Admera Health, LLC Page 6 of 21 Cardiogxone Report For: P.Ser532pro

SAMPLE REPORT

PATIENT INFORMATION SAMPLE REFERRING PHYSICIAN Patient Id Sample number Name Sex Male Source Blood Institution DOB Date received Date of report

Test results of:

Reason for the study: Dilated cardiomyopathy.

Test(s) requested: Dilated cardiomyopathy (81 genes)

RESULT: POSITIVE

We have identified a variant in MYH7 gene that we consider likely to be pathogenic, and it could explain the phenotype of dilated cardiomyopathy (DCM) described in this patient.

Population Number of Gene Variant Result Pathogenicity frequency references MYH7 NP_000248.2:p.Thr544Pro Heterozygosis Likely to be Mutation (not 0 NM_000257.2:c.1630A>C pathogenic or disease- found in controls) NC_000014.8:g.23897052T>G causing

Clinical interpretation

We consider the mutation Thr544Pro in the MYH7 gene likely to be pathogenic. It has never been described, but affects a relevant region of the gene where most of the mutations have been associated with dilated cardiomyopathy (DCM), the phenotype described in this patient. In some of the informative families, some of the carriers developed severe cardiac failure requiring transplantation. Although the pathogenicity of this variant must be confirmed, we suggest close follow-up of the carriers.

Cosegregation study of the variant with the disease in other relatives could be useful to confirm the pathogenicity of this variant. Once confirmed, it could be used for predictive testing.

Technical aspects of the study

This sample has been studied by massive parallel sequencing method using a library that included 81 genes related to dilated cardiomyopathy.

Signatures

James Dermody, PhD Doc. Lorenzo Monserrat Iglesias Laboratory Director Cardiologist and Scientific director ABMG Certified, Clinical Molecular Genetics

• CLIA ID 31D2038676

Page 1 of 21 CardioGxOne Report for: DETAILED RESULTS

Gene: MYH7 (Encoding the protein myosin-7) NP_000248.2:p.Thr544Pro/NC_000014.8:g.23897052T>G

A point mutation in heterozygosis has been identified: NM_000257.2:c.1630A>C NC_000014.8:g.23897052T>G (nucleotide code), NP_000248.2:p.Thr544Pro (amino acid code), dbSNP rs397516119. Alternative names at the protein level: NP_000248.2:p.T544P. Exon: 16.

Pathogenicity: likely to be pathogenic or disease-causing

Population frequency: mutation (not found in controls)

The NP_000248.2:p.Thr544Pro variant in the MYH7 gene has not been previously described in any paper or communication.

Clinical information

- Reported in public databases: The Thr544Pro variant of the MYH7 gene has not been previously described in any scientific publication nor reported in public databases such as Ensembl, dbSNP, HGMD, NHLBI GO Exome Sequencing Project (ESP), or Exome Aggregation Consortium (ExAC). Another variant affecting the same nucleotide position (Thr544Ala, rs397516119) can be found in the ExAC database in two heterozygous individuals. ClinVar considers Thr544Ala as of “uncertain significance”.

- Clinical information of missense variants affecting the same region: This variant is located in the first actin-binding site, a very specific and relevant region of the protein (see bioinformatics study). There are several missense variants in this region of the gene that have been associated with disease. They are described in table 1 in the supplemental information: in 59 carriers of 22 families, the most commonly associated phenotype was dilated cardiomyopathy (DCM), followed by left ventricular non-compaction (LVNC) or an overlapping phenotype between the two. Some cases of hypertrophic cardiomyopathy (HCM) are also described. The variants cosegregated with the disease with an elevated penetrance in two big informative families (carrying mutations Ser532Pro and Asp545Asn). Mutations in this region could be associated with early diagnosis of the phenotype (in childhood) and a bad prognosis: progression to cardiac heart failure is frequent, and there are cases of heart failure death or cardiac transplantation in almost half of the families.

Functional study / Animal model

A murine model of the nearby DCM-causing missense mutation Ser532Pro has been reported (Schmitt et al., 2006). The authors found that the contractile function of isolated myocytes was depressed and preceded left ventricular dilation and reduced fractional shortening. In an in vitro motility assay, mutant cardiac myosin exhibited a reduced ability to translocate actin and actin-activated ATPase activity was also reduced. In another work (Palmer et al., 2013), Ca(2+)-activated skinned myocardial strips from a Ser532Pro heterozygous mouse model were found to have a Mg-ATP binding rate higher than those from the controls. In addition, the rate constant of force development (2πb) was also significantly higher compared to controls.

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Bioinformatics study

The Thr544 residue is located at the myosin motor domain (amino acids 85-778) of the MYH7 protein. Within this domain, it is located in the first actin-binding peptide (amino acids 526-557) that, together with a second actin-binding peptide (amino acids 652-661) is a helical segment that constitutes the primary hydrophobic actin contact. Mutations in this region have been associated with the development of dilated or left ventricular non-compaction cardiomyopathy. Thr544Pro affects a highly conserved amino acid up to opossum (considering 11 species). The mutation causes a change from an amino acid with neutral polar side chain (threonine, THR) (ACC) to another amino acid with cyclic hydrophobic side chain (proline, PRO) (CCC). There are small modifications in the physicochemical properties, e.g. polarity, charge and volume, between threonine and proline (Grantham distance: 38 [0-215]).

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

Predictor Prediction Score Version

SIFT Damaging 0 Computed from ENSEMBL 55 (September, 2014)

Polyphen-2 (HumVar) Benign 0.44 v2.2.2 (September, 2014)

Polyphen-2 (HumDiv) Benign 0.03 v2.2.2 (September, 2014)

MutationTaster Polymorphism 0.966

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.

