Forensic Science International 236 (2014) 38–45

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Is sudden unexplained nocturnal death syndrome in Southern China a cardiac sodium channel dysfunction disorder? Chao Liu a,1, David J. Tester b,1, Yiding Hou c,1, Wen Wang c, Guoli Lv a, Michael J. Ackerman b, Jonathan C. Makielski d, Jianding Cheng c,* a

Guangzhou Institute of Forensic Science, Guangzhou 510030, China Departments of Medicine, Pediatrics, and Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, United States Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China d Division of Cardiovascular Medicine, Department of Medicine, University of Wisconsin, Madison, WI 53792, United States b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 September 2013 Received in revised form 27 November 2013 Accepted 27 December 2013 Available online 7 January 2014

Sudden unexplained nocturnal death syndrome (SUNDS) remains an enigma to both forensic pathologists and physicians. Previous epidemiological, clinical, and pilot genetic studies have implicated that SUNDS is most likely a disease allelic to Brugada syndrome (BrS). We have performed postmortem genetic testing to address the spectrum and role of genetic abnormalities in the SCN5A-encoded cardiac sodium channel and its several associated proteins in SUNDS victims from Southern China. Genomic DNA extracted from the blood samples of 123 medico-legal autopsy-negative SUNDS cases and 104 sex-, age- and ethnic-matched controls from Southern China underwent comprehensive amino acid coding region mutational analysis for the BrS associated genes SCN5A, SCN1B, SCN2B, SCN3B, SCN4B, MOG1, and GPD1-L using PCR and direct sequencing. We identified a total of 7 unique (4 novel) putative pathogenic mutations (all in SCN5A; V95I, R121Q [2 cases], R367H, R513H, D870H, V1764D, and S1937F) in 8/123 (6.5%) SUNDS cases. Three SCN5A mutations (V95I, R121Q, and R367H) have been previously implicated in BrS. An additional 8 cases hosted rare variants of uncertain clinical significance (SCN5A: V1098L, V1202M, R1512W; SCN1B: V138I [3 cases], T189M [2 cases]; SCN3B: A195T). There were no nonsynonymous mutations found in SCN2B, SCN4B, MOG1, or GPD1-L. This first comprehensive genotyping for SCN5A and related genes in the Chinese Han population with SUNDS discovered 13 mutations, 4 of them novel, in 16 cases, which suggests cardiac sodium channel dysfunction might account for the pathogenesis of 7–13% of SUNDS in Southern China. ß 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Sudden unexplained nocturnal death syndrome (SUNDS) SCN5A gene Cardiac sodium channel b subunits MOG1 gene GPD1-L gene

1. Introduction As an ethnic and region specific natural death, sudden unexplained nocturnal death syndrome (SUNDS) or sudden unexplained death during sleep (SUDS), is a disorder that prevails predominantly in Southeast Asia [1] and has various synonyms in different countries such as the Philippines (bangungut) [2], Thailand (lai-tai) [1], Japan (pokkuri) [3], and China (sudden manhood death syndrome) [4]. The annual incidence of SUNDS has been reported to be as high as 43 per 100,000 people aged 20–40 years in the Philippines [5] and 38 per 100,000 people aged 20–49 years in Thailand [6]. In Southern China, the incidence is about 1

* Corresponding author at: Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, No. 74, Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China. Tel.: +86 20 87330704; fax: +86 20 87334353. E-mail address: [email protected] (J. Cheng). 1 These authors contributed equally to this work. 0379-0738/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.12.033

per 100,000 people [4]. These reported worldwide syndromes have an unusual clinical phenotype [1–4] in common: the vast majority of victims were apparently healthy young males between 20 and 50 years old; death mostly occurred at night during sleep with symptoms of moaning, tachypnea, and abrupt tic of limbs; gross autopsy and microscopic findings showed no morphological changes to elucidate the cause of death. Since its initial description in 1917 in the Philippines [2], SUNDS has remained an enigma to both forensic pathologists and clinicians. The ECG characteristics, as well as clinical phenotype among SUNDS survivors [1,7], strongly suggested that SUNDS is similar to Brugada syndrome (BrS), which is associated with loss-of-function mutations in the SCN5A-encoded cardiac sodium channel a subunit [8,9]. The linkage of SCN5A gene variation and SUNDS was originally established by Vatta et al. with the identification of three SCN5A mutations in 3 out of 10 probands with clinical evidence of SUNDS [10]. This association was subsequently confirmed by us with the identification of a reported BrS associated genetic variant in a sporadic SUNDS victim from Southern China following a

