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.

Development of a residents' needs assessment-based novel pathology residency boot camp designed on the graduated release of responsibility model.

Boot camps in medical education have emerged as effective tools for addressing knowledge gaps and facilitating the integration of incoming residents into clinical training. We developed a pathology boot camp designed on a needs assessment survey, the graduated release of responsibility model, and active involvement of senior residents as educators. A comprehensive survey was administered to current first-year residents to assess educational needs to identify significant knowledge gaps and difficult areas. Based on these findings, a four-week, three-phase curriculum was developed, comprising introductory didactics, hands-on training, and mentorship under senior residents. Pre- and post-tests, as well as resident surveys, were used to evaluate the efficacy of the boot camp curriculum. Senior residents unanimously expressed the need for a boot camp, expressing challenges in histology, workflow, autopsy, and laboratory operations. Pre- and post-boot camp assessment tests showed a 15% knowledge improvement. Most pathology programs across the country have implemented structured pathology boot camps. The strengths of our boot camp included the emphasis on the previous year's residents' feedback, and the challenges that they encountered, serving as a comparison group; a graduated release of responsibility model; and the involvement of senior residents as designated mentors for the duration of the boot camp. The pathology boot camp presented here serves as a promising model for addressing foundational knowledge deficits among incoming residents. By tailoring the curriculum to meet specific educational needs and leveraging senior residents as educators, institutions can better prepare residents for the challenges of clinical training.

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.

Gastric bypass after liver transplantation.

Few data are available for assessing the outcomes of bariatric surgery for patients who have undergone orthotopic liver transplantation (OLT). The University of Minnesota bariatric surgery database and transplant registry were retrospectively reviewed to identify patients who had undergone OLT and then open Roux-en-Y gastric bypass (RYGB) surgery between 2001 and 2009. Comorbidity-appropriate laboratory values, body mass indices (BMIs), histopathology reports, and immunosuppressive regimens were collected. Seven patients were identified with a mean age of 55.4 ± 8.64 years and a mean follow-up of 59.14 ± 41.49 months from the time of RYGB. The mean time between OLT and RYGB was 26.57 ± 8.12 months. The liver disease etiologies were hepatitis C (n = 4), jejunoileal bypass surgery (n = 1), hemangioendothelioma (n = 1), and alcoholic cirrhosis (n = 1). There were 2 deaths for patients with hepatitis C 6 and 9 months after bariatric surgery due to multiple-organ dysfunction syndrome and metastatic esophageal squamous carcinoma, respectively. One patient with hepatitis C required a reversal of the RYGB because of malnutrition and an inability to tolerate oral intake. Four of the 7 patients had type 2 diabetes mellitus (T2DM), 4 had hypertension, and 6 patients had dyslipidemia. All patients were on immunosuppressive medications, but only 4 were on corticosteroids. Glycemic control was improved in all surviving patients with T2DM. The mean BMI was 34.27 ± 5.51 kg/m(2) before OLT and 44.34 ± 6.08 kg/m(2) before RYGB; it declined to 26.47 ± 5.53 kg/m(2) after RYGB. In conclusion, in this case series of patients undergoing RYGB after OLT, we observed therapeutic weight loss, improved glycemic control, and improved high-density lipoprotein levels in the presence of continued dyslipidemia. RYGB may have contributed to the death of 1 patient due to multiple-organ dysfunction syndrome.

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.

The next phase in patient safety education: Towards a standardized, tools-based pathology patient safety curriculum: A call to action from the Association of Pathology Chairs' Residency Program Directors Section Training Residents in Patient Safety Workgroup.

Patient safety education is a mandated Common Program Requirement of the Accreditation Council for Graduate Medical Education and for the Royal College of Physicians and Surgeons of Canada in all medical residency and fellowship programs. Although many hospitals and healthcare environments have general patient safety education tools for trainees, few to none focus on the unique training milieu of pathologists, including a mix of highly automated and manual error-prone processes, frequent multiplicity of events, and lack of direct patient relationships for error disclosure. We established a national Association of Pathology Chairs-Program Directors Section Workgroup focused on patient safety education for pathology trainees entitled Training Residents in Patient Safety (TRIPS). TRIPS included diverse representatives from across the United States, as well as representatives from pathology organizations including the American Board of Pathology, the American Society for Clinical Pathology, the United States and Canadian Academy of Pathology, the College of American Pathologists, and the Society to Improve Diagnosis in Medicine. Objectives of the workgroup included developing a standardized patient safety curriculum, designing teaching and assessment tools, and refining them with pilot sites. Here we report the establishment of TRIPS as well as data from national needs assessment of Program Directors across the country, who confirmed the need for a standardized patient safety curriculum.

