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Postmortem review and genetic analysis in sudden infant death syndrome: an 11-year review
Human Pathology (2013) xx, xxx–xxx
www.elsevier.com/locate/humpath
Original contribution
Postmortem review and genetic analysis in sudden infant death syndrome: an 11-year review☆ Angharad Evans BSc a , Richard D. Bagnall PhD a,b , Johan Duflou MBCHB b,c , Christopher Semsarian MBBS, PhD a,b,d,⁎ a
Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Sydney, NSW 2042, Australia Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia c Deparment of Forensic Medicine, Glebe, Sydney, NSW 2037, Australia d Department of Cardiology, Royal Prince Alfred Hospital, Sydney, NSW 2050, Australia b
Received 3 December 2012; revised 22 January 2013; accepted 31 January 2013
Keywords: Sudden infant death syndrome; HCN gene; Arrhythmia; Ion channel
Summary Sudden infant death syndrome (SIDS) is the unexpected death of a child younger than 1 year that remains unexplained after thorough evaluation. The possibility of an underlying primary arrhythmogenic disorder has been proposed as a potential cause of SIDS. This study sought to review SIDS deaths and to perform genetic analysis in key genes that may contribute to sudden death. From 2000 to 2010, all postmortem records from the Department of Forensic Medicine in Sydney, Australia, were reviewed. Cases that gave the cause of death as “SIDS” or “undetermined” but consistent with SIDS were included. In a subset of cases, the hyperpolarization-activated cyclic nucleotide (HCN)–gated channel family of genes (HCN2 and HCN4) was analyzed. A total of 226 SIDS cases were identified; 61% were male, 41% occurred while bed sharing, and there was a peak in deaths between 2 and 4 months old. The incidence did not decrease over the study period. In a subgroup of SIDS cases (n = 46), genetic analysis identified 2 likely pathogenic variants (2/46; 4%). A novel nonsynonymous variant, HCN4-Ala195Val, predicted to be pathogenic, was identified in a female infant who died at age 4 months. A female infant aged 5 weeks carried a rare nonsynonymous variant, HCN4-Val759Ile, which is similar to previously described variants associated with cardiac arrhythmias. In conclusion, the incidence of SIDS remains constant, with no apparent decline in the last decade. The underlying cause of SIDS remains largely unknown. Mutations in cardiac ion channel genes including rare nonsynonymous HCN gene variants may play a role in the pathogenesis of some SIDS cases. © 2013 Elsevier Inc. All rights reserved.
☆
A.E. is the recipient of a Splash of Red Foundation Scholarship. C.S. is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship. This study was also supported, in part, by a Heart Kids NSW project grant. ⁎ Corresponding author. Agnes Ginges, Centre for Molecular Cardiology, Centenary Institute, Locked Bag 6, Newtown, NSW 2042, Australia. E-mail address: [email protected] (C. Semsarian). 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.01.024
1. Introduction Sudden infant death syndrome (SIDS) is the unexpected death of a child younger than 1 year that remains unexplained after a detailed medical autopsy, death scene investigation, and clinical history evaluation [1]. This
2 profoundly tragic event is the leading cause of postneonatal infant death in Australia [2]. The incidence of SIDS in developed countries has dramatically reduced since the late 1980s owing to public awareness campaigns of modifiable environmental risk factors [3,4]. However, there remain more than 80 SIDS cases a year in Australia, a number that has not declined in the past decade [2]. The triple-risk hypothesis proposes that SIDS is a complex event by which an environmental stressor triggers an aberrant response in a genetically predisposed infant at a critical stage of development [5]. Exposure to secondhand smoke, prone sleeping position, bed sharing, and viral and bacterial infections have all been identified as environmental risk factors that may contribute to SIDS [6]. A number of genetic factors likely to predispose an infant to a fatal event, such as faults in genes encoding serotonin transporters, those vital to early development of the autonomic nervous system, and those that regulate inflammation and immune response or energy production, have been associated with SIDS; however, the mechanisms are poorly understood [7]. Pathogenic mutations in genes that encode cardiac ion channels are believed to account for up to 10% of SIDS cases [8–10]. Mutations that are responsible for heritable arrhythmias such long-QT syndrome (LQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic ventricular tachycardia (CPVT) may cause a fatal arrhythmia that leaves no structural damage identifiable at postmortem [11]. These mutations, which occur in cardiac ion channel genes, have more recently been described in a proportion of SIDS cases. The hyperpolarization-activated cyclic nucleotide (HCN)–gated channel family of genes (HCN1-4) are voltage-gated ion channels permeable to both potassium and sodium that are activated by both hyperpolarization and binding of cyclic adenosine monophosphate (cAMP) at a cyclic nucleotide-binding domain. Unlike the cardiacspecific channels that are associated with LQTS, BrS, and CPVT, HCN channels are responsible for generating spontaneous rhythmic activity at varying levels in both cardiac pacemaker cells and neurons in the brain [12–14]. Genetic variants in HCN4, expressed in the sinoatrial node of the heart, have been associated with abnormal cardiac rhythms, namely, sinus bradycardia, sinus node dysfunction, and sudden unexplained death in epilepsy (SUDEP) [15–17]. Genetic variants in HCN2, expressed ubiquitously throughout the heart, have been associated with generalized epilepsy and SUDEP, which may be resultant of a cardiac arrhythmia [17,18]. Genetic variants in HCN channel isoforms HCN2 and HCN4, because of their critical role in the cardiac pacemaker, may therefore contribute to a subset of SIDS deaths through deregulation of the heart rhythm and predisposition to a fatal cardiac arrest. This study sought to characterize a large cohort of SIDS cases from a review of postmortem reports and to perform genetic analysis of the HCN2 and HCN4 genes
