Identification of Mutations of the RYR2 in Sudden Infant Death Syndrome

Abstract

Background

Despite efforts by the National Education on Sleeping Environment to reduce sudden infant death syndrome (SIDS), it remains the leading cause of post-neonatal mortality. In Korea, the incidence of SIDS was estimated at 0.4 per 1,000 infants in 2022. Mutations in the ryanodine receptor 2 (RYR2) gene, known to be associated with catecholaminergic polymorphic ventricular tachycardia, have been implicated in cases of sudden death. However, genetic studies investigating the link between RYR2 mutations and SIDS have not been conducted in Korea.

Methods

We extracted DNA from archived formalin-fixed, paraffin-embedded myocardial tissues from 249 SIDS cases autopsied between 2005 and 2017. DNA analysis focused on sequencing key exons (3, 8, 14, 15, 37, 42, 44–47, 49, 50, 83, 87–91, 93–95, 97, 99, and 100–105) of the RYR2 gene, critical for its functional role.

Results

Among the 249 SIDS cases, 62% were male infants, with an average age of 124 days, all of Asian-Korean descent. We identified two previously unreported RYR2 variants in two Korean patients with SIDS, namely c.13175A>G (p.Lys4392Arg) and c.4652A>G (p.Asn1551Ser).

Conclusion

Our study identified two RYR2 variants (c.13175A>G/p.Lys4392Arg and c.4652A>G/p.Asn1551Ser) associated with SIDS through postmortem genetic analysis. Given the limited diagnostic yield, our findings underscore the importance of selectively performing molecular autopsies in cases with documented familial clinical history. This approach aims to enhance the quality of genetic counseling available to affected families.

Graphical Abstract

INTRODUCTION

The rates of SIDS have significantly declined, largely attributed to the "Back to Sleep" campaign promoting safe infant sleep environments. However, SIDS remains the leading cause of post-neonatal death, with an estimated incidence of 0.4 per 1,000 infants in Korea in 2022.1 Post-"Back to Sleep" campaigns, SIDS cases may be influenced by genetic factors. Studies indicate that subsequent siblings have a 4- to 5-fold increased relative risk of SIDS and that rates are higher in monozygotic twins compared to dizygotic twins.2,3

Ryanodine receptor 2 (RYR2) is predominantly expressed in cardiac muscle cells, where it mediates calcium release from the sarcoplasmic reticulum, essential for muscle contraction.4 The RYR2 gene spans 105 exons, making it one of the largest human genes, and produces an mRNA approximately 15 kb in length. Mutations in the cardiac RYR2 gene are implicated in 55% of catecholaminergic polymorphic ventricular tachycardia (CPVT) cases, manifesting as bidirectional and polymorphic ventricular tachycardia during physical and emotional stress.5 CPVT, a rare genetic disorder (prevalence: 1/10,000), is characterized by life-threatening arrhythmias in individuals with structurally normal hearts.5,6,7 Approximately 50–65% of CPVT type 1 cases are linked to RYR2 gene mutations.5 Several studies have also associated CPVT with sudden infant death syndrome (SIDS) and sudden unexpected death in infancy.8,9,10

In 2018, a comprehensive mutational analysis of long QT syndrome (LQTS)-associated genes (KCNQ1, SCN5A, KCNE1, KCNE2, KCNJ2, and CAV3) in 200 cases of Korean SIDS identified fifteen infants with R1193Q variants in SCN5A associated with LQTS.11 In this study, we hypothesized that genetic mutations in the RYR2 gene might contribute to some SIDS cases within a cohort of 249 cases. To our knowledge, this represents the first genetic study of a large cohort of SIDS cases investigating CPVT in Korea.

METHODS

Study participants

This retrospective study investigated genetic risk factors for CPVT among SIDS cases in Korea. Cases were sourced from nationwide infant death records spanning January 2005 to December 2017. SIDS is diagnosed by exclusion, defined as "the sudden death of an infant under one year of age that remains unexplained after a thorough investigation, including a complete autopsy, death scene examination, and clinical history review."12 Identified risk factors include male sex, prematurity, maternal alcohol or tobacco exposure, and sleeping positions.13,14,15,16,17

After comprehensive review of police documents, autopsy reports, and histological re-examination, 249 cases were selected and reclassified as SIDS IA and IB following the criteria defined by Krous et al.18 Molecular genetic screening was performed as described by Son et al.11 Genomic DNA was extracted from paraffin-embedded myocardial tissue blocks using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany). A targeted mutational analysis covered 33 exons of the RYR2 gene (3, 8, 10, 12, 14, 15, 37, 41, 42, 44–47, 49, 50, 83, 87–91, 93–97, 99–105), selected based on prior research.19,20 Real-time polymerase chain reaction (PCR) was employed for single nucleotide polymorphism detection, validated via multiplex genotyping strategies. Sequence analysis PCR utilized Prime STAR™ HS premix (TAKARA, Shiga, Japan), adhering to manufacturer guidelines.