Conclusions

We consider this mutation likely to be pathogenic. It is present in a relevant region of the gene where most of the mutations are considered disease-causing. The most common phenotype is dilated cardiomyopathy (which is the one described in this patient). Although we need more information to establish the clinical course associated to this variant, some carriers of similar mutations have evolved to an early and severe phenotype that included end-stage cardiac failure and cardiac transplantation.

We suggest to include this variant in familial genetic screening to evaluate cosegregation with the phenotype. Once the pathogenicity is confirmed, it could be used for predictive testing.

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Information about other mutations described affecting the same region of the gene: first actin-binding site

This section describes available data about the following 12 variants: NP_000248.2:p.Glu525Lys/NC_000014.8:g.23897714C>T NP_000248.2:p.Met528Ile/NC_000014.8:g.23897098C>T NP_000248.2:p.Gly529Asp/NC_000014.8:g.23897096C>T NP_000248.2:p.Met531Arg/NC_000014.8:g.23897090A>C NP_000248.2:p.Ser532Pro/NC_000014.8:g.23897088A>G NP_000248.2:p.Met539Leu/NC_000014.8:g.23897067T>G NP_000248.2:p.Phe540Leu/NC_000014.8:g.23897064A>G NP_000248.2:p.Lys542Arg/NC_000014.8:g.23897057T>C NP_000248.2:p.Ala543Thr/NC_000014.8:g.23897055C>T NP_000248.2:p.Thr544Pro/NC_000014.8:g.23897052T>G NP_000248.2:p.Asp545Asn/NC_000014.8:g.23897049C>T NP_000248.2:p.Ala550Val/NC_000014.8:g.23897033G>A

Selected variants have been described in 22 families. Information is available about 59 carriers (46 affected or possibly affected, 5 not affected or healthy, 8 without phenotypic study). We also have information about 28 non-carriers (19 not affected or healthy, 9 without phenotypic study). We have also found 14 relatives without genetic study (2 patients with left ventricular non-compaction hypertrabeculation, 1 patient with hypertrophic cardiomyopathy, 4 patients with dilated cardiomyopathy, 1 not affected or healthy, 6 without phenotypic study).

Affected Affected Major phenotypes patients families Dilated cardiomyopathy 28 11 Hypertrophic cardiomyopathy 9 8 Left ventricular non-compaction / hypertrabeculation 7 2 Dilated cardiomyopathy + Left ventricular non-compaction / 2 2 hypertrabeculation

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Population Affected Healthy Variant Pathogenicity Phenotypes (patients) Families Events (patients) References frequency carriers carriers

MYH7 p.Glu525Lys + 0% Dilated cardiomyopathy(2) 2 2 0 Cardiac transplantation (1) 5 MYH7 ++ 0% Left ventricular non-compaction / hypertrabeculation(2) 1 2 0 2 p.Met528Ile MYH7 + 0% Dilated cardiomyopathy(1) 1 1 0 2 p.Gly529Asp MYH7 ++ 0% Dilated cardiomyopathy(2) 1 2 0 Heart failure death (2) 4 p.Met531Arg Left ventricular non-compaction / hypertrabeculation(1) MYH7 +++ 0% Dilated cardiomyopathy(17) 1 17 0 Cardiac transplantation (1) 21 p.Ser532Pro Sudden death (1) MYH7 ++ 0% Hypertrophic cardiomyopathy(7) 7 7 1 Cardiac transplantation (1) 6 p.Met539Leu MYH7 ++ 0% Dilated cardiomyopathy(2) 2 2 0 2 p.Phe540Leu Left ventricular non-compaction / hypertrabeculation(1) MYH7 ++ 0% Hypertrophic cardiomyopathy(2) 2 2 2 2 p.Lys542Arg MYH7 p.Ala543Thr + 0% Dilated cardiomyopathy(1) 1 1 0 3 MYH7 ++ 0% Left ventricular non-compaction / hypertrabeculation(5) 3 8 2 Stroke-related death (2) 7 p.Asp545Asn Dilated cardiomyopathy(3) MYH7 p.Ala550Val ++ 0% Dilated cardiomyopathy(2) 1 2 0 Heart failure death (1) 8 Cardiac transplantation (1)

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Selected references p.Glu525Lys

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. http://www.ncbi.nlm.nih.gov/pubmed/25125180

Pugh TJ, Kelly MA, Gowrisankar S, Hynes E, Seidman MA, Baxter SM, Bowser M, Harrison B, Aaron D, Mahanta LM, Lakdawala NK, McDermott G, White ET, Rehm HL, Lebo M, Funke BH. The landscape of genetic variation in dilated cardiomyopathy as surveyed by clinical DNA sequencing. Genet Med. 2014 Aug;16(8):601-8. http://www.ncbi.nlm.nih.gov/pubmed/24503780

Lakdawala NK, Funke BH, Baxter S, Cirino AL, Roberts AE, Judge DP, et al. Genetic Testing for Dilated Cardiomyopathy in Clinical Practice. J Card Fail. 2012 Apr;18(4):296-303. http://www.ncbi.nlm.nih.gov/pubmed/22464770

p.Gly529Asp

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. http://www.ncbi.nlm.nih.gov/pubmed/25125180

Waldmüller S, Erdmann J, Binner P, Gelbrich G, Pankuweit S, Geier C, et al. Novel correlations between the genotype and the phenotype of hypertrophic and dilated cardiomyopathy: results from the German Competence Network Heart Failure. Eur J Heart Fail. 2011 Nov;13(11):1185-92. http://www.ncbi.nlm.nih.gov/pubmed/21750094

p.Met531Arg

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. http://www.ncbi.nlm.nih.gov/pubmed/25125180

Spudich JA. The myosin mesa and a possible unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy. Biochem Soc Trans. 2015 Feb;43(1):64-72. http://www.ncbi.nlm.nih.gov/pubmed/25619247

Kaneda T, Naruse C, Kawashima A, Fujino N, Oshima T, Namura M, et al. A novel beta-myosin heavy chain gene mutation, p.Met531Arg, identified in isolated left ventricular non-compaction in humans, results in left ventricular hypertrophy that progresses to dilation in a mouse model. Clin Sci (Lond). 2008 ;114(6):431-40.