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targeted analysis of SCN5A [4]. Based on these studies, SUNDS and BrS are now considered to be most likely phenotypically, genetically, and functionally the same allelic disorder [10]. However, only limited genetic studies involving small SUNDS cohorts, single BrS susceptible candidate gene, and noncomprehensive ‘‘hot-spot’’ screening have been completed. Here, we performed postmortem genetic testing for 123 SUNDS cases from Southern China to determine the spectrum and prevalence of genetic abnormalities of the cardiac sodium channel (Nav1.5) macromolecular complex that have been implicated in the pathogenesis of BrS, including the SCN5A-encoded cardiac sodium channel a subunit [8,9], the SCN1B, SCN2B, SCN3B, and SCN4B-encoded cardiac sodium channel b subunits [11], MOG1encoded multicopy suppressor of Gspl protein [12], and GPD1-Lencoded glycerol-3-phosphate dehydrogenase like protein [13]. 2. Materials and methods 2.1. Subjects From 2005 to 2012, 123 sporadic SUNDS cases (mean age = 30.9  7.6 years, range 18–52 years) diagnosed by the Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University were collected. The inclusion criteria for SUNDS were as follows: (1) a Chinese Han male  17 years of age that was (2) previously healthy without any significant disease, (3) prior to experiencing a sudden unexpected death during sleep (4) and had a negative autopsy, toxicology, histology, and death-scene investigation resulting in an unexplained death. Genomic DNA collected from 104 unrelated healthy males (mean age = 33.7  8.2 years, range 20– 57 years) from Southern China served as controls. None of the control subjects had a history of syncope or cardiovascular disease. All participants or agents gave informed consent and the principles outlined in the Declaration of Helsinki were followed. The project was approved for human study by the ethics committee of Sun Yat-sen University. 2.2. Mutation analysis Genomic DNA was extracted from peripheral blood samples using the DNA Blood Midi Kit (Bo kun, Changchun, China). Using previously reported primers (some of primers were modified or designed by Primer Premier 5.0), all coding region exons and relevant exon-intron boundaries for SCN5A [14] (GenBank NM_198056.1), MOG1 (NM_016492.4), GPD1-L (NM_015141.3), SCN1B (NM_001037.4), SCN2B (NM_004588.4), SCN3B (NM_001040151.1), and SCN4B (NM_174934.3) were PCR amplified. The PCR products were then sequenced in both directions on an ABI 3730XL Automated DNA Sequencer (Applied Biosystems, Foster City, CA). The obtained sequence data were compared with the corresponding normal cDNA sequence. To assess the allele frequency of each identified genetic variant, 104 unrelated ethnically matched and region specific healthy controls were also sequenced. In order to be considered a putative pathogenic mutation, the genetic variant needed to be non-synonymous and absent in all ethnically matched controls as well as absent in four publicly available exome databases including the 1000 Genome Project [15] (http://www. 1000genomes.org/ensembl-browser, n = 1094 subjects; 381 Caucasian, 246 African-American, 286 Asians, and 181 Hispanics), the NHLBI GO Exome Sequencing Project [16] (http://evs.gs.washington.edu/EVS/, n = 6500 subjects; 4300 Caucasians and 2203 African-Americans), the Exome Chip Design [17] (http://genome. sphumich.edu/wiki/Exome_Chip_Design, n = 12,000 subjects), and the International HapMap Project (http://hapmap.ncbi. nlm.nih.gov/) [18].

