James S. Ware

Analysis of protein-coding genetic variation in 60,706 humans

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human ‘knockout’ variants in protein-coding genes.

De novo mutations in congenital heart disease with neurodevelopmental and other congenital anomalies

Putting both heart and brain at risk For reasons that are unclear, newborns with congenital heart disease (CHD) have a high risk of neurodevelopmental disabilities. Homsy et al. performed exome sequence analysis of 1200 CHD patients and their parents to identify spontaneously arising (de novo) mutations. Patients with both CHD and neurodevelopmental disorders had a much higher burden of damaging de novo mutations, particularly in genes with likely roles in both heart and brain development. Thus, clinical genotyping of patients with CHD may help to identify those at greatest risk of neurodevelopmental disabilities, allowing surveillance and early intervention. Science, this issue p. 1262 Genotyping of children with congenital heart disease may identify those at high risk of neurodevelopmental disorders. Congenital heart disease (CHD) patients have an increased prevalence of extracardiac congenital anomalies (CAs) and risk of neurodevelopmental disabilities (NDDs). Exome sequencing of 1213 CHD parent-offspring trios identified an excess of protein-damaging de novo mutations, especially in genes highly expressed in the developing heart and brain. These mutations accounted for 20% of patients with CHD, NDD, and CA but only 2% of patients with isolated CHD. Mutations altered genes involved in morphogenesis, chromatin modification, and transcriptional regulation, including multiple mutations in RBFOX2, a regulator of mRNA splicing. Genes mutated in other cohorts examined for NDD were enriched in CHD cases, particularly those with coexisting NDD. These findings reveal shared genetic contributions to CHD, NDD, and CA and provide opportunities for improved prognostic assessment and early therapeutic intervention in CHD patients.

Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples

Purpose:The accurate interpretation of variation in Mendelian disease genes has lagged behind data generation as sequencing has become increasingly accessible. Ongoing large sequencing efforts present huge interpretive challenges, but they also provide an invaluable opportunity to characterize the spectrum and importance of rare variation.Methods:We analyzed sequence data from 7,855 clinical cardiomyopathy cases and 60,706 Exome Aggregation Consortium (ExAC) reference samples to obtain a better understanding of genetic variation in a representative autosomal dominant disorder.Results:We found that in some genes previously reported as important causes of a given cardiomyopathy, rare variation is not clinically informative because there is an unacceptably high likelihood of false-positive interpretation. By contrast, in other genes, we find that diagnostic laboratories may be overly conservative when assessing variant pathogenicity.Conclusions:We outline improved analytical approaches that evaluate which genes and variant classes are interpretable and propose that these will increase the clinical utility of testing across a range of Mendelian diseases.Genet Med 19 2, 192–203.

Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease

Truncating variants of the giant protein titin cause dilated cardiomyopathy when they occur toward the protein’s carboxyl terminus and in highly expressed exons. What Happens When Titins Are Trimmed? The most common form of inherited heart failure, dilated cardiomyopathy, can be caused by mutations in a mammoth heart protein, appropriately called titin. Now, Roberts et al. sort out which titin mutations cause disease and why some people can carry certain titin mutations but remain perfectly healthy. In an exhaustive survey of more than 5200 people, with and without cardiomyopathy, the authors sequenced the titin gene and measured its corresponding RNA and protein levels. The alterations in titin were truncating mutations, which cause short nonfunctional versions of the RNA or protein. These defects produced cardiomyopathy when they occurred closer to the protein’s carboxyl terminus and in exons that were abundantly transcribed. The titin-truncating mutations that occur in the general population tended not to have these characteristics and were usually benign. This new detailed understanding of the molecular basis of dilated cardiomyopathy penetrance will promote better disease management and accelerate rational patient stratification. The recent discovery of heterozygous human mutations that truncate full-length titin (TTN, an abundant structural, sensory, and signaling filament in muscle) as a common cause of end-stage dilated cardiomyopathy (DCM) promises new prospects for improving heart failure management. However, realization of this opportunity has been hindered by the burden of TTN-truncating variants (TTNtv) in the general population and uncertainty about their consequences in health or disease. To elucidate the effects of TTNtv, we coupled TTN gene sequencing with cardiac phenotyping in 5267 individuals across the spectrum of cardiac physiology and integrated these data with RNA and protein analyses of human heart tissues. We report diversity of TTN isoform expression in the heart, define the relative inclusion of TTN exons in different isoforms (using the TTN transcript annotations available at http://cardiodb.org/titin), and demonstrate that these data, coupled with the position of the TTNtv, provide a robust strategy to discriminate pathogenic from benign TTNtv. We show that TTNtv is the most common genetic cause of DCM in ambulant patients in the community, identify clinically important manifestations of TTNtv-positive DCM, and define the penetrance and outcomes of TTNtv in the general population. By integrating genetic, transcriptome, and protein analyses, we provide evidence for a length-dependent mechanism of disease. These data inform diagnostic criteria and management strategies for TTNtv-positive DCM patients and for TTNtv that are identified as incidental findings.