Chang B, Nishizawa T, Furutani M, Fujiki A, Tani M, Kawaguchi M, et al. Identification of a novel TPM1 mutation in a family with left ventricular noncompaction and sudden death. Mol Genet Metab. 2011 Feb;102(2):200-6. http://www.ncbi.nlm.nih.gov/pubmed/20965760

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McNally EM, Golbus JR, Puckelwartz MJ. Genetic mutations and mechanisms in dilated cardiomyopathy. J Clin Invest. 2013 Jan 2;123(1):19-26. http://www.ncbi.nlm.nih.gov/pubmed/23281406

Sivaramakrishnan S, Ashley E, Leinwand L, Spudich JA. Insights into human beta-cardiac myosin function from single molecule and single cell studies. J Cardiovasc Transl Res. 2009;2(4):426-40. http://www.ncbi.nlm.nih.gov/pubmed/20560001

Frazier AH, Ramirez-Correa GA, Murphy AM. Molecular mechanisms of sarcomere dysfunction in dilated and hypertrophic cardiomyopathy. Prog Pediatr Cardiol. 2011 Jan 1;31(1):29-33. http://www.ncbi.nlm.nih.gov/pubmed/21297871

Alves ML, Gaffin RD, Wolska BM. Rescue of familial cardiomyopathies by modifications at the level of sarcomere and Ca2+ fluxes. J Mol Cell Cardiol. 2010;48(5):834-42.

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. http://www.ncbi.nlm.nih.gov/pubmed/25125180

Lakdawala NK, Thune JJ, Colan SD, Cirino AL, Farrohi F, Rivero J, McDonough B, Sparks E, Orav EJ, Seidman JG, Seidman CE, Ho CY. Subtle Abnormalities in Contractile Function Are an Early Manifestation of Sarcomere Mutations in Dilated Cardiomyopathy. J Electrocardiol. 2012 Nov;45(6):770-3. http://www.ncbi.nlm.nih.gov/pubmed/22949430

Moore JR, Leinwand L, Warshaw DM. Understanding cardiomyopathy phenotypes based on the functional impact of mutations in the Myosin motor. Circ Res. 2012 Jul 20;111(3):375-85.

Zimmerman RS, Cox S, Lakdawala NK, Cirino A, Mancini-DiNardo D, Clark E, et al. A novel custom resequencing array for dilated cardiomyopathy. Genet Med. 2010 ;12(5):268-78.

Buvoli M, Hamady M, Leinwand LA, Knight R. Bioinformatics assessment of beta-myosin mutations reveals myosin's high sensitivity to mutations. Trends Cardiovasc Med 2008;18(4):141-9. http://www.ncbi.nlm.nih.gov/pubmed/18555187?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Results Panel.Pubmed_RVDocSum

p.Met539Leu

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. http://www.ncbi.nlm.nih.gov/pubmed/25125180

Frisso G, Limongelli G, Pacileo G, Del Giudice A, Forgione L, Calabrò P, Iacomino M, et al. A child cohort study from southern Italy enlarges the genetic spectrum of hypertrophic cardiomyopathy. Clin Genet. 2009 Jul;76(1):91-101.

Coppini R, Ho CY, Ashley E, Day S, Ferrantini C, Girolami F, et al. Clinical Phenotype and Outcome of Hypertrophic Cardiomyopathy Associated With Thin-Filament Gene Mutations. J Am Coll Cardiol. 2014 Dec 23;64(24):2589-600. http://www.ncbi.nlm.nih.gov/pubmed/25524337

E.A. Arbustini, M.L. Rossi, N. Marziliano, A. Pilotto, M. Grasso, M. Pasotti, G. Tocco, P. Presbitero. Gene Symbol: LDB3. Disease: Dilated cardiomyopathy with left ventricular noncompaction (NCLV DCM). Hum Genet (2007) 120:910.

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Biagini E, Olivotto I, Iascone M, Parodi MI, Girolami F, Frisso G, et al. Significance of sarcomere gene mutations analysis in the end-stage phase of hypertrophic cardiomyopathy. Am j cardiol. 2014;114(5):769-76. http://www.ncbi.nlm.nih.gov/pubmed/25037680

p.Lys542Arg

Coto E, Reguero JR, Palacin M, Gomez J, Alonso B, Iglesias S, et al. Resequencing the Whole MYH7 Gene (Including the Intronic, Promoter, and 3 ' UTR Sequences) in Hypertrophic Cardiomyopathy. J Mol Diagn. 2012 Sep;14(5):518-24. http://www.ncbi.nlm.nih.gov/pubmed?term=22765922

p.Ala543Thr

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. http://www.ncbi.nlm.nih.gov/pubmed/25125180

p.Asp545Asn

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. http://www.ncbi.nlm.nih.gov/pubmed/25125180

Hoedemaekers YM, Caliskan K, Majoor-Krakauer D, van de Laar I, Michels M, Witsenburg M, et al. Cardiac beta-myosin heavy chain defects in two families with non-compaction cardiomyopathy: linking non-compaction to hypertrophic, restrictive, and dilated cardiomyopathies. Eur Heart J 2007;28(22):2732-7. http://www.ncbi.nlm.nih.gov/pubmed/17947214?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Results Panel.Pubmed_RVDocSum van Spaendonck-Zwarts KY, van Rijsingen IA, van den Berg MP, Lekanne Deprez RH, Post JG, et al. Genetic analysis in 418 index patients with idiopathic dilated cardiomyopathy: overview of 10 years' experience. Eur J Heart Fail. 2013 Jun;15(6):628-36. http://www.ncbi.nlm.nih.gov/pubmed/23349452

Dooijes D. Noncompaction Cardiomyopathy- mutation spectrum , distribution of disease genes and implications for diagnostic strategies. European Society of Cardiology Congress Dennis (Utrecht), 30 Aug 2009. http://spo.escardio.org/eslides/view.aspx?eevtid=33&fp=1210

Zaragoza MV, Arbustini E, Narula J. Noncompaction of the left ventricle: primary cardiomyopathy with an elusive genetic etiology. Curr Opin Pediatr. 2007 Dec;19(6):619-27.