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2.3. Statistical analysis Statistical analyses were conducted using SPSS (version 19.0) and a P value 12,000 subjects), the V1098L-SCN5A variant identified in a 26year-old SUNDS case, was previously seen in 1/131 ostensibly healthy Asian control subjects and the R1512W-SCN5A identified in a 38-year-old SUNDS case, was previously identified in 1/148 Hispanic controls [21]. Interestingly, R1512W-SCN5A was initially identified in a BrS patient by Rook [22] and was subsequently found in another BrS patient by Descheˆnes [23], and electrophysiological studies showed a slowing of both inactivation and recovery from inactivation in this mutant channel [23]. The SCN5A variant V1202M (Fig. 1) seen in a 32-year-old SUNDS case, was seen in 1/4300 European American exomes of the NHLBI exome sequencing project. The V138I-SCN1B variant was detected in 3/123 (2.4%) SUNDS cases but was also identified in 11/12,000 (0.09%) subjects belonging to the exome chip design and in 3/2203 (0.14%) African American subjects from the NHLBI exome sequencing project. The T189M-SCN1B variant was detected in 2/123 (1.6%) SUNDS cases but also identified in 12/869 (1.5%) unrelated individuals of diverse ethnicities from the international HapMap project. The A195T-SCN3B variant was detected in a

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Fig. 1. Sequencing chromatograms of the wild type and novel mutant in SCN5A. (A) Reverse sequencing chromatograms of R513 and R513H:C ! T transition at nucleotide position 1538 that led to the replacement of Arginine (R) by Histidine (H) at codon 513. (B) Forward sequencing chromatograms of D870 and D870H; G ! C transition at nucleotide position 2608 that led to the replacement of Aspartic acid (D) by Histidine (H) at codon 870. (C) Forward sequencing chromatograms of V1202 and V1202 M:G ! A transition at nucleotide position 3604 that led to the replacement of Valine (V) by Methionine (M) at codon 1202. (D) Forward sequencing chromatograms of V1764 and V1764D:T ! A transition at nucleotide position 5291 that led to the replacement of Valine (V) by Aspartic acid (D) at codon 1764. (E) Reverse sequencing chromatograms of S1937 and S1937F: G ! A transition at nucleotide position 5810 that led to the replacement of Serine (S) by Phenylalanine (F) at codon 1937.

31-year-old SUNDS victim, but was seen in 1/4300 European American exomes of the NHLBI exome sequencing project. 3.3. Common single nucleotide polymorphisms (SNPs) identified in Chinese SUNDS sporadic cases Nine common SCN5A single nucleotide polymorphisms (SNPs) were also identified including 3 non-synonymous coding region

(c.1673A > G, H558R, rs1805124; c.3269C > T, P1090L, rs1805125; and c.3578G > A, R1193Q, rs41261344), 3 synonymous coding region (c.87G > A, A29A, rs6599230; c.3183A > G, E1061E, rs7430407; and c.5457C > T, D1819D, rs1805126), and 4 intronic (c.1141-3C > A, rs41312433; c.3840 + 17G > A, rs45466091; c.4245 + 82A > G, rs6799868; and c.4299 + 53T > C, rs41312393) variants (Table 2). The genotype distributions of all the variants (except c.3183 A > G) were in Hardy–Weinberg equilibrium.

Table 1 Rare non-synonymous variants of the cardiac sodium channel genes in SUNDS. Case no.