A. Evans et al. to identify pathogenic DNA variants that may predispose to SIDS.
2. Materials and methods 2.1. Study populations Postmortem reports over an 11-year period from 2000 to 2010 from the Department of Forensic Medicine, Sydney, Australia, were reviewed. Reports of infants younger than 1 year with the cause of death given as “SIDS” or “undetermined” were reviewed in detail. The cohort included all categories of SIDS, and bed-sharing cases were not excluded. Demographic, clinical, and autopsy data were collected in all cases. This study was approved by the Office of the NSW State Coroner and performed in accordance with institutional human ethics guidelines.
2.2. Postmortem evaluation Although studies were performed over an 11-year period, in all cases, every effort was made to complete a comprehensive postmortem examination. This included detailed macroscopic and microscopic analysis, with particular attention to the heart and brain, careful histologic analysis, evidence of intrathoracic petechiae, toxicology and microbiology analysis, and, in some cases, metabolic testing. This study included all categories of SIDS, which incorporated both SIDS, as well as cases where the cause of death was undetermined but was consistent with SIDS.
2.3. Genetic analysis In a representative subgroup of SIDS cases (the most recent 50 cases), DNA was extracted from postmortem blood or liver tissue, as previously described [19]. Primers flanking the protein-coding regions and exon/intron junctions were used to amplify the 8 exons of HCN2 and HCN4, as previously described [17]. Polymerase chain reaction product for each case was visualized on a 2% agarose gel, sequenced, and analyzed for nucleotide variants using Sequencher v4.8 (Gene Codes Corp, Ann Arbor, MI). The following criteria were used to determine pathogenicity for nonsynonymous variants found in the SIDS population: (i) frequency of the variant in the National Heart, Lung and Blood Institute Exome Variant Server (EVS) (http://evs.gs. washington.edu/EVS/), a database of more than 6500 human exome sequences; (ii) conservation of the amino acid residue among orthologous proteins; and (iii) prediction of pathogenicity using the online resource PolyPhen2, which predicts the impact of an amino acid substitution on the structure and function of a human protein (http://genetics.bwh.harvard. edu/pph2/).
Postmortem review and genetic analysis in SIDS
3
3. Results
Table 1 Comparison of whole cohort vs most recent cases for genetic analysis
3.1. Cohort characteristics
Characteristic
Whole cohort Most recent cases P (for genetic analysis)
A total of 226 cases consistent with SIDS were identified. The characteristics of the SIDS cases are summarized in Fig. 1. The ages ranged from 2 days to 12 months, with 61% of the cohort being male and a peak in cases in the 2- to 4month period. Forty-one percent of cases occurred while bed sharing, and the number of deaths while bed sharing decreased as infants got older. There was no decrease in the incidence of SIDS over the 11-year period (Fig. 1).
n Age (mo), mean Age (mo), range Male sex, n (%) Bed sharing, n (%)
226 3.6 0-12 138 (61) 93 (41)
3.2. Genetic analysis In a subgroup of SIDS cases (n = 46), genetic analysis of the HCN2 and HCN4 genes was performed. This subgroup of cases was representative of the total SIDS group in terms of age, sex, and frequency of bed sharing (Table 1). The findings of the genetic analysis in this subgroup of cases are summarized in Table 2. DNA sequencing of all 8 exons and intron/exon junctions of the HCN4 gene identified 2 potentially pathogenic nonsynonymous DNA variants, that is, 1 novel (Ala195Val) and 1 rare (Val759Ile) nonsynonymous variant. The HCN4-Ala195Val variant, a c.1578CNT
50 3.3 0-11 32 (64) 21 (42)
NS NS NS NS
Abbreviations: n, number; NS, not statistically significant.
substitution, falls in the N-terminal cytosolic domain of the protein (Fig. 2), has not been reported in the EVS, and is predicted by Polyphen2 to be probably damaging (Polyphen2 score of 0.98). The Genomic Evolutionary Rate Profiling (GERP) score, measuring conservation of the individual nucleotide c.1578CNT, is 2.49. The amino acid is conserved in mammals. The HCN4-Val759Ile variant, a c.3269GNA, is a rare variant located in the C-terminal cytosolic domain following the cyclic nucleotide-binding domain (Fig. 2). It is reported in EVS at a frequency of 0.3% in a European population and 0.06% in an African American population and has a GERP nucleotide conservation score of 3.45, and Polyphen2 predicts this variant to be benign. The amino acid is conserved to the level of reptile. DNA
Fig. 1 SIDS cases in NSW from 2000 to 2010. A, Incidence of SIDS over the study period 2000 to 2010. B, Number of SIDS cases categorized by age at death. C, Number of SIDS cases categorized by age at death and whether the death occurred while bed sharing. D, Number of SIDS categorized by age at death and sex.