Ethics statement

The present study protocol was reviewed and approved by the Institutional Review Board of Seoul National University Hospital (approval No. 2018-001). All data were analyzed anonymously. The requirement for informed consent was waived by the board.

RESULTS

We enrolled 249 patients with SIDS, among whom 62% were male infants, comprising 154 male and 95 female patients. The mean age was 124 days (median, 108 days; range, 31–238 days), and all infants were of Asian ethnicity (Fig. 1). SIDS IA category criteria were met in 25.3% of cases (n = 45), with the remaining classified as SIDS IB (Table 1). Among the 186 cases (74.7%) where a sleeping position before death was reported, 30.6% were in a prone or side position. Of those with a reported position at death, 38.4% were found in the prone or side position (Table 1).

Fig. 1. Age and sex distribution of patients with sudden infant death syndrome.

M = male, F = female.

Table 1. Classification of the sudden infant death syndrome cohort.

Characteristics			No. (%) of cases
Sex
	Male
		IAa	18
		IBa	136
	Female
		IAa	27
		IBa	68
Sleeping position
	Position placed to sleep (missing = 63)
		Supine	129 (69.3)
		Prone	23 (12.4)
		Side	34 (18.3)
	Position found at death (missing = 12)
		Supine	146 (61.6)
		Prone	65 (27.4)
		Side	26 (10.9)

^aSan Diego criteria.

Two variants in RYR2 associated with CPVT-related genes were identified in the patients with SIDS. Demographics, sleeping environments, and details of these RYR2 variants are presented in Table 2. The c.13175A>G variant, located in exon 90 of RYR2, causes a conserved amino acid change within the Ryanodine Receptor TM 4-6 domain (IPR009460) of the encoded protein sequence. This variant has been previously reported in adults with CPVT.15 The c.4652A>G variant, located in exon 34 of RYR2, confers gain-of-function properties and is classified as a variant of uncertain significance associated with CPVT. While observed in CPVT cases, it has not been reported in SIDS previously.21,22

Table 2. Demographics, history, investigations, and cardiac testing in two cases with genetic variants of RYR2 .

Variables		Case 1	Case 2
ID number		213	76
Sex		M	M
Age, days		60	53
Position placed to sleep		Supine	Supine
Position found at death scene		Supine	Supine
SIDS category		IA	IA
RYR2 variants
	Amino acid change	p.Lys4392Arg	p.Asn1551Ser
	Nucleotide change	c.13175A>G	c.4652A>G
	Region	90	34
	PolyPhen-2a	Possibly damaging	Possibly damaging
	SIFTb	Neutral	Neutral
	ACMG	Variant of uncertain significance	Variant of uncertain significance

RYR2 = ryanodine receptor 2, M = male, SIDS = sudden infant death syndrome, ACMG = American College of Medical Genetics and Genomics.

^aPolyPhen-2: http://genetics.bwh.harvard.edu/pph2; ^bSIFT: http://sift.jcvi.org.

DISCUSSION

CPVT is characterized by bidirectional and polymorphic ventricular tachycardia, typically triggered by physical activity or emotional stress. CPVT, a fatal arrhythmia, is predominantly caused by autosomal-dominant inheritance of RYR2 variants, although de novo variants have been observed in sporadic cases. Over 170 RYR2 mutations associated with skeletal and cardiac diseases have been documented.16,23 Tester et al.,10 in a study of 134 SIDS cases, identified two RYR2 variants displaying gain-of-function effects consistent with CPVT-associated variants.7 Similarly, a 2016 study involving 47 SIDS cases reported three RYR2 variants exhibiting similar gain-of-function effects observed in CPVT.5