Gene

Nucleotide change

Amino acid change

Age (years)

Vocation

Clinical data

Putative pathogenic mutations 43 SCN5A c.283 G > A 27 SCN5A c.362 G > A 62 SCN5A c.362 G > A 19 SCN5A c.1100 G > A 78 SCN5A c.1538 G > A 99 SCN5A c.2608 G > C 121 SCN5A c.5291 T > A 2 SCN5A c.5810 C > T

V95I R121Q R121Q R367H R513H* D870H* V1764D* S1937F*

21 31 28 32 38 27 42 39

Spark machine operator Unknown White collar General worker Cleaner Unknown Unknown Maintenance

Dead in bed, morning Died after convulsions during middle night sleep Dead in bed, morning Dead in bed, morning Dead in bed, morning Dead in bed, morning Died after convulsions during his nap Dead in bed, morning

Variants of uncertain clinical significance 12 SCN5A c.3292 G > T 92 SCN5A c.3604 G > A 46 SCN5A c.4534 C > T 34 SCN1B c.412 G > A 91 SCN1B c.412 G > A 94 SCN1B c.412 G > A 43 SCN1B c.566 C > T 77 SCN1B c.566 C > T 14 SCN3B c.583 G > A

V1098L V1202M R1512 W V138I V138I V138I T189M T189M A195T

26 32 38 34 21 40 21 38 31

Electrician Unknown Unknown Construction worker Unknown Unknown Spark machine operator Unknown Mason

Dead in bed, morning Dead in bed, morning Spasm during sleep prior to death in the morning Dead in bed, morning Dead in bed, morning Dead in bed, morning Dead in bed, morning Dyspnea during sleep in the morning prior to death Dead in bed, morning

This table represents non-synonymous variants identified in our SUNDS cases but absent among our 104 age-, sex-, and ethnicity-matched healthy controls. In order to meet our strict definition of putative pathogenic mutation, the variants must be absent in all publically available exome (n > 12,000 subjects) databases and published controls. * Novel genetic variants. Case# 43 hosted both an SCN5A mutation and SCN1B variant of uncertain clinical significance.

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Fig. 2. Predicted topology of SCN5A with the location of each mutation. The SCN5A-encoded cardiac voltage-gated sodium channel (Nav1.5), consists of 4 homologous domains (DI-DIV) that are connected by intracellular linkers. Each domain contains 6 transmembrane segments (S1–S6) and a reentrant loop that descends into the transmembrane region of the protein between segments S5 and S6 (pore). SCN5A mutations identified from the SUNDS cases in this study are indicated in the predicted topology of SCN5A. Putative pathogenic mutations are designated in yellow and rare variants of uncertain clinical significance are designated in white. The mutation R121Q was identified in 2 cases (2) The * represents novel genetic variants.

To assess the latent effect of these common polymorphisms on Chinese SUNDS, the genotype and allele frequency of theses 9 SNPs identified in 123 SUNDS cases were compared with that in 104 age, sex-, region-, and ethnically matched healthy controls of this study as well as their reported frequency in previously published Han Chinese BrS cases and controls (Table 3) [24–26]. In regards to the three common non-synonymous SCN5A polymorphisms (H558R [c.1673 A > G], P1090L [c.3269 C > T], and R1193Q [c.3578 G > A]) that were identified in our SUNDS population, only P1090L showed a statistically significant difference in allelic frequency between SUNDS and healthy controls. Interestingly, the healthy controls (CC = 98, CT = 7, TT = 1; C = 203, T = 9; minor allele

frequency [MAF] = 0.042) were more likely to have the c.3269 T (1090L) minor allele compared to the SUNDS (CC = 106, CT = 2, TT = 0; C = 214, T = 2; MAF = 0.0093) cases (p < 0.05, Table 3). Despite the previous reports of over-representation of the R558SCN5A (c.1673 G) minor allele among Han Chinese BrS patients (MAF = 0.26) compared with healthy controls (MAF = 0.104) [27], we did not observe a significant difference in genotype or allele frequency between our SUNDS cases (AA = 98, AG = 11, GG = 1; A = 207, G = 13; MAF = 0.059) and our controls (AA = 85, AG = 15, GG = 0; A = 185, G = 15; MAF = 0.075). Similarly, despite R1193QSCN5A (c.3578 G > A) as being previously reported as causal for BrS and SUNDS [10], we did not observe a significant difference in

