Tester, David J.

Background: Heterologous functional validation studies of putative long-QT syndrome subtype 2–associated variants clarify their pathological potential and identify disease mechanism(s) for most variants studied. The purpose of this study is to clarify the pathological potential for rare nonsynonymous KCNH2 variants seemingly associated with sudden infant death syndrome. Methods: Genetic testing of 292 sudden infant death syndrome cases identified 9 KCNH2 variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. Previous studies show R181Q-, P967L-, and R1047L-Kv11.1 channels function similar to wild-type Kv11.1 channels, whereas Q1068R-Kv11.1 channels accelerate inactivation gating. We studied the biochemical and biophysical properties for E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels expressed in human embryonic kidney 293 cells; examined the electronic health records of patients who were genotype positive for the sudden infant death syndrome–linked KCNH2 variants; and simulated their functional impact using computational models of the human ventricular action potential. RESULTS: Western blot and voltage-clamping analyses of cells expressing E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels demonstrated these variants express and generate peak Kv11.1 current levels similar to cells expressing wild-type-Kv11.1 channels, but R791W- and R1005W-Kv11.1 channels accelerated deactivation and activation gating, respectively. Electronic health records of patients with the sudden infant death syndrome–linked KCNH2 variants showed that the patients had median heart rate–corrected QT intervals <480 ms and none had been diagnosed with long-QT syndrome or experienced cardiac arrest. Simulating the impact of dysfunctional gating variants predicted that they have little impact on ventricular action potential duration. Conclusions: We conclude that these rare Kv11.1 missense variants are not long-QT syndrome subtype 2–causative variants and therefore do not represent the pathogenic substrate for sudden infant death syndrome in the variant-positive infants.

ABSTRACT Reduced levels of the cardiac human (h)ERG ion channel protein and the corresponding repolarizing current IKr can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. Here, we identified TRIOBP-1, an F-actin-binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction by performing Förster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 colocalized predominantly with myosin-binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native IKr and disrupted action potential repolarization. Ca2+ currents were also somewhat reduced and cell capacitance was increased. These findings establish that TRIOBP-1 interacts directly with hERG and can affect protein levels, IKr magnitude and cardiac membrane excitability.

The SCN5A-encoded voltage-gated mechanosensitive Na+channel NaV1.5 is expressed in human gastrointestinal smooth muscle cells and interstitial cells of Cajal. NaV1.5 contributes to smooth muscle electrical slow waves and mechanical sensitivity. In predominantly Caucasian irritable bowel syndrome (IBS) patient cohorts, 2–3% of patients have SCN5A missense mutations that alter NaV1.5 function and may contribute to IBS pathophysiology. In this study we examined a racially and ethnically diverse cohort of IBS patients for SCN5A missense mutations, compared them with IBS-negative controls, and determined the resulting NaV1.5 voltage-dependent and mechanosensitive properties. All SCN5A exons were sequenced from somatic DNA of 252 Rome III IBS patients with diverse ethnic and racial backgrounds. Missense mutations were introduced into wild-type SCN5A by site-directed mutagenesis and cotransfected with green fluorescent protein into HEK-293 cells. NaV1.5 voltage-dependent and mechanosensitive functions were studied by whole cell electrophysiology with and without shear force. Five of 252 (2.0%) IBS patients had six rare SCN5A mutations that were absent in 377 IBS-negative controls. Six of six (100%) IBS-associated NaV1.5 mutations had voltage-dependent gating abnormalities [current density reduction (R225W, R433C, R986Q, and F1293S) and altered voltage dependence (R225W, R433C, R986Q, G1037V, and F1293S)], and at least one kinetic parameter was altered in all mutations. Four of six (67%) IBS-associated SCN5A mutations (R225W, R433C, R986Q, and F1293S) resulted in altered NaV1.5 mechanosensitivity. In this racially and ethnically diverse cohort of IBS patients, we show that 2% of IBS patients harbor SCN5A mutations that are absent in IBS-negative controls and result in NaV1.5 channels with abnormal voltage-dependent and mechanosensitive function.NEW & NOTEWORTHY The voltage-gated Na+channel NaV1.5 contributes to smooth muscle physiology and electrical slow waves. In a racially and ethnically mixed irritable bowel syndrome cohort, 2% had mutations in the NaV1.5 gene SCN5A. These mutations were absent in irritable bowel syndrome-negative controls. Most mutant NaV1.5 channels were loss of function in voltage dependence or mechanosensitivity.

