Katherine G. Spoonamore

Dysfunction in the βII Spectrin–Dependent Cytoskeleton Underlies Human Arrhythmia

Background— The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. Methods and Results— Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that &bgr;II spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. &bgr;II spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/&bgr;II spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac &bgr;II spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, &bgr;II spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and &agr;II spectrin. Finally, we observe accelerated heart failure phenotypes in &bgr;II spectrin–deficient mice. Conclusions— Our findings identify &bgr;II spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

Genetic testing and genetic counseling in patients with sudden death risk due to heritable arrhythmias

Sudden cardiac death due to heritable ventricular arrhythmias is an important cause of mortality, especially in young healthy individuals. The identification of the genetic basis of Mendelian diseases associated with arrhythmia has allowed the integration of this information into the diagnosis and clinical management of patients and at-risk family members. The rapid expansion of genetic testing options and the increasing complexity involved in the interpretation of results creates unique opportunities and challenges. There is a need for competency to incorporate genetics into clinical management and to provide appropriate family-based risk assessment and information. In addition, disease-specific genetic knowledge is required to order and correctly interpret and apply genetic testing results. Importantly, genetic diagnosis has a critical role in the risk stratification and clinical management of family members. This review summarizes the approach to genetic counseling and genetic testing for inherited arrhythmias and highlights specific genetic principles that apply to long QT syndrome, short QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia.

Adaptation and validation of the ACMG/AMP variant classification framework for MYH7 -associated inherited cardiomyopathies: recommendations by ClinGen’s Inherited Cardiomyopathy Expert Panel

PurposeIntegrating genomic sequencing in clinical care requires standardization of variant interpretation practices. The Clinical Genome Resource has established expert panels to adapt the American College of Medical Genetics and Genomics/Association for Molecular Pathology classification framework for specific genes and diseases. The Cardiomyopathy Expert Panel selected MYH7, a key contributor to inherited cardiomyopathies, as a pilot gene to develop a broadly applicable approach.MethodsExpert revisions were tested with 60 variants using a structured double review by pairs of clinical and diagnostic laboratory experts. Final consensus rules were established via iterative discussions.ResultsAdjustments represented disease-/gene-informed specifications (12) or strength adjustments of existing rules (5). Nine rules were deemed not applicable. Key specifications included quantitative frameworks for minor allele frequency thresholds, the use of segregation data, and a semiquantitative approach to counting multiple independent variant occurrences where fully controlled case-control studies are lacking. Initial inter-expert classification concordance was 93%. Internal data from participating diagnostic laboratories changed the classification of 20% of the variants (n = 12), highlighting the critical importance of data sharing.ConclusionThese adapted rules provide increased specificity for use in MYH7-associated disorders in combination with expert review and clinical judgment and serve as a stepping stone for genes and disorders with similar genetic and clinical characteristics.

Use of genetic testing to identify sudden cardiac death syndromes.

Sudden cardiac death (SCD) is a leading cause of mortality worldwide. Although coronary artery disease remains the most common substrate for SCD, primary cardiac genetic diseases, presenting with or without structural heart abnormalities, play a significant role. In the last 30 years, the study of large family pedigrees allowed the discovery of causative genes unveiling the genetic basis of diseases such as primary cardiomyopathies and arrhythmia syndromes, which are known to increase the risk of SCD. However, recent technological advancement with the ability to perform massive parallel sequencing and analyze the entire genome has uncovered a higher level of complexity in the genetic predisposition for cardiac diseases, which are usually characterized by Mendelian inheritance patterns. Clinical genetic testing, historically shaped around a monogenic Mendelian disorder paradigm, is now facing the challenge to adopt and adapt to a more complex model in which a significant portion of subjects may present with multi-allelic inheritance involving additional genes that could modulate the severity and type of disease-related phenotypes. Here, we will try to provide a viewpoint that will hopefully foster further debate in the field.

Evidence for replicative mechanism in a CHD7 rearrangement in a patient with CHARGE syndrome.

Haploinsufficiency of CHD7 (OMIM# 608892) is known to cause CHARGE syndrome (OMIM# 214800). Molecular testing supports a definitive diagnosis in approximately 65–70% of cases. Most CHD7 mutations arise de novo, and no mutations affecting exon‐7 have been reported to date. We report on an 8‐year‐old girl diagnosed with CHARGE syndrome that was referred to our laboratory for comprehensive CHD7 gene screening. Genomic DNA from the subject with a suspected diagnosis of CHARGE was isolated from peripheral blood lymphocytes and comprehensive Sanger sequencing, along with deletion/duplication analysis of the CHD7 gene using multiplex ligation‐dependent probe amplification (MLPA), was performed. MLPA analysis identified a reduced single probe signal for exon‐7 of the CHD7 gene consistent with potential heterozygous deletion. Long‐range PCR breakpoint analysis identified a complex genomic rearrangement (CGR) leading to the deletion of exon‐7 and breakpoints consistent with a replicative mechanism such as fork stalling and template switching (FoSTeS) or microhomology‐mediated break‐induced replication (MMBIR). Taken together this represents the first evidence for a CHD7 intragenic CGR in a patient with CHARGE syndrome leading to what appears to be also the first report of a mutation specifically disrupting exon‐7. Although likely rare, CGR may represent an overlooked mechanism in subjects with CHARGE syndrome that can be missed by current sequencing and dosage assays.

