Cystathionine-b-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.T HE first complete human genome sequence seeded the defining challenge of human genetics for the foreseeable future: interpreting the impact of variations in the sequences of individual human genomes. Comparative genome sequencing reveals an average of one single-nucleotide change per 1200 bp between any two individuals. In the absence of strong Mendelian inheritance and linkage, confirming that any human genotype actually caused a phenotype is a significant challenge given the approximately 3 million genetic variants per person. Indeed, 4000 traits of medical interest show evidence for inheritance but lack a clear determinant (Online Mendelian Inheritance in Man 2012). Next-generation sequencing within small pedigrees (Ng et al. 2010a,b;Fan et al. 2011), or a more narrowly defined clinical phenotype (Schubert et al. 1997), can sometimes disentangle the underlying contribution of a gene to disease. In this work we have taken an approach that complements both increased sequencing capacity and expanded phenotypic description. We used surrogate genetics to assay directly the function of allelic variants and then evaluate their potential contribution to phenotypes of clinical importance.Homocystinuria, elevated levels of the sulfur-containing metabolite homocystine in the urine, illustrates several Reference numbers for publicly available data; GenBank: L14577.1 (CBS); dbSNP: rs17849313 (A69P), rs2229413 (P70L), rs11700812 (R369P); SGD: YGR155W (CYS4) and YDR232W (HEM1 challenges inherent to elucidating the molecular bases of human geneti...

Atherosclerosis is a complex disease resulting from the interaction of multiple genes. We have used the Ldlr knockout mouse model in an interspecific genetic cross to map atherosclerosis susceptibility loci. A total of 174 (MOLF͞Ei ؋ B6.129S7-Ldlr tm1Her ) ؋ C57BL͞ 6J-Ldlr tm1Her backcross mice, homozygous for the Ldlr null allele, were fed a Western-type diet for 3 months and then killed for quantification of aortic lesions. A genome scan was carried out by using DNA pools and microsatellite markers spaced at Ϸ18-centimorgan intervals. Quantitative trait locus analysis of individual backcross mice confirmed linkages to chromosomes 4 (Athsq1, logarithm of odds ‫؍‬ 6.2) and 6 (Athsq2, logarithm of odds ‫؍‬ 6.7). Athsq1 affected lesions in females only whereas Athsq2 affected both sexes. Among females, the loci accounted for Ϸ50% of the total variance of lesion area. The susceptible allele at Athsq1 was derived from the MOLF͞Ei genome whereas the susceptible allele at Athsq2 was derived from C57BL͞6J. Inheritance of susceptible alleles at both loci conferred a 2-fold difference in lesion area, suggesting an additive effect of Athsq1 and Athsq2. No associations were observed between the quantitative trait loci and levels of plasma total cholesterol, high density lipoprotein cholesterol, non-high density lipoprotein cholesterol, insulin, or body weight. We provide strong evidence for complex inheritance of atherosclerosis in mice with elevated plasma low density lipoprotein cholesterol and show a major influence of nonlipoprotein-related factors on disease susceptibility. Athsq1 and Athsq2 represent candidate susceptibility loci for human atherosclerosis, most likely residing on chromosomes 1p36 -32 and 12p13-12, respectively.

Objective-Susceptibility to atherosclerosis is genetically complex, and modifier genes that do not operate via traditional risk factors are largely unknown. A mouse genetics approach can simplify the genetic analysis and provide tools for mechanistic studies. Methods and Results-We previously identified atherosclerosis susceptibility QTL (Athsq1) on chromosome 4 acting independently of systemic risk factors. We now report confirmation of this locus in congenic strains carrying the MOLF-derived susceptibility allele in the C57BL/6J-Ldlr Ϫ/Ϫ genetic background. Homozygous congenic mice exhibited up to 4.5-fold greater lesion area compared to noncongenic littermates (PϽ0.0001). Analysis of extracellular matrix composition revealed prominent accumulation of versican, a presumed proatherogenic matrix component abundant in human lesions but almost absent in the widely-used C57BL/6 murine atherosclerosis model. The results of a bone marrow transplantation experiment suggested that both accelerated lesion development and versican accumulation are mediated, at least in part, by macrophages. Interestingly, comparative mapping revealed that the Athsq1 congenic interval contains the mouse region homologous to a widely-replicated CHD locus on human chromosome 9p21. Key Words: atherosclerosis Ⅲ congenic strain Ⅲ genetics Ⅲ extracellular matrix Ⅲ mapping S usceptibility to atherosclerosis is influenced by both genetic and environmental factors, with approximately 40% to 60% of interindividual variation attributed to genetic factors. 1 The complex etiology has hampered genetic studies of atherosclerosis in humans per se until recently. Early studies used the candidate gene approach to identify rare genetic variants contributing to traditional risk factors including plasma levels of LDL/VLDL, HDL, lipoprotein (a), homocysteine, and blood pressure. [2][3][4][5][6] Now many of the genes underlying Mendelian forms of dyslipidemia have been shown to contribute to population variation of plasma lipids using genome-wide association (GWA) studies. [7][8][9] Attempts to identify genes directly underlying coronary artery disease (CAD)/ myocardial infarct (MI) were first pursued through linkage studies of families enriched for disease 10 -14 and large-scale association studies. [15][16][17][18] However, the first locus to be widely-replicated for CHD was recently revealed through a series of GWA studies. 19 -22 A common variant was localized to chromosome (chr) 9p21, but the underlying gene and mechanism of action are unknown. Thus, while a number of genes contributing to traditional risk factors have been identified, few of the genes underlying nontraditional risk factors are known. Conclusion-These studies confirm the proatherogenic activity of a novel gene(s) in the MOLF-derivedAnimal models offer an alternative approach for the genetic analysis of complex diseases such as atherosclerosis. In early work, the existence of atherosclerosis susceptibility loci in the mouse model was suggested using recombinant inbred strains of mice. [23]...

Background-The formation of an occluding thrombus on a ruptured or eroded atherosclerotic plaque is the hallmark event leading to acute coronary syndromes, myocardial infarction, and sudden death in humans. However, other species are highly resistant to plaque complications, and the specific processes predisposing to plaque destabilization and thrombosis are poorly understood. Methods and Results-Mice carrying a null mutation of a gene regulating intracellular cholesterol transport (the Niemann-Pick C1 [Npc1] gene) were crossed with apolipoprotein E (Apoe) knockout mice to examine the effect of Npc1 on atherosclerotic lesion formation. Double-mutant mice showed greater lesion area compared with Apoe Ϫ/Ϫ littermates. Remarkably, the double mutants also developed large, protruding thrombi associated with the plaques and prominent medial degradation with inflammatory cell infiltration into the adventitia. Genetic studies suggested that the BALB background was permissive for plaque complications compared with C57BL/6J, and a BALB susceptibility locus was mapped by linkage analysis to chromosome 6. Examination of clotting parameters in double-knockout mice revealed that native clotting times were shortened and thrombin-antithrombin complex and soluble CD40 ligand levels were elevated compared with wild-type controls. In addition, cathepsin K was induced in Npc1 Ϫ/Ϫ macrophages, and cathepsin K immunostaining and elastase activity were increased in proximal aortas of double-mutant mice compared with controls. Conclusions-A defect in intracellular cholesterol trafficking caused by the Npc1 null mutation predisposes to increased lesion formation, atherothrombosis, and medial degradation. Plaque complications may require a procoagulant state and an increased protease activity, leading to plaque destabilization.