Cardiac Genetic Predisposition in Sudden Infant Death Syndrome

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JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY VOL. 71, NO.

11, 2018

ª 2018 THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION.

PUBLISHED BY ELSEVIER. ALL RIGHTS RESERVED.

Cardiac Genetic Predisposition in


Sudden Infant Death Syndrome
David J. Tester, BS,a Leonie C.H. Wong, MBBCHIR,b,c Pritha Chanana, MS,a Amie Jaye, MSC,d Jared M. Evans, MS,a
David R. FitzPatrick, MD,e Margaret J. Evans, MBCHB,f Peter Fleming, PHD,g Iona Jeffrey, MBCHB,h,i
Marta C. Cohen, MD,j,k Jacob Tfelt-Hansen, MD, DMSC,l,m Michael A. Simpson, PHD,d Elijah R. Behr, MD,b,c
Michael J. Ackerman, MD, PHDa

ABSTRACT

BACKGROUND Sudden infant death syndrome (SIDS) is a leading cause of postneonatal mortality. Genetic heart dis-
eases (GHDs) underlie some cases of SIDS.

OBJECTIVES This study aimed to determine the spectrum and prevalence of GHD-associated mutations as a potential
monogenic basis for SIDS.

METHODS A cohort of 419 unrelated SIDS cases (257 male; average age 2.7  1.9 months) underwent whole exome
sequencing and a targeted analysis of 90 GHD-susceptibility genes. The yield of “potentially informative,” ultra-rare
variants (minor allele frequency <0.00005) in GHD-associated genes was assessed.

RESULTS Overall, 53 of 419 (12.6%) SIDS cases had $1 “potentially informative,” GHD-associated variant. The yield was
14.9% (21 of 141) for mixed-European ancestry cases and 11.5% (32 of 278) for European ancestry SIDS cases.
Infants older than 4 months were more likely to host a “potentially informative” GHD-associated variant. There was
significant overrepresentation of ultra-rare nonsynonymous variants in European SIDS cases (18 of 278 [6.5%]) versus
European control subjects (30 of 973 [3.1%]; p ¼ 0.013) when combining all 4 major cardiac channelopathy genes
(KCNQ1, KCNH2, SCN5A, and RYR2). According to the American College of Medical Genetics guidelines, only 18 of
419 (4.3%) SIDS cases hosted a “pathogenic” or “likely pathogenic” variant.

CONCLUSIONS Less than 15% of more than 400 SIDS cases had a “potentially informative” variant in a
GHD-susceptibility gene, predominantly in the 4- to 12-month age group. Only 4.3% of cases possessed immediately
clinically actionable variants. Consistent with previous studies, ultra-rare, nonsynonymous variants within the major
cardiac channelopathy-associated genes were overrepresented in SIDS cases in infants of European ethnicity. These
findings have major implications for the investigation of SIDS cases and families. (J Am Coll Cardiol 2018;71:1217–27)
© 2018 the American College of Cardiology Foundation. Published by Elsevier. All rights reserved.

From the aDepartments of Cardiovascular Medicine (Division of Heart Rhythm Services), Pediatrics (Division of Pediatric Cardi-
ology), and Molecular Pharmacology & Experimental Therapeutics (Windland Smith Rice Sudden Death Genomics Laboratory),
Mayo Clinic, Rochester, Minnesota; bMolecular and Clinical Sciences Research Institute, St. George’s, University of London,
London, United Kingdom; cCardiology Clinical Academic Group, St. George’s University Hospitals’ NHS Foundation Trust, Lon-
don, United Kingdom; dMedical and Molecular Genetics, Guy’s Hospital, King’s College London, London, United Kingdom; eMRC
Human Genetics Unit, University of Edinburgh, Edinburgh, United Kingdom; fRoyal Infirmary of Edinburgh, Edinburgh, United
Kingdom; gCentre for Child and Adolescent Health, Bristol Medical School, University of Bristol, Bristol, United Kingdom;
h
Department of Cellular Pathology, St George’s, University of London, London, United Kingdom; iDepartment of Cellular
Pathology’, St. George’s University Hospitals’ NHS Foundation Trust, London, United Kingdom; jHistopathology Department,
Sheffield Children’s Hospital, Sheffield, United Kingdom; kHonorary Senior Lecturer, University of Sheffield, Sheffield, United
Kingdom; lDepartment of Cardiology, The Heart Centre, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark;
and the mDepartment of Forensic Medicine, Faculty of Medical Sciences, University of Copenhagen, Copenhagen, Denmark. This
work was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National In-
Listen to this manuscript’s
stitutes of Health (grant no. R01HD042569 to Dr. Ackerman) and by the British Heart Foundation (BHF Clinical Research Training
audio summary by
Fellowship FS/13/78/30520 to Drs. Wong and Behr). The content is solely the responsibility of the authors and does not necessarily
JACC Editor-in-Chief
represent the official views of the National Institutes of Health. Mr. Tester is supported by the Mayo Clinic Windland Smith Rice
Dr. Valentin Fuster.
Comprehensive Sudden Cardiac Death Program. Dr. Wong is supported by additional funds from Biotronik and Cardiac Risk in the
Young. Dr. Behr is supported by the Higher Education Funding Council for England; is a consultant for Medtronic; has received
research funding from Biotronik; and has received funds from The Robert Lancaster Memorial Fund sponsored by McColl’s RG Ltd.
Dr. Ackerman is a consultant for Audentes Therapeutics, Boston Scientific, Gilead Sciences, Invitae, Medtronic, MyoKardia, and
St. Jude Medical; is supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program; and the Mayo
Clinic have an equity- or royalty-based relationship with AliveCor, Blue Ox Health, and StemoniX, although none of these entities

ISSN 0735-1097/$36.00 https://doi.org/10.1016/j.jacc.2018.01.030


1218 Tester et al. JACC VOL. 71, NO. 11, 2018

Cardiac Genetic Analyses in SIDS MARCH 20, 2018:1217–27

S METHODS
ABBREVIATIONS udden infant death syndrome (SIDS) is
AND ACRONYMS the sudden unexpected death of an in-
fant <1 year of age that remains unex- STUDY GROUP. The SIDS cohort (N ¼ 427) consisted
GHD = genetic heart disease
plained despite comprehensive clinical and of 95 coroners’ cases from the United Kingdom
gnomAD = Genome
pathological investigations (1). SIDS repre- (London, Sheffield, Edinburgh, and Bristol) and 332
Aggregation Database
sents 70% to 80% of all sudden unexpected coroner-, medical examiner–, or forensic pathologist–
LQTS = long QT syndrome
infant deaths with an incidence of 0.4 in referred cases collected from 6 ethnically and
MAF = minor allele frequency
1,000 live births in the United Kingdom and geographically diverse U.S. population groups.
NSV = nonsynonymous variant
0.5 in 1,000 live births in the United States Because of the lack of uniformity in procedures and
PCA = principal component
(2,3). The peak incidence occurs between 2 reporting among medical examiner offices in the
analysis
and 4 months of age and is more common United States, minor differences in protocol may
SIDS = sudden infant death
syndrome
in boys. Such infant deaths are commonly exist. Nonetheless, both gross and histological ex-
associated with environmental risk factors aminations of all major organs were performed, and
WES = whole exome
sequencing such as co-sleeping or prone sleeping posi- all cases satisfied our enrollment criteria, which
tion (4). Despite successful targeted risk included the following: 1) sudden unexplained death
reduction campaigns, the number of SIDS cases has of an infant <1 year of age; 2) European descent; and
plateaued, and SIDS remains the leading cause of 3) a comprehensive negative medicolegal autopsy
postneonatal death (4). including a negative toxicology screen result and
death scene investigation. Infants with asphyxia or a
SEE PAGE 1228
specific disease causing death were excluded.

