Francesco Mazzarotto

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.

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.

Titin-truncating variants affect heart function in disease cohorts and the general population

Titin-truncating variants (TTNtv) commonly cause dilated cardiomyopathy (DCM). TTNtv are also encountered in ∼1% of the general population, where they may be silent, perhaps reflecting allelic factors. To better understand TTNtv, we integrated TTN allelic series, cardiac imaging and genomic data in humans and studied rat models with disparate TTNtv. In patients with DCM, TTNtv throughout titin were significantly associated with DCM. Ribosomal profiling in rat showed the translational footprint of premature stop codons in Ttn, TTNtv-position-independent nonsense-mediated degradation of the mutant allele and a signature of perturbed cardiac metabolism. Heart physiology in rats with TTNtv was unremarkable at baseline but became impaired during cardiac stress. In healthy humans, machine-learning-based analysis of high-resolution cardiac imaging showed TTNtv to be associated with eccentric cardiac remodeling. These data show that TTNtv have molecular and physiological effects on the heart across species, with a continuum of expressivity in health and disease.

FineSplice, enhanced splice junction detection and quantification: a novel pipeline based on the assessment of diverse RNA-Seq alignment solutions

Alternative splicing is the main mechanism governing protein diversity. The recent developments in RNA-Seq technology have enabled the study of the global impact and regulation of this biological process. However, the lack of standardized protocols constitutes a major bottleneck in the analysis of alternative splicing. This is particularly important for the identification of exon–exon junctions, which is a critical step in any analysis workflow. Here we performed a systematic benchmarking of alignment tools to dissect the impact of design and method on the mapping, detection and quantification of splice junctions from multi-exon reads. Accordingly, we devised a novel pipeline based on TopHat2 combined with a splice junction detection algorithm, which we have named FineSplice. FineSplice allows effective elimination of spurious junction hits arising from artefactual alignments, achieving up to 99% precision in both real and simulated data sets and yielding superior F1 scores under most tested conditions. The proposed strategy conjugates an efficient mapping solution with a semi-supervised anomaly detection scheme to filter out false positives and allows reliable estimation of expressed junctions from the alignment output. Ultimately this provides more accurate information to identify meaningful splicing patterns. FineSplice is freely available at https://sourceforge.net/p/finesplice/.

Comparison of long-term outcome in anthracycline-related versus idiopathic dilated cardiomyopathy: a single centre experience: Outcome of anthracycline-related DCM

Cardiac dysfunction is a severe complication of anthracycline‐containing anticancer therapy. The outcome of anthracycline‐induced cardiomyopathy (AICM) compared with other non‐ischaemic causes of heart failure (HF), such as idiopathic dilated cardiomyopathy (IDCM), is unresolved. The aim of this study was to compare the survival of AICM patients with an IDCM cohort followed at our centre from 1990 to 2016.

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.

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.

Defining the diagnostic effectiveness of genes for inclusion in panels: the experience of two decades of genetic testing for hypertrophic cardiomyopathy at a single center

PurposeGenetic testing in hypertrophic cardiomyopathy (HCM) has long relied on Sanger sequencing of sarcomeric genes. The advent of next-generation sequencing (NGS) has catalyzed routine testing of additional genes of dubious HCM-causing potential. We used 19 years of genetic testing results to define a reliable set of genes implicated in Mendelian HCM and assess the value of expanded NGS panels.MethodsWe dissected genetic testing results from 1,198 single-center HCM probands and devised a widely applicable score to identify which genes yield effective results in the diagnostic setting.ResultsCompared with early panels targeting only fully validated sarcomeric HCM genes, expanded NGS panels allow the prompt recognition of probands with HCM-mimicking diseases. Scoring by “diagnostic effectiveness” highlighted that PLN should also be routinely screened besides historically validated genes for HCM and its mimics.ConclusionThe additive value of expanded panels in HCM genetic testing lies in the systematic screening of genes associated with HCM mimics, requiring different patient management. Only variants in a limited set of genes are highly actionable and interpretable in the clinic, suggesting that larger panels offer limited additional sensitivity. A score estimating the relative effectiveness of a given gene’s inclusion in diagnostic panels is proposed.