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Shared Genetic Predisposition in Peripartum and Dilated Cardiomyopathies

Background Peripartum cardiomyopathy shares some clinical features with idiopathic dilated cardiomyopathy, a disorder caused by mutations in more than 40 genes, including TTN, which encodes the sarcomere protein titin. Methods In 172 women with peripartum cardiomyopathy, we sequenced 43 genes with variants that have been associated with dilated cardiomyopathy. We compared the prevalence of different variant types (nonsense, frameshift, and splicing) in these women with the prevalence of such variants in persons with dilated cardiomyopathy and with population controls. Results We identified 26 distinct, rare truncating variants in eight genes among women with peripartum cardiomyopathy. The prevalence of truncating variants (26 in 172 [15%]) was significantly higher than that in a reference population of 60,706 persons (4.7%, P=1.3×10(-7)) but was similar to that in a cohort of patients with dilated cardiomyopathy (55 of 332 patients [17%], P=0.81). Two thirds of identified truncating variants were in TTN, as seen in 10% of the patients and in 1.4% of the reference population (P=2.7×10(-10)); almost all TTN variants were located in the titin A-band. Seven of the TTN truncating variants were previously reported in patients with idiopathic dilated cardiomyopathy. In a clinically well-characterized cohort of 83 women with peripartum cardiomyopathy, the presence of TTN truncating variants was significantly correlated with a lower ejection fraction at 1-year follow-up (P=0.005). Conclusions The distribution of truncating variants in a large series of women with peripartum cardiomyopathy was remarkably similar to that found in patients with idiopathic dilated cardiomyopathy. TTN truncating variants were the most prevalent genetic predisposition in each disorder.

Safety and Efficacy of Switching From Either Unfractionated Heparin or Enoxaparin to Bivalirudin in Patients With Non-ST-Segment Elevation Acute Coronary Syndromes Managed With an Invasive Strategy Results From the ACUITY (Acute Catheterization and Urgent Intervention Triage strategY) Trial

OBJECTIVES The aim of this study was to compare outcomes in patients receiving consistent unfractionated heparin (UFH)/enoxaparin (ENOX) therapy and in those switched at randomization to bivalirudin monotherapy. BACKGROUND Crossover between UFH and ENOX has been associated with increased adverse outcomes in patients with acute coronary syndromes. The ACUITY (Acute Catheterization and Urgent Intervention Triage strategY) trial demonstrated superior net clinical outcomes with similar rates of ischemia and significantly less major bleeding with bivalirudin monotherapy compared with UFH/ENOX plus a glycoprotein (GP) IIb/IIIa inhibitor. It is unknown if these results would be preserved in patients switched from UFH/ENOX to bivalirudin monotherapy. METHODS We compared composite ischemia, major bleeding, and net clinical outcomes at 30 days in patients receiving consistent UFH/ENOX therapy and in those switched at randomization from pre-treatment with UFH/ENOX to bivalirudin monotherapy. We also compared outcomes in patients naive to antithrombin therapy who were randomized to UFH/ENOX or bivalirudin monotherapy. RESULTS Two thousand one hundred thirty-seven patients received consistent UFH/ENOX (UFH n = 1,294, ENOX n = 843), and 2,078 patients pre-treated with UFH/ENOX were switched to bivalirudin. Patients switching to bivalirudin had similar rates of ischemia (6.9% vs. 7.4%, p = 0.52), less major bleeding (2.8% vs. 5.8%, p < 0.01), and improved net clinical outcomes (9.2% vs. 11.9%, p < 0.01) than those on consistent UFH/ENOX plus a GP IIb/IIIa inhibitor. Patients naive to antithrombin therapy who were administered bivalirudin (n = 1,427) had similar rates of ischemia (6.2% vs. 5.5%, p = 0.47), less major bleeding (2.5% vs. 4.9%, p < 0.001), and similar net clinical outcomes (8.0% vs. 9.4%, p = 0.17) compared with naive patients administered UFH/ENOX plus a GP IIb/IIIa inhibitor (n = 1,462). CONCLUSIONS Switching from UFH/ENOX to bivalirudin monotherapy results in comparable ischemic outcomes and an approximately 50% reduction in major bleeding compared with consistent UFH/ENOX plus a GP IIb/IIIa inhibitor. Patients naive to antithrombin therapy administered bivalirudin monotherapy had a significant reduction in bleeding and similar rates of ischemia compared with naive patients initiated with UFH or ENOX plus a GP IIb/IIIa inhibitor.