A triple-risk model for SIDS suggests the conver- Ethnicity was self-reported by the referring coroner

gence of the vulnerable infant in the setting of or medical examiner. This anonymous autopsy study

exogenous stressors during a critical development had only limited medical information available such

period (5) (Central Illustration). Although many path- as the sex, ethnicity, age at the time of death, and

ophysiological theories have been proposed, decisive sleep position. This study complies with the Decla-

pathogenic substrates or mechanisms triggering an ration of Helsinki; locally appointed ethics commit-

infant’s sudden demise remain unclear (6–9). Several tees including Mayo Clinic’s Institutional Review

studies have implicated both common and rare ge- Board approved the research protocol. Some of the

netic variants involved in autonomic function, 332 samples from the United States were included in

neurotransmission, energy metabolism, response to previous publications that involved hypothesis-

infection, and cardiac repolarization (10–14). In driven, specific candidate gene mutational analysis

addition, potentially lethal genetic heart diseases (10,18,19,23–28). Of the 332 U.S. cases, 58 had been

(GHDs) including long QT syndrome (LQTS), Brugada analyzed previously for variants in SCN5A (10),

syndrome, catecholaminergic polymorphic ventricu- KCNQ1 (18), KCNH2 (18), RYR2 (19), SNTA1 (23),

lar tachycardia, and hypertrophic cardiomyopathy KCNJ8 (24), Cx43 (25), GPD1L (26), CAV3 (27), SCN1B

have been implicated as monogenic causes for a small (28), SCN2B (28), SCN3B (28), and SCN4B (28). An

proportion of SIDS cases (10,13,15–27). additional 25 of the 332 cases were also analyzed for

However, fewer than 100 investigations of genetic RYR2 (19), and an additional 145 of the 332 cases were

variations in population-based SIDS cohorts have also analyzed for SNTA1 (23), KCNJ8 (24), Cx43 (25),

been published to date, largely on the basis of GPD1L (26), CAV3 (27), SCN1B (28), SCN2B (28), SCN3B

hypothesis-driven, candidate gene– or pathway- (28), and SCN4B (28). None of the 95 cases from the

based approaches that recognize established patho- United Kingdom have been published previously.

biological risk factors for SIDS, with an average cohort CONTROL GROUP. A total of 973 control exomes (509
size of just 125 SIDS cases (13). In the present study, female, 464 male) from the ICR1000 U.K. exome se-
using whole exome sequencing (WES), we conducted ries and the 1958 Birth Cohort study were included for
a GHD-associated gene-specific analysis on a cohort case-control analysis (29). As previously reported,
of more than 400 unrelated SIDS cases. exome sequencing was performed using the Illumina

were involved in this study in any way. Dr. Simpson has a part-time contract of service with Genomics. All other authors have
reported that they have no relationships relevant to the contents of this paper to disclose. Mr. Tester and Dr. Wong contributed
equally to this work and are joint first authors. Drs. Simpson, Behr, and Ackerman contributed equally to this work and are joint
senior authors.

Manuscript received November 8, 2017; revised manuscript received December 15, 2017, accepted January 8, 2018.
JACC VOL. 71, NO. 11, 2018 Tester et al. 1219
MARCH 20, 2018:1217–27 Cardiac Genetic Analyses in SIDS

C ENTR AL I LL U STRA T I O N Whole Exome Sequencing and a Targeted Analysis of 90 GHD-Susceptibility Genes

Tester, D.J. et al. J Am Coll Cardiol. 2018;71(11):1217–27.

The triple-risk hypothesis for SIDS highlighting genetic heart disease (GHD) as a potential explanation for infant vulnerability, and our whole exome sequencing
strategy to detect American College of Medical Genetics and Genomics (ACMG) guideline–predicated “pathogenic” or “likely pathogenic” variants in SIDS cases.
MAF ¼ minor allele frequency; SIDS ¼ sudden infant death syndrome.

TruSeq and Illumina instruments (Illumina, San index DNA adaptors (Agilent) with a single “T” base
Diego, California) (29). overhang at the 3 0 end were ligated, and the resulting
constructs were purified using AMPure SPRI (solid-
WHOLE EXOME SEQUENCING. Genomic DNA iso- phase reversible immobilization) beads (Agencourt
lated from each SIDS case underwent WES at the KCL- Bioscience Corporation, Beverly, Massachusetts). The
GSTT Biomedical Research Centre Genomics Platform adapter-modified DNA fragments were enriched by 4
in London, United Kingdom or Mayo Clinic’s Medical cycles of polymerase chain reaction using SureSelect
Genome Facility in Rochester, Minnesota. forward and SureSelect ILM Pre-Capture Indexing
Paired-end libraries were prepared following the reverse (Agilent) primers. The concentration and size
manufacturer’s protocol (Agilent Technologies, Inc., distribution of the libraries was determined on an
Santa Clara, California) using the Bravo Automated Agilent Bioanalyzer DNA 1000 chip.
Liquid Handling Platform from Agilent. Briefly, 1 to Whole exon capture was performed using the
3 m g of genomic DNA was fragmented to 150 to 200 bp protocol for Agilent’s Sure SelectXT Human All Exon
using the Covaris E210 sonicator (Covaris, Inc., V5þUTR kit. Briefly, 750 ng of the prepared library
Woburn, Massachusetts). The ends were repaired, was incubated with whole exon biotinylated RNA
and an “A” base was added to the 3 0 ends. Paired-end capture baits supplied in the kit for 24 h at 65  C.
1220 Tester et al. JACC VOL. 71, NO. 11, 2018

