1. Supplemental Methods and Expanded Results

Appendix

1. Supplemental Methods and Expanded Results

2. Supplemental Tables

3. Supplemental Figures and Figure Legends

4. Supplemental References

1. Supplemental Methods and Expanded Results

1.1Variant classification criteria

Pathogenic variants: 1) The variant resides in a validated cardiac channelopathy or cardiomyopathy-associated gene;2) The mutation is a null variant in a gene where loss of function is a known mechanism of disease or is a missense variant already reported in literature as clearly associated with an arrhythmic disease phenotype; 3) The variant is absent in the general population, identified with the ExAC and the gnomAD databases 14) The variant maps to an important domain of the protein and/or in a region where pathogenic mutations have been previously reported; 5) The affected amino acid residue is highly conserved across species (>95% in 100 Vertebrates);6)The variant obtains adamaging or possibly damaging effect predictionby at least 3 bioinformatics tools (PolyPhen-2,2 SIFT,3 SIFT Human Protein,4 Mutation Taster,5 M-CAP6); 7) The clinical phenotype of the subject matches the phenotype expected from the associatedvariant;8) De novo status of thevariant, provided the parents are not affected, supports pathogenicity;9) The variant co-segregates with the clinical phenotype in the family members (when available).

Likely pathogenic variants:Variants fulfilling all above criteria except criterion number 2.

Variants of Unknown Significance (VUS):Variants fulfilling some of the criteria 1-9, as above.

Variants with functional effects: Variants fulfilling partly the criteria 1-2 and 4-9 as above with a MAF < 0.5% and with a demonstrated functional effect in vitro.

Likely benign:Variants that do not fulfill most of the pathogenicity criteria or variants in known channelopathy and cardiomyopathy-associated genes clearly reported in literature as benign.

1.2 Case summaries for IVF pathogenic and likely pathogenic variant carriers

Case description summaries of clinical and genetic data for the 7 IVF cases carrying pathogenic and likely pathogenic variants are provided below. The respective family pedigrees are shown in Fig.A.1. The sequencing coverage for the variants identified in the Finnish patients ranged from 69x for RYR2p.Gln3774Arg to 120x for CACNA1Cp.Gly402Ser, and all the variants identified in the Italian cohort had a sequencing coverage > 20x. The existence of all the pathogenic/likely pathogenic variants was confirmed by Sanger sequencing.

Case F10 is a boy resuscitated from VF at age 12after bickering at the school yard. During the initial clinical workup he manifested PVCs during the exercise stress test. The test was prematurely interrupted due to the premature ventricular complexes, but a CPVT diagnosis was not made at the time.Except for an isolated ventricular extrasystole, the next exercise stress test after molecular testing at the age of 20 years did not provoke any arrhythmias, although the patient was not under beta-blockermedication during the test. However, at this time the patient was also severely obese (BMI>40), with likely limited exercise capacity. Whole-exome sequencing and subsequent sequencing of the relatives revealed that the patient carried a novel RYR2 mutation (p.Leu575Phe) that seems to have arisen de novo. The variant is absent from the general population (also from the gnomAD database1 of 126,216 exomes), has concordant 5 bioinformatics tools support on impact on function and affects a highly conserved amino acid (Table 3A).Family history was unremarkable for SCD or cardiac disease. The patient has remained free of symptoms till the end of the follow-up.

Case F27 is a female who experienced an epileptic seizure at the age of 10. She was resuscitated from VF after sexual intercourse at the age of 19. Resting ECG, flecainide test, MRI and molecular genetic testing for LQTS were not diagnostic. Initially, the patient refused to undergo exercise stress testing. Low-dose beta-blocker treatment was initiated and an ICD was implanted. The mother of the patient had died suddenly at the age of 35 years during the night, having previously experienced syncopal spells with convulsions. Her death was classified as of unknown cause after autopsy. The WES analyses showed that the index patient was a heterozygous carrier of a novel RYR2 variant (p.Gln3774Arg). The variant is absent from the general population (including the gnomAD database of 126,216 exomes1), has concordant 5 bioinformatics tools support on impact on function and affects an amino acid residue fully conserved among 100 vertebrates (Table 3A). Since there was no possibility to confirm the presence of the variant in the patient’s mother, we classified it as likely pathogenic. After receiving the molecular genetic diagnosis suggestive of CPVT, she agreed to undergo an exercise stress test. The test provoked bigeminal and paired PVCs,appearingat a heart rate exceeding90 beats/min. During the follow-up of 5 years, no ICD therapies havebeen delivered.