4
A. Evans et al. Table 2
Genetic variants identified in HCN2 and HCN4
Gene
HCN2
HCN4
Variant
rs56342526 rs56131056 rs55659726 rs56170955 rs56180027 rs55780677 rs56279131 rs12981860 rs3752158 rs34397648 rs2301778 rs1054786 rs55691080 Novel rs143090627 Novel Novel rs12909882 rs62641689 rs11781925 Novel Novel rs14273518
Exon
2 2 2 2 2 2 3 3 4 5 6 7 8 1 1 1 3 4 8 8 8 8 8
Amino acid change
MAF SIDS (n = 46)
ESV a
Asp238Asp Thr241Thr Tyr286Tyr Phe305Phe IIe307IIe Arg321Arg Ser362Ser Pro389Pro Leu413Leu Glu484Glu Ala548Ala Ala624Ala Ala751Ala Val19Val Glu36Gly Ala195Val a Ala414Ala Leu520Leu Val759Ile a Pro852Pro Ala913Ala Pro1042Pro Met1113Val a
0.109 0.109 0.109 0.109 0.109 0.109 0.011 0.163 0.032 0.108 0.283 0.283 0.217 0.011 0.065 0.011 0.011 0.087 0.011 0.022 0.011 0.011 0.022
0.100 0.101 0.105 0.105 0.105 0.103 0.023 0.360 0.064 0.122 0.300 0.425 NA NA 0.051 NA NA 0.091 0.003 0.049 NA NA 0.012
Abbreviations: MAF, minor allele frequency; EVS, exome variant server; NA, not available. a Indicates a nonsynonymous (amino acid changing) variant.
sequencing of all 8 exons and intron/exon junctions of the HCN2 gene revealed no nonsynonymous variants in the HCN2 gene.
4. Discussion This study describes a large cohort of SIDS cases with genetic screening for variants in the 2 HCN isoforms most expressed in the heart, HCN2 and HCN4, which have been previously associated with cardiac arrhythmias and sudden death. A total of 226 SIDS deaths were identified in the 11year review. The incidence of SIDS failed to decline from 2000 onward, in line with other recent reports that incidence of SIDS has remained constant in the past decade [3]. Our data show a clear male bias and a peak in cases in the 2- to 4-month age group. This is a trend seen in most SIDS cohorts and, as stated in the triple-risk hypothesis, suggests that there is a critical development period between 2 and 4 months, resulting in increased vulnerability of the infant. Genetic analysis identified a novel nonsynonymous variant, HCN4-Ala195Val, in a female infant who died at age 4 months and a rare nonsynonymous variant, HCN4Val759Ile, in a female infant aged 5 weeks. Collectively, these findings provide further support for the potential role of mutations in cardiac ion channel genes in the pathogenesis of some SIDS cases.
The precise mechanisms underlying the pathogenesis of SIDS are currently unclear. In Australia and other developed countries, strong campaigns aimed at encouraging reduction of modifiable risk factors for SIDS have seen a dramatic reduction of SIDS deaths from the late 1980s onward, with a 76% decrease in cases in Australia between 1986 and 2000 [20]. Bed sharing has been a major focus of SIDS awareness campaigns, and despite the efforts of such campaigns, 41% of deaths in our study occurred while bed sharing. The practice of bed sharing is controversial; however, safe-sleeping environment recommendations state that infants should not share a bed with any other person because of risk of accidental asphyxiation [21]. Interestingly, the number of deaths that occurred while bed sharing in our study fell dramatically as infants grew older, with 79% of SIDS deaths occurring while bed sharing in 0- to 2-month-old babies falling to 14% of cases in 10- to 12-month-old babies (Fig. 1C). This finding may be attributed to the likelihood that parents are more likely to bed share only with younger infants. Because SIDS is a diagnosis of exclusion, determining an underlying cause is extremely difficult and it is unlikely that there is one single cause common to all cases. A genetic susceptibility to the development of cardiac arrhythmias could be a potential cause in some cases because the negative autopsy mirrors adult sudden unexplained death stemming from fatal arrhythmias such as LQTS, BrS, and CPVT [11].
Postmortem review and genetic analysis in SIDS
5
Fig. 2 A, Protein schematic of HCN4 channel with 6 transmembrane domains (S1-S6) and the location of variants associated with disease. B, Location of DNA variants associated with SIDS (current study), SUDEP, and familial bradycardia is shown. C, Sequence chromatograms of SIDS HCN4 variants and conservation of the respective amino acids that are altered.