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Hoedemaekers YM, Caliskan K, Michels M, Frohn-Mulder I, van der Smagt JJ, Phefferkorn JE, Wessels MW, ten Cate FJ, Sijbrands EJ, Dooijes D, Majoor-Krakauer DF. The importance of genetic counseling, DNA diagnostics, and cardiologic family screening in left ventricular noncompaction cardiomyopathy. Circ Cardiovasc Genet. 2010 Jun 1;3(3):232-9. http://circgenetics.ahajournals.org/content/3/3/232.long

p.Ala550Val

Sanbe A. Dilated Cardiomyopathy: A Disease of the Myocardium. Biol Pharm Bull. 2013;36(1):18-22.

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. http://www.ncbi.nlm.nih.gov/pubmed/25125180

Zimmerman RS, Cox S, Lakdawala NK, Cirino A, Mancini-DiNardo D, Clark E, et al. A novel custom resequencing array for dilated cardiomyopathy. Genet Med. 2010 ;12(5):268-78.

Marston B. How Do Mutations in Contractile Proteins Cause the Primary Familial Cardiomyopathies?. J Cardiovasc Transl Res. 2011 Jun;4(3):245-55. http://www.ncbi.nlm.nih.gov/pubmed/21424860

Villard E, Duboscq-Bidot L, Charron P, Benaiche A, Conraads V, et al. Mutation screening in dilated cardiomyopathy: prominent role of the beta myosin heavy chain gene. Eur Heart J 2005 ;26(8):794-803. http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=15769782

Chang AN, Potter JD. Sarcomeric protein mutations in dilated cardiomyopathy. Heart Fail Rev. 2005 Sep;10(3):225-35. http://www.ncbi.nlm.nih.gov/pubmed/16416045

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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 identified in healthy controls or because they do not affect protein structure and function.

Region Variants found Exonic 164 Synonymous 92 Nonsynonymous 70 Insertion 2 Intronic 160 Intronic splicing 17 UTR 21 Total 345

Exonic 143 UTR 164 Intron splicing Intron non-splicing

17 21

Only good quality variants were included (QUAL ≥ 170)

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

Exonic dbSNP 1000G 5000G HiC DP Freq. Gene Variant Function dbSNP AF1 Qual function freq. freq. freq. freq. Qual alt.

ALMS1 NP_055935.4:p.Glu25_Glu26insGlu; NM_015120.4:c.74_75insGGA; Exon Insertion 64.37 Het. 183 255 48.6 NC_000002.11:g.73613070_73613071insGGA

ALMS1 NP_055935.4:p.Ser524_Leu525insPro; NM_015120.4:c.1573_1574insCTC; Exon Insertion 44.92 87.27 Het. 346 255 45.1 NC_000002.11:g.73675230_73675231insCTC

ALMS1 NP_055935.4:p.Val671Gly; NM_015120.4:c.2012T>G; Exon Nonsynonymous 12.78 11.94 98.86 Het. 310 255 51 NC_000002.11:g.73675669T>G

ALMS1 NP_055935.4:p.Ser2574Asn; NM_015120.4:c.7721G>A; Exon Nonsynonymous 12.68 12.01 21.39 Het. 345 255 51.3 NC_000002.11:g.73716810G>A

ALMS1 NP_055935.4:p.Asp2672His; NM_015120.4:c.8014G>C; Exon Nonsynonymous 52.04 13.6 12.89 22.04 Het. 370 255 49.2 NC_000002.11:g.73717103G>C

ALMS1 NP_055935.4:p.Arg4029Lys; NM_015120.4:c.12086G>A; Exon Nonsynonymous 41.77 46.34 86.53 Het. 345 255 48.7 NC_000002.11:g.73828538G>A

BAG3 NP_004272.2:p.Cys151Arg; NM_004281.3:c.451T>C; Exon Nonsynonymous 13.31 9.64 15.11 36.31 Het. 315 255 53 NC_000010.10:g.121429633T>C

DMD NP_003997.1:p.Arg2937Gln; NM_004006.2:c.8810G>A; Exon Nonsynonymous 16.41 11.81 4.32 96.11 Hem. 132 170 100 NC_000023.10:g.31496350C>T

DMD NP_003997.1:p.Lys2366Gln; NM_004006.2:c.7096A>C; Exon Nonsynonymous 4.25 18.33 20.46 23.32 Hem. 245 170 99.6 NC_000023.10:g.31893307T>G splicing

DSG2 NP_001934.2:p.Ile293Val; NM_001943.3:c.877A>G; NC_000018.9:g.29104714A>G Exon Nonsynonymous 4.77 3.23 6.36 17.79 Het. 369 255 51

DSP NP_004406.2:p.Arg1738Gln; NM_004415.2:c.5213G>A; Exon Nonsynonymous 20.77 24.04 19.41 26.54 Het. 408 255 53.4 NC_000006.11:g.7581636G>A

