Lisa Dellefave-Castillo

Population-Based Variation in Cardiomyopathy Genes

Background—Hypertrophic cardiomyopathy and dilated cardiomyopathy arise from mutations in genes encoding sarcomere proteins including MYH7, MYBPC3, and TTN. Genetic diagnosis of cardiomyopathy relies on complete sequencing of the gene coding regions, and most pathogenic variation is rare. The 1000 Genomes Project is an ongoing consortium designed to deliver whole genome sequence information from an ethnically diverse population and, therefore, is a rich source to determine both common and rare genetic variants. Methods and Results—We queried the 1000 Genomes Project database of 1092 individuals for exonic variants within 3 sarcomere genes MHY7, MYBPC3, and TTN. We focused our analysis on protein-altering variation, including nonsynonymous single nucleotide polymorphisms, insertion/deletion polymorphisms, or splice site altering variants. We identified known and predicted pathogenic variation in MYBPC3 and MYH7 at a higher frequency than what would be expected based on the known prevalence of cardiomyopathy. We also found substantial variation, including protein-disrupting sequences, in TTN. Conclusions—Cardiomyopathy is a genetically heterogeneous disorder caused by mutations in multiple genes. The frequency of predicted pathogenic protein-altering variation in cardiomyopathy genes suggests that many of these variants may be insufficient to cause disease on their own but may modify phenotype in a genetically susceptible host. This is suggested by the high prevalence of TTN insertion/deletions in the 1000 Genomes Project cohort. Given the possibility of additional genetic variants that modify the phenotype of a primary driver mutation, broad-based genetic testing should be employed.

S100A12 expression in thoracic aortic aneurysm is associated with increased risk of dissection and perioperative complications

OBJECTIVES The purpose of this study was to determine the relevance of S100A12 expression to human thoracic aortic aneurysms and type A thoracic aortic aneurysm dissection and to study mechanisms of S100A12-mediated dysfunction of aortic smooth muscle cells. BACKGROUND Transgenic expression of proinflammatory S100A12 protein in murine aortic smooth muscle causes thoracic aneurysm in genetically modified mice. METHODS Immunohistochemistry of aortic tissue (n = 50) for S100A12, myeloperoxidase, and caspase 3 was examined and S100A12-mediated pathways were studied in cultured primary aortic smooth muscle cells. RESULTS We found S100A12 protein expressed in all cases of acute thoracic aortic aneurysm dissection and in approximately 25% of clinically stable thoracic aortic aneurysm cases. S100A12 tissue expression was associated with increased length of stay in patients undergoing elective surgical repair for thoracic aortic aneurysm, despite similar preoperative risk as determined by European System for Cardiac Operative Risk Evaluation. Reduction of S100A12 expression in human aortic smooth muscle cells using small hairpin RNA attenuates gene and protein expression of many inflammatory- and apoptosis-regulating factors. Moreover, genetic ablation of the receptor for S100A12, receptor for advanced glycation end products (RAGE), in murine aortic smooth muscle cells abolished cytokine-augmented activation of caspase 3 and smooth muscle cell apoptosis in S100A12-expressing cells. CONCLUSIONS S100A12 is enriched in human thoracic aortic aneurysms and dissections. Reduction of S100A12 or genetic ablation of its cell surface receptor, the receptor for advanced glycation end products (RAGE), in aortic smooth muscle resulted in decreased activation of caspase 3 and in reduced apoptosis. By establishing a link between S100A12 expression and apoptosis of aortic smooth muscle cells, this study identifies novel S100A12 signaling pathways and indicates that S100A12 may be a useful molecular marker and possible target for treatment for human aortic diseases.

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of >50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated

Supercomputing for the parallelization of whole genome analysis.

1000 genome, it is expected that genetic testing will shift toward comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused on 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1 to 14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and segregation analysis, where available. Three of 3 previously identified primary mutations were detected by this analysis. In 6 subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and had additional pathological correlation to provide evidence for causality. For 2 subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. Conclusions—These pilot data demonstrate that ≈30 to 40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.

Direct reprogramming of urine-derived cells with inducible MyoD for modeling human muscle disease

MOTIVATION The declining cost of generating DNA sequence is promoting an increase in whole genome sequencing, especially as applied to the human genome. Whole genome analysis requires the alignment and comparison of raw sequence data, and results in a computational bottleneck because of limited ability to analyze multiple genomes simultaneously. RESULTS We now adapted a Cray XE6 supercomputer to achieve the parallelization required for concurrent multiple genome analysis. This approach not only markedly speeds computational time but also results in increased usable sequence per genome. Relying on publically available software, the Cray XE6 has the capacity to align and call variants on 240 whole genomes in ∼50 h. Multisample variant calling is also accelerated. AVAILABILITY AND IMPLEMENTATION The MegaSeq workflow is designed to harness the size and memory of the Cray XE6, housed at Argonne National Laboratory, for whole genome analysis in a platform designed to better match current and emerging sequencing volume.

Genotype-Specific Interaction of Latent TGFβ Binding Protein 4 with TGFβ.

