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Robert’s Program for Sudden Unexpected Death in Pediatrics, Boston Children’s Hospital, Boston, MAF.M. Kirby Neurobiology Center, Boston Children’s Hospital, Boston, MAEpilepsy Genetics Program, Department of Neurology, Boston Children’s Hospital and Harvard Medical School, Boston, MADivision of Genetics and Genomics, Department of Pediatrics and Manton Center for Orphan Diseases Research, Boston Children’s Hospital, MA
Department of Genetics, Harvard Medical School, Boston, MADepartment of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MABroad Institute of MIT and Harvard, Cambridge, MA
Robert’s Program for Sudden Unexpected Death in Pediatrics, Boston Children’s Hospital, Boston, MADepartments of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA
Robert’s Program for Sudden Unexpected Death in Pediatrics, Boston Children’s Hospital, Boston, MADivision of Genetics and Genomics, Department of Pediatrics and Manton Center for Orphan Diseases Research, Boston Children’s Hospital, MADepartment of Pediatrics, Harvard Medical School, Boston, MA
Robert’s Program for Sudden Unexpected Death in Pediatrics, Boston Children’s Hospital, Boston, MADepartments of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA
Robert’s Program for Sudden Unexpected Death in Pediatrics, Boston Children’s Hospital, Boston, MADivision of Genetics and Genomics, Department of Pediatrics and Manton Center for Orphan Diseases Research, Boston Children’s Hospital, MADepartment of Pediatrics, Harvard Medical School, Boston, MA
Robert’s Program for Sudden Unexpected Death in Pediatrics, Boston Children’s Hospital, Boston, MADivision of Genetics and Genomics, Department of Pediatrics and Manton Center for Orphan Diseases Research, Boston Children’s Hospital, MABroad Institute of MIT and Harvard, Cambridge, MADepartment of Pediatrics, Harvard Medical School, Boston, MA
Robert’s Program for Sudden Unexpected Death in Pediatrics, Boston Children’s Hospital, Boston, MAF.M. Kirby Neurobiology Center, Boston Children’s Hospital, Boston, MAEpilepsy Genetics Program, Department of Neurology, Boston Children’s Hospital and Harvard Medical School, Boston, MABroad Institute of MIT and Harvard, Cambridge, MADepartment of Neurology, Harvard Medical School, Boston, MA
Correspondence and requests for materials should be addressed to Richard D. Goldstein, Department of Pediatrics, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
Robert’s Program for Sudden Unexpected Death in Pediatrics, Boston Children’s Hospital, Boston, MABroad Institute of MIT and Harvard, Cambridge, MADepartment of Pediatrics, Harvard Medical School, Boston, MADivision of General Pediatrics, Department of Pediatrics, Boston Children’s Hospital, Boston, MA
This study aimed to evaluate genetic contributions to sudden unexpected death in pediatrics (SUDP).
Methods
We phenotyped and performed exome sequencing for 352 SUDP cases. We analyzed variants in 294 "SUDP genes" with mechanisms plausibly related to sudden death. In a subset of 73 cases with parental data (trios), we performed exome-wide analyses and conducted cohort-wide burden analyses.
Results
In total, we identified likely contributory variants in 37 of 352 probands (11%). Analysis of SUDP genes identified pathogenic/likely pathogenic variants in 12 of 352 cases (SCN1A, DEPDC5 [2], GABRG2, SCN5A [2], TTN [2], MYBPC3, PLN, TNNI3, and PDHA1) and variants of unknown significance–favor-pathogenic in 17 of 352 cases. Exome-wide analyses of the 73 cases with family data additionally identified 4 de novo pathogenic/likely pathogenic variants (SCN1A [2], ANKRD1, and BRPF1) and 4 de novo variants of unknown significance–favor-pathogenic. Comparing cases with controls, we demonstrated an excess burden of rare damaging SUDP gene variants (odds ratio, 2.94; 95% confidence interval, 2.37-4.21) and of exome-wide de novo variants in the subset of 73 with trio data (odds ratio, 3.13; 95% confidence interval, 1.91-5.16).
Conclusion
We provide strong evidence for a role of genetic factors in SUDP, involving both candidate genes and novel genes for SUDP and expanding phenotypes of disease genes not previously associated with sudden death.
