Congenital Structural Heart Disease Panel

Test code: CA1501

Is a 114 panel that includes assessment of non-coding variants.

Is ideal for patients with congenital heart disease, particularly those with features of hereditary disorders.

Is not ideal for patients suspected to have a or a rasopathy. For those patients, please consider our Primary Ciliary Dyskinesia Panel and our Panel, respectively.

About Congenital Structural Heart Disease

There are many types of congenital heart disease (CHD) ranging from simple asymptomatic defects to complex defects with severe, life-threatening symptoms. CHDs are the most common type of and affect at least 8 out of every 1,000 newborns. Annually, more than 35,000 babies in the United States are born with CHDs. Many of these CHDs are simple conditions and need no treatment or are easily repaired. Some babies are born with complex CHD requiring special medical care. The diagnosis and treatment of complex CHDs has greatly improved over the past few decades. As a result, almost all children who have complex heart defects survive to adulthood and can live active, productive lives. However, many patients who have complex CHDs continue to need special heart care throughout their lives. In the United States, more than 1 million adults are living with congenital heart disease.

Availability

4 weeks

Gene Set Description

Genes in the Congenital Structural Heart Disease Panel and their clinical significance Gene Associated phenotypes Inheritance ClinVar HGMD

ABL1 Congenital heart defects and skeletal malformations syndrome AD 30 5 (CHDSKM)

ACTA2 Aortic , familial thoracic, Moyamoya disease, Multisystemic AD 20 76 smooth muscle dysfunction syndrome

ACTB* Baraitser-Winter syndrome AD 55 60

ACTC1 Left ventricular noncompaction, Hypertrophic cardiomyopathy (HCM), AD 23 63 Cardiomyopathy, restrictive, Atrial septal defect, Dilated cardiomyopathy (DCM)

ACTG1* Deafness, Baraitser-Winter syndrome AD 27 47

ACVR1 Fibrodysplasia ossificans progressiva AD 14 19

ACVR2B Heterotaxy, visceral, 4, autosomal AD 1 2

ADAMTS10 Weill-Marchesani syndrome AR 8 14

ADAMTS17 Weill-Marchesani-like syndrome AR 6 7

AFF4 Cognitive impairment, coarse facies, Heart defects, , Pulmonary AD 3 3 involvement, , and skeletal dysplasia (CHOPS syndrome)

https://blueprintgenetics.com/ AMMECR1 Midface hypoplasia, hearing impairment, elliptocytosis, and XL 4 5 nephrocalcinosis

ARHGAP31 Adams-Oliver syndrome AD 3 6

ARID1A Coffin-Siris syndrome, Mental retardation AD 27 35

ARID1B Coffin-Siris syndrome, Mental retardation AD 153 185

B3GAT3* Multiple joint dislocations, short stature, craniofacial dysmorphism, and AR 6 13 congenital heart defects

BCOR Microphthalmia, syndromic, Oculofaciocardiodental syndrome XL 40 53

BMPR2 Pulmonary , primary, Pulmonary venoocclusive disease AD 391 572

BRAF* LEOPARD syndrome, Noonan syndrome, Cardiofaciocutaneous syndrome AD 134 65

C12ORF57 Corpus callosum hypoplasia, recessive, Temtamy syndrome AR 7 6

CBL Noonan syndrome-like disorder with or without juvenile myelomonocytic AD 24 43 leukemia

CDK13 Congenital heart defects, dysmorphic facial features, and intellectual AD 13 20 developmental disorder

CDK9 AR 1

CFAP53 Heterotaxy, visceral, 6, autosomal recessive AR 4 4

CHD4 Sifrim-Hitz-Weiss syndrome AD 14 21

CHD7 Isolated gonadotropin-releasing hormone deficiency, CHARGE syndrome AD 276 860

CHRM2 Dilated cardiomyopathy (DCM) AD/AR 1

CREBBP Rubinstein-Taybi syndrome AD 175 362

CRELD1 Atrioventricular septal defect, partial, with or without heterotaxy AD 2 16

