Functional reclassification of variants of uncertain significance in the HCN4 gene identified in sudden unexpected death.

The HCN4 gene encodes a subunit of the hyperpolarization-activated cyclic nucleotide-gated channel, type 4 that is essential for the proper generation of pacemaker potentials in the sinoatrial node. The HCN4 gene is often present in targeted genetic testing panels for various cardiac conduction system disorders and there are several reports of HCN4 variants associated with conduction disorders. Here, we report the in vitro functional characterization of four rare variants of uncertain significance (VUS) in HCN4, identified through testing a cohort of 296 sudden unexpected natural deaths. The variants are all missense alterations, leading to single amino acid changes: p.E66Q in the N-terminus, p.D546N in the C-linker domain, and both p.S935Y and p.R1044Q in the C-terminus distal to the CNBD. We also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which is utilized here as a negative control. Three of the HCN4 VUS (p.E66Q, p.S935Y, and p.R1044Q) had electrophysiological characteristics similar to the wild-type channel, suggesting that these variants are benign. In contrast, the p.D546N variant in the C-linker domain exhibited a larger current density, slower activation, and was unresponsive to cyclic adenosine monophosphate (cAMP) compared to wild-type. With functional assays, we reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time-consuming further study. Our studies also provide a new lead to investigate how a VUS located in the C-linker connecting the pore to the cAMP binding domain may affect the channel open state probability and cAMP response.

Genetic testing in sudden unexpected natural death in the young: New York City Office of Chief Medical Examiner's experience and perspective.

Postmortem genetic testing is a diagnostic tool that is becoming increasingly utilized. The benefits and limitations of genetic testing in cases of sudden, unexpected death in the young (≤ 40 years old) are reviewed from the perspective of the Office of Chief Medical Examiner of the City of New York, whose Molecular Genetics Laboratory, accredited by College of American Pathologists, has had 15 years of postmortem testing experience. Challenges to the interpretation and communication of testing results are highlighted, and opportunities for improving testing yield are discussed for age groups across the lifespan, from infancy to adulthood.

Sudden Death in Pediatric Patient With Dilated Cardiomyopathy Due to Founder Variant in NKX2-5: Case Report.

Background: The NKX2-5 gene encodes a transcription factor that plays a role in atrioventricular nodal and myocardial development. Pathogenic variants of NKX2-5 are associated with congenital heart disease and sudden cardiac death. The missense variant in this case is one of the more common ones in Northern Europe and has high penetrance in familial cases. To our knowledge, this is the youngest person who died due to this variant. Case summary: This was a healthy, asymptomatic 14-year-old male with well-managed mild congenital dilated cardiomyopathy who died unexpectedly in his home. Postmortem examination revealed the NKX2-5 pathogenic missense variant, p.Phe145Leu, as the only explicable cause of death. Discussion: We propose that immediate family members of those who die suddenly due to NKX2-5 disease undergo genetic counseling and longitudinal screening to include this gene, as pathogenic variants in the NKX2-5 gene may manifest in a time-dependent manner.

Cardiac genetic test yields and genotype-phenotype correlations from large cohort investigated by medical examiner's office.