Cardiac Genetic Analyses in SIDS MARCH 20, 2018:1217–27

The captured DNA:RNA hybrids were recovered using common variants located outside of regions of the
Dynabeads MyOne Streptavidin T1 (Invitrogen Dynal, genome where there is extensive linkage disequilib-
Thermo Fisher Scientific, Waltham, Massachusetts). rium was used to estimate relatedness within the
The DNA was eluted from the beads and purified us- study cohort and ethnic ancestry alongside the con-
ing Ampure XP beads (Agencourt). The purified cap- trol group (32). Estimation was undertaken using the
ture products were then amplified using the first 2 dimensions of a multidimensional scaling using
SureSelect Post-Capture Indexing forward and index Euclidean distance undertaken with the King soft-
polymerase chain reaction reverse primers (Agilent) ware package.
for 12 cycles. ANCESTRY CONFIRMATION. To avoid potential con-
Libraries were pooled at equimolar concentrations founding secondary to population stratification
and loaded onto paired-end flow cells at concentra- resulting from genetic admixture, a principal compo-
tions of 7 to 8 pM to generate cluster densities of nent analysis (PCA) was performed. The PCA served
600,000 to 800,000/mm 2 following Illumina’s stan- only for the rare variant analysis between European
dard protocol using the Illumina cBot and HiSeq SIDS cases and European control subjects. The PCA
paired-end cluster kit version 3. Each lane of a HiSeq data were not used for attributing causality to iden-
flow cell produced 21 to 39 Gb of sequence. The level tified variants where ethnically matched control
of sample pooling was controlled by the size of the subjects would not be necessary for variant adjudi-
capture region and the desired depth of coverage. cation. SIDS cases and control subjects forming a ho-
The flow cells were sequenced as 101  2 paired- mogeneous cluster on the first 2 components were
end reads on an Illumina HiSeq 2000 using TruSeq included in the case-control rare variant analysis.
SBS sequencing kit version 3 and HiSeq data collec-
tion version 2.0.12.0 software. Base-calling was per- GENETIC HEART DISEASE GENE-SPECIFIC VARIANT

formed using Illumina’s RTA version 1.17.21.3. ANALYSIS. Genes known to be associated with car-

The FASTQ files underwent quality control checks diac channelopathy (LQTS, catecholaminergic poly-
using FASTQC (Babraham Bioinformatics, Babraham morphic ventricular tachycardia, Brugada syndrome)
Institute, Cambridge, United Kingdom). The Illumina susceptibility and cardiomyopathy (hypertrophic car-
paired-end reads were aligned to the GRCh37 (hg19) diomyopathy, dilated cardiomyopathy, arrhythmo-
human reference genome using Novoalign (Novocraft genic cardiomyopathy) susceptibility (N ¼ 90) (Online
Technologies, Selandor, Malaysia). Single-sample Table 1) were evaluated for the presence of “ultra-rare”
variant calling with the Genome Analysis Toolkit nonsynonymous variants (NSVs) with a minor allele
(GATK version 3.2-2, Broad Institute, Cambridge, frequency (MAF) <0.00005 (1 in 20,000 alleles)
Massachusetts) (30), and the resulting gVCFs (genomic derived from the Genome Aggregation Database (33). A
variant call format) subsequently underwent multi- comparison of yield was undertaken for ultra-rare
sample genotyping and variant quality score recali- NSVs in SIDS cases of PCA-determined European
bration. Genotypes were excluded if the quality ancestry versus European control subjects across all 90
control was <15 or there were fewer than 4 reads GHD-susceptibility genes and the 4 “major” channel-
supporting the call. Further filtering of variant sites opathy genes (KCNQ1, KCNH2, SCN5A, and RYR2).
was performed to exclude sites with missingness >0.1 All putative loss of function variants (i.e., a “radical”
in cases or control subjects. Variants were annotated variant: frameshift, nonsense, and essential splice-site
with respect to the genes in which they reside with variants) or missense variants with a previously
Annovar, allele frequencies were obtained from established abnormal in vitro function characteriza-
the Exome Aggregation Consortium database, and tion that resided within any of the 90 GHD-associated
Combined Annotation Dependent Depletion (CADD) genes and all ultra-rare, missense variants residing in
scores were derived from the CADD server (31). any of the 4 “major” channelopathy genes were
considered to be “potentially informative” variants
QUALITY CONTROL COVERAGE ANALYSIS AND that would be appropriate for investigation of their
PRINCIPAL COMPONENT ANALYSIS FOR RELATEDNESS significance in a family. Such variants were confirmed
AND ETHNICITY. Coverage across the exome was using standard Sanger sequencing techniques. The
assessed using the bedtools software package (Quin- American College of Medical Genetics and Genomics
lan Laboratory, University of Utah, Salt Lake City, standards and guidelines for the interpretation of
Utah), and cases were excluded from further analysis sequence variants were used to assist in the classifi-
if <75% of the GENCODE (GENCODE Project, an in- cation of our genetic findings further among all ultra-
ternational collaboration)–defined protein coding rare (MAF <0.00005) NSVs identified across the 90
exome was covered by <20 reads. A set of 3,847 GHD-associated genes (34).
JACC VOL. 71, NO. 11, 2018 Tester et al. 1221
MARCH 20, 2018:1217–27 Cardiac Genetic Analyses in SIDS

T A B L E 1 Summary of the Sudden Infant Death Syndrome F I G U R E 1 Yield of Ultra-Rare and “Potentially Informative” GHD-Associated
Cohort Demographics Gene Variants

European Mixed European


Overall Ancestry Ancestry 60
(N ¼ 419) (n ¼ 278) (n ¼ 141)

Male 257 (61.3) 173 (62.2) 84 (59.6) 49.6


50 46.3
Female 162 (38.7) 105 (37.8) 57 (40.4) 44.6
Age, months

Percent (%) Yield


40
Average 2.7  1.9 2.7  1.98 2.8  2.3
Range 0.1–12 0.1–12 0.25–9.7
Age group, months 30
<2 117 (27.9) 81 (29.1) 36 (25.5)
2–4 237 (56.6) 154 (55.4) 83 (58.9) 20
>4 65 (15.5) 43 (15.5) 22 (15.6)
14.9
12.6 11.5
Sleep position
10
Supine 113 (27) 85 (30.6) 28 (19.8)
Prone 73 (17.4) 52 (18.7) 21 (14.9)
0
Side 35 (8.4) 29 (10.4) 6 (4.2) Ultra-Rare Variants Potentially Informative Variants
Seated 5 (1.2) 2 (0.72) 3 (2.1)
Overall (n = 419) European (n = 278) Mixed-European (n = 141)
Unknown 193 (46) 110 (39.6) 83 (58.9)
Co-sleeping
Yes 95 (22.7) 66 (23.7) 29 (20.6) A bar graph depicting the percentage of yield of ultra-rare (minor allele
No 141 (33.6) 106 (38.1) 35 (24.8) frequency <0.00005), nonsynonymous variants and the “potentially informative”
Unknown 183 (43.7) 106 (38.1) 77 (54.6) variants that were identified among the 90 genetic heart disease (GHD)–associated genes
for the overall, European Caucasian, and mixed-European ancestry cohorts.
Values are n (%) or mean  SD.