CTC1 Cerebroretinal with calcifications and cysts AR 21 33

DHCR7 Smith-Lemli-Opitz syndrome AR 88 217

DLL4 Adams-Oliver syndrome AD 13 14

DOCK6 Adams-Oliver syndrome AR 21 21

EFTUD2 Mandibulofacial dysostosis with , , AD 45 99 syndromic

EHMT1 Kleefstra syndrome AD 86 89

EIF2AK4 Pulmonary venoocclusive disease AR 27 84

ELN Cutis laxa, Supravalvular aortic AD 78 113

ENG Juvenile polyposis syndrome, Hereditary hemorrhagic AD 158 491

EOGT Adams-Oliver syndrome AR 8 5

https://blueprintgenetics.com/ EP300 Rubinstein-Taybi syndrome AD 63 101

EVC Weyers acrofacial dysostosis, Ellis-van Creveld syndrome AD/AR 58 83

EVC2 Ellis-van Creveld syndrome, Weyers acrodental dysostosis AD/AR 78 75

FLNA Frontometaphyseal dysplasia, Osteodysplasty Melnick-Needles, XL 133 257 Otopalatodigital syndrome type 1, Otopalatodigital syndrome type 2, Terminal osseous dysplasia with pigmentary defects

FOXC1 Axenfeld-Rieger syndrome, Iridogoniodysgenesis, Peters anomaly AD 46 135

FOXF1 Alveolar dysplasia with misalignment of pulmonary AD 10 102

FOXH1 Congenital heart malformations, Holoprosencephaly AD 33

FOXP1 Mental retardation with language impairment and autistic features, AD 48 76 Congenital heart malformations

GATA4 , Atrioventricular septal defect, Testicular anomalies AD 37 140 with or without congenital heart disease, Ventricular septal defect, Atrial septal defect

GATA5 Familial atrial fibrillation, Tetralogy of Fallot, Single ventricular septal AD 5 32 defect

GATA6 Heart defects, congenital, and other congenital anomalies, Atrial septal AD 16 82 defect 9, atrioventricular septal defect 5, Persistent truncus arteriosus, Tetralogy of Fallot

GDF1 Transposition of the great , dextro-looped 3, Double-outlet right AR 11 15 ventricle

GJA1* Oculodentodigital dysplasia mild type, Oculodentodigital dysplasia severe AD/AR 31 107 type, type 3

GJA5 Progressive familial heart block, Atrial standstill, digenic, Atrial fibrillation AD/Digenic 8 34

GPC3 Simpson-Golabi-Behmel syndrome XL 33 75

HAND1 Congenital heart defects, Dilated cardiomyopathy AD 9

HAND2 Dilated cardiomyopathy (DCM), Congenital heart malformations AD 2 5

HDAC8 Cornelia de Lange syndrome XL 41 50

HNRNPK Au-Kline syndrome AD 14 10

HOXA1 Athabaskan brainstem dysgenesis syndrome, Bosley-Salih-Alorainy AR 4 7 syndrome

HRAS , Congenital myopathy with excess of muscle spindles AD 43 31

JAG1 Alagille syndrome AD 131 610

KDM6A XL 40 69

KMT2D Kabuki syndrome AD 350 670

KRAS* Noonan syndrome, Cardiofaciocutaneous syndrome AD 63 35

https://blueprintgenetics.com/ KYNU Hydroxykynureninuria, Vertebral, cardiac, renal, and limb defects AR 4 7 syndrome 2

LEFTY2* Left-right axis malformations AD 1 3

MED12 Ohdo syndrome, Mental retardation, with Marfanoid habitus, FG XL 29 30 syndrome, Opitz-Kaveggia syndrome, Lujan-

MED13L Transposition of the great arteries, dextro-looped 1, Mental retardation AD/AR 92 78 and distinctive facial features with or without cardiac defects, Congenital heart defects and , Intellectual disability, autosomal recessive

MEIS2 Cleft palate, cardiac defects, and mental retardation (CPCMR) AD 10 18

MMP21 Heterotaxy, visceral, 7 AR 4 18

MYCN AD 27 41

MYO18B Klippel-Feil syndrome 4, autosomal recessive, with myopathy and facial AR 2 4 dysmorphism