More than 10% of infant and child deaths in the United States occur suddenly, unexpectedly, and without an established cause, exceeding pediatric mortality from cancer and cardiac disease.
Typically affecting apparently healthy children during sleep, these deaths are certified as sudden infant death syndrome (SIDS), sudden unexpected infant death (SUID), or sudden unexplained death in childhood (SUDC) and can be conceptualized together as sudden unexpected death in pediatrics (SUDP).
SIDS is the leading cause of postneonatal mortality. Reductions in SIDS have been associated with changes in infant sleep practices, yet the reductions mirror declines in non-SIDS rates over the same time, indicating that other improvements in prenatal and infant health may have contributed.
TASK FORCE ON SUDDEN INFANT DEATH SYNDROME SIDS and other sleep-related infant deaths: evidence base for 2016 updated recommendations for a safe infant sleeping environment.
SUDC, which is less familiar to medical and lay communities and lacking an International Classification of Diseases designation, is estimated to affect 1.3 in 100,000 children.
There is emerging consensus that SUDP represents a heterogeneous grouping of rare and undiagnosed diseases presenting with death and sometimes involve genetic mechanisms.
Genetic contributions to SUDP are supported by studies of families whose children died from SIDS during the “safe sleep” era, which showed an increased risk for recurrence in subsequent siblings (odds ratio, [OR], 4.2) and within 3 generations (OR, 9.3).
The prevailing etiologic model of SUDP postulates that modest extrinsic threats become fatal in infants and children who harbor intrinsic vulnerabilities.
Early research described intrinsic vulnerabilities as biologically mediated risk factors such as prematurity, male sex, and prenatal alcohol and/or tobacco exposure. Although these children are often diagnosed with cardiac arrest when they present in a hospital setting and estimates of deaths attributable to cardiac channelopathies and cardiomyopathy in SUDP vary widely,
Although the detection of medium chain acyl-CoA deficiency in some cases led to optimism that metabolic diseases explained a significant component of SIDS cases, current estimates suggest that undetected metabolic disease accounts for only 1% to 2% of SIDS cases.
there is a substantial body of research demonstrating brainstem-mediated and epilepsy-like changes in SUDP cases. The brainstem model for SIDS is based on serotonergic deficiencies of the neurotransmitter itself and of its precursors and transporters in the ventral medulla in 40% of affected infants;
Hippocampal malformation associated with sudden death in early childhood: a neuropathologic study: part 2 of the investigations of The San Diego SUDC Research Project.
The age overlap in this neuropathologic finding also challenges the traditional notion that SIDS and SUDC have different mechanisms and instead suggests that a substantial portion of SIDS and SUDC share mechanisms that present over the pediatric age continuum.
However, recent findings of pathogenic variants in SCN1A, a gene associated with sudden unexpected death in epilepsy (SUDEP), suggest that epilepsy-related mechanisms of sudden death may play a role in SUDP.
These genetic findings provide further evidence for a relationship between SUDP and epilepsy and for the role of neurologic disease genes in SUDP pathogenicity, which has been relatively unexplored.
Although exome sequencing studies have been reported for SIDS,
they have been limited to sequencing of probands; we are unaware of previous genetic reports on trio-based (proband and parents) cohorts. The proband-only design, often a result of the way SUDP cases have been ascertained for research, limits the ability to interpret whether variants are de novo or inherited; trio analysis identifying de novo variants provides additional supportive evidence for pathogenicity, as reflected in the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines.
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
In addition, in-depth phenotyping, especially critical for interpreting genomic information in a heterogeneous condition such as SUDP, has not been consistently or systematically reported in genetic studies of SUDP. When evaluating a variant in a specific disease-associated gene that has not yet been associated with SUDP, phenotypic detail may support variant pathogenicity, thereby suggesting that the phenotype of the gene should be expanded to include sudden death. These limitations become more substantial given the relatively undeveloped concepts of infants and children at risk for SIDS or SUDC or the SUDP phenotype.