STATISTICS. Categorical variables were expressed as


absolute numbers and percentages and were GENETIC HEART DISEASE GENE-SPECIFIC ANALYSIS.

compared with the Fisher exact or chi-square tests. Overall, a total of 285 unique, ultra-rare NSVs (256
Probability values were determined on the basis of missense, 23 putative loss of function [12 frameshift,
2-sided tests considered significant at p < 0.05. 8 splice errors, 3 nonsense], and 6 in-frame indels)
Analysis was conducted with SPSS version 18.0 were identified in 194 of 419 (46.3%) SIDS cases
software (SPSS, Chicago, Illinois). overall (Figure 1). Further, 45 of 278 (16.2%) European
ancestry cases and 25 of 141 (17.7%) mixed-European
RESULTS ancestry cases hosted >1, ultra-rare NSVs.
These ultra-rare NSVs resided in 68 of the 90 GHD-
DEMOGRAPHICS. WES was performed in 427 SIDS associated genes (21 of 31 channelopathy-associated
cases. However, quality control metrics excluded 7 genes and 47 of 59 cardiomyopathy-associated
cases because of insufficient exome coverage and 1 genes). The gene-specific yields for the overall
individual from a half-sibling pair (Online Figure 1). cohort and the European and mixed European sub-
The cohort therefore consisted of 419 cases (257 male, sets are shown in Online Table 2.
162 female; average age 2.7  1.9 months) with a Of the 285 unique, ultra-rare NSVs identified, 57
skewed bell-shaped distribution of age (Online (20%) were considered “potentially informative.” A
Figure 2). The epidemiologically higher-risk age total of 25 of 57 (43.9%) were missense variants in the
group of 2 to 4 months (58.9%) and male sex (61%) 4 major channelopathy genes, 23 of 57 (40.3%) were
accounted for the majority of the cases. The PCA putative loss of function variants, and 10 of 57 (17.5%)
demonstrated a wide distribution of ancestral origins were variants previously reported in published pa-
with 278 cases (173 male, 105 female) considered to be pers as having an abnormal in vitro functional
of European ancestry and 141 cases (84 male, 57 phenotype (Table 2). Overall, 53 of 419 (12.6%) SIDS
female) considered to be of mixed-European ancestry cases hosted at least 1 “potentially informative”
(Online Figure 3, Table 1). Sleep characteristics were variant (Figure 1). Four of 419 cases (0.95%) had 2
known in 54% of the cohort (Table 1). There were no “potentially informative” variants. The yield was
significant differences in demographics and sleep 14.9% (21 of 141) for mixed-European ancestry cases
characteristics between the European and mixed- and 11.5% (32 of 278) for European ancestry SIDS cases
European ancestry cases. (Figure 1).
1222 Tester et al. JACC VOL. 71, NO. 11, 2018

Cardiac Genetic Analyses in SIDS MARCH 20, 2018:1217–27

T A B L E 2 Summary of “Potentially Informative,” Ultra-Rare NSVs (MAF <0.00005) Identified in SIDS

Age Sleep Amino Acid Novel or ACMG Variant


Case Ethnicity Sex (months) Position Co-Sleeping Gene Nucleotide Change Ref. # Interpretation

1 European F 4.2 — — CALR3 c.179_180delAA p.K60Rfs*4 Novel Likely pathogenic


2 European F 0.1 — — CASQ2 c.546delT p.F182Lfs*28 Reference (42) Pathogenic
3 European M 4 Side No CRYAB c.325-2A>G Splice error VUS
4 European F 5.8 Supine — CSRP3 c.415-1G>T Splice error Novel Likely pathogenic
5 Mixed-European F 4.1 Prone No CTNNA3 c.843þ1G>T Splice error Novel VUS
6 European M 3 — — DSG2 c.523þ2T>C Splice error Reference (43) Likely pathogenic
7 Mixed-European M 2 Supine Yes DSP c.6540delG p.V2181Sfs*6 Novel Likely pathogenic
8 Mixed-European M 1 — — GPD1L c.817C>T p.R273C Reference (26) VUS
9 European M 3 Supine Yes KCNE2 c.369_370delCT p.P123fs*14 Reference (44) Likely pathogenic
10 Mixed-European F 1 Supine No KCNH2 c.865G>A p.E289K Novel VUS
11 Mixed-European F 0.75 Supine Yes KCNH2 c.2903C>T p.P968L Reference (45) VUS
12 European F 5 Prone No KCNH2 c.3013C>T p.R1005W Novel VUS
13 European M 1.1 Supine No KCNH2 c.3347C>T p.A1116V Reference (46) VUS
14 European M 5 — — KCNJ8 c.995_997delAAG p.E332del Reference (24) Likely pathogenic
15 European F 3 — — KCNQ1 c.230C>T p.S77F Novel VUS
16 European M 6 Side No KCNQ1 c.1553G>A p.R518Q Reference (44) VUS
17 European M 6. 1 Side No LAMA4 c.2174- Splice error Novel Likely pathogenic
8_2182delGTGTAAAGGGGATGCCC
18 Mixed-European M 1.73 — — MIB1 c.2245delA p.K749Rfs*18 Novel Likely pathogenic
19 European M 3.5 Prone No MTO1 c.1966delA p.K656Nfs*19 Novel Likely pathogenic
20 Mixed-European F 0.5 Supine Yes NEBL c.1560þ1G>A Splice error Novel Likely pathogenic
21 Mixed-European M 1 — NO NEBL c.2148þ1G>A Splice error Novel VUS
22 Mixed-European F 3.2 Prone No PDLIM3 c.650-2A>G Splice error Novel Likely pathogenic
23 European M 5.5 Supine No PLN c.36_38delAAG p.R14del Reference (47) Likely pathogenic
24 European M 4.9 Prone — RYR2 c.950T>C p.M317T Novel VUS
25 European F 3.1 Prone No RYR2 c.2626C>A p.P876T Novel VUS
26 Mixed-European M 6 Side No RYR2 c.3245G>A p.G1082E Novel VUS
27 Mixed-European F 5 Prone Yes RYR2 c.6203G>A p.R2068Q Novel VUS
28 European F 7.6 Prone Yes RYR2 c.6252G>A p.M2084I Novel VUS
29 European M 11 Prone No RYR2 c.6490G>A p.A2164T Novel VUS
30 European M 3.1 Supine Yes RYR2 c.7528A>G p.T2510A Reference (48) VUS
31 European M 2 Supine Yes RYR2 c.7946T>C p.F2649S Novel VUS
32 Mixed-European M 1 — — RYR2 c.9626C>T p.P3209L Novel VUS
33 European F 2 Supine No RYR2 c.12692C>T p.P4231L Novel VUS
34 European M 2 Supine Yes RYR2 c.12713T>G p.I4238S Novel VUS
35 European M 3 — — RYR2 c.13624G>A p.A4542T Novel VUS
36 Mixed-European F 1.5 — — SCN3B c.106G>A p.V36M Reference (28) VUS
37 Mixed-European M 6 Supine No SCN3B c.161T>G p.V54G Reference (28) Likely pathogenic
38 European M 3.1 Prone Yes SCN5A c.997G>C p.G333A Novel VUS
39 Mixed-European M 4 — — SCN5A c.2251G>A p.V751I Novel VUS
40 European F 3 — — SCN5A c.4870G>A p.V1624I Novel VUS
41 Mixed-European M 3 — — SCN5A c.5359A>G p.S1787G Novel VUS
42 European M 4.1 — — SDHA c.98_107delTTCACTTCAC p.F33Lfs*22 Novel Likely pathogenic
43 Mixed-European F 0.75 — — SNTA1 c.861C>G p.S287R Reference (23) Pathogenic
44 Mixed-European M 2 — — SNTA1 c.1115C>T p.T372M Reference (23) VUS
45 European F 1.2 — — SNTA1 c.1378G>A p.G460S Reference (23) VUS
46 Mixed-European M 3 — — TMPO c.1244_1245insT p.K416fs*0 Novel VUS
47 European M 0.6 Prone No TTN c.13774G>T p.E4592* Novel VUS
48 European M 0.5 Supine No TTN c.26011C>T p.R8671* Novel VUS
49 Mixed-European M 2 — — TTN c.47653delA p.R15885Efs*9 Novel VUS
50 Mixed European M 7 — — TTN c.37432_ p.N12478Sfs*19 Novel VUS
37433insGTGGTTACTACAGCCTC
TTN c.37437_37438insG p.S12480Vfs*24 Novel VUS
Continued on the next page
JACC VOL. 71, NO. 11, 2018 Tester et al. 1223
MARCH 20, 2018:1217–27 Cardiac Genetic Analyses in SIDS