Table 2 Characters of SCN5A SNPs in SUNDS cases. Region

Nucleotide (AA) change*

Genotype frequency

Allele frequency

Homo/Het/Case/Sample size**

HWx2

Exon-2

c.87 G > A (A29A)

GG/GA/AA 0.465/0.426/0.109

G/A 0.678/0.322

11/42/53/99

0.09

Intron-10

c.1141-3 C > A

CC/CA/AA 0.918/0.072/0.010

C/A 0.954/0.046

1/7/8/97

3.30

AA/AG/GG 0.883/0.099/0.018

A/G 0.932/0.068

1/11/12/110

1.12

Exon-12

c.1673 A > G (H558R)

Exon-17

c.3183 A > G (E1061E)

AA/AG/GG 0.000/0.000/1.000

A/G 0.000/1.000

105/0/105/105

–

Exon-18

c.3269 C > T (P1090L)

CC/CT/TT 0.981/0.019/0.000

C/T 0.991/0.009

0/2/2/108

0.01

Exon-20

c.3578 G > A (R1193Q)

GG/GA/AA 0.901/0.089/0.010

G/A 0.946/0.054

1/9/10/101

1.83

Intron-21

c.3840 + 17 G > A

GG/GA/AA 0.991/0.009/0.000

G/A 0.995/0.005

0/1/1/110

0

Intron-23

c.4245 + 82 A > G

AA/AG/GG 0.375/0.500/0.125

A/G 0.625/0.375

11/44/55/88

0.39

Intron-24

c.4299 + 53 T > C

TT/TC/CC 0.550/0.378/0.072

T/C 0.739/0.261

8/42/50/111

0.04

Exon-28

c.5457 C > T (D1819D)

CC/TC/TT 0.369/0.447/0.184

C/T 0.592/0.408

19/46/65/103

0.58

*

AA = Amino acid. Due to the quality and quantity limit of the blood samples, the sample size of each site that could be successfully analyzed is different. Homo = homozygote; Het = heterozygote; HW = Hardy Weinberg distribution. **

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Table 3 Distributions of Genotype and Allele frequencies of SNPs in SCN5A gene genotyped in SUNDS cases, healthy controls, reported Chinese BrS cases, and Chinese Han population. Nucleotide variant c.87 G > A (A29A) SUNDS cases Chinese BrS cases [24] Chinese Han population [24]

n

Genotype frequency

Allele frequency

GG 46 27 58

GA 42 18 55

AA 11 3 5

P Value

99 48 118

CC 89 73

CA 7 20

AA 1 1

P Value

97 94

AA 98 85 28 97

AG 11 15 15 21

GG 1 0 5 2

P Value

105 103

AA 0 0

AG 0 0

c.3269 C > T (P1090L) SUNDS cases Healthy controls

108 106

CC 106 98

c.3578 G > A (R1193Q) SUNDS cases Healthy controls Chinese Han population [26]

101 102 94

c.3840 + 17 G > A SUNDS cases Healthy controls

110 –

c.4245 + 82 A > G SUNDS cases Chinese BrS cases Chinese Han population [26]

88 48 116

c.4299 + 53 T > C SUNDS cases Chinese Han population [25]

111 101

c.1141-3 C > A SUNDS cases Healthy controls c.1673 A > G (H558R) SUNDS cases Healthy controls Chinese BrS cases [24] Chinese Han population [24] c.3183 A > G (E1061E) SUNDS cases Chinese Han population [25]

c.5457 C > T (D1819D) SUNDS cases Chinese BrS cases [24] Chinese Han population [24]