MYRF Congenital heart malformations, Congenital abnormalities of the kidney AD 1 1 and urinary tract

NAA15 Congenital heart malformations AD 10 32

NF1* , Neurofibromatosis, Neurofibromatosis-Noonan AD 1157 2901 syndrome

NIPBL Cornelia de Lange syndrome AD 311 425

NKX2-5 Conotruncal heart malformations, Hypothyroidism, congenital AD 45 108 nongoitrous,, Atrial septal defect, Ventricular septal defect 3, Conotruncal heart malformations, variable, Tetralogy of Fallot

NKX2-6 Persistent truncus arteriosus, Conotruncal heart malformations AD/AR 2 9

NODAL Heterotaxy, visceral AD 4 15

NONO Mental retardation, X-linked, syndrome 34, Left ventricular non- XL 10 4 compaction cardiomyopathy (LVNC)

NOTCH1 Aortic valve disease, Adams-Oliver syndrome AD 56 96

NOTCH2* Alagille syndrome, Hajdu-Cheney syndrome AD 37 70

NR2F2 Congenital heart defects, multiple types, 4 AD 12 16

NSD1 , , Beckwith-Wiedemann syndrome AD 329 517

PITX2 Axenfeld-Rieger syndrome, Ring dermoid of cornea, AD 23 101 Iridogoniodysgenesis, Peters anomaly

PKD1L1 Heterotaxy, visceral, 8, autosomal AR 2 6

PPP1CB# Noonan syndrome-like disorder with loose anagen hair 2 AD 8 11

PRDM6 Patent ductus arteriosus 3, Congenital heart malformations AD 4 3

PRKD1 Congenital heart defects and ectodermal dysplasia AD 2 7

https://blueprintgenetics.com/ PTPN11 Noonan syndrome, AD 135 140

PUF60 Short stature, Microcephaly AD 24 30

RAB23# 1 AR 5 15

RAF1 LEOPARD syndrome, Noonan syndrome, Dilated cardiomyopathy (DCM) AD 45 53

RBM10 TARP syndrome XL 12 10

RERE Neurodevelopmental disorder with or without anomalies of the brain, eye, AD 24 24 or heart (NEDBEH)

RIT1 Noonan syndrome AD 23 26

SALL4 Acro-renal-ocular syndrome, Duane-radial ray/Okohiro syndrome AD 21 56

SMARCB1 Schwannomatosis, Rhabdoid tumor predisposition syndrome, Coffin-Siris AD 36 118 syndrome 3

SMC1A Cornelia de Lange syndrome XL 73 87

SMC3 Cornelia de Lange syndrome AD 25 21

SOS1 Noonan syndrome AD 44 71

SOS2 Noonan syndrome 9 AD 4 6

STAG2 Congenital heart defects, dysmorphic facial features, and intellectual XL 6 14 developmental disorder

STRA6 Microphthalmia, syndromic, Microphthalmia, isolated, with coloboma AR 22 33

TAB2 Congenital heart defects, multiple types, 2 AD 13 31

TBX1 Conotruncal anomaly face syndrome AD 17 72

TBX20* Atrial septal defect 4 AD 4 28

TBX5 Holt-Oram syndrome AD 61 127

TFAP2B Patent ductus arteriosus, nonsyndromic, Char syndrome AD 10 12

TGDS Catel-Manzke syndrome AR 6 7

TLL1 Atrial septal defect AD 3 7

TMEM94 Neurodevelopmental disorder with or without anomalies of the brain, eye, AR 3 or heart (NEDBEH)

ZEB2* Mowat-Wilson syndrome AD 154 287

ZFPM2 46,XY sex reversal, Diaphragmatic hernia 3, Tetralogy of Fallot AD/AR 9 50

ZIC3 Heterotaxy, visceral, VACTERL association, Congenital heart defects, XL 15 41 nonsyndromic

*Some regions of the gene are duplicated in the genome. Read more.