T A B L E 2 Continued

Age Sleep Amino Acid Novel or ACMG Variant


Case Ethnicity Sex (months) Position Co-Sleeping Gene Nucleotide Change Ref. # Interpretation

51 European M 2.1 — — GJA1 c.124G>A p.E42K Reference (25) Likely pathogenic


KCNH2 c.2654G>A p.R885H Novel VUS
52 European F 2 Supine No CSRP3 c.286_287delCC p.P96Kfs*35 Novel Likely pathogenic
SCN5A c.3079C>T p.R1027W Novel VUS
53 European M 1.5 — — NEBL c.1639C>T p.R547* Novel VUS
SCN5A c.2989G>T p.A997S Reference (10) VUS

ACMG ¼ American College of Medical Genetics and Genomics; MAF ¼ minor allele frequency; NSV ¼ nonsynonymous variant; SIDS ¼ sudden infant death syndrome; VUS ¼ variant of uncertain significance.

There were no significant differences in the yield of European (12 of 278 [4.3%]) and the mixed-European
either ultra-rare NSVs among all 90 GHD genes or cohorts (6 of 141 [4.3%]).
“potentially informative” variants when comparing CASE-CONTROL ANALYSIS. Consistent with previ-
sex, sleep position (supine vs. prone), or co-sleeping ous studies, there was significant overrepresentation
(yes vs. no) in either the overall or stratified study of ultra-rare NSVs in European SIDS cases (18 of 278
groups (Table 3). However, there was a significantly [6.5%]) versus European control subjects (30 of 973
higher yield of “potentially informative” GHD- [3.1%]; p ¼ 0.013) when combining all 4 major cardiac
associated genetic variants in those infants who channelopathy genes (KCNQ1, KCNH2, SCN5A, and
died at >4 months of age (15 of 65 [23.1%]) compared RYR2). (Figure 3). However, there was no significant
with those younger than 4 months of age (37 of 354 difference in yield between cases and control subjects
[10.4%]; p ¼ 0.0075) (Figure 2). for any specific gene.
Following further vetting using the strict American
College of Medical Genetics and Genomics guidelines, DISCUSSION
only 18 of the 285 ultra-rare NSVs achieved a “path-
ogenic” or “likely pathogenic” designation and were This paper reports results derived from a whole
identified in 18 (4.3%) of the 419 SIDS cases (Table 2, exome molecular autopsy with GHD gene-specific
Figure 1). There was no difference in yield of “path- analysis of a large cohort of unrelated SIDS cases.
ogenic” or “likely pathogenic” variants between the Previous postmortem genetic studies implicated

T A B L E 3 The Effect of Various Demographics on the Yield of Genetic Heart Disease–Associated Gene Variants

All Ultra-Rare, Nonsynonymous Variants “Potentially Informative” Variants

Overall European Mixed European Overall European Mixed European


Demographics (n ¼ 419) p Value (n ¼ 278) p Value (n ¼ 141) p Value (n ¼ 419) p Value (n ¼ 278) p Value (n ¼ 141) p Value

Age 0.76 0.63 0.17 0.03 0.35 0.015


2–4 months 108/237 (45.6) 71/154 (46.1) 37/83 (44.6) 22/237 (9.3) 15/154 (9.7) 7/83 (8.4)
Other 86/182 (47.3) 53/124 (42.7) 33/58 (56.9) 31/182 (17.0) 17/124 (13.7) 14/58 (24.1)
Sex 0.84 0.90 0.61 0.76 0.85 1.00
Male 118/257 (45.9) 78/173 (45.1) 40/84 (47.6) 34/257 (13.2) 21/173 (12.1) 13/84 (15.5)
Female 76/162 (46.9) 46/105 (43.8) 30/57 (52.6) 19/162 (11.7) 11/105 (10.5) 8/57 (14.0)
Sleep position 0.25 0.65 0.30 0.98 0.67 0.91
Prone 31/73 (42.5) 24/52 (46.2) 7/21 (33.3) 10/73 (13.7) 8/52 (45.4) 2/21 (9.5)
Supine 48/113 (42.5) 36/85 (42.3) 12/28 (42.9) 15/113 (13.3) 10/85 (11.8) 5/28 (17.9)
Side 14/35 (40.0) 10/29 (34.5) 4/6 (66.7) 4/35 (11.4) 3/29 (10.3) 1/6 (16.7)
Seated 1/5 (20.0) 0/2 (0) 1/3 (33.3) 1/5 (20.0) 0/2 (0) 0/3 (0)
Unknown 100/193 (51.8) 54/110 (49.1) 46/83 (55.4) 24/193 (12.4) 11/110 (10.0) 13/83 (15.7)
Co-sleeping 0.16 0.21 0.8 0.57 0.78 0.73
Yes 38/95 (40.0) 25/66 (37.9) 13/29 (44.8) 9/95 (9.5) 6/66 (9.1) 3/29 (10.3)
No 62/141 (44.0) 45/106 (42.5) 17/35 (48.6) 19/141 (13.5) 13/106 (12.3) 6/35 (17.1)
Unknown 94/183 (51.4) 54/106 (50.9) 40/77 (51.9) 25/183 (13.7) 13/106 (12.3) 12/77 (15.6)

Values are n/N (%). Bold indicates a significant p value (<0.05)


1224 Tester et al. JACC VOL. 71, NO. 11, 2018

Cardiac Genetic Analyses in SIDS MARCH 20, 2018:1217–27

variants were implicated recently in 3.5% of SIDS


F I G U R E 2 The Effect of Age at the Time of Death on the Yield of “Potentially
Informative” GHD-Associated Gene Variants
cases (21). However, on the basis of their prevalence
in the Genome Aggregation Database (gnomAD),
30 only 1.4% of these SIDS cases hosted variants rare
P = 0.0038
enough to be considered pathogenic.
P = 0.0075 25.8
25 11 Because of the rarity of SIDS and any likely
23.1 22.7
causative disorders, we used a strict minor allele
15 5
20 frequency cutoff equivalent to a heterozygous
Percent (%) Yield

frequency of <1 in 10,000 individuals in gnomAD.