Robert’s Program on SUDP at the Boston Children’s Hospital (BCH) is a translational research program that takes a novel, multidisciplinary undiagnosed diseases approach to discover and understand intrinsic vulnerabilities underlying SUDP by combining a detailed phenotypic analysis of premortem cases and autopsy data with genetic analysis to identify genetic contributions to SUDP. Here, we present the results from our approach to the genetics of SUDP, focusing on neurologic and other systemic/syndromic conditions not previously interrogated, as well as cardiac and metabolic disease.
Materials and Methods
Case ascertainment
From 2012 to 2020, we identified SUDP probands from the San Diego County Medical Examiner’s Office and proband-parent trios referred to the Robert’s Program on SUDP at BCH.
DNA samples were obtained from probands and available parents. We obtained postmortem brain specimens and additional tissues, as indicated, to investigate specific phenotypes associated with genetic findings. Informed consent was obtained from the parents of participants for trio cases. Consent for remaining probands was obtained from parents or, in cases obtained from the San Diego Office of the Medical Examiners, in accordance with the California statute (SB 1067) for research in SIDS. Research was conducted with approval from the BCH Institutional Review Board.
SUDP proband phenotyping
Detailed phenotypic analysis of each case was conducted by a multidisciplinary team with expertise in pediatrics, genetics, metabolism, neurology, cardiac genetics, pathology, and neuropathology. We obtained data from parent interviews, autopsy reports, investigative reports, and medical records regarding the circumstances of death, coincident illnesses, obstetrical, birth, and medical history, 3-generation family history, and physical findings. Histologic analysis with an emphasis on neuropathologic review was conducted according to the published methods to identify specific abnormalities associated with SUDP, specifically bilamination of the dentate gyrus and/or other abnormalities of the hippocampal architecture (eg, hyperconvolution).
Exome sequencing and variant identification and classification
Exome sequencing was performed using Agilent SureSelectXT Human All Exon V4 (Agilent) or Nextera Rapid Capture Exome (Illumina) enrichment on Illumina platforms. We conducted exome sequencing for all 352 probands and their parents when available; in total, we sequenced 279 proband-only cases and 73 trios. We analyzed exome sequencing data for potentially pathogenic variants using the WuXi NextCode platform (currently Genuity Science, https://genuitysci.com) with standard filtering for rare damaging variants (details in Supplemental Materials and Methods).
For all probands, we analyzed variants in 294 genes plausibly related to SUDP (SUDP genes) (Supplemental Table 1). The SUDP genes list was curated from the Online Mendelian Inheritance in Man and Human Gene Mutation Database and grouped into the following 3 categories of conditions: neurologic (epilepsy, neurodevelopmental, neuromuscular), cardiac (arrhythmia, cardiomyopathy), and systemic/syndromic (inborn errors of metabolism, multisystem syndromes).
For the subset of probands for whom we had trio data, we additionally performed an exome-wide analysis for rare, damaging variants.
We prioritized variants using the following criteria: (1) minor allele frequency <0.005% in the Genome Aggregation Database for dominant inheritance and <0.1% for recessive inheritance; (2) absence of homozygous/hemizygous variants in the Genome Aggregation Database; (3) location (exonic or splicing regions); (4) genotype quality = 99 and mean allele read depth >10; (5) conservation (across >7 species for missense variants); (6) deleteriousness of missense variants according to a Combined Annotation Dependent Depletion score >20 and Variant Effect Predictor max score >0.9; and (7) predicted splicing effects on cryptic splicing variants based on a SpliceAI delta score ≥0.2. We confirmed variants of interest using an independent variant analysis in a second platform (Codified Genomics) and direct inspection using Integrative Genomics Viewer. We used the database Mutalyzer to review whether variants were compliant with the Human Genome Variation Society guidelines. We defined the following as damaging variants: loss-of-function (stop-gain, frameshift, altered canonical splice site), deleterious missense, nonframeshift insertions/deletions, and cryptic splicing variants.
We classified variants as pathogenic, likely pathogenic (P/LP), or variant of unknown significance (VUS) according to the ACMG/AMP guidelines.
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
We classified variants as VUS-favor-pathogenic (VUS-FP) if published functional data demonstrated altered function, if another substitution affecting the same amino acid has been reported as pathogenic, or if a cryptic splice was affected. The VUS-FP designation is consistent with the ACMG/AMP guidelines that support the use of additional tiers in sequence variant classification and is in use in some molecular laboratory settings (personal communication, Heidi Rehm).