# The gene has suboptimal coverage (means <90% of the gene’s target nucleotides are covered at >20x with mapping quality score (MQ>20) reads), and/or the gene has listed under Test limitations section that are not included in the panel as

https://blueprintgenetics.com/ they are not sufficiently covered with high quality sequence reads.

The sensitivity to detect variants may be limited in marked with an asterisk (*) or number sign (#). Due to possible limitations these genes may not be available as single gene tests.

Gene refers to the HGNC approved gene symbol; Inheritance refers to inheritance patterns such as autosomal dominant (AD), autosomal recessive (AR), mitochondrial (mi), X-linked (XL), X-linked dominant (XLD) and X-linked recessive (XLR); ClinVar refers to the number of variants in the gene classified as pathogenic or likely pathogenic in this database (ClinVar); HGMD refers to the number of variants with possible disease association in the gene listed in Human Gene Database (HGMD). The list of associated, gene specific phenotypes are generated from CGD or Mitomap databases.

Non-coding disease causing variants covered by the panel

Gene Genomic HGVS RefSeq RS-number location HG19

ACTC1 Chr15:35080829 c.*1784T>C NM_005159.4

BMPR2 Chr2:203241251 c.-947_-946delGCinsAT NM_001204.6 rs1085307144

BMPR2 Chr2:203241851 c.-347C>T NM_001204.6

BMPR2 Chr2:203241919 c.-279C>A NM_001204.6

BMPR2 Chr2:203242106 c.-92C>A NM_001204.6

BMPR2 Chr2:203395505 c.968-12T>G NM_001204.6

CHD7 Chr8:61734568 c.2836-15C>G NM_017780.3

CHD7 Chr8:61757794 c.5051-15T>A NM_017780.3

CHD7 Chr8:61763034 c.5405-18C>A NM_017780.3 rs199981784

CHD7 Chr8:61763035 c.5405-17G>A NM_017780.3 rs794727423

CHD7 Chr8:61763039 c.5405-13G>A NM_017780.3 rs1131690787

CREBBP Chr16:3788684 c.4281-11C>G NM_004380.2 rs587783493

EHMT1 Chr9:140678546 c.2382+1697T>G NM_024757.4 rs786205602

ELN Chr7:73480347 c.2272+20C>G NM_001278939.1

ENG Chr9:130578354 c.1742-22T>C NM_001114753.2

ENG Chr9:130588962 c.361-11T>A NM_001114753.2

ENG Chr9:130616692 c.-58G>A NM_001114753.2 rs971268057

ENG Chr9:130616761 c.-127C>T NM_001114753.2

ENG Chr9:130616776 c.-142A>T NM_001114753.2

EP300 Chr22:41537040 c.1879-12A>G NM_001429.3

EVC Chr4:5749725 c.940-150T>G NM_153717.2

FLNA ChrX:153581587 c.6023-27_6023-16delTGACTGACAGCC NM_001110556.1

https://blueprintgenetics.com/ FOXC1 Chr6:1610252 c.-429C>G NM_001453.2 rs77888940