110 10,048 120

103 48 115

G 134 72 171

A 64 24 65

P Value

C 185 166

A

P Value

G 13 15 25 25

P Value

0.293 T (G117G, 5/111, rs3746255) in SCN1B, the intronic SNP c.237 + 27A > G (42/109, rs645675) and 30 UTR SNP c.*38C > T (1/102, rs8192614) in SCN2B, the intronic SNPs c.55 + 44C > T (27/114, rs3851102) and c.55 + 66T > C (17/114, rs8192616) in SCN3B, and the synonymous SNP c.174C > T (C58C, 3/105, rs45539032) and 30 UTR SNP c.*7C > T (2/113, rs79071006) in SCN4B. The previously reported intronic MOG1 SNP c.437 + 16C > T (41/104, rs869733) and GPD1-L 30 UTR SNP c.*18G > T (14/112, rs12629676) were also identified. There were no significant differences in genotype distributions or allele

frequencies for any of these common polymorphisms between our SUNDS and control groups. 4. Discussion For nearly 100 years, the pathogenic perplexity of SUNDS has remained a clinical conundrum for forensic pathologist and physicians. While various hypotheses, including nocturnal sleep respiratory disorders [28] and structural or functional abnormalities of the coronary arteries have been postulated [3], Brugada syndrome (BrS), appears to play a prominent role in the pathogenesis of SUNDS. In fact, in a study performed on 27 Thai men, including 17 SUDS survivors and 10 probable SUDS patients, 59% had a unique Right bundle branch block (RBBB) and STsegment elevation in leads V1 through V3, very similar to the type 1-BrS ECG pattern [1]. Furthermore, in a study by Sangwatanoroj, et al., 69% of their 13 SUD survivors displayed a spontaneous type-1 BrS ECG pattern using a standard 12 lead ECG and this prevalence increased to 92% when using the higher intercostal space leads V1– V3 [1]. SUNDS appears to be endemic to Southeast Asia and in particular the prevalence appears to be highest in the Philippines where annually as many as 43 per 100,000 young (20–40 years of age) Filipinos suffer a sudden unexplained death during sleep [5]. Interestingly, compared to the estimated prevalence of BrS in Europe of 1–5 per 10,000 [29] and 12 per 10,000 people in Japan

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[30], the prevalence of an individual with a type-1 BrS ECG pattern is approximately 20 per 10,000 people in the general Filipino population [31]. Collectively, these data strongly support the hypothesis that BrS serves as the underlying pathogenic basis for the majority of SUNDS. However, genetic studies examining the relationship between SUNDS and BrS have been primarily lacking. Loss-of-function mutations in the SCN5A-encoded cardiac sodium channel accounts for approximately 20–30% of BrS, the most common BrS genotype discovered to date [20]. In 2002, Vatta and colleagues were the first to establish a causal genetic link between SCN5A and SUNDS with discovery of three SCN5A missense mutations (R367H, A735 V, and R1192Q [now annotated as R1193Q]) in 3 out of 10 probands with clinical evidence of SUNDS; a prevalence strikingly similar to the established yield of SCN5A mutations in BrS [10]. Electrophysiological studies using heterologous expression and patch-clamp technique revealed a complete loss of sodium current for the R367H-SCN5A mutation, channels exhibiting currents with steady state activation voltage shifted to more positive potentials for the A735V-SCN5A mutation, and channels with an accelerated inactivation of the sodium channel current for the R1192Q-SCN5A mutation, all of which exemplify a BrS-like SCN5A loss-function electrophysiological signature [10]. It’s important to note that R1193Q has since been established as a common Asian polymorphism [26]. In fact, in our study this particular polymorphism was observed in approximately 10% of both our SUNDS cases and ethnically matched controls. Based on the previous electrocardiographic studies and their genetic interrogation of only 10 SUNDS cases followed by electrophysiological characterization of the SCN5A mutations identified, Vatta and colleagues suggested that ‘‘SUNDS and Brugada syndrome are phenotypically, genetically, and functionally the same disorder’’ [10]. Here in this study, we performed a comprehensive postmortem genetic analysis of SCN5A for 123 SUNDS cases. Overall, we identified a putative pathogenic SCN5A mutation in 6.5% of our SUNDS cases. Importantly, non-synonymous variants present in any of the publically available exome databases or cited in the literature as being identified in an ostensibly healthy individual, were not considered pathogenic but rather a variant of uncertain clinical significance. In total, 7 SCN5A mutations (V95I, R121Q [2 cases], R367H, R513H, D870H, V1764D, and S1937F) were identified in 8 cases. Although the biophysical characterization for V95I-SCN5A (originally found by Liang [19] in a Chinese male BrS patient) and R121Q-SCN5A (reported by Kapplinger [20] in 2 BrS patients) have not been performed, both have been indicated previously as BrS causing variants. The R367H-SCN5A mutation in the pore-linking region between segments S5 and S6 in DI, was initially reported in a SUNDS patient by Vatta [10] and failed to generate any sodium current [10,32,33]. This mutation was also reported by Kapplinger in 6 unrelated BrS cases [20]. The remaining four SCN5A mutations identified are novel. The R513H-SCN5A mutation localizes to the DI and DII intracellular linker adjacent to T512I and G514 C, two mutations that have been reported to cause cardiac conduction disease [34,35] and electrophysiologically present as mutant channels with a slower recovery from inactivation and more rapid transient inward current decay, respectively. In addition, this arginine residue at amino acid position 513 has been shown to undergo posttranslational methylation suggesting a role for this residue in posttranslational regulation of the cardiac sodium channel [36]. The D870H mutant in the pore-linking region between segments S5 and S6 in DII, is very close to the reported BrS related E867Q, R878C, and R878H mutations [20]. The V1764D-SCN5A mutation is in segment S6 of DIV. Interestingly, an alternative mutation of this site, V1764M was confirmed to result in LQTS through increased persistent sodium current [37]. The C-terminal S1937F-SCN5A

