeq

platform. Clinically relevant variants and low-coverage regions were tested in parallel by standard Sanger sequencing. The

analytical sensitivity and accuracy of this assay are greater than 99% for single nucleotide variants (SNVs) and small

insertions/deletions (INDELs).

CardioGxOne was developed and assessed for accuracy and precision by Admera Health. The design of the custom capture

library is property of Health in Code and includes the following 90 genes related to hypertrophic cardiomyopathy:

AARS2, ACAD9, ACADVL, ACTA1, ACTC1, ACTN2, AGK, AGL, AGPAT2, ANK2, ANKRD1, ATPAF2, BAG3, BRAF, BSCL2, CALR3,

CASQ2, CAV3, COQ2, COX15, COX6B1, CRYAB, CSRP3, CTF1, DES, DLD, FAH, FHL1, FHL2, FHOD3, FLNC, GAA, GFM1, GLA,

GLB1, GNPTAB, GUSB, HRAS, JPH2, KCNJ8, KLF10, KRAS, LAMP2, LDB3, LIAS, LMNA, MAP2K1, MAP2K2, MLYCD, MRPL3,

MRPS22, MTO1, MURC, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOZ2, MYPN, NEXN, NRAS, OBSL1, PDHA1, PDLIM3,

PHKA1, PLN, PMM2, PRKAG2, PTPN11, RAF1, RYR2, SHOC2, SLC22A5, SLC25A4, SOS1, SURF1, TAZ, TCAP, TMEM70, TNNC1,

TNNI3, TNNT2, TPM1, TRIM63, TSFM, TTN, TTR, VCL.

The genes included in this test have been selected on a clinical basis according to their relation with a particular phenotype

and classified taking in consideration the level of evidence of this relation (priority genes, secondary genes, candidate

genes).

Probes were designed to cover all coding exons and 30 bp at intronic or UTR flanking regions. Those regions with suboptimal

quality coverage were sequenced by dideoxy Sanger technique. This test is not able to identify genetic variants located at

deep intronic/UTR regions.

CardioGxOne is aimed at identifying single nucleotide variants (SNVs) and small insertions/deletions (INDELs) up to 20 bp.

Genetic variants are described following the Human Genome Variation Society (HGVS) recommendations (www.hgvs.org).

Those selected genetic variants that were considered potentially associated with the patient’s phenotype or constitute

relevant incidental findings are reported in the main table of the report on the first page. Please note that clinical

interpretation of variants could be subject to changes as new scientific evidence appears.

Confirmation by dideoxy Sanger sequencing will be performed in those selected variants included in the main table that

meet the following conditions:

▪ Point mutations identified with suboptimal quality parameters: coverage \<30x, alternative allele frequency

different from 40%-60% / 80%-100%, or quality score \<170

▪ Point mutations affecting regions/genes with high homology with other genomic regions (i.e., pseudogenes)

▪ Insertions or deletions

We have also developed an alternative bioinformatics pipeline that is able to identify gross deletions/insertions affecting

one or more exons of a gene/s included in the panel (CNVs: Copy Number Variations). This complementary analysis is

possible when bioinformatics data is adequate and might not be available in some cases. An alternative method is used to

confirm this kind of variants.

http://www.hgvs.org/


Page 36

Frequently, our test is not able to identify the phase (same/different alleles) of more than one variant affecting the same

gene. This limitation should be considered in cases of recessive disorders that need both alleles of the gene to be mutated.

Although CardioGxOne has an analytical sensitivity and specificity of over 99%, some genotyping errors could occur in

specific situations:

▪ Pre-arrival contamination of samples

▪ Mosaic mutations

▪ Monosomies and trisomies

▪ Genetic paternity problems

▪ Genetic variants producing allelic drop-outs

▪ Studies performed on paraffin-embedded tissues

▪ Presence of pseudogenes

▪ Incorrect identification of variants in homo-polymers or high GC-content zones

▪ Errors in the reference sequence

We have developed an efficient method that ensures tracking of samples after arrival, guaranteeing their proper

identification once they arrive at our laboratory. However, we cannot take responsibility for labeling errors in samples prior

to their arrival.

The clinical report: Admera, powered by Health in Code, provides a detailed report with all relevant existing clinical data

on the detected mutations. This information has been evaluated by experts on the disease and includes a description of all

families with reported cases of each mutation along with information from our own research and existing information on

in vitro and in vivo (animal models) studies for the different mutations. To handle all this information, Health in Code has

developed a computerized database that includes records of more than 106,500 individuals from the existing literature on

inherited cardiovascular diseases and from our own research.

Comments, recommendations and disclaimers

It is highly recommended that the interpretation of this genetic report is done with the help/counseling from a physician

with enough expertise in genetic conditions. Our test is not designed in a direct-to-consumer fashion. The results of this

test must be interpreted in the clinical context of each patient. This test does not replace clinical assessment of patients

and must not to be used as the only tool to decide on treatment, diagnosis, and/or pre-implantation/pre-natal studies.