15 Despite using this very conservative rarity filter,
13.4
46% of our SIDS cohort harbored novel or ultra-rare,
10.4 16
protein-altering genetic variants within 68 of the
10 8.9
37 90 GHD-susceptibility genes. Unfortunately, despite
21
their rarity, most of these ultra-rare variants still
5
remain variants of uncertain significance stuck in
genetic purgatory (35,36). In fact, about 25% of
0
354 65 235 43 119 22 these 90 GHD-associated genes have a negative
Overall (n = 419) European (n = 278) Mixed European (n = 141) z-score, suggesting that they tolerate variation
≤4 >4 (33,37).
Because of ambiguity surrounding the pathogenic
A bar graph comparing the percentage of yield of “potentially informative” variants nature of many GHD genes that play a “minor” role
between those infants who died at an age younger than or older than 4 months. in their respective diseases, we examined the yield
GHD ¼ genetic heart disease. of ultra-rare NSVs with the highest likelihood of
being true pathogenic mutations. Overall, about 13%
of our SIDS cases hosted “potentially informative”
pathogenic mutations in cardiac channelopathy- variants regarded as having the greatest probability
associated genes as a monogenic cause for up to of being causative for the infant’s sudden death and
15% of SIDS (13,16–19,23). Furthermore, rare hyper- having potential clinical utility for assessing a
trophic cardiomyopathy–associated sarcomere gene family for genetic risk. Unfortunately, the anony-
mous nature of the cohort prevents us from veri-
fying the presence of these variants among family
F I G U R E 3 Yield of Ultra-Rare Major Cardiac Channelopathy Gene Variants in European members for the purpose of potential genotype-
SIDS Cases Versus European Control Subjects phenotype co-segregation analysis or to determine
the frequency of de novo status of the variants of
8 interest. Importantly, not all of these variants have
P = 0.013
7 been characterized functionally, and great caution
6.5
must still be exercised, even when interpreting
6
ultra-rare variants residing within the major chan-
Percent (%) Yield

5 nelopathy genes.
Recently, Hertz et al. (22) reported a 34% yield of
4
3.1 3.2 “variants with likely functional effects” following a
3 genetic analysis of GHD-associated genes in only 47
2 1.7 sudden unexpected deaths in infancy cases. Howev-
1.1 1.4
er, given the rarity of GHDs in the general population,
1 0.7 0.5 0.7
0.2 we believe that their definition of rarity (MAF <1%)
0 was unacceptably and erroneously high, thus causing
4 Major KCNQ1 KCNH2 SCN5A RYR2
Genes an overestimated burden of potentially pathogenic
European SIDS (n = 278) European Controls (n = 973) variants in their SIDS cohort. In fact, of their 16
“pathogenic” variants, only 1 novel RYR2 variant
would have been deemed “potentially informative”
A bar graph depicting the percentage of yield of ultra-rare (minor allele
frequency <0.00005) variants in major cardiac channelopathy genes (KCNQ1, KCNH2, by our robust criteria.
SCN5A, and RYR2). SIDS ¼ sudden infant death syndrome. In 2017, Neubauer et al. (38) reported a yield
of “potentially causative” variants in 20% of their 155
JACC VOL. 71, NO. 11, 2018 Tester et al. 1225
MARCH 20, 2018:1217–27 Cardiac Genetic Analyses in SIDS

European SIDS cases following WES and genetic bases that are largely different genetically and
interrogation of their 192-gene focused panel that mechanistically from sudden death occurring after
comprised both cardiovascular-associated and meta- the age of 1 year.
bolic disorder–associated genes. The majority of their Several risk factors for SIDS have been established.
seemingly genotype-positive infants had a variant One could hypothesize that vulnerable infants dying
with “likely functional effects” in genes associated of SIDS without the presence of additional risk factors
with a cardiac channelopathy (9%) or cardiomyopathy are more likely to host a highly penetrant monogenic
(7%). However, most of these variants represent cause for their death compared with infants exposed
missense variants within “minor” genes (38). In fact, to additional environmental risk factors. Yet no sig-
only 2.6% of their cases hosted what we would nificant differences in the yield of “potentially
consider a “potentially informative” variant by our informative” GHD gene variants associated with sex,
strict definition. sleep position, or bed sharing were observed. How-
Although our study supports the utility of WES to ever, a significant age effect on the yield, where 23%
identify potential sudden death-causing variants of those infants older than 4 months of age hosted a
within established or potential sudden death– “potentially informative” GHD-associated variant
susceptibility genes, the challenge of the WES-based compared with only 10% of the infants younger than
molecular autopsy does not lie in the identification 4 months of age, was observed. These data support
of variants, but rather in the adjudication of their the potential stratification of those SIDS cases that
potential pathogenicity. Accurate variant classifica- may benefit most from postmortem genetic testing of
tion is crucial to enable proper counseling of surviv- the major channelopathy- or cardiomyopathy-
ing family members. Erroneously or prematurely associated genes.
adjudicating ambiguous variants as pathogenic has The significant overrepresentation of ultra-rare
the potential to harm patients and their families. NSVs within the 4 major channelopathy genes asso-
Tragically, this became a reality for 1 family described ciated with either inheritable LQTS (KCNQ1, KCNH2,
by Ackerman et al. (36) recently, as they dealt with SCN5A) or catecholaminergic polymorphic ventricular
the disastrous consequences of unnecessary treat- tachycardia (RYR2) observed in our European Cauca-
ment on the basis of an erroneously interpreted sian SIDS cases compared with ethnically matched
variant in KCNQ1. Thus, overattribution of SIDS control subjects (6.5% vs. 3.1%; p ¼ 0.013) supports
deaths to GHDs has significant implications for the that cardiac channelopathies may represent the un-
immediate family, and we urge extreme caution in derlying pathogenic basis for some SIDS cases and
variant interpretation. When such cautionary advice that post-mortem genetic testing of the 4 major
was heeded, <5% of more than 400 SIDS cases had channelopathy-associated genes may be warranted in
either a “pathogenic” or “likely pathogenic” variant cases of SIDS.
in 1 of 90 GHD-susceptibility genes, a percentage that
is substantially lower than in previous extrapolations STUDY LIMITATIONS. In our study, we used a strict
of the prevalence of either channelopathic or MAF cutoff of 0.005% (i.e., 1 in 20,000 alleles or 1
cardiomyopathic SIDS. This parallels our experience in 10,000 individuals). Although using a stringent
of the “molecular autopsy” in unexplained threshold could reduce the possibility of identifying
sudden death where stringent variant evaluation variants that would be deemed too common in the
results in a significant reduction of numbers of population to cause a rare disease such as LQTS, it
“likely pathogenic” and “pathogenic” variants of could also prevent the identification of potentially
clinical utility (39). important functionally significant variants that
Using a similarly stringent variant analysis, we could play a role in SIDS pathogenesis. For example,
observed previously a 13% (40 of 302) yield of ultra- the p.R176W-KCNH2 variant was identified in a
rare “pathogenic” or “likely pathogenic” variants single European SIDS case in our cohort. This
within sudden death–susceptibility genes among variant has been associated with LQTS previously, it
302 autopsy-negative cases of sudden arrhythmic has been demonstrated to have a functional effect
death syndrome in persons who died at an age >1 by in vitro assays, and it has been considered a
year (median age 24 years), compared with a founder LQT2 mutation in the Finnish population
significantly (p ¼ 0.00002) lower yield of 4.3% (18 (40,41). Although this variant meets the current
of 419) in our SIDS cohort (39). These data suggest American College of Medical Genetics and Genomics
that most SIDS cases stem from pathobiological guideline classification of “pathogenic” variant, its
1226 Tester et al. JACC VOL. 71, NO. 11, 2018

