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Background-A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation. Methods and Results-We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified 6 TNNT2 protein-altering variants in 9 probands, all who had early onset, aggressive disease. Additional family members of mutation carriers were then studied when available. Four of the 9 probands had DCM without a family history, and 5 probands had familial DCM. Only 1 mutation (Lys210del) could be attributed as definitively causative from previous reports. Four of the 5 missense mutations were novel (Arg134Gly, Arg151Cys, Arg159Gln, and Arg205Trp), and one was previously reported with hypertrophic cardiomyopathy (Glu244Asp). Based on the clinical, pedigree, and molecular genetic data, these 5 mutations were considered possibly or likely disease causing. To further clarify their potential pathophysiologic impact, we undertook functional studies of these mutations in cardiac myocytes reconstituted with mutant troponin T proteins. We observed decreased Ca 2ϩ sensitivity of force development, a hallmark of DCM, in support of the conclusion that these mutations are disease causing. Conclusions-We

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Our insight into the diverse and complex nature of dilated cardiomyopathy (DCM) genetic architecture continues to evolve rapidly. The foundations of DCM genetics rest on marked locus and allelic heterogeneity. While DCM exhibits a Mendelian, monogenic architecture in some families, preliminary data from our studies and others suggests that at least 20% to 30% of DCM may have an oligogenic basis, meaning that multiple rare variants from different, unlinked loci, determine the DCM phenotype. It is also likely that low-frequency and common genetic variation contribute to DCM complexity, but neither has been examined within a rare variant context. Other types of genetic variation are also likely relevant for DCM, along with gene-by-environment interaction, now established for alcohol- and chemotherapy-related DCM. Collectively, this suggests that the genetic architecture of DCM is broader in scope and more complex than previously understood. All of this elevates the impact of DCM genetics research, as greater insight into the causes of DCM can lead to interventions to mitigate or even prevent it and thus avoid the morbid and mortal scourge of human heart failure.

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Background— Mutations in the LMNA gene, encoding lamins A/C, represent a significant cause of dilated cardiomyopathy. We recently identified 18 protein-altering LMNA variants in a cohort of 324 unrelated patients with dilated cardiomyopathy. However, at least one family member with dilated cardiomyopathy in each of 6 pedigrees lacked the LMNA mutation (nonsegregation), whereas small sizes of 5 additional families precluded definitive determinations of segregation, raising questions regarding contributions by those variants to disease. Methods and Results— We have consequently expressed, in COS7 cells, GFP-prelamin A (GFPLaA) fusion constructs incorporating the 6 variants in pedigrees with nonsegregation (R101P, A318T, R388H, R399C, S437Hfsx1, and R654X), the 4 variants in pedigrees with unknown segregation (R89L, R166P [in 2 families], I210S, R471H), and 3 additional missense variants (R190Q, E203K, and L215P) that segregated with disease. Confocal immunofluorescence microscopy was used to characterize GFP-lamin A localization and nuclear morphology. Abnormal phenotypes were observed for 10 of 13 (77%) variants (R89L, R101P, R166P, R190Q, E203K, I210S, L215P, R388H, S437Hfsx1, and R654X), including 4 of 6 showing nonsegregation and 3 of 4 with uncertain segregation. All 7 variants affecting coil 1B and the lamin A-only mutation, R654X, exhibited membrane-bound GFP-lamin A aggregates and nuclear shape abnormalities. Unexpectedly, R388H largely restricted GFP-lamin A to the cytoplasm. Equally unexpected were unique streaked aggregates with S437Hfsx1 and giant aggregates with both S437Hfsx1 and R654X. Conclusions— This work expands the recognized spectrum of lamin A localization abnormalities in dilated cardiomyopathy. It also provides evidence supporting pathogenicity of 10 of 13 tested LMNA variants, including some with uncertain or nonsegregation.

In this emerging era of cardiovascular genetic medicine, increasing responsibility will be placed on cardiovascular practitioners to be aware of the latest clinical genetic testing methods and the knowledge base needed to interpret genetic test results. Some cardiovascular specialists will develop the expertise within the field to order genetic testing and interpret results, while other practitioners will refer patients to centers of excellence in cardiovascular genetic medicine. A previous article in the Cardiovascular Genetic Medicine: Clinical Perspectives and Future Applications series1 highlighted an increasing recognition of the cardiomyopathies (hypertrophic [HCM], dilated [DCM], arrhythmogenic right ventricular dysplasia [ARVD]) and channelopathies (long QT syndrome [LQTS] and others) as genetic diseases, and focused on the importance of a targeted family history as a critical part of patient evaluation. The goal of this article, second in the series, is to provide a general framework for understanding the principles of genetic testing and genetic counseling. We review the growing number of genetic tests currently available to cardiac specialists, the selection of an appropriate test, and the numerous genetic counseling issues raised by the testing process. We also provide our preliminary recommendations for genetic testing in cardiovascular genetic medicine.

Mutations in zinc-finger in cerebellum 3 (ZIC3) result in heterotaxy or isolated congenital heart disease (CHD). The majority of reported mutations cluster in zinc-finger domains. We previously demonstrated that many of these lead to aberrant ZIC3 subcellular trafficking. A relative paucity of N- and C-terminal mutations has, however, prevented similar analyses in these regions. Notably, an N-terminal polyalanine expansion was recently identified in a patient with VACTERL, suggesting a potentially distinct function for this domain. Here, we report ZIC3 sequencing results from 440 unrelated patients with heterotaxy and CHD, the largest cohort yet examined. Variants were identified in 5.2% of sporadic male cases. This rate exceeds previous estimates of 1% and has important clinical implications for genetic testing and risk-based counseling. Eight of 11 were novel, including 5 N-terminal variants. Subsequent functional analyses included 4 additional reported but untested variants. Aberrant cytoplasmic localization and decreased luciferase transactivation were observed for all zinc-finger variants, but not for downstream or in-frame upstream variants, including both analyzed polyalanine expansions. Collectively, these results expand the ZIC3 mutational spectrum, support a higher than expected prevalence in sporadic cases, and suggest alternative functions for terminal mutations, highlighting a need for further study of these domains.