Functionally relevant mutations in cardiac ion channel genes that commonly cause LQTS (such as KCNQ1, KCNH2, and SCN5A) have been identified in up to 10% of SIDS cases [9,22,23]. Mutations in genes associated with BrS
(KCND3, SCN1Bb) [24,25] and CPVT (RyR2) [26] have been identified in a smaller subset of SIDS, supporting the role of cardiac channelopathies in predisposing to sudden death in SIDS. The HCN family of ion channels, particularly
6 HCN4, is what predominantly regulates the spontaneous rhythmic activity in the cardiac pacemaker cells at the sinoatrial node. Variants in these genes have previously been associated with negative postmortem deaths that may involve disturbances of the heart's electrical system, and as shown in this study, rare variants in these genes may contribute to a SIDS event [11]. The novel genetic findings in the current study provide further evidence of the role of ion channel genes in the pathogenesis of cardiac arrhythmias in SIDS cases. The HCN4-Ala195Val was carried on one allele in a 4-month-old female infant who was found deceased sleeping in the supine position alone in her crib. She was a healthy infant who had a slight fever 2 days prior. The postmortem examination failed to reveal a definitive cause of death, and no intrathoracic petechiae were noted. This variant has not been reported in any online databases and is located at a conserved nucleotide and amino acid residue. It occurs in the N-terminal cytosolic tail of the protein, an area not previously associated with disease-causing variants. Very little is known about the function of the N-terminus, although Greene et al [27] have shown in a recent study that there is a β2-adrenergic receptor binding site located in the N-terminal tail. β2-Adrenergic receptors are at a high density in the sinoatrial node where they colocalize with cardiac ion channels and are essential for proper cardiac function [28]. The alanine-to-valine substitution at amino acid position 195 may affect the ability of HCN4 to bind strongly to β2-adrenergic receptor, rendering it ineffective at responding to adrenergic stimulation. Because SIDS is hypothesized to be the inability of a predisposed infant to cope with an exogenous stressor, the inability for the cardiac pacemaker to increase its rate in response to such a stressor may have resulted in the death of this infant. The HCN4-Val759Ile variant was found on one allele of a 5-week-old female infant who was found deceased sharing a bed with her parents and whose postmortem revealed no cause of death, with no evidence of intrathoracic petechiae. HCN4-Val759Ile has been reported in European populations at a frequency of 0.3% and in an African American population at 0.06%, making it a rare allele. This infant, however, was an indigenous Australian for which there are no genetic data available. This variant has previously been reported in a SUDEP population at a frequency of 2% (Fig. 2) [17]. Apart from age, SUDEP cases mimic SIDS cases in many ways, including the demonstration of a negative autopsy and sudden death often occurring during sleep. The HCN4-Val759Ile variant is located in the Cterminal cytoplasmic tail of the protein. Mutations located at the C-terminal tail that have been shown to reduce HCN4 ion channel activity have previously been associated with familial sinus bradycardia (Fig. 2) [15]. The clinical significance of HCN4-Val759Ile remains unknown. However, its presence in this SIDS case, as well as in 2% of SUDEP cases, and its similarity to other variants associated with cardiac disease suggest that the variant may have a
A. Evans et al. modifying affect on ion channel activity, thereby altering susceptibility to sudden death. A genetic predisposition to SIDS may also have implications for the family and may warrant basic cardiac investigation of the parents including an electrocardiogram [29]. Although this is currently not routine practice, the possibility of genetic predisposition to SIDS implies that family evaluation at some level needs to be considered.
5. Conclusions SIDS is a profoundly tragic event, and despite attempts to reduce the environmental risk factors, it is not on the decline. Bed sharing accounts for a large proportion of all SIDS cases; however in many cases, the precise cause of SIDS remains unknown. Genetic alterations in ion channels that regulate cardiac rhythms may account for some cases of SIDS, as demonstrated by this report of 2 potentially pathogenic novel or rare variants in the HCN4 gene. Further studies are required to understand the exact mechanisms that predispose an otherwise healthy infant to develop SIDS.
References [1] Krous HF, Beckwith JB, Byard RW, et al. Sudden infant death syndrome and unclassified sudden infant deaths: a definitional and diagnostic approach. Pediatrics 2004;114:234-8. [2] ABS. Causes of death, Australia, 2010. Canberra, Australia: Australian Bureau of Statistics; 2012 [cat. no. 3303.0]. [3] Hauck FR, Tanabe KO. International trends in sudden infant death syndrome: stabilization of rates requires further action. Pediatrics 2008;122:660-6. [4] Changing concepts of sudden infant death syndrome: implications for infant sleeping environment and sleep position. American Academy of Pediatrics. Task Force on Infant Sleep Position and Sudden Infant Death Syndrome. Pediatrics 2000;105:650-6. [5] Guntheroth WG, Spiers PS. The triple risk hypotheses in sudden infant death syndrome. Pediatrics 2002;110:e64. [6] Ostfeld BM, Esposito L, Perl H, Hegyi T. Concurrent risks in sudden infant death syndrome. Pediatrics 2010;125:447-53. [7] Weese-Mayer DE, Ackerman MJ, Marazita ML, Berry-Kravis EM. Sudden infant death syndrome: review of implicated genetic factors. Am J Med Genet A 2007;143A:771-88. [8] Schwartz PJ, Priori SG, Dumaine R, et al. A molecular link between the sudden infant death syndrome and the long-QT syndrome. N Engl J Med 2000;343:262-7. [9] Arnestad M, Crotti L, Rognum TO, et al. Prevalence of long-QT syndrome gene variants in sudden infant death syndrome. Circulation 2007;115:361-7. [10] Tester DJ, Ackerman MJ. Sudden infant death syndrome: how significant are the cardiac channelopathies? Cardiovasc Res 2005;67: 388-96. [11] Semsarian C, Hamilton RM. Key role of the molecular autopsy in sudden unexpected death. Heart Rhythm 2012;9:145-50. [12] Ludwig A, Zong X, Jeglitsch M, Hofmann F, Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature 1998;393:587-91. [13] DiFrancesco D. Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 1993;55:455-72.