EYA4 NP_004091.3:p.Gly277Ser; NM_004100.4:c.829G>A; Exon Nonsynonymous 40.02 40.95 38.3 57.36 Het. 352 255 49.2 NC_000006.11:g.133789728G>A

FKTN NP_001073270.1:p.Arg203Gln; NM_001079802.1:c.608G>A; Exon Nonsynonymous 24.48 15.81 26.46 49.32 Hom. 300 255 100 NC_000009.11:g.108366734G>A

FOXD4 NP_997188.2:p.Ile152Val; NM_207305.4:c.454A>G; NC_000009.11:g.117666T>C Exon Nonsynonymous 16.87 18.31 40.87 Het. 357 255 39.8

FOXD4 NP_997188.2:p.Asp136Gly; NM_207305.4:c.407A>G; NC_000009.11:g.117713T>C Exon Nonsynonymous 62.13 41.75 87.5 Het. 346 255 37

GAA NP_000143.2:p.Asp91Asn; NM_000152.3:c.271G>A; Exon Nonsynonymous 2.1 1.16 2.31 5.2 Het. 395 170 50.6 NC_000017.10:g.78078656G>A

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Exonic dbSNP 1000G 5000G HiC DP Freq. Gene Variant Function dbSNP AF1 Qual function freq. freq. freq. freq. Qual alt.

GAA NP_000143.2:p.His199Arg; NM_000152.3:c.596A>G; Exon Nonsynonymous 65.72 39.92 32.75 93.44 Hom. 274 255 100 NC_000017.10:g.78079597A>G

GAA NP_000143.2:p.Arg223His; NM_000152.3:c.668G>A; Exon Nonsynonymous 64.56 39.76 32.76 93.34 Hom. 274 255 100 NC_000017.10:g.78079669G>A

GAA NP_000143.2:p.Val780Ile; NM_000152.3:c.2338G>A; Exon Nonsynonymous 72.27 28.81 26.68 94.23 Hom. 345 255 100 NC_000017.10:g.78091405G>A splicing

GLB1 NP_000395.2:p.Cys521Arg; NM_000404.2:c.1561T>C; Exon Nonsynonymous 93.63 7.29 6.74 100 Hom. 207 255 100 NC_000003.11:g.33055721A>G

GLB1 NP_000395.2:p.Pro10Leu; NM_000404.2:c.29C>T; NC_000003.11:g.33138549G>A Exon Nonsynonymous 47.24 43.19 47.4 90.14 Het. 393 255 49.9

JUP NP_068831.1:p.Met697Leu; NM_021991.2:c.2089A>T; Exon Nonsynonymous 38.02 41.27 30.3 91.86 Het. 276 255 49.3 NC_000017.10:g.39912145T>A splicing

LAMA2 NP_000417.2:p.Arg619His; NM_000426.3:c.1856G>A; Exon Nonsynonymous 27.1 26.97 28.12 Het. 255 255 45.9 NC_000006.11:g.129571330G>A

LAMA2 NP_000417.2:p.Ala2587Val; NM_000426.3:c.7760C>T; Exon Nonsynonymous 62.81 41.61 36.14 91.23 Hom. 292 255 100 NC_000006.11:g.129807629C>T splicing

LAMA4 NP_001098676.2:p.Pro1119Arg; NM_001105206.2:c.3356C>G; Exon Nonsynonymous 63.44 21.05 22.21 45.74 Het. 316 255 49 NC_000006.11:g.112457383G>C

LAMA4 NP_001098676.2:p.Gly1117Ser; NM_001105206.2:c.3349G>A; Exon Nonsynonymous 77.46 16.01 24.87 91.81 Het. 313 255 49.8 NC_000006.11:g.112457390C>T

LAMA4 NP_001098676.2:p.Tyr498His; NM_001105206.2:c.1492T>C; Exon Nonsynonymous 24.16 35.12 87.95 Het. 154 255 51.3 NC_000006.11:g.112493872A>G

LAMA4 NP_001098676.2:p.Ala283Glu; NM_001105206.2:c.848C>A; Exon Nonsynonymous 95.19 0 99.6 Hom. 288 255 100 NC_000006.11:g.112508770G>T

MYOT NP_006781.1:p.Lys74Gln; NM_006790.2:c.220A>C; Exon Nonsynonymous 98.38 1.48 99.97 Hom. 278 255 100 NC_000005.9:g.137206560A>C

MYPN NP_115967.2:p.Phe628Leu; NM_032578.3:c.1884C>G; Exon Nonsynonymous 37.42 31.65 39.6 69.88 Hom. 328 255 100 NC_000010.10:g.69926334C>G

MYPN NP_115967.2:p.Ser691Asn; NM_032578.3:c.2072G>A; Exon Nonsynonymous 37 33.59 39.24 67.53 Hom. 338 255 100 NC_000010.10:g.69933921G>A

MYPN NP_115967.2:p.Ser707Asn; NM_032578.3:c.2120G>A; Exon Nonsynonymous 36.21 32.51 39.24 67.46 Hom. 321 255 100 NC_000010.10:g.69933969G>A

MYPN NP_115967.2:p.Ser803Arg; NM_032578.3:c.2409C>G; Exon Nonsynonymous 49.95 47.14 47.7 78.6 Hom. 320 255 99.7 NC_000010.10:g.69934258C>G

• CLIA ID 31D2038676 Page 12 of 21 CardioGxOne Report for:

Exonic dbSNP 1000G 5000G HiC DP Freq. Gene Variant Function dbSNP AF1 Qual function freq. freq. freq. freq. Qual alt.