Who Pays? Coverage Challenges for Cardiovascular Genetic Testing in U.S. Patients.

Inherited cardiovascular (CV) conditions are common, and comprehensive care of affected families often involves genetic testing. When the clinical presentations of these conditions overlap, genetic testing may clarify diagnoses, etiologies, and treatments in symptomatic individuals and facilitate the identification of asymptomatic, at-risk relatives, allowing for often life-saving preventative care. Although some professional society guidelines on inherited cardiac conditions include genetic testing recommendations, they quickly become outdated owing to the rapid expansion and use of such testing. Currently, these guidelines primarily discuss the benefits of targeted genetic testing for identifying at-risk relatives. Although most insurance policies acknowledge the benefit and the necessity of this testing, many exclude coverage for testing altogether or are vague about coverage for testing in probands, which is imperative if clinicians are to have the best chance of accurately identifying pathogenic variant(s) in a family. In response to uncertainties about coverage, many commercial CV genetic testing laboratories have shouldered the burden of working directly with commercial payers and protecting patients/institutions from out-of-pocket costs. As a result, many clinicians are unaware that payer coverage policies may not match professional recommendations for CV genetic testing. This conundrum has left patients, clinicians, payers, and laboratories at an impasse when determining the best path forward for meaningful and sustainable testing. Herein, we discuss the need for all involved parties to recognize their common goals in this process, which should motivate collaboration in changing existing frameworks and creating more sustainable access to genetic information for families with inherited CV conditions.

MIB2 variants altering NOTCH signalling result in left ventricle hypertrabeculation/non-compaction and are associated with Ménétrier-like gastropathy.

We performed whole exome sequencing in individuals from a family with autosomal dominant gastropathy resembling Ménétrier disease, a premalignant gastric disorder with epithelial hyperplasia and enhanced EGFR signalling. Ménétrier disease is believed to be an acquired disorder, but its aetiology is unknown. In affected members, we found a missense p.V742G variant in MIB2, a gene regulating NOTCH signalling that has not been previously linked to human diseases. The variant segregated with the disease in the pedigree, affected a highly conserved amino acid residue, and was predicted to be deleterious although it was found with a low frequency in control individuals. The purified protein carrying the p.V742G variant showed reduced ubiquitination activity in vitro and white blood cells from affected individuals exhibited significant reductions of HES1 and NOTCH3 expression reflecting alteration of NOTCH signalling. Because mutations of MIB1, the homolog of MIB2, have been found in patients with left ventricle non-compaction (LVNC), we investigated members of our family with Ménétrier-like disease for this cardiac abnormality. Asymptomatic left ventricular hypertrabeculation, the mildest end of the LVNC spectrum, was detected in two members carrying the MIB2 variant. Finally, we identified an additional MIB2 variant (p.V984L) affecting protein stability in an unrelated isolated case with LVNC. Expression of both MIB2 variants affected NOTCH signalling, proliferation and apoptosis in primary rat cardiomyocytes.In conclusion, we report the first example of left ventricular hypertrabeculation/LVNC with germline MIB2 variants resulting in altered NOTCH signalling that might be associated with a gastropathy clinically overlapping with Ménétrier disease.

Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization

RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20S637A mouse as a good model for in vivo study.

Erratum to: At the Heart of the Pregnancy: What Prenatal and Cardiovascular Genetic Counselors Need to Know about Maternal Heart Disease

1 Department of Internal Medicine, The Ohio State University, Columbus, OH, USA 2 Human Genetics Division, The Ohio State University, 306 BRT, 460 W. 12th Ave, Columbus, OH 43210, USA 3 Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA 4 Allegheny General Hospital, Pittsburgh, PA, USA 5 Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA 6 Division of Cardiology, Johns Hopkins University, Baltimore, MD, USA 7 Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

Lamin-A/C variants found in patients with cardiac conduction disease reduce sodium currents

Variants in the LMNA gene, which encodes Lamin-A/C, have been commonly associated with cardiac conduction system diseases usually accompanying cardiomyopathy. We have seen two unrelated patients who presented with atrioventricular block (AVB) with or without cardiomyopathy. Genetic testing identified the LMNA missense variant c.1634G>A (p.R545H) and the single nucleotide deletion c.859delG (p.A287Lfs*193). The deletion leads to a shift in the reading frame and subsequent protein truncation. Since impaired Nav1.5 function has been reported to cause AVB, we sought to investigate the effects of abnormal Lamins on Nav1.5 in HEK-293 cells using patch-clamp methods. Patch-clamp studies showed that p.R545H decreased the peak INa by approximately 70%. The voltage-dependency of steady state inactivation was rightward shifted in the cells transfected with p.R545H. The p.A287Lfs*193 also decreased the peak INa by approximately 62%. The voltagedependency of steady state inactivation was rightward shifted in the cells transfected with p.A287Lfs*193. Variants of the LMNA gene caused significant reduction of the peak INa in HEK-293 cells, which may account for