Postmortem review and genetic analysis in SIDS [14] Pape HC. Queer current and pacemaker: the hyperpolarizationactivated cation current in neurons. Annu Rev Physiol 1996;58: 299-327. [15] Milanesi R, Baruscotti M, Gnecchi-Ruscone T, DiFrancesco D. Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel. N Engl J Med 2006;354:151-7. [16] Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest 2003;111:1537-45. [17] Tu E, Waterhouse L, Duflou J, Bagnall RD, Semsarian C. Genetic analysis of hyperpolarization-activated cyclic nucleotide–gated cation channels in sudden unexpected death in epilepsy cases. Brain Pathol 2011;21:692-8. [18] Dibbens LM, Reid CA, Hodgson B, et al. Augmented currents of an HCN2 variant in patients with febrile seizure syndromes. Ann Neurol 2010;67:542-6. [19] Tu E, Bagnall RD, Duflou J, Semsarian C. Post-mortem review and genetic analysis of sudden unexpected death in epilepsy (SUDEP) cases. Brain Pathol 2011;21:201-8. [20] ABS. SIDS in Australia 1981-2000: A statistical overview. Canberra, Australia: Australian Bureau of Statistics; 2003. [21] The changing concept of sudden infant death syndrome: diagnostic coding shifts, controversies regarding the sleeping environment, and new variables to consider in reducing risk. Pediatrics 2005;116: 1245-55. [22] Tan BH, Pundi KN, Van Norstrand DW, et al. Sudden infant death syndrome–associated mutations in the sodium channel beta subunits. Heart Rhythm 2010;7:771-8.
7 [23] Otagiri T, Kijima K, Osawa M, et al. Cardiac ion channel gene mutations in sudden infant death syndrome. Pediatr Res 2008;64: 482-7. [24] Giudicessi JR, Ye D, Kritzberger CJ, et al. Novel mutations in the KCND3-encoded Kv4.3 K+ channel associated with autopsy-negative sudden unexplained death. Hum Mutat 2012; 33:989-97. [25] Hu D, Barajas-Martinez H, Medeiros-Domingo A, et al. A novel rare variant in SCN1Bb linked to Brugada syndrome and SIDS by combined modulation of Na(v)1.5 and K(v)4.3 channel currents. Heart Rhythm 2012;9:760-9. [26] Larsen MK, Berge KE, Leren TP, et al. Postmortem genetic testing of the ryanodine receptor 2 (RYR2) gene in a cohort of sudden unexplained death cases. Int J Legal Med 2013;127:139-44. [27] Greene D, Kang S, Kosenko A, Hoshi N. Adrenergic regulation of HCN4 channel requires protein association with beta2-adrenergic receptor. J Biol Chem 2012;287:23690-7. [28] Rodefeld MD, Beau SL, Schuessler RB, Boineau JP, Saffitz JE. Beta-adrenergic and muscarinic cholinergic receptor densities in the human sinoatrial node: identification of a high beta 2adrenergic receptor density. J Cardiovasc Electrophysiol 1996;7: 1039-49. [29] Ackerman MJ, Priori SG, Willems S, et al. HRS/EHRA expert consensus statement on the state of genetic testing for the channelopathies and cardiomyopathies this document was developed as a partnership between the Heart Rhythm Society (HRS) and the European Heart Rhythm Association (EHRA). Heart Rhythm 2011;8: 1308-39.
www.elsevier.com/locate/humpath
Original contribution
Postmortem review and genetic analysis in sudden infant death syndrome: an 11-year review☆ Angharad Evans BSc a , Richard D. Bagnall PhD a,b , Johan Duflou MBCHB b,c , Christopher Semsarian MBBS, PhD a,b,d,⁎ a
Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Sydney, NSW 2042, Australia Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia c Deparment of Forensic Medicine, Glebe, Sydney, NSW 2037, Australia d Department of Cardiology, Royal Prince Alfred Hospital, Sydney, NSW 2050, Australia b
Received 3 December 2012; revised 22 January 2013; accepted 31 January 2013
Keywords: Sudden infant death syndrome; HCN gene; Arrhythmia; Ion channel
Summary Sudden infant death syndrome (SIDS) is the unexpected death of a child younger than 1 year that remains unexplained after thorough evaluation. The possibility of an underlying primary arrhythmogenic disorder has been proposed as a potential cause of SIDS. This study sought to review SIDS deaths and to perform genetic analysis in key genes that may contribute to sudden death. From 2000 to 2010, all postmortem records from the Department of Forensic Medicine in Sydney, Australia, were reviewed. Cases that gave the cause of death as “SIDS” or “undetermined” but consistent with SIDS were included. In a subset of cases, the hyperpolarization-activated cyclic nucleotide (HCN)–gated channel family of genes (HCN2 and HCN4) was analyzed. A total of 226 SIDS cases were identified; 61% were male, 41% occurred while bed sharing, and there was a peak in deaths between 2 and 4 months old. The incidence did not decrease over the study period. In a subgroup of SIDS cases (n = 46), genetic analysis identified 2 likely pathogenic variants (2/46; 4%). A novel nonsynonymous variant, HCN4-Ala195Val, predicted to be pathogenic, was identified in a female infant who died at age 4 months. A female infant aged 5 weeks carried a rare nonsynonymous variant, HCN4-Val759Ile, which is similar to previously described variants associated with cardiac arrhythmias. In conclusion, the incidence of SIDS remains constant, with no apparent decline in the last decade. The underlying cause of SIDS remains largely unknown. Mutations in cardiac ion channel genes including rare nonsynonymous HCN gene variants may play a role in the pathogenesis of some SIDS cases. © 2013 Elsevier Inc. All rights reserved.