BACKGROUND: Few reports describe the yield of postmortem genetic testing from medical examiners' offices or correlate genetic test results with autopsy-confirmed phenotypes from a large cohort. OBJECTIVES: To report results from cardiomyopathy- and cardiac arrhythmia-associated genetic testing in conjunction with autopsy findings of cases investigated at the United States' largest medical examiner office. METHODS: Postmortem cases tested from 2015 to 2022 with a cardiomyopathy- and cardiac arrhythmia-associated gene panel were reviewed. American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines were used to classify variant pathogenicity. Correlations of pathogenic/likely pathogenic variants (P/LPVs) with cardiac pathology were evaluated. RESULTS: The cohort included 1107 decedents of diverse ages and ethnicities. P/LPVs were detected in 87 (7.9%) cases, with 73 and 14 variants in cardiomyopathy and cardiac arrhythmia genes, respectively. Variants of uncertain significance were detected in 437 (39.5%) cases. The diagnostic yield (percentage of P/LPV) in decedents with cardiomyopathy (26.1%) was significantly higher than those without (P<.0001). The diagnostic yield was significantly lower in infants (0.7%) than older age groups (ranging from 1 to 74 years old, 5.7%-25.9%), which had no statistical difference between their yields. The diagnostic yields by cardiac autopsy findings were 54.0% for hypertrophic cardiomyopathy, 47.1% for arrhythmogenic cardiomyopathy, 20.0% for myocardial fibrosis, 19.0% for dilated cardiomyopathy, and 11.3% for myocarditis. Most P/LPVs were in MYBPC3, TTN, PKP2, SCN5A, MYH7, and FLNC. Ten P/LPVs were novel. CONCLUSIONS: Our results support the importance of performing postmortem genetic testing on decedents of all ages with cardiomyopathy, cardiac lesions insufficient to diagnosis a specific cardiomyopathy (e.g., myocardial fibrosis), and myocarditis. Combined postmortem cardiac examination and genetic analysis are advantageous in accurately determining the underlying cause of death and informing effective clinical care of family members.

Researching COVID to enhance recovery (RECOVER) tissue pathology study protocol: Rationale, objectives, and design.

IMPORTANCE: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or organ dysfunction after the acute phase of infection, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are poorly understood. The objectives of the Researching COVID to Enhance Recovery (RECOVER) tissue pathology study (RECOVER-Pathology) are to: (1) characterize prevalence and types of organ injury/disease and pathology occurring with PASC; (2) characterize the association of pathologic findings with clinical and other characteristics; (3) define the pathophysiology and mechanisms of PASC, and possible mediation via viral persistence; and (4) establish a post-mortem tissue biobank and post-mortem brain imaging biorepository. METHODS: RECOVER-Pathology is a cross-sectional study of decedents dying at least 15 days following initial SARS-CoV-2 infection. Eligible decedents must meet WHO criteria for suspected, probable, or confirmed infection and must be aged 18 years or more at the time of death. Enrollment occurs at 7 sites in four U.S. states and Washington, DC. Comprehensive autopsies are conducted according to a standardized protocol within 24 hours of death; tissue samples are sent to the PASC Biorepository for later analyses. Data on clinical history are collected from the medical records and/or next of kin. The primary study outcomes include an array of pathologic features organized by organ system. Causal inference methods will be employed to investigate associations between risk factors and pathologic outcomes. DISCUSSION: RECOVER-Pathology is the largest autopsy study addressing PASC among US adults. Results of this study are intended to elucidate mechanisms of organ injury and disease and enhance our understanding of the pathophysiology of PASC.

Molecular genetic characterization of sudden deaths due to thoracic aortic dissection or rupture.

BACKGROUND: Sudden deaths due to thoracic aortic dissection or rupture (TADR) are often investigated by forensic pathologists in the United States. Up to a quarter of reported TADR result from a highly penetrant autosomal dominant single gene variant. Testing genes associated with familial TADR provides an underlying etiology for the cause of death and informs effective sudden death prevention for at-risk family members. At the New York City Office of Chief Medical Examiner (NYC-OCME), TADR cases are routinely tested by the in-house, CAP-accredited Molecular Genetics Laboratory. In this retrospective study, TADR and cardiovascular cases were reviewed to understand the burden of TADR in sudden deaths, value of molecular diagnostic testing in TADR, and genotype-phenotype correlations in a demographically diverse TADR cohort. METHODS: Between July 2019 and June 2022, cases with in-house cardiovascular genetic testing at NYC-OCME were retrospectively reviewed. Twenty genes associated with familial TADR were analyzed using high throughput massive parallel sequencing on postmortem tissues or bloodspot cards. Variant interpretation was conducted according to ACMG/AMP guidelines. RESULTS: A total of 1078 cases were tested for cardiovascular genetic conditions, of which 34 (3%) had TADR. Eight of those TADR cases had a pathogenic or likely pathogenic variant (P/LPV), 4 had a variant of uncertain significance (VUS), and 22 cases were negative for variants in TADR genes. The molecular diagnostic yield using the TADR subpanel was 23.5%. The genes with the greatest prevalence of P/LPV were FBN1 (6), followed by TGFBR2 (2), TGFBR1 (1), and MYLK (1). Highly penetrant P/LPV in TGFBR2, FBN1, and TGFBR1 were found in TADR individuals who died younger than 34 years old. Two P/LPV in FBN1 were secondary findings unrelated to cause of death. P/LPV in FBN1 included five truncating variants located in the N-terminal domains and one missense variant involved in the disulfide bonds of the EGF-like domain. All P/LPV in TGFBR1 and TGFBR2 were missense or in-frame deletion variants located in the protein kinase catalytic domain. Three variants were first reported in this study. CONCLUSIONS: Molecular testing of familial TADR-associated genes is a highly effective tool to identify the genetic cause of TADR sudden deaths and benefits surviving at-risk families.