Case F30 is a girl that had experienced a syncopal spell with urinary incontinence twice at the age of 11 and was resuscitated from VF two years later. Resting ECG, exercise stress test, epinephrine test, flecainide test, coronary artery angiography and EP study did not result in a diagnosis. Family history forSCD was negative. Whole-exome sequencing identified the known p.Gly402Ser pathogenic mutation in the CACNA1Cgene (Table 3A). This mutation has been previously consistently associated with Timothy syndrome type 2 and occurs inexon 8 of the cardiac isoform of the voltage-gated Ca2+ channel.7Despite the cardiac features, albeit a borderline QTc interval prolongation, the subject lacks other severe symptoms associated with Timothy syndrome.The same patient also carried novel variants in CRYAB(c.137C>T, p.Pro46Leu, NM_001289808.1) and DMD (c.8376G>T, p.Leu2792Phe, NM_000109.3), classified as likely benign. Independently from our study, the case of this patient has been recently described in detail by Hiippalaet al.8The 6-year follow-up period has been uneventful.

Case I12 is a young male resuscitated from VF at age 21 whilst playing soccer. Echocardiogram showed mild impaired ejection fraction (EF;40%) that improved during first admission (last EF 50%). Coronary angiogram was normal. MRI showed edema considered compatible with trauma due to CPR. An EP study was performed and no arrhythmias were induced. An ICD was implanted. During the initial clinical workup, he manifested PVCs during the exercise stress test and 24-hr HolterECG. Next-generation sequencing revealed that the patient carrieda novel DSP nonsense mutation(p.Gln620*). The mutation is absent from all databases of genetic variation and from the general population (including the gnomAD database1) while it is expected to result in a truncated protein. Desmoplakin (DSP) truncations are a known disease mechanism in ARVC.9During follow-up, the patient has developed negative T waves in the lateral leads and inferior leads with low QRS voltages. He also had an episode of non-sustained VT (NSVT) at 210 bpm and an episode of sustained VT terminated with ICD shock. Left ventricular systolic function has remained unchanged. Multiple VEs (>2000) have been recorded during 24-hour Holter recording.

Case I28is a male child, resuscitated from VF at age 2 whilst playing in the nursery during a moment of great positive excitation. Unfortunately, he had important neurological sequelae after the event and therefore an exercise stress test couldnot and cannot be performed. Echocardiogram and cardiac MRI were normal. ICD was implanted and beta-blocker therapy started. Parents were fully investigated and had a completely normal cardiac phenotype. During follow-up, he was free of symptoms until the age of nine, when during fever he had 5 appropriate shocks on polymorphic VTs at 290 bpm. Subsequently, he had further episodes of VTs until flecainide was associated to beta-blocker. Next-generation sequencing revealed that the patient carried the previously described CPVT-associated p.Met4002Ilemutation inRYR2 (Table 3A).10The mutation has arisen de novo in the patient explaining the parents’ normal cardiac phenotype.