☆
A.E. is the recipient of a Splash of Red Foundation Scholarship. C.S. is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship. This study was also supported, in part, by a Heart Kids NSW project grant. ⁎ Corresponding author. Agnes Ginges, Centre for Molecular Cardiology, Centenary Institute, Locked Bag 6, Newtown, NSW 2042, Australia. E-mail address: [email protected] (C. Semsarian). 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.01.024
1. Introduction Sudden infant death syndrome (SIDS) is the unexpected death of a child younger than 1 year that remains unexplained after a detailed medical autopsy, death scene investigation, and clinical history evaluation [1]. This
2 profoundly tragic event is the leading cause of postneonatal infant death in Australia [2]. The incidence of SIDS in developed countries has dramatically reduced since the late 1980s owing to public awareness campaigns of modifiable environmental risk factors [3,4]. However, there remain more than 80 SIDS cases a year in Australia, a number that has not declined in the past decade [2]. The triple-risk hypothesis proposes that SIDS is a complex event by which an environmental stressor triggers an aberrant response in a genetically predisposed infant at a critical stage of development [5]. Exposure to secondhand smoke, prone sleeping position, bed sharing, and viral and bacterial infections have all been identified as environmental risk factors that may contribute to SIDS [6]. A number of genetic factors likely to predispose an infant to a fatal event, such as faults in genes encoding serotonin transporters, those vital to early development of the autonomic nervous system, and those that regulate inflammation and immune response or energy production, have been associated with SIDS; however, the mechanisms are poorly understood [7]. Pathogenic mutations in genes that encode cardiac ion channels are believed to account for up to 10% of SIDS cases [8–10]. Mutations that are responsible for heritable arrhythmias such long-QT syndrome (LQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic ventricular tachycardia (CPVT) may cause a fatal arrhythmia that leaves no structural damage identifiable at postmortem [11]. These mutations, which occur in cardiac ion channel genes, have more recently been described in a proportion of SIDS cases. The hyperpolarization-activated cyclic nucleotide (HCN)–gated channel family of genes (HCN1-4) are voltage-gated ion channels permeable to both potassium and sodium that are activated by both hyperpolarization and binding of cyclic adenosine monophosphate (cAMP) at a cyclic nucleotide-binding domain. Unlike the cardiacspecific channels that are associated with LQTS, BrS, and CPVT, HCN channels are responsible for generating spontaneous rhythmic activity at varying levels in both cardiac pacemaker cells and neurons in the brain [12–14]. Genetic variants in HCN4, expressed in the sinoatrial node of the heart, have been associated with abnormal cardiac rhythms, namely, sinus bradycardia, sinus node dysfunction, and sudden unexplained death in epilepsy (SUDEP) [15–17]. Genetic variants in HCN2, expressed ubiquitously throughout the heart, have been associated with generalized epilepsy and SUDEP, which may be resultant of a cardiac arrhythmia [17,18]. Genetic variants in HCN channel isoforms HCN2 and HCN4, because of their critical role in the cardiac pacemaker, may therefore contribute to a subset of SIDS deaths through deregulation of the heart rhythm and predisposition to a fatal cardiac arrest. This study sought to characterize a large cohort of SIDS cases from a review of postmortem reports and to perform genetic analysis of the HCN2 and HCN4 genes
A. Evans et al. to identify pathogenic DNA variants that may predispose to SIDS.
2. Materials and methods 2.1. Study populations Postmortem reports over an 11-year period from 2000 to 2010 from the Department of Forensic Medicine, Sydney, Australia, were reviewed. Reports of infants younger than 1 year with the cause of death given as “SIDS” or “undetermined” were reviewed in detail. The cohort included all categories of SIDS, and bed-sharing cases were not excluded. Demographic, clinical, and autopsy data were collected in all cases. This study was approved by the Office of the NSW State Coroner and performed in accordance with institutional human ethics guidelines.
2.2. Postmortem evaluation Although studies were performed over an 11-year period, in all cases, every effort was made to complete a comprehensive postmortem examination. This included detailed macroscopic and microscopic analysis, with particular attention to the heart and brain, careful histologic analysis, evidence of intrathoracic petechiae, toxicology and microbiology analysis, and, in some cases, metabolic testing. This study included all categories of SIDS, which incorporated both SIDS, as well as cases where the cause of death was undetermined but was consistent with SIDS.
2.3. Genetic analysis In a representative subgroup of SIDS cases (the most recent 50 cases), DNA was extracted from postmortem blood or liver tissue, as previously described [19]. Primers flanking the protein-coding regions and exon/intron junctions were used to amplify the 8 exons of HCN2 and HCN4, as previously described [17]. Polymerase chain reaction product for each case was visualized on a 2% agarose gel, sequenced, and analyzed for nucleotide variants using Sequencher v4.8 (Gene Codes Corp, Ann Arbor, MI). The following criteria were used to determine pathogenicity for nonsynonymous variants found in the SIDS population: (i) frequency of the variant in the National Heart, Lung and Blood Institute Exome Variant Server (EVS) (http://evs.gs. washington.edu/EVS/), a database of more than 6500 human exome sequences; (ii) conservation of the amino acid residue among orthologous proteins; and (iii) prediction of pathogenicity using the online resource PolyPhen2, which predicts the impact of an amino acid substitution on the structure and function of a human protein (http://genetics.bwh.harvard. edu/pph2/).
