Olson, Timothy M

Background: Spontaneous coronary artery dissection (SCAD) is an uncommon idiopathic disorder predominantly affecting young, otherwise healthy women. Rare familial cases reveal a genetic predisposition to disease. The aim of this study was to identify a novel susceptibility gene for SCAD. Methods: Whole-exome sequencing was performed in a family comprised of 3 affected individuals and filtered to identify rare, predicted deleterious, segregating variants. Immunohistochemical staining was used to evaluate protein expression of the identified candidate gene. The prevalence and spectrum of rare (<0.1%) variants within binding domains was determined by next-generation sequencing or denaturing high-performance liquid chromatography in a sporadic SCAD cohort of 675 unrelated individuals. Results: We identified a rare heterozygous missense variant within a highly conserved β-integrin–binding domain of TLN1 segregating with familial SCAD. TLN1 encodes talin 1—a large cytoplasmic protein of the integrin adhesion complex that links the actin cytoskeleton and extracellular matrix. Consistent with high mRNA expression in arterial tissues, robust immunohistochemical staining of talin 1 was demonstrated in coronary arteries. Nine additional rare heterozygous missense variants in TLN1 were identified in 10 sporadic cases. Incomplete penetrance, suggesting genetic or environmental modifiers of this episodic disorder, was evident in the familial case and 5 individuals with sporadic SCAD from whom parental DNA was available. Conclusions: Our findings reveal TLN1 as a disease-associated gene in familial and sporadic SCAD and, together with abnormal vascular phenotypes reported in animal models of talin 1 disruption, implicate impaired structural integrity of the coronary artery cytoskeleton in SCAD susceptibility.

Background Genetic causes of dilated cardiomyopathy (DCM) are incompletely understood. LRRC10 (leucine‐rich repeat–containing 10) is a cardiac‐specific protein of unknown function. Heterozygous mutations in LRRC10 have been suggested to cause DCM, and deletion of Lrrc10 in mice results in DCM. Methods and Results Whole‐exome sequencing was carried out on a patient who presented at 6 weeks of age with DCM and her unaffected parents, filtering for rare, deleterious, recessive, and de novo variants. Whole‐exome sequencing followed by trio‐based filtering identified a homozygous recessive variant in LRRC10 , I195T. Coexpression of I195T LRRC10 with the L‐type Ca 2+ channel (Ca v 1.2, β 2CN2 , and α 2 δ subunits) in HEK293 cells resulted in a significant ≈0.5‐fold decrease in I Ca,L at 0 mV, in contrast to the ≈1.4‐fold increase in I Ca,L by coexpression of LRRC10 (n=9–12, P <0.05). Coexpression of LRRC10 or I195T LRRC10 did not alter the surface membrane expression of Ca v 1.2. LRRC10 coexpression with Ca v 1.2 in the absence of auxiliary β 2CN2 and α 2 δ subunits revealed coassociation of Ca v 1.2 and LRRC10 and a hyperpolarizing shift in the voltage dependence of activation (n=6–9, P <0.05). Ventricular myocytes from Lrrc10 −/− mice had significantly smaller I Ca,L , and coimmunoprecipitation experiments confirmed association between LRRC10 and the Ca v 1.2 subunit in mouse hearts. Conclusions Examination of a patient with DCM revealed homozygosity for a previously unreported LRRC10 variant: I195T. Wild‐type and I195T LRRC10 function as cardiac‐specific subunits of L‐type Ca 2+ channels and exert dramatically different effects on channel gating, providing a potential link to DCM.

ABSTRACTNext‐generation sequencing technologies have afforded unprecedented characterization of low‐frequency and rare genetic variation. Due to low power for single‐variant testing, aggregative methods are commonly used to combine observed rare variation within a single gene. Causal variation may also aggregate across multiple genes within relevant biomolecular pathways. Kernel‐machine regression and adaptive testing methods for aggregative rare‐variant association testing have been demonstrated to be powerful approaches for pathway‐level analysis, although these methods tend to be computationally intensive at high‐variant dimensionality and require access to complete data. An additional analytical issue in scans of large pathway definition sets is multiple testing correction. Gene set definitions may exhibit substantial genic overlap, and the impact of the resultant correlation in test statistics on Type I error rate control for large agnostic gene set scans has not been fully explored. Herein, we first outline a statistical strategy for aggregative rare‐variant analysis using component gene‐level linear kernel score test summary statistics as well as derive simple estimators of the effective number of tests for family‐wise error rate control. We then conduct extensive simulation studies to characterize the behavior of our approach relative to direct application of kernel and adaptive methods under a variety of conditions. We also apply our method to two case‐control studies, respectively, evaluating rare variation in hereditary prostate cancer and schizophrenia. Finally, we provide open‐source R code for public use to facilitate easy application of our methods to existing rare‐variant analysis results.