We reviewed variants with reference to case-specific phenotypic data in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and the Human Gene Mutation Database and including these only when the gene-associated clinical phenotypes were consistent with those of the respective probands. Because of the high prevalence of loss-of-function TTN variants in the general population and according to accepted practice,
we only considered variants in TTN exons constitutively expressed in the heart (proportion spliced in >0.9). Principal component analysis (PCA) was performed to delineate the ancestry of the subjects.
Burden analysis
Next-generation sequencing provides data and novel statistical approaches to disease gene discovery, including a burden analysis approach. In this approach, the proportion of individuals carrying variants in a given gene, group of genes, or exome-wide is compared between case and control subjects.
To determine whether there is an excess of rare damaging variants in the SUDP gene list in SUDP cases, we conducted a gene list burden analysis as follows: we compared the proportion of SUDP probands with 1433 healthy BCH controls
in terms of rare variants in (1) any gene on the SUDP gene list and in genes on the list related to (2) neurologic, (3) cardiac, and (4) systemic/syndromic diseases. Furthermore, we assessed the burden of rare, exome-wide de novo variants in SUDP, comparing the proportion of SUDP trios with 2317 control trios (from the Simons Foundation Autism Research Initiative)
with a rare de novo variant exome-wide. Proportions of variants in cases vs controls were compared using the 2-tailed Pearson’s chi-squared test. For families with >1 affected sibling who died from SUDP, only the oldest proband was included in the analysis.
Results
Study cohort and phenotypic features
Our cohort included 320 SIDS and 32 SUDC probands (total 352). The majority of probands were 2 to 6 months old at death (average, 6.0 ± 10.9 months; range, 1 day to 11 years) and male (57%). Comparable numbers were found prone (42%) and supine (40%) position at death. Death was associated with a sleep period in 346 of the children. The 6 deaths that were reported to have occurred during an awake period were in infants, 4 of them during or immediately following a feeding. A history of febrile seizures was reported in 14%. Three-generational family histories revealed SIDS or SUDC in 12% of families, febrile seizures in 41% of families, and 2 families with more than 1 child who died from SIDS. No consanguinity was reported (Table 1). Of the 162 cases with adequate neuropathologic tissue for examination, 93 had one or more abnormalities of hippocampal architecture (84 with bilamination of the dentate gyrus and 41 with other abnormalities).
Table 1Demographics and phenotypes of the SUDP cohort
Total SUDP cohort (n = 352)
Demographics
Number of Probands
Proportion
Age at death
352
<2 mo
79
22%
2-<6 mo
181
51%
6-<12 mo
60
17%
≥12 mo
32
9%
Sex
352
Male
199
57%
Female
153
43%
Ancestry
347
European
263
76%
African
33
10%
East Asian
32
9%
Mixed race
19
5%
Gestational age
337
≥37 wk
283
83%
34-37 wk
33
10%
<34 wk
21
6%
Position found
275
Prone
116
42%
Supine
110
40%
Side
37
13%
Upright/partially upright
12
4%
Sleep site
307
Crib
103
34%
Adult bed
137
45%
Couch
26
8%
Car seat
5
2%
Held
9
3%
Other
27
9%
Sleeping circumstances
344
Shared sleep surface
127
37%
Sleeping alone
217
63%
Additional phenotyping in trios (n = 73)
Personal history
73
Antecedent fever
11
15%
Antecedent minor illness
35
48%
Febrile seizures
10
14%
Other seizures
4
5%
Low birthweight (<2500 g)
3
4%
ROSC
11
15%
Family history
73
SIDS or SUDC
9
12%
SUDEP
1
1%
SCD < 50 y old
11
15%
Febrile seizures
30
41%
Seizures
19
26%
Syncope (excluding vasovagal)
8
11%
Details regarding age of death, demographic information, and circumstances of death for the 352 SUDP probands are provided.
PCA, principal component analysis; ROSC, return of spontaneous circulation; SCD, sudden cardiac death; SIDS, sudden infant death syndrome. SUDC, sudden unexplained death in childhood; SUDEP, sudden unexpected death in epilepsy.