MYPN NP_115967.2:p.Pro1135Thr; NM_032578.3:c.3403C>A; Exon Nonsynonymous 37.75 34.03 40.59 67.43 Hom. 326 255 100 NC_000010.10:g.69959242C>A

NEXN NP_653174.3:p.Gly245Arg; NM_144573.3:c.733G>A; Exon Nonsynonymous 19.83 15.08 15.78 35.22 Het. 305 255 50.2 NC_000001.10:g.78392446G>A

PRDM16 NP_071397.3:p.Ser533Pro; NM_022114.3:c.1597T>C; Exon Nonsynonymous 41.13 5.49 12.23 96.63 Hom. 271 255 100 NC_000001.10:g.3328358T>C

RBM20 NP_001127835.2:p.Trp768Ser; NM_001134363.1:c.2303G>C; Exon Nonsynonymous 66.74 0.52 99.52 Hom. 350 255 100 NC_000010.10:g.112572458G>C

RBM20 NP_001127835.2:p.Glu1223Gln; NM_001134363.1:c.3667G>C; Exon Nonsynonymous 68.09 30.29 24.16 95.95 Het. 233 255 46.3 NC_000010.10:g.112595719G>C

RYR2 NP_001026.2:p.Gln2958Arg; NM_001035.2:c.8873A>G; Exon Nonsynonymous 17.21 9.92 22.49 47.7 Het. 335 255 51.6 NC_000001.10:g.237841390A>G

SCN5A NP_932173.1:p.His558Arg; NM_198056.2:c.1673A>G; Exon Nonsynonymous 23.04 24.63 42.36 Het. 351 255 53.3 NC_000003.11:g.38645420T>C

TMPO NP_003267.1:p.Gln599Glu; NM_003276.2:c.1795C>G; Exon Nonsynonymous 6.55 5.89 7.11 15.78 Het. 257 255 47.5 NC_000012.11:g.98927830C>G

TTN NP_003310.4:p.Arg25529His; NM_003319.4:c.76586G>A; Exon Nonsynonymous 20.87 12.14 24.7 Het. 339 255 47.8 NC_000002.11:g.179397561C>T

TTN NP_003310.4:p.Arg23473His; NM_003319.4:c.70418G>A; Exon Nonsynonymous 20.93 12.21 24.77 Het. 288 255 46.5 NC_000002.11:g.179406191C>T

TTN NP_003310.4:p.Ile20331Thr; NM_003319.4:c.60992T>C; Exon Nonsynonymous 45.1 49.28 31.83 44.61 Het. 344 255 47.4 NC_000002.11:g.179421694A>G

TTN NP_003310.4:p.Ile18710Val; NM_003319.4:c.56128A>G; Exon Nonsynonymous 49.16 32 44.93 Het. 363 255 51.2 NC_000002.11:g.179427536T>C

TTN NP_003310.4:p.Thr17556Met; NM_003319.4:c.52667C>T; Exon Nonsynonymous 12.89 34.23 21.76 34.22 Het. 375 255 52.8 NC_000002.11:g.179430997G>A

TTN NP_003310.4:p.Arg15882Cys; NM_003319.4:c.47644C>T; Exon Nonsynonymous 20.81 12.21 24.51 Het. 384 255 51.3 NC_000002.11:g.179436020G>A

TTN NP_003310.4:p.Ala13351Pro; NM_003319.4:c.40051G>C; Exon Nonsynonymous 99.47 0.54 0.44 99.42 Hom. 378 255 100 NC_000002.11:g.179444768C>G

TTN NP_003310.4:p.Val13294Ile; NM_003319.4:c.39880G>A; Exon Nonsynonymous 35.12 22.39 35.93 Het. 321 255 46.4 NC_000002.11:g.179444939C>T

TTN NP_003310.4:p.Thr12338Ile; NM_003319.4:c.37013C>T; Exon Nonsynonymous 49.24 31.23 44.61 Het. 280 255 45.7 NC_000002.11:g.179451420G>A

Page 13 of 21 CardioGxOne Report for:

Exonic dbSNP 1000G 5000G HiC DP Freq. Gene Variant Function dbSNP AF1 Qual function freq. freq. freq. freq. Qual alt.

TTN NP_003310.4:p.Pro10797Leu; NM_003319.4:c.32390C>T; Exon Nonsynonymous 19.13 21.31 12.53 24.9 Het. 363 255 49.3 NC_000002.11:g.179457147G>A

TTN NP_003310.4:p.Arg10414His; NM_003319.4:c.31241G>A; Exon Nonsynonymous 21.25 12.52 24.93 Het. 332 255 46.7 NC_000002.11:g.179458591C>T splicing

TTN NP_003310.4:p.Asn9636Asp; NM_003319.4:c.28906A>G; Exon Nonsynonymous 48.72 32.21 45.22 Het. 351 255 45.6 NC_000002.11:g.179464527T>C

TTN NP_001254479.2:p.Ile10522Val; NM_001267550.1:c.31564A>G; Exon Nonsynonymous 47.78 32.97 44.64 Het. 334 255 48.5 NC_000002.11:g.179558366T>C

TTN NP_001254479.2:p.Asn8803Ser; NM_001267550.1:c.26408A>G; Exon Nonsynonymous 23.53 24.7 15.29 29.88 Het. 343 255 47.8 NC_000002.11:g.179579093T>C

TTN NP_001254479.2:p.Ser8425Asn; NM_001267550.1:c.25274G>A; Exon Nonsynonymous 22.58 24.38 15.46 30.2 Het. 322 255 49.7 NC_000002.11:g.179582327C>T

TTN NP_001254479.2:p.Ala8355Glu; NM_001267550.1:c.25064C>A; Exon Nonsynonymous 44.49 26.58 40.24 Het. 357 255 50.7 NC_000002.11:g.179582537G>T splicing

TTN NP_001254479.2:p.Glu8144Ala; NM_001267550.1:c.24431A>C; Exon Nonsynonymous 22.19 23.88 15.06 30.33 Het. 373 255 49.3 NC_000002.11:g.179583496T>G