Ethnic differences in out-of-hospital fatal pulmonary embolism.

BACKGROUND: In-hospital pulmonary embolism (PE) has been extensively studied in large populations; however, out-of-hospital fatal PE studies are rare. Here, we systematically evaluated a large number of decedents who suffered fatal PE outside of hospitals and were subsequently investigated by the New York City Office of Chief Medical Examiner. METHODS AND RESULTS: A total of 578 consecutive out-of-hospital fatal PE cases were analyzed. All underwent autopsy, toxicology, microbiology, and genetic testing. Incidence rates and baseline characteristics were analyzed. Race-adjusted incidence rates of out-of-hospital fatal PE (per 100 000 people per year) were as follows: blacks, 3.73 (95% confidence interval, 3.31 to 4.11); whites, 1.15 (95% confidence interval, 0.96 to 1.33); and Hispanics, 0.93 (95% confidence interval, 0.72 to 1.10). Overall, obesity (body mass index ≥30 kg/m(2)) was 2.5- to 3-fold higher in fatal PE cases than in the New York City population as a whole. Carrier frequencies for prothrombin G20210A in fatal PE were 2- to 10-fold higher than reported frequencies in ethnically matched controls. Cumulative distribution curves showed that compared with whites, blacks and Hispanics died at significantly younger ages (P<0.001). Univariate and multiple linear regression analyses showed that in addition to nonwhite ethnicity, heterozygous carriers for factor V Leiden (P=0.001) and obesity (P=0.002) are significantly associated with younger age at death. CONCLUSION: There are unique epidemiological differences in out-of-hospital fatal PE between ethnic groups in New York City.

Case-matched outcomes in bariatric surgery for treatment of type 2 diabetes in the morbidly obese patient.

OBJECTIVE: To compare the relative efficacy of medical management, the duodenal switch (DS), and the laparoscopic adjustable gastric band (LAGB) to the Roux-en-Y gastric bypass (RYGB) for treatment of type 2 diabetes mellitus (T2DM). BACKGROUND: The RYGB resolves T2DM in a high proportion of patients and is considered the standard operation for T2DM resolution in morbidly obese patients. However, no data exist comparing the efficacy of medical management and other bariatric operations to the RYGB for treatment of T2DM in comparable patient populations. METHODS: We performed a retrospective case-matched study of morbidly obese patients with T2DM who had undergone medical management (nonsurgical controls [NSC]; N = 29), LAGB (N = 30), or DS (N = 27) and were compared with matched T2DM patients who had undergone RYGB. Matching was performed with respect to age, sex, body mass index, and hemoglobin A1C (HbA1C). Outcomes assessed were changes in body mass index, HbA1C, and diabetes medication scores at 1 year. RESULTS: The Roux-en-Y gastric bypass produced greater weight loss, HbA1C normalization, and medication score reduction compared to both NSC and LAGB-matched cohorts. Duodenal switch produced greater reductions in HbA1C and medication score than RYGB, despite no greater weight loss at 1 year. Surgical complications were rarely life threatening. CONCLUSIONS: This study provides an important perspective about the comparative efficacy of LAGB, DS, and NSC to the RYGB for treatment of T2DM among obese patients. After 1 year of follow-up, RYGB is superior to NSC and LAGB with respect to weight loss and improvement in diabetes whereas DS is superior to RYGB in reducing HbA1C and medication score.

Genetic screening of relatives of decedents experiencing sudden unexpected death: medical examiner's office referrals to a multi-disciplinary cardiogenetics program.

Currently, no standardized system exists for evaluating and testing at-risk family members of decedents with abnormal post-mortem genetic testing in cases of sudden unexpected death (SUD). The goal of this study was to evaluate the outcomes of referrals made by an urban medical examiner's office to a multi-disciplinary cardiogenetics clinic. Relatives of decedents with pathogenic/likely pathogenic (P/LP) variants or variants of unknown significance (VUS) in genes known to be associated with cardiomyopathies and/or arrhythmias were identified by the New York City Office of Chief Medical Examiner and referred to the Cardiogenetics Clinic at Montefiore Medical Center. Familial referrals of 15 decedents (median 15 years, range 2 days to 57 years) were evaluated. Variants in 13 genes were identified among decedents (9 arrhythmia, 5 cardiomyopathy). P/LP variants were identified in both arrhythmia (RYR2, SCN5A) and cardiomyopathy syndrome (MYBPC3 (2), MYH7) genes. Thirty-two family members were referred, and 14 variants were detected. One pathogenic (MYBPC3) and two likely pathogenic (RYR2, MYH7) mutations were identified. Referral of at-risk family members of decedents who experienced SUD based on informative post-mortem genetic testing for cardiac and genetic evaluation is warranted, as family studies help to reclassify variants and prevent additional sudden death.