Proband analysis for contribution of rare damaging variants in genes on the SUDP gene list
We took a candidate-gene approach using the 294 genes on the SUDP gene list for all of our proband cases because we had a substantial number (279) of proband-only cases for whom we could thus not determine whether variants were inherited or de novo. Analysis of the proband-only data for variants on the SUDP gene list identified 109 rare damaging variants in 98/352 probands (28%) (Supplemental Table 2). Of these 109 variants, we classified 12 variants as P/LP in genes related to neurologic disease (SCN1A, DEPDC5 [2], and GABRG2), cardiac disease (SCN5A [2], TTN [2], MYBPC3, PLN, TNNI3), and systemic/syndromic disease (PDHA1 [1 male]). We classified 17 variants as VUS-FP in genes associated with neurologic disease (CACNA1A, DYRK1A, GABRB3, SCN1A, SCN4A, SCN8A), cardiac disease (SCN5A [2], TTN [3], CAV3, FLNC, KCNE1, MYBPC3, TNNI3), and systemic/syndromic disease (KCNJ2) (Table 2). The remaining 80 variants were classified as VUS.
Table 2Summary of genes with variants identified in our SUDP cohort and associated phenotypes
We present each case with P/LP variants and VUS-FP implicated in our SUDP cohort by disease categories.
ACMG, American College of Medical Genetics and Genomics; AMP, The Association of Molecular Pathology; AF, allele frequency; F, female; GI, gastrointestinal symptoms; M, male; LP, likely pathogenic; P, pathogenic; VUS, variant of unknown significance; URI, upper respiratory infection.
VUS-FP are indicated with notes regarding the reasons for classifying them as such (column indicated with asterisk).
a functional evidence.
b validated in an exome-wide approach.
c deleterious splicing variant.
d different missense substitution at the same amino acid position that is established as pathogenic.
Burden analysis of rare damaging variants in the 294 genes on the SUDP gene list demonstrated an excess of variants in the 352 SUDP probands when compared with 1433 controls (OR, 2.94; 95% CI, 2.20-3.94) (Figure 1). We further demonstrated an excess of variants in cases when compared with controls for each of the following disease groups: neurologic (OR, 3.91; 95% CI, 2.54-6.02), cardiac (OR, 2.16; 95% CI, 1.47-3.16), and systemic/syndromic (OR, 2.54; 95% CI, 1.29-4.99) (Figure 1, Supplemental Table 3).
Figure 1Burden analysis reveals excess of rare damaging variants in SUDP. A. By comparing all rare damaging variants in our cohort (n = 352) with those in controls (n = 1433), we demonstrate an excess of rare damaging variants in the entire SUDP gene list (odds ratio [OR], 2.94; 95% confidence interval [CI], 2.20-3.91), as well as within each of the following disease-related groups: neurologic, cardiac, and systemic/syndromic disease. B. Considering all genes exome-wide, we observed an excess of rare damaging de novo variants in SUDP cohort trios (n = 73) when compared with control trios (n = 2317) (OR, 3.13; 95% CI, 1.91-5.16; Pearson chi-squared 2-tailed P = 2.56 × 10−6).
Trio analysis for contribution of de novo variants to SUDP
For the 73 cases with trio data, we conducted exome-wide analysis to determine the presence of de novo or X-linked variants. The analysis of the 73 trios revealed 50 de novo variants (34 probands with 1 de novo variant, 5 probands with 2, and 2 probands with 3 variants), 13 X-linked maternally inherited variants, 3 homozygous variants (1 case each), and 3 compound heterozygous rare damaging variants (Figure 2, Supplemental Table 4). Eight de novo variants were classified as P/LP or VUS-FP. Initially, 6 of these were classified in proband-only analyses as VUS and their de novo status led to reclassification, with 2 variants being reclassified as LP (SCN1A) and 4 as VUS-FP (ALG13, AKAP10, FLNA, TCF4). Two de novo variants in genes not on the SUDP list (BRPF1 and ANKRD11) were identified and classified as LP.
Figure 2Rare damaging de novo and maternally inherited X-linked variants among 73 trios. Analysis of 73 SUDP trios identified rare damaging de novo and maternally inherited X-linked variants in 16 genes with known associations with neurologic (blue), cardiac (red), and systemic/syndromic (orange) disease (left) and in 46 additional genes without known disease relevance (right). Genes found in the SUDP gene list analysis are indicated by asterisks.