GATA4 Chr8:11561282 c.-989C>T NM_002052.3

GATA4 Chr8:11561369 c.-902G>T NM_002052.3

GATA4 Chr8:11561399 NM_002052.3 rs1195641788

GATA4 Chr8:11612500 c.910-55T>C NM_002052.3

GATA4 Chr8:11612745 c.997+103G>T NM_002052.3 rs113049875

GATA4 Chr8:11614418 c.998-26G>A NM_002052.3

GATA5 Chr20:61051165 c.-201A>G NM_080473.4

GATA5 Chr20:61051462 NM_080473.4 rs1193866928

GATA6 Chr18:19749151 c.-530A>T NM_005257.4

GATA6 Chr18:19749272 c.-409C>G NM_005257.4

HNRNPK Chr9:86590455 c.214-35A>G NM_002140.3

JAG1 Chr20:10629767 c.1349-12T>G NM_000214.2

KMT2D Chr12:49428461 c.10356-12G>A NM_003482.3

NF1 Chr17:29422055 c.-273A>C NM_001042492.2

NF1 Chr17:29422056 c.-272G>A NM_001042492.2

NF1 Chr17:29431417 c.60+9031_60+9035delAAGTT NM_001042492.2

NF1 Chr17:29475515 c.61-7486G>T NM_001042492.2

NF1 Chr17:29488136 c.288+2025T>G NM_001042492.2

NF1 Chr17:29508426 c.587-14T>A NM_001042492.2

NF1 Chr17:29508428 c.587-12T>A NM_001042492.2

NF1 Chr17:29510334 c.888+651T>A NM_001042492.2

NF1 Chr17:29510427 c.888+744A>G NM_001042492.2

NF1 Chr17:29510472 c.888+789A>G NM_001042492.2

NF1 Chr17:29527428 c.889-12T>A NM_001042492.2

NF1 Chr17:29530107 c.1260+1604A>G NM_001042492.2

NF1 Chr17:29533239 c.1261-19G>A NM_001042492.2

NF1 Chr17:29534143 c.1392+754T>G NM_001042492.2

NF1 Chr17:29540877 c.1393-592A>G NM_001042492.2

NF1 Chr17:29542762 c.1527+1159C>T NM_001042492.2

NF1 Chr17:29548419 c.1642-449A>G NM_001042492.2 rs863224655

https://blueprintgenetics.com/ NF1 Chr17:29549489 c.*481A>G NM_001128147.2

NF1 Chr17:29553439 c.2002-14C>G NM_001042492.2

NF1 Chr17:29554225 c.2252-11T>G NM_001042492.2

NF1 Chr17:29556025 c.2410-18C>G NM_001042492.2

NF1 Chr17:29556027 c.2410-16A>G NM_001042492.2

NF1 Chr17:29556028 c.2410-15A>G NM_001042492.2

NF1 Chr17:29556031 c.2410-12T>G NM_001042492.2

NF1 Chr17:29556839 c.2851-14_2851-13insA NM_001042492.2

NF1 Chr17:29557267 c.2991-11T>G NM_001042492.2

NF1 Chr17:29558777 c.3198-314G>A NM_001042492.2

NF1 Chr17:29563299 c.3974+260T>G NM_001042492.2

NF1 Chr17:29577082 c.4110+945A>G NM_001042492.2

NF1 Chr17:29580296 c.4173+278A>G NM_001042492.2

NF1 Chr17:29588708 c.4578-20_4578-18delAAG NM_001042492.2

NF1 Chr17:29588715 c.4578-14T>G NM_001042492.2

NF1 Chr17:29654479 c.5269-38A>G NM_001042492.2

NF1 Chr17:29656858 c.5610-456G>T NM_001042492.2

NF1 Chr17:29657848 c.5812+332A>G NM_001042492.2 rs863224491

NF1 Chr17:29661577 c.5813-279A>G NM_001042492.2

NF1 Chr17:29664375 c.6428-11T>G NM_001042492.2

NF1 Chr17:29664618 c.6642+18A>G NM_001042492.2

NF1 Chr17:29676126 c.7190-12T>A NM_001042492.2

NF1 Chr17:29676127 c.7190-11_7190-10insGTTT NM_001042492.2

NF1 Chr17:29685177 c.7971-321C>G NM_001042492.2

NF1 Chr17:29685481 c.7971-17C>G NM_001042492.2

NF1 Chr17:29685665 c.8113+25A>T NM_001042492.2

NIPBL Chr5:36877039 c.-321_-320delCCinsA NM_133433.3 rs724159980

NIPBL Chr5:36877266 c.-94C>T NM_133433.3

NIPBL Chr5:36953718 c.-79-2A>G NM_133433.3

NIPBL Chr5:37022138 c.5329-15A>G NM_133433.3 rs587783968

NIPBL Chr5:37026318 c.5710-13_5710-12delCTinsAA NM_133433.3

https://blueprintgenetics.com/ NKX2-5 Chr5:172662741 NM_004387.3

NKX2-5 Chr5:172672291 c.-10205G>A .

NKX2-5 Chr5:172672303 c.-10217G>C .