TTN NP_001254479.2:p.Asp7462His; NM_001267550.1:c.22384G>C; Exon Nonsynonymous 23.53 24.7 15.72 30.43 Het. 351 255 49.3 NC_000002.11:g.179587130C>G

TTN NP_596870.2:p.Asp3747Gly; NM_133379.4:c.11240A>G; Exon Nonsynonymous 80.17 26.7 18.19 97.33 Hom. 342 255 100 NC_000002.11:g.179615887T>C

TTN NP_596870.2:p.Leu3732Phe; NM_133379.4:c.11196G>C; Exon Nonsynonymous 97.5 2.5 2.75 99.2 Hom. 342 255 100 NC_000002.11:g.179615931C>G

TTN NP_001254479.2:p.Gly3751Asp; NM_001267550.1:c.11252G>A; Exon Nonsynonymous 83.2 19.55 9.72 98.84 Hom. 274 255 100 NC_000002.11:g.179620951C>T splicing

TTN NP_001254479.2:p.Ala3576Thr; NM_001267550.1:c.10726G>A; Exon Nonsynonymous 99.97 0.1 0.08 99.97 Hom. 264 255 100 NC_000002.11:g.179621477C>T

TTN NP_003310.4:p.Ser3373Asn; NM_003319.4:c.10118G>A; Exon Nonsynonymous 87.38 19.11 9.76 98.46 Hom. 332 255 100 NC_000002.11:g.179623758C>T

TTN NP_003310.4:p.Val3215Met; NM_003319.4:c.9643G>A; Exon Nonsynonymous 86.72 19.51 10.35 98.81 Hom. 247 255 100 NC_000002.11:g.179629461C>T

TTN NP_003310.4:p.His2462Arg; NM_003319.4:c.7385A>G; Exon Nonsynonymous 0.09 0.08 0.1 Het. 333 255 49 NC_000002.11:g.179638260T>C

TTN NP_003310.4:p.Ser1249Leu; NM_003319.4:c.3746C>T; Exon Nonsynonymous 7.99 4.91 99.49 Hom. 288 255 100 NC_000002.11:g.179644035G>A

Page 14 of 21 CardioGxOne Report for:

Exonic dbSNP 1000G 5000G HiC DP Freq. Gene Variant Function dbSNP AF1 Qual function freq. freq. freq. freq. Qual alt.

TTN NP_003310.4:p.Lys1155Glu; NM_003319.4:c.3463A>G; Exon Nonsynonymous 55.49 49.98 26.51 95.85 Het. 310 255 47.7 NC_000002.11:g.179644855T>C

TTN NP_003310.4:p.Thr765Ile; NM_003319.4:c.2294C>T; Exon Nonsynonymous 10.04 18.97 44.29 Het. 290 255 50.3 NC_000002.11:g.179650408G>A

TTN NP_003310.4:p.Arg328Cys; NM_003319.4:c.982C>T; Exon Nonsynonymous 15.48 23.6 8.81 12.35 Het. 362 255 55.2 NC_000002.11:g.179659912G>A

TXNRD2 NP_006431.2:p.Ile370Thr; NM_006440.4:c.1109T>C; Exon Nonsynonymous 73.93 28.17 20.39 92.19 Het. 247 255 46.2 NC_000022.10:g.19868218A>G Function: location of the variant according to RefSeq annotation database: exonic, intronic, splicing, UTR. dbSNP: identification of the Single Nucleotide Polymorphism Database (dbSNP). dbSNP freq. variant frequency taken from dbSNP (%). 1000G freq. frequency of variant taken from 1000 Genomes project (%). 5000G freq. minor allele frequency of variant taken from 5000 Genomes project (%). HiC freq. variant frequency taken from our HiC database (%). AF1: heterozygous, homozygous or hemizygous. 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 so has low probabilities of being a true called variant). Frequency alt: the frequency of alternative allele in high quality fragments (%). Only good quality variants were included (QUAL ≥ 170).

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

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

ABCC9 NM_005691.3:c.574-5C>A; NC_000012.11:g.22068849G>T 65.09 35.52 41.21 86.72 Het. 213 255 44.6

ACTN2 NM_001103.3:c.877-8C>G; NC_000001.10:g.236902594C>G 76.53 23.24 22.25 94.24 Het. 236 255 52.1

GAA NM_000152.3:c.547-4C>G; NC_000017.10:g.78079544C>G 44.95 39.72 32.73 93.47 Hom. 233 255 100

GAA NM_000152.3:c.858+7_858+8insAGCGGGC; NC_000017.10:g.78081528_78081529insAGCGGGC 40.37 92.18 Hom. 274 255 100

NEBL NM_006393.2:c.1671+9T>C; NC_000010.10:g.21120116A>G 47.13 46.9 43.02 64.56 Het. 275 255 44.4

NEBL NM_006393.2:c.1008+5A>G; NC_000010.10:g.21141469T>C 96.6 4.75 0.72 100 Hom. 142 255 100

RBM20 NM_001134363.1:c.3452-9G>C; NC_000010.10:g.112590810G>C 97.18 3.04 2.87 99.52 Hom. 182 255 100

RYR2 NM_001035.2:c.1477-11_1477-10insT; NC_000001.10:g.237619889_237619890insT 55.52 Hom. 230 223 80

RYR2 NM_001035.2:c.3599-9delT; NC_000001.10:g.237753084delT 53.43 Het. 259 191 45.6

RYR2 NM_001035.2:c.13783-6A>G; NC_000001.10:g.237957161A>G 71.93 24.24 34.86 86.23 Het. 330 255 52.7

SCN5A NM_198056.2:c.1141-3C>A; NC_000003.11:g.38647642G>T 14.92 15.12 16.64 32.16 Het. 192 255 51