BackgroundCellular models of muscle disease are taking on increasing importance with the large number of genes and mutations implicated in causing myopathies and the concomitant need to test personalized therapies. Developing cell models relies on having an easily obtained source of cells, and if the cells are not derived from muscle itself, a robust reprogramming process is needed. Fibroblasts are a human cell source that works well for the generation of induced pluripotent stem cells, which can then be differentiated into cardiomyocyte lineages, and with less efficiency, skeletal muscle-like lineages. Alternatively, direct reprogramming with the transcription factor MyoD has been used to generate myotubes from cultured human fibroblasts. Although useful, fibroblasts require a skin biopsy to obtain and this can limit their access, especially from pediatric populations.ResultsWe now demonstrate that direct reprogramming of urine-derived cells is a highly efficient and reproducible process that can be used to establish human myogenic cells. We show that this method can be applied to urine cells derived from normal individuals as well as those with muscle diseases. Furthermore, we show that urine-derived cells can be edited using CRISPR/Cas9 technology.ConclusionsWith progress in understanding the molecular etiology of human muscle diseases, having a readily available, noninvasive source of cells from which to generate muscle-like cells is highly useful.

Experimental Modeling Supports a Role for MyBP-HL as a Novel Myofilament Component in Arrhythmia and Dilated Cardiomyopathy

Latent TGFβ binding proteins are extracellular matrix proteins that bind latent TGFβ to form the large latent complex. Nonsynonymous polymorphisms in LTBP4, a member of the latent TGFβ binding protein gene family, have been linked to several human diseases, underscoring the importance of TGFβ regulation for a range of phenotypes. Because of strong linkage disequilibrium across the LTBP4 gene, humans have two main LTBP4 alleles that differ at four amino acid positions, referred to as IAAM and VTTT for the encoded residues. VTTT is considered the “risk” allele and associates with increased intracellular TGFβ signaling and more deleterious phenotypes in muscular dystrophy and other diseases. We now evaluated LTBP4 nsSNPs in dilated cardiomyopathy, a distinct disorder associated with TGFβ signaling. We stratified based on self-identified ethnicity and found that the LTBP4 VTTT allele is associated with increased risk of dilated cardiomyopathy in European Americans extending the diseases that associate with LTBP4 genotype. However, the association of LTBP4 SNPs with dilated cardiomyopathy was not observed in African Americans. To elucidate the mechanism by which LTBP4 genotype exerts this differential effect, TGFβ’s association with LTBP4 protein was examined. LTBP4 protein with the IAAM residues bound more latent TGFβ compared to the LTBP4 VTTT protein. Together these data provide support that LTBP4 genotype exerts its effect through differential avidity for TGFβ accounting for the differences in TGFβ signaling attributed to these two alleles.

Genetic Counselors’ Approach To Postmortem Genetic Testing After Sudden Death: An Exploratory Study

Background: Cardiomyopathy and arrhythmias are under significant genetic influence. Here, we studied a family with dilated cardiomyopathy and associated conduction system disease in whom prior clinical cardiac gene panel testing was unrevealing. Methods: Whole-genome sequencing and induced pluripotent stem cells were used to examine a family with dilated cardiomyopathy and atrial and ventricular arrhythmias. We also characterized a mouse model with heterozygous and homozygous deletion of Mybphl. Results: Whole-genome sequencing identified a premature stop codon, R255X, in the MYBPHL gene encoding MyBP-HL (myosin-binding protein-H like), a novel member of the myosin-binding protein family. MYBPHL was found to have high atrial expression with low ventricular expression. We determined that MyBP-HL protein was myofilament associated in the atria, and truncated MyBP-HL protein failed to incorporate into the myofilament. Human cell modeling demonstrated reduced expression from the mutant MYBPHL allele. Echocardiography of Mybphl heterozygous and null mouse hearts exhibited a 36% reduction in fractional shortening and an increased diastolic ventricular chamber size. Atria weight normalized to total heart weight was significantly increased in Mybphl heterozygous and null mice. Using a reporter system, we detected robust expression of Mybphl in the atria, and in discrete puncta throughout the right ventricular wall and septum, as well. Telemetric electrocardiogram recordings in Mybphl mice revealed cardiac conduction system abnormalities with aberrant atrioventricular conduction and an increased rate of arrhythmia in heterozygous and null mice. Conclusions: The findings of reduced ventricular function and conduction system defects in Mybphl mice support that MYBPHL truncations may increase risk for human arrhythmias and cardiomyopathy.

Response to Letter Regarding Article, “Population-Based Variation in Cardiomyopathy Genes”

A significant portion of sudden death cases result from an underlying genetic etiology, which may be determined through postmortem genetic testing. The National Association of Medical Examiners (NAME) recommends that an appropriate postmortem sample is saved on all sudden death cases under the age of 40. Genetic counselors (GCs) play an important role in this process by working with medical examiners and coroners (ME/Cs) to recommend and interpret specific testing and to guide family members. A survey sent to the National Society of Genetic Counselors was designed and implemented to learn more about the experiences of genetic counselors who had considered or ordered postmortem genetic testing. Results showed that cardiovascular GCs were significantly more willing to recommend genetic testing in younger age decedents (ages 10, 18, 30, 40, and 50) compared to other specialty GCs (p<0.05, Chi-square). Thirty-seven percent (7 of 19) of GCs reported insurance covering some portion of genetic testing. Participants also reported highest success for DNA extractions with fresh and frozen blood, reinforcing NAME recommendations for appropriate sample collection for postmortem genetic testing. Overall, participating GCs demonstrated a very good understanding for the appropriate use of postmortem genetic testing and did identify suspected barriers of cost and lack of insurance coverage as deterrents. With the rapid decrease in costs for diagnostic genetic testing, ME/C awareness of NAME recommendations for sample collection and storage remain important to facilitate postmortem genetic testing.

Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy

We thank Iascone et al for their thoughtful comments on our survey of potentially pathological or reported pathological genetic variation found in the 1000 Genomes database. …