Women have less severe and extensive coronary atherosclerosis in fatal cases of ischemic heart disease: an autopsy study.

OBJECTIVE: The study aims to evaluate sex differences in extent and severity of coronary artery disease (CAD) and myocardial findings at autopsy among young people with fatal ischemic heart disease (IHD). BACKGROUND: Women with acute coronary syndrome are less likely than men to display obstructive CAD at angiography. This suggests unique mechanisms of acute coronary syndrome exist in women or may reflect prehospital death of women with the most severe CAD. METHODS: Reports of autopsies by the Office of the Chief Medical Examiner of New York City on people aged 21 to 54 years who died between January 1, 2006, and December 31, 2008, were reviewed. A total of 639 cases of death due to atherosclerotic or arteriosclerotic cardiovascular disease according to the medical examiner were analyzed. Significant CAD was defined as ≥75% cross-sectional area stenosis in an epicardial vessel or ≥50% left main. RESULTS: Women were less likely to have obstructive CAD (63% vs 77% of men, P = .002). There was pathologic evidence of myocardial infarction (MI) in 43% of cases, 17% of which had nonobstructive CAD. Frequency of MI did not vary by sex overall (38% of women vs 45% of men, P = .18) or among those without significant CAD (23% vs 29%, P = .45). CONCLUSIONS: Among young people determined at autopsy to have died of IHD, fewer women had obstructive CAD, consistent with angiographic data in other IHD syndromes. Pathologic evidence of MI may exist in the absence of obstructive CAD.

Using postmortem formalin fixed paraffin-embedded tissues for molecular testing of sudden cardiac death: A cautionary tale of utility and limitations.

For archived cases of previously young healthy individuals where cause of sudden death remains undetermined, formalin fixed paraffin-embedded tissues (FFPE) samples are often the only biological resource available for molecular testing. We aim to ascertain the validity of postmortem molecular analysis of 95 cardiac genes using the FFPE samples routinely processed in the offices of medical examiners - typical fixation time in formalin ranges from days to months. The study was conducted in the College of American Pathologists accredited Molecular Genetics Laboratory within the City of New York Office of Chief Medical Examiner. Twelve cases, with FFPE samples and corresponding non-formalin fixed samples (RNAlater-preserved tissues or bloodstain card), were chosen for testing results comparison. The methods of extracting DNA from FFPE samples using Covaris, Qiagen, and Promega products showed comparable results. The quality of the extracted DNA, the target-enriched DNA libraries of 95 cardiac genes using HaloPlex Target Enrichment system by Agilent Technologies, and sequencing results using Illumina Miseq instrument were evaluated. Compared to the sequencing results of the nonfixed samples, the FFPE samples were categorized into three groups: 1) Group 1 samples fixed in formalin 2-6 days, had greater than 55 % sequencing regions ≥30x and 94%-100% variant concordance. 2) Group 2 samples fixed in formalin for 8 days, showed intra-sample sequencing variations: the surface tissues showed 25%-27% extra variants (false positive) and 8.1%-9.7% missing variants (false negative), whereas the repeated core tissues showed reduced extra variants to 1.6 % and the false negative error was unchanged. 3) Group 3 samples fixed in formalin 29-136 days, had 2-55 % sequencing regions ≥30x, up to 52.2 % missed variants and up to 6.3 % extra variants. All reportable variants (pathogenic, likely pathogenic or variant of uncertain significance) identified in the nonfixed samples were also identified in FFPE, albeit three variants had low confidence variant calling. In summary, our study showed that postmortem molecular diagnostic testing using FFPE samples routinely processed by the medical examiners should be cautioned, as they are replete with false positive and negative results, particularly when sample fixation time is longer than 8 days. Saving non-formalin fixed samples for high fidelity molecular analysis is strongly encouraged.