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mutation resides near the previously associated BrS mutations G1935S and E1938K [20]. All these relevant studies, together with their absence in controls, implicate these variants as putative pathogenic mutations responsible for SUNDS. Electrophysiological functional studies are being performed by our research team to elucidate the underlying biophysical mechanisms of these mutations. In addition to those cases hosting a putative pathogenic mutation, 8 SUNDS cases (6.5%) hosted rare non-synonymous variants of uncertain clinical significance. Whether or not these rare genetic variants may have contributed to tragic sudden death of these individuals is difficult to conclude. However, some of these variants have been previously associated with disease. For example, R1512W-SCN5A was originally identified in BrS patients [22,23] and showed abnormal biophysical phenotype. Yet, R1512W-SCN5A has been reported as being present in healthy controls [21]. While our 6.5% yield is clearly less than the 20% yield of SCN5A mutations in SUNDS reported by Vatta and the approximate 20– 30% reported yield of SCN5A mutations in BrS, one must recognize that the mutation detection yield may significantly differ depending on the ethnic and regional differences between cohorts as well as differences in mutation calling. In 2009, Kapplinger reported on an international compendium of SCN5A mutations in patients referred for BrS genetic testing from 9 different centers from around the world. While the overall yield was 21%, this yield ranged from 11–28% [20]. Interestingly, the lowest yield was among Japanese BrS referral cases (14 out of 130, 10.8%). However, Nakajima recently reported an SCN5A mutation detection yield of 28% in their 30 Japanese BrS patients [3]. In a 2004 study by Mok and Colleagues involving 36 Chinese BrS probands, SCN5A mutations were identified in only 5.5% [38]; the inclusion of two common SCN5A polymorphisms (R1193Q and V1951L) had prompted the original reporting of an erroneous yield of 14%. In addition, while our lower than expected SCN5A yield may suggest that SUNDS is not as strongly related to BrS as previously thought, one must keep in mind that there are now 14 different genes that have been implicated to cause BrS. Besides sodium channel dysfunction, mutations in genes resulting in either a loss of cardiac L-type calcium channel function (CACNA1C and CACN2B) [39], an increase in the transient outward potassium (Ito) current (KCND3) [40] or an increase in IKATP current (KCNJ8) [41] have been described to be causative for BrS. The extent to which mutations in these genes contribute to SUNDS is currently unknown. However, despite the identification of 14 BrS-susceptibility genes discovered to date, more than two-thirds of BrS remains unexplained genetically [42]. Because mutations in SCN5A currently account for the majority of genotype-positive BrS cases, we analyzed key components of the cardiac sodium channel macromolecular complex that have been previously shown to be attributed to the pathogenesis of BrS, including all four sodium channel beta subunits (SCN1B-4B) [11]. Interestingly, 4% of our SUNDS cases (3 with V138I and 2 with T189M) hosted rare SCN1B genetic variants. While V138I-SCN1B was observed in 3/123 (2.4%) of our SUNDS cohort, this variant was identified in only 11/12,000 (0.09%) subjects belonging to the exome chip design and in 3/2203 (0.14%) African American subjects from the NHLBI exome sequencing project. Unfortunately, these large exome databases vastly under-represent the Han Chinese population and therefore it is possible that V138I-SCN1B may be more common among Han Chinese than other ethnicities. The T189M-SCN1B variant, identified here in two SUNDS cases, was initially found in two unrelated patients with paroxysmal atrial fibrillation [43] and has recently been associated with epilepsy in Chinese [44]. These findings not only implicate SCN1B