Background— WEMA (Whole-Exome Molecular Autopsy) and surveillance of cardiac channelopathy and cardiomyopathy genes represents the latest molecular autopsy for sudden death in the young (SDY). To date, the majority of WEMA has been performed on the SDY case only. Methods and Results— We performed whole-exome sequencing and nucleotide-level coverage analysis on 28 SDY cases (18.4±7.8 years) and their parents to determine the inheritance patterns of ultrarare, nonsynonymous variants in 99 sudden death–susceptibility genes. Nonsynonymous variants were adjudicated using the American College of Medical Genetics guidelines. Overall, 17 sudden death–susceptibility gene variants were identified in 12 of 28 (43%) SDY cases. On the basis of the American College of Medical Genetics guidelines, 6 of 28 (21%) cases had a pathogenic or likely pathogenic nonsynonymous variant with 3 (50%) being de novo. Two nonsynonymous variants would not have been elevated to likely pathogenic status without knowing their de novo status. Whole-exome sequencing reached a read depth of 10× across 90% of nucleotides within sudden death–susceptibility genes in 100% of parental exomes from fresh blood draw, compared with only 82% of autopsy-sourced SDY exomes. Conclusions— An SDY-parent, trio-based WEMA may be an effective way of elucidating a monogenic cause of death and bringing clarity to otherwise ambiguous variants. If other studies confirm this relatively high rate of SDY cases stemming from de novo mutations, then the WEMA should become even more cost-effective given that the decedent’s first-degree relatives should only need minimal cardiological evaluation. In addition, autopsy-sourced DNA demonstrated strikingly lower whole-exome sequencing coverage than DNA from fresh blood draw.

AbstractObjectiveTo identify the underlying genetic cause for recurrent intrauterine fetal death (IUFD) of males.MethodsWhole genome sequencing was performed on DNA from five healthy obligatory carrier females and an unaffected male offspring of a multigenerational pedigree with recurrent second‐trimester IUFD of males (n = 19). When documented, all deaths occurred at ≤20 weeks of gestation. Hydrops fetalis was diagnosed at death in the most recent case.ResultsFollowing variant filtering based on a recessive X‐linked inheritance pattern, a rare FOXP3 frameshift mutation (p.D303fs*87) that results in a premature truncation of the protein was discovered. Sanger sequencing confirmed the mutation in the affected fetus. The FOXP3 gene encodes for a transcriptional regulator critical to the function of regulatory T cells. FOXP3 mutations are associated with immune dysregulation, polyendocrinopathy, enteropathy, and X‐linked (IPEX) syndrome which exclusively affects males and may present with a potentially life‐threatening complex autoimmune disorder in early childhood.ConclusionsHere, we demonstrate the utility of whole genome sequencing‐based pedigree analysis to identify the genetic cause for recurrent IUFD when chromosome studies, including microarray analysis, are normal. Further studies are needed to determine the prevalence of FOXP3‐mediated IUFD in males. © 2017 John Wiley & Sons, Ltd.

Noonan syndrome (NS; MIM 163950) is an autosomal dominant disorder and a member of a family of developmental disorders termed “RASopathies,” which are caused mainly by gain-of-function mutations in genes encoding RAS/MAPK signaling pathway proteins. Whole exome sequencing (WES) and trio-based genomic triangulation of a 15-year-old female with a clinical diagnosis of NS and concomitant cardiac hypertrophy and her unaffected parents identified a de novo variant in MRAS -encoded RAS-related protein 3 as the cause of her disease. Mutation analysis using in silico mutation prediction tools and molecular dynamics simulations predicted the identified variant, p.Gly23Val-MRAS, to be damaging to normal protein function and adversely affect effector interaction regions and the GTP-binding site. Subsequent ectopic expression experiments revealed a 40-fold increase in MRAS activation for p.Gly23Val-MRAS compared with WT-MRAS. Additional biochemical assays demonstrated enhanced activation of both RAS/MAPK pathway signaling and downstream gene expression in cells expressing p.Gly23Val-MRAS. Mutational analysis of MRAS in a cohort of 109 unrelated patients with phenotype-positive/genotype-negative NS and cardiac hypertrophy yielded another patient with a sporadic de novo MRAS variant (p.Thr68Ile, c.203C>T). Herein, we describe the discovery of mutations in MRAS in patients with NS and cardiac hypertrophy, establishing MRAS as the newest NS with cardiac hypertrophy-susceptibility gene.

Background Variable expressivity and incomplete penetrance between individuals with identical long QT syndrome (LQTS) causative mutations largely remain unexplained. Founder populations provide a unique opportunity to explore modifying genetic effects. We examined the role of a novel synonymous KCNQ1 p.L353L variant on the splicing of exon 8 and on heart rate corrected QT interval (QTc) in a population known to have a pathogenic LQTS type 1 (LQTS1) causative mutation, p.V205M, in KCNQ1-encoded Kv7.1. Methods 419 adults were genotyped for p.V205M, p.L353L and a previously described QTc modifier (KCNH2-p.K897T). Adjusted linear regression determined the effect of each variant on QTc, alone and in combination. In addition, peripheral blood RNA was extracted from three controls and three p.L353L-positive individuals. The mutant transcript levels were assessed via qPCR and normalised to overall KCNQ1 transcript levels to assess the effect on splicing. Results For women and men, respectively, p.L353L alone conferred a 10.0 (p=0.064) ms and 14.0 (p=0.014) ms increase in QTc and in men only a significant interaction effect in combination with the p.V205M (34.6 ms, p=0.003) resulting in a QTc of ∼500 ms. The mechanism of p.L353L's effect was attributed to approximately threefold increase in exon 8 exclusion resulting in ∼25% mutant transcripts of the total KCNQ1 transcript levels. Conclusions Our results provide the first evidence that synonymous variants outside the canonical splice sites in KCNQ1 can alter splicing and clinically impact phenotype. Through this mechanism, we identified that p.L353L can precipitate QT prolongation by itself and produce a clinically relevant interactive effect in conjunction with other LQTS variants.