PRISM-E: Comparison of Integrated Care and Enhanced Specialty Referral Models in Depression Outcomes

OBJECTIVE This study, entitled Primary Care Research in Substance Abuse and Mental Health for the Elderly, examined six-month outcomes for older primary care patients with depression who received different models of treatment. METHODS Clinical outcomes were compared for patients who were randomly assigned to integrated care or enhanced specialty referral. Integrated care consisted of mental health services co-located in primary care in collaboration with primary care physicians. Enhanced specialty referral consisted of referral to physically separate, clearly identified mental health or substance abuse clinics. RESULTS A total of 1,531 patients were included; their mean age was 73.9 years. Remission rates and symptom reduction for all depressive disorders were similar for the two models at the three- and six-month follow-ups. For the subgroup with major depression, the enhanced specialty referral model was associated with a greater reduction in depression severity than integrated care, but rates of remission and change in function did not differ across models of care for major depression. CONCLUSIONS Six-month outcomes were comparable for the two models. For the subgroup with major depression, reduction in symptom severity was superior for those randomly assigned to the enhanced specialty referral group.

Three-year Results of the Prospective Evaluation of Radial Keratotomy (PERK) Study

The Prospective Evaluation of Radial Keratotomy (PERK) study is a nine-center clinical trial of a standardized technique of radial keratotomy in 435 patients who had simple myopia with a preoperative refractive error between -2.00 and -8.00 diopters (D). We report results for one eye of each patient. The surgical technique consisted of eight incisions using a diamond micrometer knife with the blade length determined by intraoperative ultrasonic pachymetry and the diameter of the central clear zone determined by the preoperative refractive error. At three years after surgery, 58% of eyes had refractive error within one diopter of emmetropia; 26% were undercorrected, and 16% were overcorrected by more than one diopter. Uncorrected visual acuity was 20/40 or better in 76% of eyes. The operation was more effective in eyes with a preoperative refractive error between -2.00 and -4.37 diopters. Between one and three years after surgery, the refractive error changed by 1.00 diopter or more in 12% of eyes, indicating a lack of stability in some eyes.

The families in recovery from stroke trial (FIRST): Primary study results

Objective: Social support and family ties are strong predictors of functional recovery after stroke; however, development of successful psychosocial intervention programs has been difficult. This study examined whether a family-systems intervention designed to influence social support and self-efficacy affects functional outcome in older stroke patients. Methods: Two hundred ninety-one community-residing survivors of ischemic stroke or nontraumatic cerebral hemorrhage from eight acute-care hospitals and rehabilitation centers were randomized to either psychosocial intervention (PSI) or usual care (UC). PSI involved up to 16 sessions conducted in the home by a mental health worker. Functional recovery (measured by the Barthel Index [BI] at 6 months postrandomization, inability to assess functioning because of illness or death) was the primary end point. Results: Functional recovery did not differ between UC and PSI in intention-to-treat analyses. In adjusted logistic regression, the odds of being functionally independent at 6 months was 60% higher in the intervention group, but this difference was not statistically significant (p = .31). Subgroup analyses revealed that PSI may be more effective in subjects with better psychologic and cognitive functioning and who required less inpatient rehabilitation. Conclusion: This study does not provide evidence for the efficacy of psychosocial intervention to improve functional recovery in stroke. Although PSI showed greater improvement than UC, the differences were not statistically significant. BASRS = Boston Aphasia Severity Rating Scale; BI = Barthel Index; CES-D = Center for Epidemiologic Studies Depression Scale; FIRST = Families in Recovery from Stroke Trial; MMSE = Mini-Mental Status Exam; NIH = National Institutes of Health; NIHSSI = National Institutes of Health Stroke Severity Index; PSI = psychosocial intervention; REFFI = recovery efficacy; RSS = received social support; SIS = Social Isolation Scale; TOAST = Trial of ORG 10172 in Acute Stroke Treatment; UC = usual care.