Case I34 is a 12 year-old boy who had a syncopal event at school and soon after he had a cardiac arrest. At the arrival of the emergency team VF was documented and the boy was immediately defibrillated. After recovery of sinus rhythm, single ventricular ectopies and mild QT prolongation were detected. An ICD was implanted and beta-blocker therapy was started. He was brought to our attention for borderline QT values. He performed an exercise stress test on full dose of beta-blocker therapy and only isolated PVCs occurred at the peak of exercise. Molecular screening of LQTS genes revealed a variant in KCNE2 (p.Ile57Thr), previously associated with drug-induced QT prolongation. Besides not meeting many of the pathogenicity classification criteria, such as MAF, the KCNE2 variant was also present in both unaffected brothers and in the mother. Furthermore the boy had a normal potassium value and was not taking QT-prolonging drugs at the time of the cardiac arrest; therefore we excluded the KCNE2 variant as the cause of the cardiac arrest and performed a more complete molecular screening. Thisled to the identification of a novel variant in RYR2 (p.Met399Leu) affecting a fully conserved residue (Table 3A) that was not present in any family member (i.e. de novo), raising suspicions of CPVT.The patient was admitted to Hospital to allow a wash-out from BB therapy and the possibility to perform an exercise stress test without therapy. The test confirmed the CPVT diagnosis, showing progressive occurrence of ventricular ectopies and a NSVT at the peak of exercise. During the follow-up he never had appropriate shocks; however, NSVTs were recorded at ICD-interrogation during a period of incomplete compliance to beta-blocker therapy. The patient, apart from thede novoRYR2 likely pathogenic variant, also carried twoinherited VUSs (Table 3B), one in DSP (p.Val2518Ile) and one in SCN5A (p.Thr1069Arg).

Case I40 is a 62 year-old female with a history of supraventricular paroxysmal tachycardia who was resuscitated from VF during dinner.A CPVTdiagnosis was not made at the time as she was on post anoxic coma and therefore an exercise stress test could not be performed.- She had a positive family history for SCD, as the son had died suddenly at 19 years whilst swimming (no autopsy available). During the clinical evaluation, she underwent an ajmaline test that resulted negative for Brugada Syndrome and had a normal echocardiogram. Genetic analysis revealed the presence of the previously described CPVT-associatedp.Glu1724Lys mutation in RYR2(Table 3A).11 A VUS in MYH7 (p.Met113Leu) was detected as well (Table 3B).

1.3Descriptions of the patients with VUSs and likely benign variants in the Finnish cohort of 36 IVF patients.

Case F16is a maleexperiencing VF at the age of 15. The patient’s AV conduction was normal. His QTc was slightly prolonged (474ms) and the maximum heart rate during exercise stress test was 88 % of the age specific expected maximum heart rate. Polymorphic VTs were inducible and degenerated into VF. The patient carried a variant in HCN4 (c.2138G>A, p.Arg713His, NM_005477.2) that is present once in the ExAC database (Table 3B). 1 The variant was also present in the patient’s asymptomatic mother, and the family history was negative for SCD or sick sinus syndrome. We classified the variant as VUS.

Case F24is a male who had lost consciousness twice at the age of 33 and 34, and was subsequently resuscitated from VF at age 34. Cardiac ultrasonography and MRI showed normal cardiac structure, dimensions and function. He carried a novel protein truncating TTN mutation (c. 74959C>T, p.Arg24987*; Table 3B)along with another rare TTN variant (rs200766837, c.88880A>C, p.Lys29627Thr, NM_001256850.2). Based on the family data, the p.Lys29627Thr variant segregates in cis with the p.Arg24987* mutation. The p.Arg24987* mutation resides in the C-terminal end of TTN, where similar variants robustly associate with dilated cardiomyopathy (DCM).12The mutation is present in four other members of the family with no VF or cardiomyopathy history. Two cousins of the patient, however, have been diagnosed with cardiomyopathy, but unfortunately they were not available for genetic testing. The patient has received an ICD shock therapy due to VF, but no findings suggestive of cardiomyopathy have been detected during the follow-up of 7 years. We therefore stringently classified the p.Arg24987*variant as VUS.

Case F14is a female patient identified as a heterozygous carrier of a novel variant in the muscular dystrophy gene DMD (c.3791T>C, p.Leu1264Ser, NM_000109). Since mutations in DMD are linked with recessive disease, and because DMD-mutations rarely cause arrhythmias but generally manifest as muscular dystrophy, we considered the mutation likely benign and did not invite relatives into the study.

Case F17 was identified as a heterozygous carrier of a SCGDvariant (c.610G>T, p.Ala204Ser NM_000337). Ambulatory ECG recording showed frequent PVCs (>11000/24 h) and the histopathologic examination of endomyocardium some fibrofatty infiltration. However, the MRI did not support the diagnosis of ARVC. The variant was also present in the father with no history of cardiac disease. We classified the variant as likely benign.

