Angharad M. Roberts

A genome-wide association study identifies two loci associated with heart failure due to dilated cardiomyopathy

AIMS Dilated cardiomyopathy (DCM) is a major cause of heart failure with a high familial recurrence risk. So far, the genetics of DCM remains largely unresolved. We conducted the first genome-wide association study (GWAS) to identify loci contributing to sporadic DCM. METHODS AND RESULTS One thousand one hundred and seventy-nine DCM patients and 1108 controls contributed to the discovery phase. Pools of DNA stratified on disease status, population, age, and gender were constituted and used for testing association of DCM with 517 382 single nucleotide polymorphisms (SNPs). Three DCM-associated SNPs were confirmed by individual genotyping (P < 5.0 10(-7)), and two of them, rs10927875 and rs2234962, were replicated in independent samples (1165 DCM patients and 1302 controls), with P-values of 0.002 and 0.009, respectively. rs10927875 maps to a region on chromosome 1p36.13 which encompasses several genes among which HSPB7 has been formerly suggested to be implicated in DCM. The second identified locus involves rs2234962, a non-synonymous SNP (c.T757C, p. C151R) located within the sequence of BAG3 on chromosome 10q26. To assess whether coding mutations of BAG3 might cause monogenic forms of the disease, we sequenced BAG3 exons in 168 independent index cases diagnosed with familial DCM and identified four truncating and two missense mutations. Each mutation was heterozygous, present in all genotyped relatives affected by the disease and absent in a control group of 347 healthy individuals, strongly suggesting that these mutations are causing the disease. CONCLUSION This GWAS identified two loci involved in sporadic DCM, one of them probably implicates BAG3. Our results show that rare mutations in BAG3 contribute to monogenic forms of the disease, while common variant(s) in the same gene are implicated in sporadic DCM.

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.

Next generation sequencing for clinical diagnostics and personalised medicine: implications for the next generation cardiologist

The fast moving field of genomic medicine is already impacting on clinical care and cardiologists are fortunate to be in a position to benefit early from the transformative advances in genomics. However, the challenges associated with genomics in the clinic in general, and with next generation sequencing technologies in particular, are significant and cardiologists need to be prepared if they wish to surf the wave of genomic opportunity. This paper presents an overview of the implications of next generation sequencing for clinical diagnostics and personalised medicine in the cardiology clinic.

Genotype–phenotype analysis of the branchio‐oculo‐facial syndrome

Branchio‐oculo‐facial syndrome (BOFS; OMIM#113620) is a rare autosomal dominant craniofacial disorder with variable expression. Major features include cutaneous and ocular abnormalities, characteristic facies, renal, ectodermal, and temporal bone anomalies. Having determined that mutations involving TFAP2A result in BOFS, we studied a total of 30 families (41 affected individuals); 26/30 (87%) fulfilled our cardinal diagnostic criteria. The original family with the 3.2 Mb deletion including the TFAP2A gene remains the only BOFS family without the typical CL/P and the only family with a deletion. We have identified a hotspot region in the highly conserved exons 4 and 5 of TFAP2A that harbors missense mutations in 27/30 (90%) families. Several of these mutations are recurrent. Mosaicism was detected in one family. To date, genetic heterogeneity has not been observed. Although the cardinal criteria for BOFS have been based on the presence of each of the core defects, an affected family member or thymic remnant, we documented TFAP2A mutations in three (10%) probands in our series without a classic cervical cutaneous defect or ectopic thymus. Temporal bone anomalies were identified in 3/5 patients investigated. The occurrence of CL/P, premature graying, coloboma, heterochromia irides, and ectopic thymus, are evidence for BOFS as a neurocristopathy. Intrafamilial clinical variability can be marked. Although there does not appear to be mutation‐specific genotype–phenotype correlations at this time, more patients need to be studied. Clinical testing for TFAP2A mutations is now available and will assist geneticists in confirming the typical cases or excluding the diagnosis in atypical cases.

Phenotype and Clinical Outcomes of Titin Cardiomyopathy.

Background Improved understanding of dilated cardiomyopathy (DCM) due to titin truncation (TTNtv) may help guide patient stratification. Objectives The purpose of this study was to establish relationships among TTNtv genotype, cardiac phenotype, and outcomes in DCM. Methods In this prospective, observational cohort study, DCM patients underwent clinical evaluation, late gadolinium enhancement cardiovascular magnetic resonance, TTN sequencing, and adjudicated follow-up blinded to genotype for the primary composite endpoint of cardiovascular death, and major arrhythmic and major heart failure events. Results Of 716 subjects recruited (mean age 53.5 ± 14.3 years; 469 men [65.5%]; 577 [80.6%] New York Heart Association function class I/II), 83 (11.6%) had TTNtv. Patients with TTNtv were younger at enrollment (49.0 years vs. 54.1 years; p = 0.002) and had lower indexed left ventricular mass (5.1 g/m2 reduction; padjusted = 0.03) compared with patients without TTNtv. There was no difference in biventricular ejection fraction between TTNtv+/− groups. Overall, 78 of 604 patients (12.9%) met the primary endpoint (median follow-up 3.9 years; interquartile range: 2.0 to 5.8 years), including 9 of 71 patients with TTNtv (12.7%) and 69 of 533 (12.9%) without. There was no difference in the composite primary outcome of cardiovascular death, heart failure, or arrhythmic events, for patients with or without TTNtv (hazard ratio adjusted for primary endpoint: 0.92 [95% confidence interval: 0.45 to 1.87]; p = 0.82). Conclusions In this large, prospective, genotype-phenotype study of ambulatory DCM patients, we show that prognostic factors for all-cause DCM also predict outcome in TTNtv DCM, and that TTNtv DCM does not appear to be associated with worse medium-term prognosis.