NR2F2 Chr15:96869479 c.-60C>T NM_001145155.1

PITX2 Chr4:111538758 c.*520_*522delTAT NM_000325.5 rs561702585,rs775662096

PITX2 Chr4:111539855 c.412-11A>G NM_000325.5

PITX2 Chr4:111559138 c.-1214_-1213delAT NM_153426.2

PTPN11 Chr12:112915602 c.934-59T>A NM_002834.3

SMARCB1 Chr22:24130008 c.93+559A>G NM_003073.3

SMARCB1 Chr22:24176316 c.1119-12C>G NM_003073.3

SMARCB1 Chr22:24176437 c.*70C>T NM_003073.3

SMARCB1 Chr22:24176449 c.*82C>T NM_003073.3

TBX1 Chr22:19743578 c.-777C>T NM_080647.1

TBX1 Chr22:19743735 c.-620A>C NM_080647.1 rs536892777

TBX20 Chr7:35293780 c.-549G>A NM_001077653.2 rs571512677

TBX5 Chr12:114704515 c.*88822C>A NM_000192.3 rs141875471

ZEB2 Chr2:145274987 c.-69-1G>A NM_014795.3

ZEB2 Chr2:145274988 c.-69-2A>C NM_014795.3

Test Strengths

The strengths of this test include:

CAP accredited laboratory CLIA-certified personnel performing clinical testing in a CLIA-certified laboratory Powerful sequencing technologies, advanced target enrichment methods and precision bioinformatics pipelines ensure superior analytical performance Careful construction of clinically effective and scientifically justified gene panels Some of the panels include the whole mitochondrial genome (please see the Panel Content section) Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level Our publicly available analytic validation demonstrating complete details of test performance ~2,000 non-coding disease causing variants in our clinical grade NGS assay for panels (please see ‘Non-coding disease causing variants covered by this panel’ in the Panel Content section) Our rigorous variant classification scheme Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data Our comprehensive clinical statements

https://blueprintgenetics.com/ Test Limitations

The following exons are not included in the panel as they are not sufficiently covered with high quality sequence reads: B3GAT3 (NM_001288722:5). Genes with suboptimal coverage in our assay are marked with number sign (#) and genes with partial, or whole gene, segmental duplications in the are marked with an asterisk (*) if they overlap with the UCSC pseudogene regions. Gene is considered to have suboptimal coverage when >90% of the gene’s target nucleotides are not covered at >20x with mapping quality score (MQ>20) reads. The technology may have limited sensitivity to detect variants in genes marked with these symbols (please see the Panel content table above).

This test does not d etect the following:

Complex inversions Gene conversions Balanced translocations Some of the panels include the whole mitochondrial genome (please see the Panel Content section) Repeat expansion disorders unless specifically mentioned Non-coding variants deeper than ±20 base pairs from -intron boundary unless otherwise indicated (please see above Panel Content / non-coding variants covered by the panel).

This test may not reliably detect the following:

Low level mosaicism in nuclear genes (variant with a minor allele fraction of 14.6% is detected with 90% probability) Stretches of mononucleotide repeats Low level heteroplasmy in mtDNA (>90% are detected at 5% level) Indels larger than 50bp Single exon deletions or duplications Variants within pseudogene regions/duplicated segments Some disease causing variants present in mtDNA are not detectable from blood, thus post-mitotic tissue such as skeletal muscle may be required for establishing molecular diagnosis.

The sensitivity of this test may be reduced if DNA is extracted by a laboratory other than Blueprint .

For additional information, please refer to the Test performance section and see our Analytic Validation.

Test Performance

The genes on the panel have been carefully selected based on scientific literature, mutation databases and our experience.

Our panels are sectioned from our high-quality, clinical grade NGS assay. Please see our sequencing and detection performance table for details regarding our ability to detect different types of alterations (Table).