Burden analysis revealed that a significantly greater proportion of SUDP trio cases (38/73) had exome-wide rare damaging de novo variants than controls (596/2317) (OR, 3.13; 95% CI, 1.91-5.16) (Figure 1).
Summary of genetic analysis in proband-only and trio analyses
Overall, we identified rare damaging P/LP or VUS-FP variants in 37 of 352 SUDP probands (11%). We identified 16 P/LP variants (12 on analysis of the genes on the SUDP gene list; 2 VUS-FP variants were reclassified to LP based on the de novo finding following exome-wide analysis; and 2 de novo variants in genes not on the SUDP list) and 21 VUS-FP variants (17 on analysis of the genes on the SUDP gene list and 4 VUS that were reclassified to VUS-FP based on the de novo finding on exome-wide analysis) (Table 2). Among the 37 variants in these cases, 13 were in genes related to neurologic disease, 18 in cardiac-related disease genes, and 6 in systemic/syndromic disease genes. The implicated genes are displayed according to disease category and age of death in Figure 3.
Figure 3Genes implicated in SUDP according to age of death. Our SUDP cohort included 320 SIDS and 32 SUDC probands (total 352) among which we identified a pathogenic/likely pathogenic (P/LP) variant or variant of unknown significance–favor-pathogenic (VUS-FP) in 37 (11%) cases. Each case with a likely contributory genetic variant is represented by a box with the associated gene name (bold for P/LP and nonbold for VUS-FP) displayed on a timeline indicating age of death. Each gene’s disease category is indicated by color: neurologic (blue), cardiac (red), and systemic/syndromic (orange).
Among the 37 cases with variants identified, relevant history or family history was observed in several cases (Table 2). Febrile seizures were reported in 3 probands with variants in genes associated with neurologic disease (epilepsy) (ALG13, GABRG2, and SCN1A); among these, the child with the SCN1A variant had a sibling with epilepsy (reported previously
). Two probands had a significant family history of cardiac disease: 1 with a TTN variant whose father had undergone a heart transplant at 29 years of age and 1 with a PLN variant and a family history of early cardiac death in 3 maternal family members.
Detailed phenotypic review revealed that the probands with de novo ANKRD11 and BRPF1
variants had features consistent with the related genetic syndromes, namely KBG syndrome and intellectual developmental disorder with dysmorphic facies and ptosis (IDDDFP), respectively. Notably, neither were recognized to have these syndromes premortem or at autopsy (Table 2). Ten of the 84 cases with dentate gyrus bilamination (12%) harbored a P/LP variant or VUS-FP: 3 cases had variants in genes associated with neurologic disease (DEPDC5, GABRB3, GABRG2), 5 in genes associated with cardiac disease (AKAP10, SCN5A, TNNI3, TTN [2 cases]), and 2 in genes associated with systemic/syndromic disease (FLNA, TCF4). Ten of the 41 cases with abnormal hippocampal architecture harbored a P/LP variant or VUS-FP (24%): these included 8 of the aforementioned cases with variants in DEPDC5, GABRB3, GABRG2, AKAP10, SCN5A, TTN (2 cases), and TCF4, an additional case with a variant in SCN5A, and 1 with a variant in ANKRD11 associated with systemic/syndromic disease (Table 2).
Discussion
Given our hypotheses and emerging evidence that heterogeneous genetic factors contribute to SUDP, we employed an undiagnosed disease approach to SUDP, which included in-depth phenotyping and analysis of exome data. We undertook a candidate-gene approach for all cases in a large SUDP cohort comprising 352 cases. Furthermore, for a subset of 73 probands, we leveraged the availability of trio data and conducted exome-wide analyses. We identified genetic contributions to SUDP in 11% of our cohort, providing specific examples of intrinsic vulnerabilities to sudden death. Our exome-wide trio analysis of the subset with parental DNA identified de novo variants in genes not previously associated with SUDP. Incorporating parental data also allowed us to reclassify several VUS, identified in the proband-only analysis, to P/LP variants or VUS-FP.
We used a genetic burden testing approach, which leverages data from unrelated probands to increase the power, to identify novel genetic associations.