Case F19 is a 44 year-old male. In the screen for recessive mutations, we observed two rare missense variants in MYH7B (c.324G>T, p.Lys108Asn and c.691C>T, p.Arg231Trp, NM_020884). MRI revealed mild septal hypertrophy in the patient (13 mm), the patient’s sister (12 mm) and the patient’s older son (17 mm). All of them and the younger son carry both variants, which seem to segregate together in cis with a mild cardiac phenotype. Any effects associated with these variants would be inherited in a dominant fashion, conflicting previously published information related to MYH7B.13Additionally, neither of the variants fulfills the frequency criteria set for the dominant mutations in this study. We classify these two variants as likely benign.

Case F21 carries a variant in FKRP (c.1253G>A, p.Trp418*, NM_001039885), a gene associated with limb-girdle muscular dystrophy type 2I (LGMD2I). The patient is a female that had suffered an isolated VF incident at the age of 43. The observed variant is present in the Finnish reference population at a low frequency (MAF=0.0004).Interestingly, thepatient’s aunt has been diagnosed with facioscapulohumeral muscular dystrophy (FSHD), with symptoms resembling LGMD2I. The pedigree analysis and the expected recessive inheritance pattern for LGMD2I suggest the variant is not a dominant pathogenic mutation causing VF nor muscular dystrophy. Thus, we considered the variant likely benign for IVF. The patient also carries additional rare variant in MYH7B (c.3286G>C, p.Asp1096His, NM_020884). We classified the variant as likely benign based on the segregation data and lack of studies linking MYH7B to dominantly inherited arrhythmias.

2. Supplemental tables

Table A.1. Clinical tests performed for the IVF patients – The Finnish cohort
DNA# / Age at VF / Sex / 24h ECG / Exercise stress test / EP study / CMR / Cardiac biopsy / Coronary angiogram / Flecainide test
F1 / 25 / M / X / X / X / X / X / X
F2 / 37 / M / X / X / X / X
F3 / 38 / F / X / X
F4 / 39 / F / X / X / X / X / X / X
F5 / 20 / M / X / X / X / X / X / X
F6 / 36 / M / X / X / X / X
F7 / 18 / F / X / X / X / X / X / X / X
F8 / 30 / M / X / X / X / X / X
F9 / 12 / F / X / X / X / X
F10 / 12 / M / X / X / X / X / X
F11 / 44 / F / X / X / X
F12 / 23 / F / X
F13 / 22 / M / X / X / X / X / X / X
F14 / 44 / F / X / X / X / X / X
F15 / 30 / M / X / X / X
F16 / 15 / M / X / X / X / X / X
F17 / 39 / M / X / X / X / X / X / X / X
F18 / 42 / F / X / X / X / X / X
F19 / 43 / M / X / X / X / X / X / X / X
F20 / 23 / F / X / X / X
F21 / 43 / F / X / X / X / X / X
F22 / 18 / M / X / X / X / X / X
F23 / 22 / M / X / X / X / X / X / X
F24 / 34 / M / X / X / X / X / X / X
F25 / 35 / F / X / X / X / X / X
F26 / 17 / F / X
F27 / 18 / F / X* / X / X
F28 / 48 / M / X / X / X / X / X
F29 / 43 / F / X / X / X / X / X / X / X
F30 / 13 / F / X / X / X / X / X
F31 / 19 / F / X / X / X
F32 / 47 / F / X / X / X
F33 / 38 / F / X / X / X / X
F34 / 31 / M / X / X / X / X / X / X
F35 / 44 / F / X / X / X / X
F36 / 39 / F / X / X / X / X / X / X
Table A.2. Clinical tests performed for the IVF patients – The Italian cohort
DNA# / Age at VF / Sex / 24h ECG / Exercise stress test / EP Study / CMR / Cardiac biopsy / Coronary angiogram / Flecainide test
I1 / 26 / M / X / X / X / X / X
I2 / 29 / F / X / X / X / X / X / X
I3 / 33 / M / X / X / X / X
I4 / 63 / F / X
I5 / 0 / M / X
I6 / 41 / M / X / X / X / X / X
I7 / 51 / M / X / X / X / X
I8 / 50 / F / X / X
I9 / 37 / F / X / X / X
I10 / 27 / F / X / X / X
I11 / 16 / F / X / X / X / X / X
I12 / 21 / M / X / X / X / X
I13 / 60 / M / X / X
I14 / 43 / M / X / X / X / X / X / X
I15 / 32 / M / X / X
I16 / 37 / F / X / X / X / X / X / X
I17 / 47 / F / X / X / X
I18 / 43 / M / X / X / X / X
I19 / 20 / F / X / X / X / X / X
I20 / 30 / F / X / X / X
I21 / 22 / F / X / X / X / X
I22 / 17 / F / X / X / X / X / X
I23 / 14 / M / X / X
I24 / 29 / F / X / X / X / X
I25 / 25 / M / X / X / X
I26 / 16 / M / X / X / X / X / X
I27 / 18 / F / X / X / X / X / X / X
I28 / 2 / M / X / X
I29 / 32 / F / X / X / X / X
I30 / 56 / F / X / X / X / X
I31 / 31 / F / X / X / X / X / X
I32 / 40 / F / X / X / X / X
I33 / 45 / F / X / X / X
I34 / 11 / M / X / X* / X
I35 / 16 / M / X / X / X / X / X / X
I36 / 47 / M / X / X / X
I37 / 22 / M / X / X / X / X / X
I38 / 20 / M / X / X / X / X / X / X
I39 / 40 / M / X
I40 / 62 / F / X / X