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as a possible new susceptible gene for SUNDS, but also suggest the possibility that some SUNDS may be correlated with epilepsy. Sudden unexpected death in epilepsy (SUDEP) often occurs during sleep [45]. Whether or not our SCN1B positive cases had experience seizure-like activity prior to their death is unknown. None of our SUNDS cases carried a prior clinical diagnosis or family history of epilepsy. While there is compelling electrocardiographic evidence strongly suggesting that the majority of SUNDS is due to BrS, this may not be the underlying cause for some of these tragic deaths. Other cardiac channelpathies, including long QT syndrome type 1 (LQT1) and catecholamiergic polymorphic ventricular tachycardia (CPVT) have been previously associated with sudden death during sleep. In 2012, Tester et al. reported that 13% of their male sudden unexplained death cases with death during sleep in a SUD cohort of US were found to be mutation positive for KCNQ1 (LQT1, 5 cases) or RYR2 (CPVT1, 1 case) [46]. Most recently, we found two novel mutations in potassium channel gene (KCNQ1, KCNH2, KCNE1, KCNE2) [47] and no non-synonymous mutation in RYR2 gene in our SUNDS cohort. The lack of Asian exome server and biophysical characterization of these mutants maybe the limitations for accurately understanding the role of all these variants found in this study in the pathogenesis of SUNDS. 5. Conclusion In conclusion, our post-mortem genetic analysis for the largest cohort of SUNDS cases to date has revealed an SCN5A mutation detection yield of 6.5%, a considerably lower yield than the previous reported yield of 20–30% observed in BrS. The 7 plausible pathogenic mutations together with 6 rare variants in 16 out of 123 SUNDS cases found in cardiac sodium channel related genes may account for the genetic cause of 7–13% of Chinese SUNDS victims in this cohort. Our data suggest that a majority of Chinese SUNDS may be due to other mechanisms that lead to an increased risk for sudden death during sleep. However, in light of the mounting evidence for association between the type-1 BrS ECG and SUNDS, it is important to be cognitive of the current elusiveness of the genetic underpinnings of BrS. We anticipate that as novel mechanisms for BrS begin to emerge, a deeper understanding of the pathogenesis of Chinese SUNDS will surely follow. The functional analyses of the plausible pathogenic variants identified in this study and the exploration of new susceptible genes for the vast majority of SUNDS cases (>87%) are currently being performed by our research team to gain deeper insight in understanding the pathogenesis of Chinese SUNDS. Conflict of interest None declared. Sources of funding This work was supported by grants 81172901 & 81373238 (J.C.) from the National Natural Science Foundation of China, grant 11ykpy04 (to J.C.) from the Fundamental Research Funds for the Central Universities, and grant 2012BAK02B02-3 (to C.L.) from the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period. Acknowledgement We thank Mr. Terry Su (Sun Yat-sen University) for this manuscript’s review.

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