When the genetic study identifies one or more genetic variants potentially associated with the development of pathology,

family screening is recommended. All first-degree relatives (parents, siblings, children; whether or not clinically affected)

should be considered for inclusion in this screening due to variable penetrance and age of onset associated with the

majority of these genetic alterations. Genetic diagnosis can identify those family members who are at risk of disease

development and need periodical clinical assessment. Moreover, testing in family members can be useful in determining

the cosegregation of the identified variants with the phenotype and the associated prognosis in carriers.

This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA), but the FDA has determined

that such clearance or approval is not necessary. The CardioGxOne test is used for clinical purposes. It should not be

regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement

Amendments (CLIA) as qualified to perform high complexity clinical laboratory testing. Health in Code provided the

professional component of clinical interpretation of the CardioGxOne results.

For additional information or comments, please contact us at [email protected].

mailto:[email protected]


Page 37

Resource references

Population databases:

▪ Exome Aggregation Consortium (ExAC), Cambridge, MA (URL: http://exac.broadinstitute.org) [version 0.3.1].

▪ Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (URL:

http://evs.gs.washington.edu/EVS/) [ESP6500SI-V2-SSA137].

▪ 1000 Genomes Project, An integrated map of genetic variation from 1,092 human genomes, McVean et al., Nature

491, 56–65 (01 November 2012) doi:10.1038/nature11632 (www.1000genomes.org/).

▪ Database of Single Nucleotide Polymorphisms (dbSNP) [Internet]. Bethesda (MD): National Center for

Biotechnology Information, National Library of Medicine (dbSNP Build ID:135) Available from:

www.ncbi.nlm.nih.gov/SNP.

▪ HGMD® [Internet]: Stenson PD et al. Genome Med. 2009;1(1):13 www.hgmd.cf.ac.uk.

▪ ClinVar: Landrum MJ, Lee JM, Riley GR, Jang W, Rubinstein WS, Church DM, Maglott DR. ClinVar: public archive of

relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014 Jan 1;42(1):D980-5. doi:

10.1093/nar/gkt1113. PubMed PMID: 24234437.

▪ Health in Code proprietary database.

Functional studies:

▪ POLYPHEN: A method and server for predicting damaging missense mutations. Adzhubei IA, Schmidt S, Peshkin L,

Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. Nat Methods. 2010 Apr;7(4):248-9.

▪ SIFT: Predicting the effects of coding nonsynonymous variants on protein function using the SIFT algorithm. Kumar

P, Henikoff S, Ng PC. Nat Protoc. 2009;4(7):1073-81.

▪ MUTATION TASTER: MutationTaster2: mutation prediction for the deep-sequencing age. Schwarz JM, Cooper DN,

Schuelke M, Seelow D. Nat Methods. 2014 Apr;11(4):361-2.

▪ NNSplice: Eeckman, FH, Kulp, D, Haussler, D, 1997. Improved Splice Site Detection in Genie. J Comp Biol 4(3), 311-

23.

▪ HSFHuman Splicing Finder: FO Desmet, Hamroun D, Lalande M, Collod-Beroud G, Claustres M, Beroud C. Human

Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acid Research, 2009.

▪ MaxEntScan: Yeo G, Burge CB. Maximum entropy modeling of short sequence motifs with applications to RNA

splicing signals. J Comput Biol. 2004;11(2-3):377-94.


Page 38

0

500

1000

1500

AA

RS2

AC

AD

9

AC

AD

VL

AC

TA1

AC

TC1

AC

TN2

AG

K

AG

L

AG

PA

T2

AN

K2

AN

KR

D1

ATP

AF2

BA

G3

BR

AF

BSC

L2

CA

LR3

CA

SQ2

CA

V3

CO

Q2

CO

X1

5

CO

X6

B1

CR

YAB

CSR

P3

CTF

1

DES

DLD

FAH

FHL1

FHL2

FHO

D3

FLN

C

GA

A

GFM

1

GLA

GLB

1

GN

PTA

B

GU

SB

HR

AS

JPH

2

KC

NJ8

KLF

10

KR

AS

LAM

P2

LDB

3

LIA

S

Average coverage per gene

Coverage 15 x

0

500

1000

1500

LMN

A

MA

P2

K1

MA

P2

K2

MLY

CD

MR

PL3

MR

PS2

2

MTO

1

MU

RC

MYB

PC

3

MYH

6

MYH

7

MYL

2

MYL

3

MYL

K2

MYO

Z2

MYP

N

NEX

N

NR

AS

OB

SL1

PD

HA

1

PD

LIM

3

PH

KA

1

PLN

PM

M2

PR

KA

G2

PTP

N1

1

RA

F1

RYR

2

SHO

C2

SLC

22

A5

SLC

25

A4

SOS1

SUR

F1

TAZ

TCA

P

TMEM

70

TNN

C1

TNN

I3

TNN

T2

TPM

1

TRIM

63

TSFM TT

N

TTR

VC

L

Coverage 15 x


Page 39

Coverage stats

Stats Studied genes Priority genes

Average coverage 1526 x 1158 x

Bases sequenced 327540 36293

% Bp with coverage ≥15 100% 100%

% Bp with coverage ≥30 100% 100%

Documents

Test results of: SAMPLE REPORT