Assays have been validated for various sample types including EDTA-blood, isolated DNA (excluding from formalin fixed paraffin embedded tissue), saliva and dry blood spots (filter cards). These sample types were selected in order to maximize the likelihood for high-quality DNA yield. The diagnostic yield varies depending on the assay used, referring healthcare professional, hospital and country. Plus analysis increases the likelihood of finding a genetic diagnosis for your patient, as large deletions and duplications cannot be detected using sequence analysis alone. Blueprint Genetics’ Plus Analysis is a combination of both sequencing and deletion/duplication (copy number variant (CNV)) analysis.

The performance metrics listed below are from an initial validation performed at our main laboratory in Finland. The performance metrics of our laboratory in Seattle, WA, are equivalent.

Performance of Blueprint Genetics high-quality, clinical grade NGS sequencing assay for panels.

Sensitivity % (TP/(TP+FN) Specificity %

Single nucleotide variants 99.89% (99,153/99,266) >99.9999%

https://blueprintgenetics.com/ Insertions, deletions and indels by sequence analysis

1-10 bps 99.2% (7,745/7,806) >99.9999%

11-50 bps 99.13% (2,524/2,546) >99.9999%

Copy number variants (exon level dels/dups)

1 exon level deletion (heterozygous) 100% (20/20) NA

1 exon level deletion (homozygous) 100% (5/5) NA

1 exon level deletion (het or homo) 100% (25/25) NA

2-7 exon level deletion (het or homo) 100% (44/44) NA

1-9 exon level duplication (het or homo) 75% (6/8) NA

Simulated CNV detection

5 exons level deletion/duplication 98.7% 100.00%

Microdeletion/-duplication sdrs (large CNVs, n=37))

Size range (0.1-47 Mb) 100% (25/25)

The performance presented above reached by Blueprint Genetics high-quality, clinical grade NGS sequencing assay with the following coverage metrics

Mean sequencing depth 143X

Nucleotides with >20x sequencing coverage (%) 99.86%

Performance of Blueprint Genetics Mitochondrial Sequencing Assay.

Sensitivity % Specificity %

ANALYTIC VALIDATION (NA samples; n=4)

Single nucleotide variants

Heteroplasmic (45-100%) 100.0% (50/50) 100.0%

Heteroplasmic (35-45%) 100.0% (87/87) 100.0%

Heteroplasmic (25-35%) 100.0% (73/73) 100.0%

Heteroplasmic (15-25%) 100.0% (77/77) 100.0%

Heteroplasmic (10-15%) 100.0% (74/74) 100.0%

Heteroplasmic (5-10%) 100.0% (3/3) 100.0%

Heteroplasmic (<5%) 50.0% (2/4) 100.0%

CLINICAL VALIDATION (n=76 samples)

https://blueprintgenetics.com/ All types

Single nucleotide variants n=2026 SNVs

Heteroplasmic (45-100%) 100.0% (1940/1940) 100.0%

Heteroplasmic (35-45%) 100.0% (4/4) 100.0%

Heteroplasmic (25-35%) 100.0% (3/3) 100.0%

Heteroplasmic (15-25%) 100.0% (3/3) 100.0%

Heteroplasmic (10-15%) 100.0% (9/9) 100.0%

Heteroplasmic (5-10%) 92.3% (12/13) 99.98%

Heteroplasmic (<5%) 88.9% (48/54) 99.93%

Insertions and deletions by sequence analysis n=40 indels

Heteroplasmic (45-100%) 1-10bp 100.0% (32/32) 100.0%

Heteroplasmic (5-45%) 1-10bp 100.0% (3/3) 100.0%

Heteroplasmic (<5%) 1-10bp 100.0% (5/5) 99,997%

SIMULATION DATA /(mitomap )

Insertions, and deletions 1-24 bps by sequence analysis; n=17

Homoplasmic (100%) 1-24bp 100.0% (17/17) 99.98%

Heteroplasmic (50%) 100.0% (17/17) 99.99%

Heteroplasmic (25%) 100.0% (17/17) 100.0%

Heteroplasmic (20%) 100.0% (17/17) 100.0%

Heteroplasmic (15%) 100.0% (17/17) 100.0%

Heteroplasmic (10%) 94.1% (16/17) 100.0%

Heteroplasmic (5%) 94.1% (16/17) 100.0%

Copy number variants (separate artifical mutations; n=1500)