X=Test performed, results not diagnostic. Empty fields=test not performed.*Patients F27 underwent an exercise stress test after the molecular genetic study. I34 underwent an exercise stress test on full dose of beta-blockers before molecular screening that was not diagnostic for CPVT. After molecular diagnosis, the boy was admitted to Hospital, a wash-out from beta-blocker therapy was performed and the exercise stress test was repeated off therapy.

1

Table A.3. List of the 100 genes under WES primary focus. The 21 genes studied in both cohorts indicated in bold
Gene / Description / Disease / Chromosome / RefSeq
ABCC9 / ATP-binding cassette, sub-family C (CFTR/MRP), member 9 / DCM / 12q12.1 / NM_005691
ACTC1 / Actin, alpha, cardiac muscle 1 / DCM, HCM / 15q14 / NM_005159
ACTN2 / Actinin, alpha 2 / DCM, HCM / 1q45-q43 / NM_001103
AKAP9 / A kinase (PRKA) anchor protein 9 / LQT11 / 7q21-q22 / NM_005751
ANK2 / Ankyrin 2, neuronal / LQT4 / 4q25-q26 / NM_001148
ANKRD1 / Ankyrin repeat domain 1 (cardiac muscle) / DCM, HCM / 10q23.33 / NM_014391
CACNA1C / Calcium channel, voltage-dependent, L type, alpha 1C subunit / BrS3, ERS / 12p13.3 / NM_000719
CACNA2D1 / Calcium channel, voltage-dependent, alpha 2/delta subunit 1 / BrS10, ERS / 7q21-22 / XM_005250570
CACNB2 / Calcium channel, voltage-dependent, beta 2 subunit / BrS4, ARS / 10p12 / NM_000724
CALM1 / Calmodulin 1 (phosphorylase kinase, delta) / CPVT5, LQT14 / 14q32.11 / NM_006888
CALM2 / Calmodulin 2 (phosphorylase kinase, delta) / CPVT5, LQT14 / 2p21.3-p21.1 / NM_001743
CALR3 / Calreticulin 3 / HCM / 19p13.11 / NM_145046
CASQ2 / Calsequestrin 2 (cardiac muscle) / CPVT2 / 1p13.3 / NM_001232
CAV3 / Caveolin 3 / HCM, LQT9 / 3p25 / NM_033337
CRYAB / Crystallin, alpha B / DCM, HCM / 11q22.3-q23.1 / NM_001885
CSRP3 / Cysteine and glycine-rich protein 3 (cardiac LIM protein) / DCM, HCM / 11p15.1 / NM_003476
DES / Desmin / DCM / 2q35 / NM_001927
DMD / Dystrophin / DCM / Xp21.2 / NM_004006
DOLK / Dolichol kinase / DCM / 9q34.13 / NM_014908
DPP6 / Dipeptyl-peptidase 6 / Idiopathic VF / 7q36.2 / NM_130797
DSC2 / Desmocollin 2 / ARVC / 18q12.1 / NM_024422
DSG2 / Desmoglein 2 / ARVC / 18q12.1 / NM_001943
DSP / Desmoplakin / ARVC, DCM / 6p24.3 / NM_004415
FHL1 / Four and a half LIM domains 1 / HCM / Xq26.3 / NM_001449
FHL2 / Four and a half LIM domains 2 / DCM / 2q12.2 / NM_201557
FKRP / Fukutin related protein / DCM / 19q13.32 / NM_024301
FKTN / Fukutin / DCM / 9q31.2 / NM_006731
FLNC / Filamin C, gamma / Myopathy, HCM / 7q32-q35 / NM_001458
FXN (FRDA) / Frataxin / HCM / 9q21.11 / NM_000144