This approach aggregates variants across a gene or group of genes to improve the discovery power by comparing the proportion of cases with variants in the gene(s) of interest with that of controls. Importantly, our cohort-wide analyses demonstrated an increased genetic burden in SUDP cases both with respect to rare damaging variants in targeted genes and exome-wide de novo variants. These results provide further evidence to support a role for genetic factors in SUDP and, moreover, to support the premise that children dying from SUDP may harbor intrinsic vulnerabilities that differentiate them from unaffected children at a population level.
Our findings support the hypothesis that diverse neurologic, cardiac, and metabolic mechanisms play a role in SUDP. Categories in our SUDP gene list were based on the predominant clinical symptoms associated with the genes; we acknowledge that some genes are expressed in multiple tissues, including both the brain and heart. Although previous studies on SUDP have focused on genes related to cardiac or metabolic conditions, our candidate approach using the SUDP gene list additionally included genes related to neurologic and other systemic/syndromic conditions not previously interrogated. Indeed, 19 of the 37 probands harboring P/LP variants or VUS-FP (51%) had variants in genes related to neurologic disease and other systemic/syndromic conditions, supporting the validity of this approach. We hypothesize that epilepsy-related mechanisms may have contributed to death in cases with variants in genes associated with epilepsy (SCN1A, DEPDC5, ALG13, CACNA1A, GABRB3, GABRG2, SCN8A). Among these, SCN1A has previously been implicated in SIDS
In addition, although simple febrile seizures have not been shown to be associated with an increased risk of death, the history of febrile seizures in some SUDP cases with variants in epilepsy-related genes suggests the possibility that these previous episodes may have been seizures that were unmasked by fever in individuals with a genetic risk for epilepsy.
Eighteen (49%) of the 37 P/LP variants and VUS-FP were in cardiac disease genes, namely TTN (5), SCN5A (4), MYBPC3 (2), CAV3, FLNC, KCNE1, and TNNI3, which were all previously reported in cases with sudden death. The presence of a VUS-FP in the additional cardiac-related gene AKAP10 in 1 proband suggests an expansion of its associated phenotype. Because the penetrance of some arrhythmia-related genes is incomplete, the identification of variants in these genes in SUDP cases may have implications for living family members unaware of their risk. In addition, genetic variants known to disrupt cardiac electrical activity may also be expressed and affect function in the brain.
Our results demonstrate the importance of conducting trio exome-wide analysis when trio data are available. Trio analysis led to reclassification in 6 cases from our initial proband-only SUDP gene list analysis. Two VUS were reclassified to LP (SCN1A) and 4 to VUS-FP (ALG13, AKAP10, FLNA, TCF4) following the discovery of de novo status. In addition, standard candidate-gene–based approaches may overlook the role of genes in SUDP because classification of variants relies, in part, on disease associations, which are largely based on phenotypes described typically in people living with disease. The risk for SUDP may not be well reflected among the known phenotypes of many disease genes because children may die of SUDP before a genetic condition is recognized. A trio approach to SUDP, not restricted to genes with known or hypothesized associations with sudden death, allows for novel genotype-phenotype discoveries for an entity that is still largely not understood and requires a broad-based approach. Our trio analysis identified new associations between syndromic disease genes and sudden death, including BRPF1,
associated with IDDDFP, and ANKRD11, associated with KBG syndrome, both in cases not recognized as such premortem.
We identified a male proband with a pathogenic variant in PDHA1, responsible for pyruvate dehydrogenase E1-alpha deficiency, a metabolic disorder with variable expressivity. The variant, assessed as pathogenic by our classification and in ClinVar, expands the phenotype of this condition to include sudden death in the absence of overt metabolic disease. We additionally observed a VUS-FP in ALG13, also related to epilepsy and metabolic disease (glycosylation disorder), in a proband whose premortem history was positive only for seizures with fever. Although we classified this gene as neurologic because of the epilepsy association, it is also possible that the child had an occult glycosylation defect.
There is growing evidence that stillbirth, SIDS, and SUDC represent a continuum with shared etiologies presenting as unexplained death over a continuum from fetal life through childhood.