Cardiac Genetic Analyses in SIDS MARCH 20, 2018:1217–27

heterozygous frequency in gnomAD (44 of 53,551


overall, 31 of 22,031 Europeans, and 10 of 4,022 ADDRESS FOR CORRESPONDENCE: Dr. Elijah R.

Finnish European individuals) exceeds our stringent Behr, St. George’s, University of London, Cranmer
cutoff and was therefore not included in our Terrace, London SW17 0RE, United Kingdom. E-mail:
analysis. [email protected]. OR Dr. Michael J. Ackerman, Mayo
Clinic, 200 First Street Southwest, Rochester,
CONCLUSIONS Minnesota 55905. E-mail: [email protected].

A whole exome molecular autopsy followed by a PERSPECTIVES


cardiac gene–specific focus reveals that <15% of more
than 400 SIDS cases had a “potentially informative” COMPETENCY IN MEDICAL KNOWLEDGE:
variant in 1 of 90 GHD-susceptibility genes. Further- Postmortem genetic testing using WES can identify
more, <5% of these infants who died possessed var- rare NSVs in heart disease susceptibility genes that may
iants that are immediately useful in a family for underlie some cases of SIDS, predominantly in the 4- to
further cascade testing. This finding represents a 12-month age group. The yield of these variants
substantial reduction of the perceived importance of involving all but the principal channelopathy genes is
monogenic cardiac genetic disease in SIDS compared similar to that in a healthy group, thus suggesting that
with previous studies. Interpretation of GHD- single-gene disorders are not the major cause of SIDS.
associated rare variants must therefore be stringent Clinically relevant variants useful for predictive testing
and careful given the implications of misattribution are identified in about 4% of infant deaths.
in families. This has important clinical implications
for the community managing SIDS cases and their TRANSLATIONAL OUTLOOK: Because the patho-
relatives. genesis of SIDS is complex and likely involves more
than 1 genetic pathway, future research should entail
ACKNOWLEDGMENTS The authors gratefully
functional studies to identify truly pathogenic variants
acknowledge both the medical examiners and coro-
and assess the degree to which each variant contrib-
ners for referring the sudden death victims to our
utes to these cases of unexpected early death.
program in an effort to find an explanation for the
infant’s sudden death.

REFERENCES

1. Krous HF, Beckwith JB, Byard RW, et al. Sudden 9. Yun AJ, Lee PY. Sudden death among infants 16. Glengarry JM, Crawford J, Morrow PL,
infant death syndrome and unclassified sudden and adults: companion disorders of maladaptive Stables SR, Love DR, Skinner JR. Long QT molec-
infant deaths: a definitional and diagnostic sympathetic bias. Med Hypotheses 2004;62: ular autopsy in sudden infant death syndrome.
approach. Pediatrics 2004;114:234–8. 857–60. Arch Dis Child 2014;99:635–40.

2. Matthews T, MacDorman MF. Infant mortality 10. Ackerman MJ, Siu BL, Sturner WQ, et al. 17. Arnestad M, Crotti L, Rognum TO, et al. Prev-
statistics from the 2010 period linked birth/infant Postmortem molecular analysis of SCN5A defects alence of long-QT syndrome gene variants in
death data set. Natl Vital Stat Rep 2013;18:1–26. in sudden infant death syndrome. JAMA 2001; sudden infant death syndrome. Circulation 2007;
3. Unexplained deaths in infancy: England and 286:2264–9. 115:361–7.
Wales: 2009. Office for National Statistics 2011. 18. Tester DJ, Ackerman MJ. Sudden infant
11. Kinney HC, Richerson GB, Dymecki SM,
Available at: http://www.ons.gov.uk/ons/ death syndrome: how significant are the cardiac
Darnall RA, Nattie EE. The brainstem and seroto-
dcp171778_227450.pdf. Accessed January 24, channelopathies? Cardiovasc Res 2005;67:
nin in the sudden infant death syndrome. Annu
2018. 388–96.
Rev Pathol 2009;4:517–50.
4. Moon RY, Horne RSC, Hauck FR. Sudden infant 19. Tester DJ, Dura M, Carturan E, et al.
death syndrome. Lancet 2007;370:1578–87. 12. Weese-Mayer DE, Ackerman MJ, Marazita ML,
A mechanism for sudden infant death syndrome
Berry-Kravis EM. Sudden infant death syndrome:
5. Filiano JJ, Kinney HC. A perspective on neuro- (SIDS): Stress-induced leak via ryanodine re-
review of implicated genetic factors. Am J Med
pathologic findings in victims of the sudden infant ceptors. Heart Rhythm 2007;4:733–9.
Genet 2007;143:771–88.
death syndrome: the triple-risk model. Biol 20. Van Norstrand DW, Ackerman MJ. Sudden in-
Neonate 1994;65:194–7. 13. Van Norstrand DW, Ackerman MJ. Genomic risk fant death syndrome: do ion channels play a role?
factors in sudden infant death syndrome. Genome Heart Rhythm 2009;6:272–8.
6. Valdes-Dapena MA. Editorial: Sudden, unex-
pected and unexplained death in infancy—a status Med 2010;2:86.
21. Brion M, Allegue C, Santori M, et al. Sarcomeric
report—1973. N Engl J Med 1973;289:1195–7. 14. Salomonis N. Systems-level perspective of gene mutations in sudden infant death syndrome
7. Schwartz PJ. Cardiac sympathetic innervation sudden infant death syndrome. Pediatr Res 2014; (SIDS). Forensic Sci Int 2012;219:278–81.
and the sudden infant death syndrome: a possible 76:220–9.
22. Hertz CL, Christiansen SL, Larsen MK, et al.
pathogenic link. Am J Med 1976;60:167–72.
15. Schwartz PJ, Stramba-Badiale M, Segantini A, Genetic investigations of sudden unexpected
8. Guntheroth WG. Theories of cardiovascular et al. Prolongation of the QT interval and the deaths in infancy using next-generation
causes in sudden infant death syndrome. J Am Coll sudden infant death syndrome. N Engl J Med sequencing of 100 genes associated with cardiac
Cardiol 1989;14:443–7. 1998;338:1709–14. diseases. Eur J Hum Genet 2016;24:817–22.
JACC VOL. 71, NO. 11, 2018 Tester et al. 1227
MARCH 20, 2018:1217–27 Cardiac Genetic Analyses in SIDS