GATAD1 / GATA zinc finger domain containing 1 / DCM / 7q21-q22 / NM_021167
GLA / Galactosidase, alpha / HCM / Xq21.3-q22 / NM_000169
GPD1L / Glycerol-3-phosphate dehydrogenase 1-like / BrS2 / 3p22.3 / NM_015141
HCN4 / Hyperpolarization activated cyclic nucleotide gated potassium channel 4 / BrS8, SSS / 15q24.1 / NM_005477
HFE / Hemochromatosis / DCM / 6p21.3 / NM_001300749
JPH2 / Junctophilin 2 / HCM / 20q13.12 / NM_175913
JUP / Junction plakoglobin / ARVC / 17q21 / NM_021991
KCND2 / Potassium channel, voltage gated ShaI related subfamily D, member 2 / - / 7q31 / NM_012281
KCND3 / Potassium channel, voltage-gated ShaI related subfamily D member 3 / BrS11 / 1p13.2 / NM_172198
KCNE1 / Potassium channel, voltage-gated subfamily E regulatory beta subunit 1 / LQT5 / 21q22.1-q22.2 / NM_001127670
KCNE2 / Potassium channel, voltage-gated subfamily E regulatory beta subunit 2 / LQT6, FAF / 21q22.1 / NM_172201
KCNE3 / Potassium channel, voltage-gated subfamily E regulatory beta subunit 3 / BrS6 / 11q13.4 / NM_005472
KCNE5 / Potassium channel, voltage-gated subfamily E regulatory beta subunit 5 / BrS15 / Xq22.3 / NM_012282
KCNH2 / Potassium channel, voltage-gated eag related subfamily H, member 2 / LQT2, SQT1 / 7q36.1 / NM_000238
KCNJ2 / Potassium channel, inwardly rectifying subfamily J, member 2 / LQT7 (CPVT-like), FAF, SQT3 / 17q24.3 / NM_000891
KCNJ5 / Potassium channel, inwardly rectifying subfamily J, member 5 / LQT13 / 11q24 / NM_000890
KCNJ8 / Potassium channel, inwardly rectifying subfamily J, member 8 / BrS9, ERS / 12p12.1 / NM_004982
KCNK17 / Potassium channel, two pore domain subfamily K, member 17 / 6p21 / NM_031460
KCNQ1 / Potassium channel, voltage-gated KQT-like subfamily Q, member 1 / LQT1, FAF, SQT2 / 11p15.5 / NM_000218
LAMA4 / Laminin, alpha 4 / DCM / 6q21 / NM_001105206
LAMP2 / Lysosomal-associated membrane protein 2 / HCM / Xq24 / NM_002294
LDB3 / LIM domain binding 3 / DCM, HCM / 10q22.3-q23.2 / NM_001171610
LMNA / Lamin A/C / ARVC, DCM / 1q22 / NM_170707
MTTL1 / Mitochondrially encoded tRNA leucine 1 (UUA/G) / HCM / mitochondria
MYBPC3 / Myosin binding protein C, cardiac / DCM, HCM / 11p11.2 / NM_000256
MYH6 / Myosin, heavy chain 6, cardiac muscle, alpha / DCM, HCM / 14q11.2-q13 / NM_002471
MYH7 / Myosin, heavy chain 7, cardiac muscle, beta / DCM, HCM / 14q11.2-q13 / NM_000257
MYL2 / Myosin, light chain 2, regulatory, cardiac, slow / HCM / 12q24.11 / NM_000432
MYL3 / Myosin, light chain 3, alkali; ventricular, skeletal, slow / HCM / 3p / NM_000258
MYLK2 / Myosin light chain kinase 2 / HCM / 20q13.31 / NM_033118