SLC22A5 NM_003060.3:c.652+6A>G; NC_000005.9:g.131719999A>G 100 0 100 Hom. 248 255 99.6

TNNI3 NM_000363.4:c.373-10T>G; NC_000019.9:g.55665584A>C 100 0 99.97 Hom. 99 255 100

TNNT2 NM_001001430.2:c.53-11_53-7delCTTCT; NC_000001.10:g.201341181_201341185delAGAAG 81.38 Hom. 282 255 100

TTN NM_003319.4:c.70903+9T>A; NC_000002.11:g.179404786A>T 19.81 23.26 14.42 25.89 Het. 376 255 51.9

TTN NM_003319.4:c.70600+6G>T; NC_000002.11:g.179406003C>A 49.54 30.12 44.55 Het. 256 255 49.2

TTN NM_003319.4:c.4342+6C>T; NC_000002.11:g.179642425G>A 91.59 8.85 5.35 99.74 Hom. 333 255 100 dbSNP: dbSNP freq. variant frequency taken from dbSNP (%). 1000G freq. frequency of variant taken from 1000 Genomes project (%). 5000G freq. minor allele frequency of variant taken from 5000 Genomes project (%). HiC freq. variant frequency taken from our HiC database (%). AF1: Heterozygous 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 so has low probabilities of being a true called variant). Frequency alt: the frequency of alternative allele in high quality fragments (%). Only good quality variants were included (QUAL ≥ 170)

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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) are subjected to automatic genomic DNA purification (QIAsymphony SP, Qiagen), and sample preparation is carried out using the SureSelect XT Target Enrichment technology for Illumina paired-end multiplexed sequencing method (Agilent). Enrichment is 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 is sequenced on either Illumina HiSeq 1500, MiSeq or NextSeq platform. Clinically relevant variants and low-coverage regions are tested in parallel by standard Sanger sequencing. The analytical sensitivity and accuracy of this assay is 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 81 genes related to dilated cardiomyopathy:

ACTC1, BAG3, DES, DMD, DSP, FLNC, LMNA, MYBPC3, MYH7, PKP2, PLN, RBM20, TAZ, TNNC1, TNNI3, TNNT2, TPM1, TTN, ABCC9, ACTA1, ACTN2, ALMS1, ANKRD1, CAV3, CRYAB, CSRP3, DNAJC19, DOLK, DSC2, DSG2, EMD, EYA4, FHL2, FHOD3, FKRP, FKTN, FOXD4, GAA, GATA4, GATA6, GATAD1, GLB1, HFE, JUP, LAMA2, LAMA4, LAMP2, LDB3, MURC, MYH6, MYL2, MYL3, MYOT, MYPN, NEBL, NEXN, PRDM16, PSEN1, PSEN2, RAF1, RYR2, SCN5A, SGCD, SLC22A5, TBX20, TCAP, TMEM43, TMPO, TTR, TXNRD2, VCL, BRAF*, CTF1*, GLA*, KCNJ2*, KCNJ8*, NKX2-5*, PDLIM3*, PTPN11*, SGCA*, SGCB*

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. Genetics 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 constitutes 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 genome regions (i.e.: pseudogenes).

• Insertions or deletions.

We have also developed an alternative bioinformatics pipeline that is able to identified 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.

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.

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Although CardioGxOne has more than 99% of analytical sensitivity and specificity, some genotyping errors could occur in specific situations:

• Pre-arrival contaminated samples

• Mosaic mutations

• Monosomies and trisomy

• 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, which ensures tracking of samples after arrival, guaranteeing their proper identification once they arrive at our lab. However, we cannot take responsibility for labelling errors in pre-arrival samples.

Comments, recommendations and disclaimers

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 86,200 individuals from the existing literature on inherited cardiovascular diseases and from our own research.

It is highly recommended that the interpretation of this genetic report is done with the help/counselling 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 (Admera contact details).

Coverage stats

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Stats Studied genes Priority genes Average coverage 388 x 420 x Bases sequenced 352750 183527 % Bp with coverage ≥ 15 99.99% 100% % Bp with coverage ≥ 30 99.98% 100%

Page 19 of 21 CardioGxOne Report for:

Average coverage per gene

Coverage 15 x

500 400 300 200 100 0 JUP HFE DES DSP GLA GAA EMD CTF1 FHL2 FKRP FLNC EYA4 LDB3 DMD GLB1 FKTN DSC2 BRAF CAV3 DSG2 DOLK BAG3 KCNJ2 KCNJ8 CSRP3 ACTC1 ABCC9 ACTA1 CRYAB ACTN2 FOXD4 GATA4 GATA6 ALMS1 FHOD3 LAMP2 LAMA2 LAMA4 GATAD1 ANKRD1 DNAJC19

Coverage 15 x

500 400 300 200 100 0 TTR TAZ VCL PLN TTN PKP2 RAF1 RYR2 NEBL TCAP SGCB SGCA SGCD MYL2 MYL3 NEXN TPM1 MYH6 MYH7 MYOT MYPN LMNA TMPO TNNI3 TBX20 MURC PSEN1 PSEN2 SCN5A TNNT2 TNNC1 RBM20 NKX2-5 PDLIM3 PTPN11 TXNRD2 MYBPC3 SLC22A5 PRDM16 TMEM43

Page 20 of 21 CardioGxOne Report for:

Resource references

Population databases:

• Exome Aggregation Consortium (ExAC), Cambridge, MA (URL: http://exac.broadinstitute.org) [version 0.3]

• 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/) [accessed 04/01/2014]

• 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.Accessed Jan 2012).

• HGMD® [Internet]: Stenson PD etal. 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 non-synonymous 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

• Splice-site Finder (SSF)

• HSF

• MaxEnt

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