Faculty and Research
Douglas Marchuk, PhD
Professor and Vice Chair
Director, Duke University Program in Genetics and Genomics
Scientific Approach. My laboratory studies the genetics of cardiovascular disease using both the human and the mouse as a model system. We are particularly interested in harnessing the power of mouse genetics to map novel genes that affect the severity and progression of disease in mouse models of disease. We begin with an animal model of the disease, such as a surgically induced or transgenic model of disease. The surgical intervention or the transgene acts as a sensitizer to create the disease in the animals. These sensitized models often exhibit drastically different rates of disease progression or outcome depending on the inbred strain employed. Using genetic crosses with the sensitizer, we can map the loci that influence disease outcome. We call these risk factors “modifier loci” since they modify disease progression or outcome. Importantly, given the ancestral relatedness of the commonly used inbred mouse strains, we can use ancestral haplotype mapping to rapidly identify the gene and sequence variant underlying the modifier locus. The orthologs of the modifier genes discovered in the mouse models are then investigated in the corresponding human disease populations.
Example 1: Heart Disease Modifier Genes. One example of this approach is the Calsequestrin model of cardiomyopathy (heart disease). The Calsequestrin (CSQ) mouse model exhibits extreme variation in the phenotype progression and severity highly dependent on the genetic background. Genetic mapping in the context of the transgenic sensitizer has yielded seven heart failure modifier (Hrtfm) loci that modify disease progression. We have now identified the gene for one of these - Hrtfm2. A single gene in the interval shows a significant difference in transcript levels between strains. Strains with a susceptible phenotype show high levels of transcript while strains that express low transcript levels are protected. In strains showing reduced message levels, we have identified an intronic SNP that activates a cryptic splice site leading to aberrant splicing, followed by nonsense-mediated decay of the message. We are currently investigating the role of the human ortholog of this gene in heart disease progression. In addition, we are seeking a small molecule inhibitor of the encoded protein as a novel therapeutic for heart disease.
Example 2: Ischemic Stroke Modifier Genes. A second project relates to genes that modify the outcome of ischemic stroke. Although epidemiological studies have provided substantial evidence for genetic influences in the development of stroke, genetic determinants of the extent of ischemic tissue damage remain unknown. Through the use of a surgically-induced mouse model of focal cerebral ischemia we have shown large, reproducible differences in infarct volume among different inbred mouse strains. A genome-wide linkage scan in a cross of phenotypic outlier strains identified a locus that accounts for the majority of the phenotypic variance in infarct volume. Measurement of infarct volume in additional inbred strains allowed an ancestral SNP haplotype-based approach to fine-mapping the locus, reducing the possible candidate genes to only six. In vitro and in vivo studies are currently underway to investigate the role of these genes in modulating tissue damage after ischemia. The results of this study may uncover novel genes that modulate the severity of human ischemic stroke, as well as provide new targets for therapeutic intervention.
Example 3: Human Molecular Genetic Study of the Pathogenesis of a Mendelian Form of Stroke. Finally, we also investigate the molecular genetic mechanisms behind a number of different Mendelian forms of vascular disease. One example is our work on Cerebral Cavernous Malformations. Cerebral cavernous malformations (CCM) are vascular lesions of the brain consisting of closely-packed, grossly-dilated vessels. The lesions have a propensity for bleeding, leading to seizures and/or hemorrhagic stroke. CCMs may occur sporadically or may be inherited as an autosomal dominant trait due to mutation in one of three genes, CCM1, CCM2, or CCM3. While the causative genes have been identified, the molecular events initiating lesion formation have yet to be elucidated.
We have hypothesized that CCM lesion genesis occurs due to second-site somatic mutations, following a “two-hit” mutation model. Patients harboring a germline mutation would require a somatic mutation in the wild-type allele to initiate lesion formation. We have been analyzing CCM lesion from human patients harboring a germline (inherited) mutation. In a number of cases, we have identified a somatic mutation in the wild-type allele, thus fulfilling the two-hit hypothesis. Laser capture microdissection of the lesion shows that the somatic mutations arise only in the endothelial cells of the grossly-dilated vessels.
However, human CCM lesion tissue availability is restricted to large, multi-vessel, late stage lesions. This limits our ability to perform molecular analyses at the earliest stages of lesion development. Therefore, we have created mouse mutant models for Ccm1 and Ccm2. Using MRI technology, we can identify and then dissect CCM lesions at the earliest stages of genesis – a single dilated capilllary. Using laser capture microscopy, we can analyze these “pre-CCM lesions” to determine if the somatic mutation occurs at this early stage in the pathogenesis of the disease.