MYOZ2 / Myozenin 2 / HCM / 4q26-q27 / NM_016599
MYPN / Myopalladin / DCM, HCM / 10q22.1 / NM_032578
NEXN / Nexilin (F actin binding protein) / DCM, HCM / 1p31.1 / NM_144573
NKX2-5 / NK2 homeobox 5 / 5q34 / NM_004387
NOS1AP / Nitric oxide synthase 1 (neuronal) adaptor protein / 1q23.3 / NM_014697
PDLIM3 / PDZ and LIM domain 3 / DCM / 4q35 / NM_014476
PKP2 / Plakophilin 2 / ARVC / 12p11 / NM_004572
PLN / Phospholamban / ARVC, DCM, HCM / 6q22.1 / NM_002667
PRDM16 / PR domain containing 16 / DCM, LVNC / 1p36.23-p33 / NM_022114
PRKAG2 / Proteine kinase, AMP-activated, gamma 2 non-catalytic subunit / HCM / 7q35-q36 / NM_016203
PSEN1 / Presenilin 1 / DCM / 14q24.3 / NM_000021
PSEN2 / Presenilin 2 / DCM / 1q42.13 / NM_000447
PTPN11 / Proteine tyrosine phosphatase, non-receptor type 11 / HCM / 12q24.1 / NM_080601
RAF1 / Raf-1 proto-oncogene, serine/threonine kinase / HCM / 3p25 / NM_002880
RANGRF (MOG1) / RAN guanine nucleotide release factor / BrS12 / 17p13 / NM_016492
RBM20 / RNA binding motif protein 20 / DCM / 10q25.3 / NM_001134363
RYR2 / Ryanodine receptor 2 (cardiac) / ARVC, CPVT1 / 1q43 / NM_001035
SCN1B / Sodium channel, voltage gated, type I beta subunit / BrS5 / 19q13.12 / NM_001037
SCN3B / Sodium channel, voltage gated, type III beta subunit / BrS7 / 11q24.1 / NM_018400
SCN4B / Sodium channel, voltage gated, type IV beta subunit / LQT10 / 11q23.3 / NM_174934
SCN5A / Sodium channel, voltage gated, type V alpha subunit / BrS1, ERS, DCM, PCCD, SSS, LQT3, MEPPC / 3p21 / NM_198056
SCN10A / Sodium channel, voltage gated, type X alpha subunit / 3p22.2 / NM_006514
SDHA / Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) / DCM / 5p15 / NM_004168
SEMA3A / Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A / 7p12.1 / NM_006080
SGCD / Sarcoglycan, delta (35kDa dystrophin-associated glycoprotein) / DCM / 5q33-q34 / NM_000337
SLMAP / Sarcolemma associated protein / BrS13 / 3p21.2-p14.3 / NM_007159
SNTA1 / Syntrophin, alpha 1 / LQT12 / 20q11.2 / NM_003098
TAZ / Tafazzin / DCM / Xq28 / NM_006724836
TBX20 / T-box 20 / DCM / 7p14.3 / NM_020417
TCAP / Titin-cap / DCM, HCM / 17q12 / NM_003673
TGFB3 / Transforming growth factor, beta 3 / ARVC / 14q24 / NM_003239
TMEM43 / Transmembrane protein 43 / ARVC / 3p25.1 / NM_024334
TMPO / Thymopoietin / DCM / 12q22 / NM_003276
TNNC1 / Troponin C type 1 (slow) / DCM, HCM / 3p21.1 / NM_003280
TNNI3 / Troponin I type 3 (cardiac) / DCM, HCM / 19q13.4 / NM_000363
TNNT2 / Troponin T type 2 (cardiac) / DCM, HCM / 1q32 / NM_000364
TPM1 / Tropomyosin 1 (alpha) / DCM, HCM / 15q22.1 / NM_001018004
TRDN / Triadin / CPVT4 / 6q22.31 / NM_001251987