Functional characterization of SCN10A variants in several cases of sudden unexplained death.

BACKGROUND: Multiple genome-wide association studies (GWAS) and targeted gene sequencing have identified common variants in SCN10A in cases of PR and QRS duration abnormalities, atrial fibrillation and Brugada syndrome. The New York City Office of Chief Medical Examiner has now also identified five SCN10A variants of uncertain significance in six separate cases within a cohort of 330 sudden unexplained death events. The gene product of SCN10A is the Nav1.8 sodium channel. The purpose of this study was to characterize effects of these variants on Nav1.8 channel function to provide better information for the reclassification of these variants. METHODS AND RESULTS: Patch clamp studies were performed to assess effects of the variants on whole-cell Nav1.8 currents. We also performed RNA-seq analysis and immunofluorescence confocal microcopy to determine Nav1.8 expression in heart. We show that four of the five rare 'variants of unknown significance' (L388M, L867F, P1102S and V1518I) are associated with altered functional phenotypes. The R756W variant behaved similar to wild-type under our experimental conditions. We failed to detect Nav1.8 protein expression in immunofluorescence microscopy in rat heart. Furthermore, RNA-seq analysis failed to detect full-length SCN10A mRNA transcripts in human ventricle or mouse specialized cardiac conduction system, suggesting that the effect of Nav1.8 on cardiac function is likely to be extra-cardiac in origin. CONCLUSIONS: We have demonstrated that four of five SCN10A variants of uncertain significance, identified in unexplained death, have deleterious effects on channel function. These data extend the genetic testing of SUD cases, but significantly more clinical evidence is needed to satisfy the criteria needed to associate these variants with the onset of SUD.

Functional characterization of ABCC9 variants identified in sudden unexpected natural death.

BACKGROUND: Genetic variation in ion channel genes ('channelopathies') are often associated with inherited arrhythmias and sudden death. Genetic testing ('molecular autopsies') of channelopathy genes can be used to assist in determining the likely causes of sudden unexpected death. However, different in silico approaches can yield conflicting pathogenicity predictions and assessing their impact on ion channel function can assist in this regard. METHODS AND RESULTS: We performed genetic testing of cases of sudden expected death in the New York City metropolitan area and found four rare or novel variants in ABCC9, which codes for the regulatory SUR2 subunit of KATP channels. All were missense variants, causing amino acid changes in the protein. Three of the variants (A355S, M941V, and K1379Q) were in cases of infants less than six-months old and one (H1305Y) was in an adult. The predicted pathogenicities of the variants were conflicting. We have introduced these variants into a human SUR2A cDNA, which we coexpressed with the Kir6.2 pore-forming subunit in HEK-293 cells and subjected to patch clamp and biochemical assays. Each of the four variants led to gain-of-function phenotypes. The A355S and M941V variants increased in the overall patch current. The sensitivity of the KATP channels to inhibitory 'cytosolic' ATP was repressed for the M941V, H1305Y and K1379Q variants. None of the variants had any effect on the unitary KATP channel current or the surface expression of KATP channels, as determined with biotinylation assays, suggesting that all of the variants led to an enhanced open state. CONCLUSIONS: All four variants caused a gain-of-function phenotype. Given the expression of SUR2-containing KATP channels in the heart and specialized cardiac conduction, vascular smooth muscle and respiratory neurons, it is conceivable that electrical silencing of these cells may contribute to the vulnerability element, which is a component of the triple risk model of sudden explained death in infants. The gain-of-function phenotype of these ABCC9 variants should be considered when assessing their potential pathogenicity.