Postmortem review and genetic analysis in SIDS
3
3. Results
Table 1 Comparison of whole cohort vs most recent cases for genetic analysis
3.1. Cohort characteristics
Characteristic
Whole cohort Most recent cases P (for genetic analysis)
A total of 226 cases consistent with SIDS were identified. The characteristics of the SIDS cases are summarized in Fig. 1. The ages ranged from 2 days to 12 months, with 61% of the cohort being male and a peak in cases in the 2- to 4month period. Forty-one percent of cases occurred while bed sharing, and the number of deaths while bed sharing decreased as infants got older. There was no decrease in the incidence of SIDS over the 11-year period (Fig. 1).
n Age (mo), mean Age (mo), range Male sex, n (%) Bed sharing, n (%)
226 3.6 0-12 138 (61) 93 (41)
3.2. Genetic analysis In a subgroup of SIDS cases (n = 46), genetic analysis of the HCN2 and HCN4 genes was performed. This subgroup of cases was representative of the total SIDS group in terms of age, sex, and frequency of bed sharing (Table 1). The findings of the genetic analysis in this subgroup of cases are summarized in Table 2. DNA sequencing of all 8 exons and intron/exon junctions of the HCN4 gene identified 2 potentially pathogenic nonsynonymous DNA variants, that is, 1 novel (Ala195Val) and 1 rare (Val759Ile) nonsynonymous variant. The HCN4-Ala195Val variant, a c.1578CNT
50 3.3 0-11 32 (64) 21 (42)
NS NS NS NS
Abbreviations: n, number; NS, not statistically significant.
substitution, falls in the N-terminal cytosolic domain of the protein (Fig. 2), has not been reported in the EVS, and is predicted by Polyphen2 to be probably damaging (Polyphen2 score of 0.98). The Genomic Evolutionary Rate Profiling (GERP) score, measuring conservation of the individual nucleotide c.1578CNT, is 2.49. The amino acid is conserved in mammals. The HCN4-Val759Ile variant, a c.3269GNA, is a rare variant located in the C-terminal cytosolic domain following the cyclic nucleotide-binding domain (Fig. 2). It is reported in EVS at a frequency of 0.3% in a European population and 0.06% in an African American population and has a GERP nucleotide conservation score of 3.45, and Polyphen2 predicts this variant to be benign. The amino acid is conserved to the level of reptile. DNA
Fig. 1 SIDS cases in NSW from 2000 to 2010. A, Incidence of SIDS over the study period 2000 to 2010. B, Number of SIDS cases categorized by age at death. C, Number of SIDS cases categorized by age at death and whether the death occurred while bed sharing. D, Number of SIDS categorized by age at death and sex.
4
A. Evans et al. Table 2
Genetic variants identified in HCN2 and HCN4
Gene
HCN2
HCN4
Variant
rs56342526 rs56131056 rs55659726 rs56170955 rs56180027 rs55780677 rs56279131 rs12981860 rs3752158 rs34397648 rs2301778 rs1054786 rs55691080 Novel rs143090627 Novel Novel rs12909882 rs62641689 rs11781925 Novel Novel rs14273518
Exon
2 2 2 2 2 2 3 3 4 5 6 7 8 1 1 1 3 4 8 8 8 8 8
Amino acid change
MAF SIDS (n = 46)
ESV a
Asp238Asp Thr241Thr Tyr286Tyr Phe305Phe IIe307IIe Arg321Arg Ser362Ser Pro389Pro Leu413Leu Glu484Glu Ala548Ala Ala624Ala Ala751Ala Val19Val Glu36Gly Ala195Val a Ala414Ala Leu520Leu Val759Ile a Pro852Pro Ala913Ala Pro1042Pro Met1113Val a
0.109 0.109 0.109 0.109 0.109 0.109 0.011 0.163 0.032 0.108 0.283 0.283 0.217 0.011 0.065 0.011 0.011 0.087 0.011 0.022 0.011 0.011 0.022
0.100 0.101 0.105 0.105 0.105 0.103 0.023 0.360 0.064 0.122 0.300 0.425 NA NA 0.051 NA NA 0.091 0.003 0.049 NA NA 0.012
Abbreviations: MAF, minor allele frequency; EVS, exome variant server; NA, not available. a Indicates a nonsynonymous (amino acid changing) variant.
sequencing of all 8 exons and intron/exon junctions of the HCN2 gene revealed no nonsynonymous variants in the HCN2 gene.
4. Discussion This study describes a large cohort of SIDS cases with genetic screening for variants in the 2 HCN isoforms most expressed in the heart, HCN2 and HCN4, which have been previously associated with cardiac arrhythmias and sudden death. A total of 226 SIDS deaths were identified in the 11year review. The incidence of SIDS failed to decline from 2000 onward, in line with other recent reports that incidence of SIDS has remained constant in the past decade [3]. Our data show a clear male bias and a peak in cases in the 2- to 4-month age group. This is a trend seen in most SIDS cohorts and, as stated in the triple-risk hypothesis, suggests that there is a critical development period between 2 and 4 months, resulting in increased vulnerability of the infant. Genetic analysis identified a novel nonsynonymous variant, HCN4-Ala195Val, in a female infant who died at age 4 months and a rare nonsynonymous variant, HCN4Val759Ile, in a female infant aged 5 weeks. Collectively, these findings provide further support for the potential role of mutations in cardiac ion channel genes in the pathogenesis of some SIDS cases.