TRPM4 / Transient receptor potential cation channel, subfamily M, member 4 / BrS14, PCCD / 19q13.3 / NM_017636
TTN / Titin / DCM, HCM / 2q31 / NM_001256850
VCL / Vinculin / DCM, HCM / 10q22.1-q23 / NM_003373, NM_014000
Table A.4. List of 21 genes analyzed in both cohorts and their coverage metrics
Gene / FI Mean coverage (X) / FI % coverage (10x) / FI % coverage (20x) / IT Mean coverage (X) / IT % coverage (20x)*
CASQ2 / 62.3 / 92.92 / 87.57 / 500 / 100
DSC2 / 72.5 / 99.97 / 97.84 / 500 / 100
DSG2 / 60.7 / 95.23 / 93.04 / 500 / 100
DSP / 58.1 / 95.94 / 91.79 / 500 / 100
JUP / 27.1 / 91.5 / 57.21 / 500 / 100
KCNE1 / 19.7 / 71.81 / 33.17 / 500 / 100
KCNE2 / 36.9 / 68.24 / 54.53 / 500 / 100
KCNH2 / 19.2 / 52.34 / 28.89 / 500 / 100
KCNJ2 / 42.4 / 60.97 / 55.09 / 500 / 100
KCNQ1 / 26.1 / 90.43 / 65.28 / 500 / 100
LDB3 / 25.7 / 78.85 / 48.6 / 500 / 100
LMNA / 21.0 / 69.82 / 33.26 / 500 / 100
MYBPC3 / 23.0 / 88.26 / 49.09 / 500 / 100
MYH7 / 34.9 / 96.87 / 75.69 / 500 / 100
PKP2 / 57.9 / 91.04 / 87.57 / 500 / 100
RYR2 / 73.0 / 97.9 / 95.78 / 500 / 100
SCN5A / 40.7 / 91.37 / 75.22 / 500 / 100
TMEM43 / 51.4 / 100 / 97.31 / 500 / 100
TNNI3 / 30.4 / 87.78 / 58.2 / 500 / 100
TNNT2 / 39.4 / 88.21 / 62.7 / 500 / 100
TPM1 / 53.9 / 93.08 / 87.8 / 500 / 100
FI=Finnish data from WES before extended Sanger sequencing for KCNQ1, KCNH2, SCN5A and RYR2. IT= Italian data from targeted amplicon-based NGS. Mean coverage = Mean coverage of target bases. % coverage = % of target bases covered with at least 10x or 20x. *For the Italian cohort, all regions with a coverage 20x (about 10%) were subjected to Sanger sequencing.
Table A.5. Exons in selected LQTS and CPVT genes subjected to extended Sanger sequencing in the Finnish cohort.
Gene / Exon / Position (GRCh37/hg19)
KCNQ1 / Exon 1 / 11:2466221-2466714
KCNQ1 / Exon 5 / 11:2593243-2593339
KCNQ1 / Exon 16 / 11:2868997-2870340
KCNH2 / Exon 1 / 7:150674926-150675402
KCNH2 / Exon 2 / 7:150671799-150672029
KCNH2 / Exon 4 / 7:150655147-150655590
KCNH2 / Exon 1 / 7:150652484-150652917
KCNH2 / Exon 9 / 7:150647256-150647508
KCNH2 / Exon 12 / 7:150644694-150644966
KCNH2 / Exon 13 / 7:150644416-150644602
SCN5A / Exon 12 / 3:38645203-38645574
SCN5A / Exon 13 / 3:38640409-38640541
SCN5A / Exon 18 / 3:38620825-38620986
RYR2 / Exon 1 / 1:237205822-237205869
CACNA1C / Exon 2 / 12:2224390-12:2224711
CACNA1C / Exon 42 / 12:2788610-12:2788962
CACNA1C / Exon 43 / 12:2789529-12:2789741
CACNA1C / Exon 47 / 12:2797613-12:2797944
CACNA1C / Exon 48 / 12:2800066-12:2800365

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