ZBTB17 (MIZ1) Is Important for the Cardiac Stress Response and a Novel Candidate Gene for Cardiomyopathy and Heart Failure.

Background—Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects. Methods and Results—We used cysteine and glycine-rich protein 3, a known cardiomyopathy gene, in a yeast 2-hybrid screen and identified zinc-finger and BTB domain-containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte–specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurin-dependent manner. Conclusions—We revealed new functions for ZBTB17 in the heart, a transcription factor that may play a role as a novel cardiomyopathy gene.

Republished review: Next generation sequencing for clinical diagnostics and personalised medicine: implications for the next generation cardiologist

The fast moving field of genomic medicine is already impacting on clinical care and cardiologists are fortunate to be in a position to benefit early from the transformative advances in genomics. However, the challenges associated with genomics in the clinic in general, and with next generation sequencing technologies in particular, are significant and cardiologists need to be prepared if they wish to surf the wave of genomic opportunity. This paper presents an overview of the implications of next generation sequencing for clinical diagnostics and personalised medicine in the cardiology clinic.

A gene-centric strategy for identifying disease-causing rare variants in dilated cardiomyopathy

PurposeWe evaluated strategies for identifying disease-causing variants in genetic testing for dilated cardiomyopathy (DCM).MethodsCardiomyopathy gene panel testing was performed in 532 DCM patients and 527 healthy control subjects. Rare variants in 41 genes were stratified using variant-level and gene-level characteristics.ResultsA majority of DCM cases and controls carried rare protein-altering cardiomyopathy gene variants. Variant-level characteristics alone had limited discriminative value. Differentiation between groups was substantially improved by addition of gene-level information that incorporated ranking of genes based on literature evidence for disease association. The odds of DCM were increased to nearly 9-fold for truncating variants or high-impact missense variants in the subset of 14 genes that had the strongest biological links to DCM (P <0.0001). For some of these genes, DCM-associated variants appeared to be clustered in key protein functional domains. Multiple rare variants were present in many family probands, however, there was generally only one “driver” pathogenic variant that cosegregated with disease.ConclusionRare variants in cardiomyopathy genes can be effectively stratified by combining variant-level and gene-level information. Prioritization of genes based on their a priori likelihood of disease causation is a key factor in identifying clinically actionable variants in cardiac genetic testing.

Titin: a phenotype-genotype descriptive comparison of dilated cardiomyopathy

Background Cardiovascular magnetic resonance (CMR) imaging is the gold standard imaging modality for characterization of dilated cardiomyopathy (DCM). We recently identified genetic variants that truncate the giant protein Titin (TTN) accounting for up to 25% of DCM cases: a discovery that will increase the utility of genetic testing and family screening in DCM. However, the clinical consequences of TTN truncating variants (TTNtv) are unknown. Here, we integrated the power of CMR and capacity of next generation sequencing (NGS) to assess the relationship between genotype and phenotype in a non-ischemic DCM population. Methods 319 patients referred for characterization of DCM were recruited and underwent quantitative CMR study with detailed phenotyping (Siemens Avanto or Siemens Sonata 1.5T scanners). Additional clinical data were collected. TTN genotypes were determined by target enrichment using SureSelect and NGS sequencing using the SOLiD 5500 platform. Results TTN truncating variants (TTNtv) were identified in 13% (n = 42) of cases, the commonest genetic cause of DCM. Table 1 compares the CMR and clinical characteristics of TTNtv-positive and TTNtv-negative cohorts. TTNtv-positive DCM patients had lower left ventricular (LV) ejection fraction (EF) (p = 0.02), thinner LV walls (p < 0.02) and higher incidence of sustained ventricular tachycardia (VT) (p = 0.001) which was independent of EF. No statistically significant difference in the prevalence of midwall fibrosis existed between the two groups. Multivariate linear regression models that considered age, sex, TTNtv presence and position (distance from the protein N-terminus) indicated that TTNtv position was significantly correlated with biventricular ejection fraction and indexed stroke volumes such that more distal truncations had worse indices of cardiac function. For example, a C-terminal TTNtv would be associated with an absolute reduction in LV EF of 18 ± 7%, (p = 0.005), as compared with an N-terminal TTNtv. Conclusions These data highlight the prevalence of TTNtv in this unselected DCM cohort and suggest that TTNtvpositive DCM patients may benefit from tailored clinical management. Sustained VT, LV wall thickness and LVEF influence DCM outcomes. The increased risk of sustained VT in TTNtv-positive DCM may warrant a lower threshold for device therapy, as recommended for DCM caused by LMNA mutations. As distal TTNtv were associated with worse LV function, more aggressive clinical management may be appropriate in patients carrying these variants. The phenotypic expression of genetic substrates can be highly variable, and CMR is central to the accurate phenotypic description of this common yet heterogenous condition. Enhanced molecular understanding may lead to strategies to alter disease course beyond current pharmacological and device based therapy.

Response to Shah et al: Using high-resolution variant frequencies empowers clinical genome interpretation and enables investigation of genetic architecture

Recent work by Shah and colleagues demonstrated that many variants in the ClinVar database are misclassified, and that disease-specific allele frequency (AF) thresholds can identify wrongly classified alleles by flagging variants that are too prevalent in the population to be causative of rare penetrant disease. While we agree with the main conclusions of this work, the authors compare their AF filtering approach to our recently published method, concluding that the method we advanced 9may be prone to removing potentially pathogenic variants9. This is incorrect. Here we demonstrate that our approach is robust, and further illustrate the power of disease-specific AF thresholds for investigating the genetic architecture of disease.