23. Cheng J, Van Norstrand DW, Medeiros- admixed populations. Am J Hum Genet 2008; the CASQ2 gene in patients with CPVT: implication
Domingo A, et al. Alpha1-syntrophin mutations 83:132–5. for genetic counselling and clinical management.
identified in sudden infant death syndrome cause Hum Mut 2011;32:995–9.
33. Exome Aggregation Consortium (ExAC), Cam-
an increase in late cardiac sodium current. Circ
bridge, MA. Available at: http://exac. 43. Fressart V, Duthoit G, Donal E, et al. Desmo-
Arrhythm Electrophysiol 2009;2:667–76.
broadinstitute.org. Accessed December 30, 2017. somal gene analysis in arrhythmogenic right ven-
24. Tester DJ, Tan B-H, Medeiros-Domingo A, tricular dysplasia/cardiomyopathy: spectrum of
34. Richards S, Aziz N, Bale S, et al. Standards and
Song C, Makielski JC, Ackerman MJ. Loss-of- mutations and clinical impact in practice. Europace
guidelines for the interpretation of sequence var-
function mutations in the KCNJ8-encoded Kir6.1 2010;12:861–8.
iants: a joint consensus recommendation of the
KATP channel and sudden infant death syndrome. 44. Kapplinger JD, Tester DJ, Salisbury BA, et al.
American College of Medical Genetics and Geno-
Circ Cardiovasc Genet 2011;4:510–5. Spectrum and prevalence of mutations from the
mics and the Association for Molecular Pathology.
25. Van Norstrand DW, Asimaki A, Rubinos C, et al. Genet Med 2015;17:405–24. first 2,500 consecutive unrelated patients referred
Connexin43 mutation causes heterogeneous gap for the FAMILION long QT syndrome genetic test.
35. Ackerman MJ. Genetic purgatory and the car-
junction loss and sudden infant death. Circulation Heart Rhythm 2009;6:1297–303.
diac channelopathies: exposing the variants of
2012;125:474–81. 45. Napolitano C, Priori SG, Schwartz PJ, et al.
uncertain/unknown significance (VUS) issue. Heart
Rhythm 2015;12:2325–31. Genetic testing in the long QT syndrome: devel-
26. Van Norstrand DW, Valdivia CR, Tester DJ,
opment and validation of an efficient approach to
et al. Molecular and functional characteriza- 36. Ackerman JP, Bartos DC, Kapplinger JD,
tion of novel glycerol-3-phosphate dehydro- genotyping in clinical practice. JAMA 2005;294:
Tester DJ, Delisle BP, Ackerman MJ. The promise
2975–80.
genase 1 like gene (GPD1-L) mutations in and peril of precision medicine: phenotyping still
sudden infant death syndrome. Circulation matters most. Mayo Clin Proc 2016;91:1606–16. 46. Crotti L, Lundquist AL, Insolia R, et al. KCNH2-
2007;116:2253–9. K897T is a genetic modifier of latent congenital
37. Lek M, Karczewski KJ, Minikel EV, et al. Anal- long-QT syndrome. Circulation 2005;112:1251–8.
27. Cronk LB, Ye B, Kaku T, et al. Novel mechanism ysis of protein-coding genetic variation in 60,706
for sudden infant death syndrome: Persistent late humans. Nature 2016;536:285–91. 47. Haghighi K, Kolokathis F, Gramolini AO, et al.
sodium current secondary to mutations in A mutation in the human phospholamban gene,
caveolin-3. Heart Rhythm 2007;4:161–6. 38. Neubauer J, Lecca MR, Russo G, et al. Post- deleting arginine 14, results in lethal, hereditary
mortem whole-exome analysis in a large sudden cardiomyopathy. Proc Natl Acad Sci U S A 2006;
28. Tan B-H, Pundi KN, Van Norstrand DW, et al. infant death syndrome cohort with a focus on 103:1388–93.
Sudden infant death syndrome-associated muta- cardiovascular and metabolic genetic diseases. Eur
tions in the sodium channel beta subunits. Heart 48. Medeiros-Domingo A, Bhuiyan ZA, Tester DJ,
J Hum Genet 2017;25:404–9.
Rhythm 2010;7:771–8. et al. The RYR2-Encoded Ryanodine Receptor/
39. Lahrouchi N, Raju H, Lodder EM, et al. Utility Calcium Release Channel in patients diagnosed
29. Ruark E, Münz M, Renwick A, et al. The of post-mortem genetic testing in cases of sudden previously with either catecholaminergic poly-
ICR1000 UK exome series: a resource of gene arrhythmic death syndrome. J Am Coll Cardiol morphic ventricular tachycardia or genotype
variation in an outbred population. F1000Res 2017;69:2134–45. negative, exercise-induced long QT Syndrome: a
2015;4:883. comprehensive open reading frame mutational
40. Marjamaa A, Salomaa V, Newton-Cheh C,
30. McKenna A, Hanna M, Banks E, et al. The et al. High prevalence of four long QT syndrome analysis. J Am Coll Cardiol 2009;54:2065–74.
Genome Analysis Toolkit: a MapReduce framework founder mutations in the Finnish population. Ann
for analyzing next-generation DNA sequencing Med 2009;41:234–40. KEY WORDS genetic heart diseases,
data. Genome Res 2010;20:1297–303.
41. Lahti AL, Kujala VJ, Chapman H, et al. Model molecular autopsy, sudden infant death
31. Combined Annotation Dependent Depletion for long QT syndrome type 2 using human iPS cells syndrome, whole exome sequencing
(CADD) scores. Available at: http://cadd.gs. demonstrates arrhythmogenic characteristics in
washington.edu. Accessed January 24, 2018. cell culture. Dis Model Mech 2012;5:220–30.
A PP END IX For supplemental tables and
32. Price AL, Weale ME, Patterson N, et al. 42. Roux-Buisson N, Rendu J, Denjoy I, et al. figures, please see the online version of this
Long-range LD can confound genome scans in Functional analysis reveals splicing mutations of paper.

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