In this study, we report two cases of Korean patients with SIDS with variants in genes associated with RYR2 mutations not previously identified. The RYR2 c.13175A>G/p. Lys4392Arg variant, found in 0.023% of East Asian populations,24 has been reported in Japanese youth and adult patients with CPVT and is considered a causative factor of CPVT.18,25

The RYR2 c.4652A>G/p. Asn1551Ser variant, which shows a slight increase in sensitivity to cytosolic Ca2+, has conflicting interpretations of pathogenicity based on in silico predictions related to CPVT26 but has also been identified in adult CPVT cases.18,19A recent review indicated that the overall diagnostic yield of gene variants in SIDS cases was substantially lower (14%) than in the Exome Aggregation Consortium (41%).27 Additionally, a study from New Zealand found no significant pathological variants in an unselected series of SIDS cases28 and identified only a few positive variants in cases with a family history of sudden death or cardiac arrhythmia, without identifying risk factors such as bed sharing. In unselected SIDS cases, the diagnostic rate of pathological variants associated with CPVT was extremely low, highlighting the importance of investigating family histories and conducting postmortem molecular studies.

Postmortem genetic analysis of sudden death, including SIDS, was introduced in forensic medicine in the mid-2000s. Despite global guidelines recommending molecular autopsies for sudden deaths,27 our 2018 study remains the only molecular autopsy of sudden death cases in Korea.8 In Korea, medicolegal autopsies are typically conducted at the discretion of public prosecutors in cases of suspected criminal activity. Consequently, many SIDS cases lack pathological investigation, hindering opportunities to explore family medical histories and limiting systematic genetic counseling for preventing sudden death among siblings. Addressing this gap requires forensic molecular autopsies, comprehensive genetic counseling, and thorough investigation of family histories by clinicians to effectively prevent SIDS.

Several limitations in interpreting our data must be addressed. Firstly, it is crucial to note that half of the RYR2 mutations associated with CPVT are known to arise de novo.29 However, the absence of parental DNA data in this SIDS cohort, due to Korea's medicolegal death investigation system as previously described, hampers our ability to fully discern the genetic contributions to SIDS. Hence, it remains unclear whether observed variants are de novo or inherited. Secondly, although the RYR2 c.13175A>G/p. Lys4392Arg and c.4652A>G/p. Asn1551Ser variants have been identified in CPVT and are classified as variants of uncertain significance based on in silico predictions, these variants may not be the sole monogenic causes of CPVT linked to SIDS. There remains a possibility that these variants are influenced by environmental or other biological factors, aligning with the triple-risk model of SIDS.30 Thirdly, we utilized formalin-fixed paraffin-embedded tissues, which may increase the likelihood of allelic dropout due to DNA fragmentation.31 To mitigate this issue, we performed DNA extraction and TaqMan SNP assays in triplicate for all point mutations, followed by sequence analysis.

Despite these limitations, our study boasts several strengths. It enabled us to elucidate the potential genetic associations between fatal cardiac diseases and post-"Back to Sleep" SIDS. Recent advancements in molecular autopsy have revealed that rare mutations associated with cardiac diseases, including CPVT, Brugada Syndrome, and LQTS, could account for up to 14% of SIDS cases.5,6,7,32 Additionally, to our knowledge, this study represents the first report focusing on genetic analysis of CPVT in Korean SIDS cases. Rare genes not previously observed in SIDS are etiological factors. This study identified two mutations in RYR2 in SIDS cases, which encode gain-of-function mutations in cardiomyocytes and appear to contribute to the cause of death, considering the sleep environments of the patients.

In conclusion, two RYR2 variants (c.13175A>G/p. Lys4392Arg and c.4652A>G/p. Asn1551Ser), previously unreported in SIDS, were identified in Korean SIDS cases through postmortem genetic analysis. Although the pathogenicity of these variants in CPVT has not been fully elucidated, our findings suggest that molecular autopsies should be selectively conducted in cases with documented familial clinical histories to enhance the quality and accessibility of genetic counseling for victims’ families. Additionally, it is crucial to investigate other factors contributing to the sudden death of vulnerable infants through comprehensive autopsy examinations, given the complex and multifactorial nature of SIDS etiology. Furthermore, establishing a national registry and investigative program for sudden young deaths, including genetic counseling, is imperative. This program aims to explore the genetic underpinnings of inherited cardiac conditions such as LQTS, Brugada Syndrome, and CPVT, as well as to identify and assess genetic risk factors for SIDS in Korea.