143 Clinical and Genetic Characteristics of Familial Dilated Cardiomyopathy in a Large UK Prospective Cohort

Background Up to fifty percent of idiopathic dilated cardiomyopathy (DCM) has a familial basis. Variants can occur in over 40 genes, though truncating variants in the sarcomeric gene titin account for the largest proportion (~20%). At least half of familial DCM cases are genetically orphan. We sought to study whether familial DCM was associated with distinct clinical characteristics, independently of the underlying genetic variant. Methods 595 prospectively recruited DCM patients underwent detailed phenotyping with cardiac MRI (Siemens scanners, 1.5T) and were sequenced using a customised panel of ~100 cardiomyopathy genes on Illumina and 5500xl platforms. Variants were identified and annotated using a customised bioinformatics pipeline. Clinical information including family pedigree data, ECG, and arrhythmia status at diagnosis (presence of confirmed ventricular or atrial arrhythmias) was collected on all patients. Familial DCM was defined as DCM occurring in 2 or more 1st or 2nd degree family members. Chi squared or Fisher’s exact test was used to compare across categorical variables and t-tests or Mann-Whitney U tests across continuous variables as appropriate. Results Overall, 16% of patients (95 out of 595) had familial DCM. Thirty individuals came from 13 families, the remaining were unrelated probands. Patients with familial DCM had an earlier age of disease onset (49.8 years vs 58.8 years, p < 0.0001). Non-familial DCM was characterised by a male preponderance (71% vs 56%, p = 0.004). Patients with familial DCM had less conduction disease at baseline (11% vs 36%, p < 0.0001). There was no difference in confirmed VT, NSVT or atrial fibrillation at baseline between groups. Patients with familial DCM had a milder intermediate phenotype of DCM (left ventricular ejection fraction 45.2% vs 38.2%, p < 0.0001). Right ventricular ejection fraction was similar in both groups (39.1% familial vs 37.1% non-familial, p = 0.14). There was no difference in the presence of mid wall fibrosis detected on late gadolinium imaging (p = 0.54). There were 44 potentially disease-causing variants in DCM genes in the familial DCM cohort (Table 1). Genetic testing had a yield of 44% in familial (n = 42), and 22% in non-familial DCM (n = 117). Five patients carried 2 variants. Truncating variants in titin were the most common variant (n = 17) and were over twice as common in patients with familial DCM compared to those without (18% vs 6.8%, p < 0.001). Truncating and missense variants in LMNA were ten times more frequent in familial DCM compared to non familial DCM (p < 0.001). Conclusions Disease causing variants in TTN and LMNA were more commonly associated with familial DCM, but 56% of patients with familial DCM remain genetically unexplained. This highlights the need for further novel DCM disease gene discovery. Our data show that familial DCM is characterised by a younger age of disease onset and less severe ventricular dysfunction as compared to non-familial DCM.Abstract 143 Table 1 Burden of variants in DCM genes in familial and non-familial DCM Gene Percentage of variants in familial DCM patients (N=95) (=total number of variants in cohort) Percentage of variants in non familial DCM patients (N=500) (=total number of variants in cohort) P value Titin (TTN) 22.1% (21) 7.6% (38) <0.001 Lamin A/C (LMNA) 6.3% (6) 1.2% (6) 0.006 Myosin heavy chain beta (MYH7) 6.3% (6) 4.2% (21) 0.42 Plakophilin 2 (PKP2) 4.2% (4) 4.0% (20) 1.0 Troponin T 2 (TNNT2) 3.2% (3) 1.2% (6) 0.16 RNA Binding Motif Protein 20 (RBM20) 2.1% (2) 4.4% (22) 0.40 Tropomyosin1 (TPM1) 1.1% (1) 0 0.16 BCL2-Associated Athanogene 3 (BAG3) 1.1% (1) 1.6% (8) 1