Shared neuropathologic changes and a gene (SCN1A) common to SIDS and SUDC provides further evidence that unexplained infant and child deaths may contribute to the continuum. A recent study reported causal variants in stillbirth in a similar proportion of cases as reported in the present study, and shared variants in genes in their cohort and ours (eg, MYBPC3)
provide a genetic connection between stillbirth and SUDP. Our observation that neurologic and syndromic cases span SIDS and SUDC, whereas cardiac genes clustered in the SIDS age range (Figure 3) may help to further delineate the mechanisms related to sudden death along this continuum.
The majority of deaths in our cohort remained genetically unexplained, paralleling results in other studies of undiagnosed disease. Additional cases will be required to validate the association between SUDP and genes implicated here in sudden death. Although we categorized genes on the SUDP gene list based on the predominant clinical symptoms associated with them, some genes are expressed in multiple tissues, including both the brain and heart, and further work will be necessary to determine the pathogenic mechanisms. Furthermore, the presence of pathogenic variants, even those deemed pathogenic, does not in itself establish causality. Despite these limitations, collectively, our findings demonstrate a genetic contribution to SUDP and highlight the need for future investigation into as yet unidentified genetic causes owing to the limited cohort size and numbers of trios thus far sequenced. Future genetic evaluation of SUDP cohorts should include trio analyses when possible to identify additional de novo causes (hypothesized to be involved given the lethal nature of the condition) and inherited causes in genes with decreased penetrance. In addition, deep sequencing of candidate genes, genome sequencing, and copy number analyses could lead to the identification of mosaic variants, noncoding variants, and structural variants, respectively.
Our finding of specific genetic contributions to SUDP in 11% of our cohort highlights the role of genetics in SUDP and indicates diverse mechanisms for the diagnosis. In addition, we demonstrate a paradigm for the genetic evaluation of SUDP that benefits from engaging parents to obtain data about the deceased infant and the family history, thereby maximizing the available phenotypic data to inform genetic analyses, as well as obtaining samples from the proband and parents so that trio analyses can be conducted. Ideally, such practice should be undertaken in collaboration with specialized teams that can deliver results in a clinical context, providing bereaved parents with an approach to search for answers to explain why their child died, medical surveillance for at-risk surviving family members, counseling about recurrence risks, and the opportunity to participate in a process that will ultimately lead to a better understanding and prevention of SUDP.
In conclusion, we provide evidence for diverse genetic contributions to SUDP through an undiagnosed disease approach. We advocate that, when resources permit, a comprehensive evaluation for SUDP should include a comprehensive genetic evaluation.
Data Availability
Data supporting the findings of this study are included in the supplementary data. The variants included in Table 2 are available in SCV002030048 to SCV002030084 at Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/).
Conflict of Interest
The authors have no financial or other interests related to the submitted work that (1) could affect or have the perception of affecting the author’s objectivity or (2) could influence or have the perception of influencing the authors or the content of the article.
Acknowledgments
The authors would like to thank Dr Hannah Kinney whose work and mentorship guided our efforts. We are grateful to the families who participated in this research, the Massachusetts Office of the Chief Medical Examiner (OCME), Boston, Massachusetts, the County Office of the Medical Examiner, San Diego, California, and team members who supported subject recruitment and sequencing. This work was supported by funds from the Robert's Program on Sudden Unexpected Death in Pediatrics, the Cooper Trewin Memorial SUDC Research Fund, Citizens United for Research in Epilepsy through the Isaiah Stone Award, Three Butterflies SIDS Foundation, The Florida SIDS Alliance, Borrowed Time 151, and The Eunice Kennedy Shriver National Institute of Child Health and Human Development under grant numbers R21 HD096355 and R01 HD090064. Alireza Haghighi was supported by the National Heart, Lung, and Blood Institute (NHLBI K8HL150284), American Heart Association, and Saving Tiny Hearts Society.
Informed consent was obtained from the parents of participants. Consent for the remaining probands was obtained from parents or, in cases obtained from the San Diego Office of the Medical Examiners, in accordance with the California statute (SB 1067) for research in sudden infant death syndrome. Research was conducted with approval from the BCH Institutional Review Board.
Hippocampal malformation associated with sudden death in early childhood: a neuropathologic study: part 2 of the investigations of The San Diego SUDC Research Project.
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
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