Abstract Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) attributable to multifactorial molecular underpinnings. Multiple genetic loci have been implicated to increase the risk of disease, yet genotype-phenotype relationships remain poorly defined. Whole genome sequencing complemented by cardiac phenotype from five individuals in an HLHS-affected family enabled the identification of NOTCH1 as a prioritized candidate gene linked to CHD in three individuals with mutant allele burden significantly impairing Notch signaling in the HLHS-affected proband. To better understand a mechanistic basis through which NOTCH1 contributes to heart development, human induced pluripotent stem cells (hiPSCs) were created from the HLHS-affected parent-proband triad and differentiated into cardiovascular cell lineages for molecular characterization. HLHS-affected hiPSCs exhibited a deficiency in Notch signaling pathway components and a diminished capacity to generate hiPSC-cardiomyocytes. Optimization of conditions to procure HLHS-hiPSC-cardiomyocytes led to an approach that compensated for dysregulated nitric oxide (NO)-dependent Notch signaling in the earliest specification stages. Augmentation of HLHS-hiPSCs with small molecules stimulating NO signaling in the first 4 days of differentiation provided a cardiomyocyte yield equivalent to the parental hiPSCs. No discernable differences in calcium dynamics were observed between the bioengineered cardiomyocytes derived from the proband and the parents. We conclude that in vitro modeling with HLHS-hiPSCs bearing NOTCH1 mutations facilitated the discovery of a NO-dependent signaling component essential for cardiovascular cell lineage specification. Potentiation of NO signaling with small therapeutic molecules restored cardiogenesis in vitro and may identify a potential therapeutic target for patients affected by functionally compromised NOTCH1 variants.

ABSTRACTRare variants (RVs) have been shown to be significant contributors to complex disease risk. By definition, these variants have very low minor allele frequencies and traditional single‐marker methods for statistical analysis are underpowered for typical sequencing study sample sizes. Multimarker burden‐type approaches attempt to identify aggregation of RVs across case‐control status by analyzing relatively small partitions of the genome, such as genes. However, it is generally the case that the aggregative measure would be a mixture of causal and neutral variants, and these omnibus tests do not directly provide any indication of which RVs may be driving a given association. Recently, Bayesian variable selection approaches have been proposed to identify RV associations from a large set of RVs under consideration. Although these approaches have been shown to be powerful at detecting associations at the RV level, there are often computational limitations on the total quantity of RVs under consideration and compromises are necessary for large‐scale application. Here, we propose a computationally efficient alternative formulation of this method using a probit regression approach specifically capable of simultaneously analyzing hundreds to thousands of RVs. We evaluate our approach to detect causal variation on simulated data and examine sensitivity and specificity in instances of high RV dimensionality as well as apply it to pathway‐level RV analysis results from a prostate cancer (PC) risk case‐control sequencing study. Finally, we discuss potential extensions and future directions of this work.

Ebstein anomaly of the tricuspid valve (EA) can be associated with left ventricular non‐compaction (LVNC), a rare congenital cardiomyopathy. We report a 2 year‐old female with EA and severe tricuspid regurgitation, LVNC, pulmonary hypertension, and chronic biventricular systolic heart failure, who died during evaluation for cardiac transplantation. Gene panel testing revealed a heterozygous de novo missense mutation in TPM1, which encodes the cardiac sarcomeric thin filament protein α‐tropomyosin. The c.475G>A variant results in a p.Asp159Asn substitution, altering a highly conserved residue predicted to be damaging to protein structure and function. TPM1 is the second gene linked to EA with LVNC in humans, implicating overlap in the molecular basis of structural and myopathic heart disease. © 2016 Wiley Periodicals, Inc.

For inherited cardiomyopathies, abnormal sensitivity to intracellular calcium (Ca2+), incurred from genetic mutations, initiates subsequent molecular events leading to pathological remodeling. Here, we characterized the effect of β‐adrenergic stress in familial dilated cardiomyopathy (DCM) using human‐induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes (CMs) from a patient with RBM20 DCM. Our findings suggest that β‐adrenergic stimulation accelerated defective Ca2+ homeostasis, apoptotic changes, and sarcomeric disarray in familial DCM hiPSC‐CMs. Furthermore, pharmacological modulation of abnormal Ca2+ handling by pretreatment with β‐blocker, carvedilol, or Ca2+‐channel blocker, verapamil, significantly decreased the area under curve, reduced percentage of disorganized cells, and decreased terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling (TUNEL)‐positive apoptotic loci in familial DCM hiPSC‐CMs after β‐adrenergic stimulation. These translational data provide patient‐based in vitro analysis of β‐adrenergic stress in RBM20‐deficient familial DCM hiPSC‐CMs and evaluation of therapeutic interventions to modify heart disease progression, which may be personalized, but more importantly generalized in the clinic.

Objective Spontaneous coronary artery dissection (SCAD) is an under-recognised but important cause of myocardial infarction and sudden cardiac death. We sought to determine the role of medical and molecular genetic screening for connective tissue disorders in patients with SCAD. Methods We performed a single-centre retrospective descriptive analysis of patients with spontaneous coronary artery disease who had undergone medical genetics evaluation 1984–2014 (n=116). The presence or absence of traits suggestive of heritable connective tissue disease was extracted. Genetic testing for connective tissue disorders and/or aortopathies, if performed, is also reported. Results Of the 116 patients (mean age 44.2 years, 94.8% women and 41.4% with non-coronary fibromuscular dysplasia (FMD)), 59 patients underwent genetic testing, of whom 3 (5.1%) received a diagnosis of connective tissue disorder: a 50-year-old man with Marfan syndrome; a 43-year-old woman with vascular Ehlers–Danlos syndrome and FMD; and a 45-year-old woman with vascular Ehlers–Danlos syndrome. An additional 12 patients (20.3%) had variants of unknown significance, none of which was thought to be a definite disease-causing mutation based on in silico analyses. Conclusions Only a minority of patients with SCAD who undergo genetic evaluation have a likely pathogenic mutation identified on gene panel testing. Even fewer exhibit clinical features of connective tissue disorder. These findings underscore the need for further studies to elucidate the molecular mechanisms of SCAD.