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.

Burden of Cardiomyopathic Genetic Variation in Lethal Pediatric Myocarditis.

BACKGROUND: Acute myocarditis (AM) is a well-known cause of sudden death and heart failure, often caused by prevalent viruses. We previously showed that some pediatric AM correlates with putatively damaging variants in genes related to cardiomyocyte structure and function. We sought to evaluate whether deleterious cardiomyopathic variants were enriched among fatal pediatric AM cases in New York City compared with ancestry-matched controls. METHODS: Twenty-four children (aged 3 weeks to 20 years) with death due to AM were identified through autopsy records; histologies were reviewed to confirm that all cases met Dallas criteria for AM and targeted panel sequencing of 57 cardiomyopathic genes was performed. Controls without cardiovascular disease were identified from a pediatric database and matched by genetic ancestry to cases using principal components from exome sequencing. Rates of putative deleterious variations (DV) were compared between cases and controls. Where available, AM tissues underwent viral analysis by polymerase chain reaction. RESULTS: DV were identified in 4 of 24 AM cases (16.7%), compared with 2 of 96 age and ancestry-matched controls (2.1%, P=0.014). Viral causes were proven for 6 of 8 AM cases (75%), including the one DV+ case where tissue was available for testing. DV+ cases were more likely to be female, have no evidence of chronic inflammation, and associate with sudden cardiac death than DV- cases. CONCLUSIONS: Deleterious variants in genes related to cardiomyocyte integrity are more common in children with fatal AM than controls, likely conferring susceptibility. Additionally, genetically mediated AM may progress more rapidly and be more severe.

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.

Low-grade myofibroblastic sarcoma of the heart causing sudden death.

A 5-year-old Asian girl collapsed at school and was dead on arrival to the hospital. At autopsy, she had a tumor arising from her aortic valve. Histology revealed a low-grade myofibroblastic sarcoma, which we believe occluded a coronary artery ostium, leading to sudden death. Primary cardiac neoplasms are rare. The most common primary cardiac neoplasm is the cardiac myxoma. Although uncommon, the most prevalent primary malignant tumors of the heart are sarcomas, and regardless of the subtype, the prognosis is typically poor. We present a case of a primary cardiac sarcoma presenting as sudden death in a 5-year-old Asian girl. Pathologic examination of the lesion confirmed that this was an atypical myofibroblastic neoplasm, consistent with a low-grade sarcoma. Pediatric cardiac sarcomas are rare in general, and this is the first reported case of a valvular low-grade myofibroblastic sarcoma presenting in a child.

Functional characterization of TRPM4 variants identified in sudden unexpected natural death.

BACKGROUND: The TRPM4 gene encodes the subunit of the Ca2+-activated nonselective cation channel, which is enriched in the specialized cardiac conduction system and Purkinje fibers. To date, several putative disease-causing variants in TRPM4 have been reported to be associated with cardiac arrhythmia and progressive conduction disease. Here, we report the functional effects of previously uncharacterized variants of uncertain significance (VUS) that we have found while performing a "genetic autopsy" in individuals who have suffered sudden unexpected death (SUD) in the New York City area. METHODS AND RESULTS: We have identified thirteen uncommon missense VUS in TRPM4 by testing 95 targeted genes implicated in channelopathy and cardiomyopathy in 330 cases of SUD. In several cases there were co-existing VUS in one or more other genes that were tested. We selected four TRPM4 VUS (C20S, A380V, L595V and I1082S) for functional characterization, since these cases lacked detectable variants in other genes of our testing panel. Two of the cases were infants, one was a child and one an adult. RNA-seq data analysis showed that the longer TRPM4b splice variant is predominantly expressed in adult and fetal human heart. We therefore used site-directed mutagenesis to introduce these variants in a TRPM4b cDNA. HEK293 cells were transfected with the cDNAs and patch clamping was performed to assess the functional consequences of the TRPM4 mutants. The TRPM4 current was recorded in excised patches and was significantly reduced by each of the mutants. The total protein level of TRPM4-C20S was markedly decreased, whereas the A380V and L595V mutants exhibited decreased surface expression. The TRPM4-A380V current rapidly desensitized following patch excision. CONCLUSIONS: Each of the VUS tested caused a defect in TRPM4 channel function via distinctly different mechanisms, hence, it lays the foundation for further co-segregation family studies and animal studies of the TRPM4 variants.