Homoplasmic (100%) 500 bp, 1kb, 5 kb 100.0% 100.0%

Heteroplasmic (50%) 500 bp, 1kb, 5 kb 100.0% 100.0%

Heteroplasmic (30%) 500 bp, 1kb, 5 kb 100.0% 100.0%

Heteroplasmic (20%) 500 bp, 1kb, 5 kb 99.7% 100.0%

Heteroplasmic (10%) 500 bp, 1kb, 5 kb 99.0% 100.0%

The performance presented above reached by following coverage metrics at assay level (n=66)

Mean of medians Median of medians

https://blueprintgenetics.com/ Mean sequencing depth MQ0 (clinical) 18224X 17366X

Nucleotides with >1000x MQ0 sequencing coverage (%) (clinical) 100%

rho zero cell line (=no mtDNA), mean sequencing depth 12X

Bioinformatics

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases such as, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as SIFT, PolyPhen, MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, the customer has an access to details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with inadequate coverage if present. This reflects our mission to build fully transparent diagnostics where customers have easy access to crucial details of the analysis process.

Clinical Interpretation

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the corner stone of clinical interpretation and resulting patient management decisions. Our classifications follow the ACMG guideline 2015.

The final step in the analysis of sequence variants is confirmation of variants classified as pathogenic or likely pathogenic using bi-directional Sanger sequencing. Variant(s) fulfilling the following criteria are not Sanger confirmed: the variant quality score is above the internal threshold for a true positive call, and visual check-up of the variant at IGV is in-line with the variant call. Reported variants of uncertain significance are confirmed with bi-directional Sanger sequencing only if the quality score is below our internally defined quality score for true positive call. Reported copy number variations with a size <10 exons are confirmed by orthogonal methods such as qPCR if the specific CNV has been seen less than three times at Blueprint Genetics.

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts and detailed information about related phenotypes. We also provide links to the references used, congress abstracts and mutation variant databases used to help our customers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification within the family. In the case of variants of uncertain significance (VUS), we do not recommend family member risk stratification based on the VUS result. Furthermore, in the case of VUS, we do not recommend the use of genetic information in patient management or genetic counseling.

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Thus, our database, and our understanding of variants and related phenotypes, is growing by leaps and bounds. Our laboratory is therefore well

https://blueprintgenetics.com/ positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering health care provider at no additional cost.

ICD Codes

Refer to the most current version of ICD-10-CM manual for a complete list of ICD-10 codes.

Sample Requirements

Blood (min. 1ml) in an EDTA tube Extracted DNA, min. 2 μg in TE buffer or equivalent Saliva (Please see Sample Requirements for accepted saliva kits)

Label the sample tube with your patient's name, date of birth and the date of sample collection.

We do not accept DNA samples isolated from formalin-fixed paraffin-embedded (FFPE) tissue. In addition, if the patient is affected with a hematological malignancy, DNA extracted from a non-hematological source (e.g. skin fibroblasts) is strongly recommended.

Please note that, in rare cases, mitochondrial genome (mtDNA) variants may not be detectable in blood or saliva in which case DNA extracted from post-mitotic tissue such as skeletal muscle may be a better option.

Read more about our sample requirements here.

For Patients

Other

Baumgartner H et al. ESC Guidelines for the management of grown-up congenital heart disease (new version 2010). Eur Heart J. 2010 Dec;31(23):2915-57. GeneReviews - Alagille's syndrome GeneReviews - Holt-Oram syndrome GeneReviews - Hypoplastic left heart syndrome Goossens E et al. Implementation of the American College of Cardiology/American Heart Association 2008 Guidelines for the Management of Adults With Congenital Heart Disease. Am J Cardiol. 2015 Aug 1;116(3):452-7. NORD - Alagille's syndrome NORD - Holt-Oram syndrome NORD - Hypoplastic Left Heart Syndrome Structural Heart Disease Australia University of Chicago University of Chicago - Congenital Heart Disease

https://blueprintgenetics.com/