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Dissecting Molecular Pathways for Cardiac Conduction System Development and Disease
Summary: Christine Seidman's laboratory discovers gene mutations that cause human disease, with a focus on cardiovascular conditions such as cardiomyopathy (hypertrophic and dilated), heart failure, and congenital heart malformations. Her laboratory also produces model organisms that carry human mutations, and uses these models to determine how responses to gene mutations perturb or influence myocardial structure and specialized heart functions.
The human genome project has spawned many new strategies that enable high-throughput, high-fidelity DNA sequencing at markedly reduced costs. We have harnessed these technologies to advance our studies of human heart disease in two ways. Employing comprehensive sequencing approaches, we have interrogated genes that are mutated in inherited forms of cardiac hypertrophy to assess their relevance to more common and sporadic forms of cardiac hypertrophy that occur within populations. In addition, we have applied novel sequencing strategies to enable comprehensive analyses of the transcriptional response of the heart to gene mutations. Together these results have provided new knowledge about genetic causes and molecular mechanisms of cardiac hypertrophy.
Genetic studies of families with hypertrophic cardiomyopathy (HCM), an inherited disorder that is clinically characterized by increased ventricular wall thickness and that causes considerable morbidity and premature mortality, have led to the identification of many disease genes and hundreds of family-specific mutations. Most HCM patients have a mutation in 1 of 10 genes that encode the protein constituents of the sarcomere (the heart's fundamental unit of contraction, which includes myosin, actin, and troponin molecules). Less commonly, these human mutations alter genes involved in myocyte metabolism, including PRKAG2 (which encodes the γ subunit of AMP-dependent protein kinase), LAMP2 (lysosome-associated membrane protein-2), or GLA (which encodes α-galactosidase A). Because these mutations alter genes that encode proteins involved in diverse aspects of myocyte biology, we have concluded that there are multiple independent mechanisms by which cardiac hypertrophy develops. This considerable molecular complexity has necessitated great effort and expense to determine the precise genetic cause for cardiac hypertrophy in any one patient and has posed substantial barriers for studying larger, unrelated populations with cardiac hypertrophy. Because of these issues, it has not been known whether there is a genetic relationship between cardiac hypertrophy that occurs as a familial condition and the more prevalent form of cardiac hypertrophy that occurs as a sporadic condition. To address this we have used high-throughput DNA-sequencing methodologies to interrogate genes that cause familial cases of hypertrophy in older adults and in young children, two populations where hypertrophy is commonly recognized as a sporadic condition of unknown etiology.
Epidemiologic studies, including the Framingham Heart Study (FHS), a longitudinal community-based study of heart disease, have demonstrated that hypertrophy develops in 3 percent of the general population. While the increase in left ventricular wall thickness is often more modest than occurs in HCM, this more prevalent form of cardiac hypertrophy also conveys an increased risk for development of heart failure and for premature death. To determine if genes that are mutated in inherited forms of cardiac hypertrophy also contribute to hypertrophy that occurs in the general adult population, we studied 1,862 FHS participants. We identified subjects with cardiac hypertrophy despite neither a personal nor a family history of HCM, and subjects without hypertrophy. The average age of these subjects was approximately 60 years, and both subject groups had many common risk factors for heart disease, including hypertension, obesity, diabetes, and tobacco abuse. We comprehensively sequenced hypertrophic disease genes, including 8 sarcomere protein genes, PRKAG2, LAMP2, GLA, and 27 mitochondrial genes in FHS subjects with hypertrophy and in a subset without hypertrophy. Among the sequence variants identified, most were synonymous (i.e., they did not change the encoded amino acids or the protein structure) or these variants were frequently found in control populations, and so were unlikely to cause hypertrophy. In contrast, we found seven variants (in sarcomere protein genes and GLA) that altered amino acid residues that have been conserved throughout mammalian evolution. These seven variants were only in FHS participants with hypertrophy. Subsequent study of these seven variants in more than 1,700 randomly selected FHS participants revealed that two variants also occurred in other unrelated subjects with abnormal cardiac dimensions and/or function. By extrapolation from these results, among the 3 percent of adults with cardiac hypertrophy in the general population, approximately 18 percent have a single gene variant, most of which alter sarcomere proteins.
We have extended these studies to children with unexplained cardiac hypertrophy. Although rare, cardiomyopathy that is diagnosed during childhood has a worse prognosis (including high rates of sudden death and cardiac transplantation for heart failure) than cardiomyopathy that is diagnosed during adulthood. In addition to these adverse clinical parameters, more than 70 percent of cases of childhood cardiomyopathy occur without a family history for this condition. Because of these striking distinctions, the causes of hypertrophy in adults and children have been presumed to be different. To test this model we employed comprehensive sequencing strategies to analyze 10 disease genes that cause hypertrophy in adults, in two cohorts of children with cardiac hypertrophy: one with sporadic disease and one with a family history of cardiomyopathy. Not unexpectedly, gene mutations were found in 75 percent of children with a positive family history of cardiomyopathy. However, 49 percent of children without a family history of cardiac hypertrophy also had gene mutations that altered amino acids in sarcomere proteins or in PRKAG2. Genetic analyses of parental samples showed that one-third of children with sporadic disease had de novo mutations, while two-thirds of these children inherited the mutations from one parent. Clinical studies of parents with mutations indicated that some had unrecognized hypertrophy. These findings indicate that both childhood-onset and adult-onset cardiac hypertrophy share a common genetic etiology, most often a mutation that alters a sarcomere protein gene. This conclusion implies that unexplained cardiac hypertrophy that occurs in childhood should prompt genetic analyses to establish a precise cause, information that can guide management strategies and family counseling.
The substantial contribution of gene mutations to inherited HCM and to sporadic cases of childhood-onset and adult-onset cardiac hypertrophy underscores the importance of understanding the consequences of these mutations on heart structure and function. To define myocyte responses to gene mutations, we have adapted a new high-throughput nucleic acid–sequencing technique that enables comprehensive transcriptional profiling. This modified procedure defines the quantity of mRNAs that are expressed in a tissue, similar to serial analysis of gene expression but with a far greater depth of interrogation. We assess approximately 2 million mRNAs in each analysis, so that even transcripts that are expressed at low abundance are detected. Harnessing this sequence-based transcriptional profiling approach, we defined the early transcriptional changes that precede pathological manifestations of HCM in mice that carry a human disease-causing mutation. In comparison to normal hearts, the expression of more than 700 genes significantly changed in these prehypertrophic hearts. Some of these genes encode proteins that are known to participate in pathways for myocardial excitation-contraction coupling, Ca2+ homeostasis, and energy metabolism. Other genes with altered expression encode transcription factors and signaling molecules that can promote myocyte growth and increase myocardial fibrosis, the prototypic histopathologic features of cardiac hypertrophy. That hearts carrying a hypertrophic gene mutation have altered RNA expression before the onset of clinical disease suggests that these molecules account for the eventual development of hypertrophic cardiomyopathy. By interrogating the temporal and spatial expression of these transcription factors and signaling molecules, we are dissecting the networks activated in cardiac hypertrophy, with the expectation that these studies will help identify new molecular targets for therapeutic intervention.
These studies were funded in part by grants from the National Institutes of Health.
Last updated: October 7, 2008
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