The precise mechanisms underlying the pathogenesis of SIDS are currently unclear. In Australia and other developed countries, strong campaigns aimed at encouraging reduction of modifiable risk factors for SIDS have seen a dramatic reduction of SIDS deaths from the late 1980s onward, with a 76% decrease in cases in Australia between 1986 and 2000 [20]. Bed sharing has been a major focus of SIDS awareness campaigns, and despite the efforts of such campaigns, 41% of deaths in our study occurred while bed sharing. The practice of bed sharing is controversial; however, safe-sleeping environment recommendations state that infants should not share a bed with any other person because of risk of accidental asphyxiation [21]. Interestingly, the number of deaths that occurred while bed sharing in our study fell dramatically as infants grew older, with 79% of SIDS deaths occurring while bed sharing in 0- to 2-month-old babies falling to 14% of cases in 10- to 12-month-old babies (Fig. 1C). This finding may be attributed to the likelihood that parents are more likely to bed share only with younger infants. Because SIDS is a diagnosis of exclusion, determining an underlying cause is extremely difficult and it is unlikely that there is one single cause common to all cases. A genetic susceptibility to the development of cardiac arrhythmias could be a potential cause in some cases because the negative autopsy mirrors adult sudden unexplained death stemming from fatal arrhythmias such as LQTS, BrS, and CPVT [11].
Postmortem review and genetic analysis in SIDS
5
Fig. 2 A, Protein schematic of HCN4 channel with 6 transmembrane domains (S1-S6) and the location of variants associated with disease. B, Location of DNA variants associated with SIDS (current study), SUDEP, and familial bradycardia is shown. C, Sequence chromatograms of SIDS HCN4 variants and conservation of the respective amino acids that are altered.
Functionally relevant mutations in cardiac ion channel genes that commonly cause LQTS (such as KCNQ1, KCNH2, and SCN5A) have been identified in up to 10% of SIDS cases [9,22,23]. Mutations in genes associated with BrS
(KCND3, SCN1Bb) [24,25] and CPVT (RyR2) [26] have been identified in a smaller subset of SIDS, supporting the role of cardiac channelopathies in predisposing to sudden death in SIDS. The HCN family of ion channels, particularly
6 HCN4, is what predominantly regulates the spontaneous rhythmic activity in the cardiac pacemaker cells at the sinoatrial node. Variants in these genes have previously been associated with negative postmortem deaths that may involve disturbances of the heart's electrical system, and as shown in this study, rare variants in these genes may contribute to a SIDS event [11]. The novel genetic findings in the current study provide further evidence of the role of ion channel genes in the pathogenesis of cardiac arrhythmias in SIDS cases. The HCN4-Ala195Val was carried on one allele in a 4-month-old female infant who was found deceased sleeping in the supine position alone in her crib. She was a healthy infant who had a slight fever 2 days prior. The postmortem examination failed to reveal a definitive cause of death, and no intrathoracic petechiae were noted. This variant has not been reported in any online databases and is located at a conserved nucleotide and amino acid residue. It occurs in the N-terminal cytosolic tail of the protein, an area not previously associated with disease-causing variants. Very little is known about the function of the N-terminus, although Greene et al [27] have shown in a recent study that there is a β2-adrenergic receptor binding site located in the N-terminal tail. β2-Adrenergic receptors are at a high density in the sinoatrial node where they colocalize with cardiac ion channels and are essential for proper cardiac function [28]. The alanine-to-valine substitution at amino acid position 195 may affect the ability of HCN4 to bind strongly to β2-adrenergic receptor, rendering it ineffective at responding to adrenergic stimulation. Because SIDS is hypothesized to be the inability of a predisposed infant to cope with an exogenous stressor, the inability for the cardiac pacemaker to increase its rate in response to such a stressor may have resulted in the death of this infant. The HCN4-Val759Ile variant was found on one allele of a 5-week-old female infant who was found deceased sharing a bed with her parents and whose postmortem revealed no cause of death, with no evidence of intrathoracic petechiae. HCN4-Val759Ile has been reported in European populations at a frequency of 0.3% and in an African American population at 0.06%, making it a rare allele. This infant, however, was an indigenous Australian for which there are no genetic data available. This variant has previously been reported in a SUDEP population at a frequency of 2% (Fig. 2) [17]. Apart from age, SUDEP cases mimic SIDS cases in many ways, including the demonstration of a negative autopsy and sudden death often occurring during sleep. The HCN4-Val759Ile variant is located in the Cterminal cytoplasmic tail of the protein. Mutations located at the C-terminal tail that have been shown to reduce HCN4 ion channel activity have previously been associated with familial sinus bradycardia (Fig. 2) [15]. The clinical significance of HCN4-Val759Ile remains unknown. However, its presence in this SIDS case, as well as in 2% of SUDEP cases, and its similarity to other variants associated with cardiac disease suggest that the variant may have a
A. Evans et al. modifying affect on ion channel activity, thereby altering susceptibility to sudden death. A genetic predisposition to SIDS may also have implications for the family and may warrant basic cardiac investigation of the parents including an electrocardiogram [29]. Although this is currently not routine practice, the possibility of genetic predisposition to SIDS implies that family evaluation at some level needs to be considered.
5. Conclusions SIDS is a profoundly tragic event, and despite attempts to reduce the environmental risk factors, it is not on the decline. Bed sharing accounts for a large proportion of all SIDS cases; however in many cases, the precise cause of SIDS remains unknown. Genetic alterations in ion channels that regulate cardiac rhythms may account for some cases of SIDS, as demonstrated by this report of 2 potentially pathogenic novel or rare variants in the HCN4 gene. Further studies are required to understand the exact mechanisms that predispose an otherwise healthy infant to develop SIDS.
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