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Metabolic Syndrome (MetS) is a collective term for a cluster of disorders, including dysglycemia, central obesity, dyslipidemia, hypertension, and eventual end organ damage. The combination of these disorders increases the risk of many kinds of end organ damages, including coronary heart disease, kidney failure, and cirrhosis. MetS is highly prevalent in the United States, affecting one third of the U.S. population in a 2009 estimate.
The Lyon strains are three rat strains selectively inbred from the same colony of outbred rats for different blood pressure levels. The Lyon Hypertensive (LH) strain, in addition to its essential hypertension phenotype, also harbors many disorders found in MetS. The Lyon Normotensive (LN) rat strain is completely devoid of these symptoms, while Lyon Low-pressure (LL) is obese but is resistant to other traits of MetS.
Rat chromosome 17 (RNO17) has previously been linked with many of MetS' phenotypes in Lyon Hypertensive (LH). In this project, we are using a mixture of genetical genomics and systems biology methods to identify genetic elements that may cause the LH phenotype.
Divergent haplotype blocks between the Lyon strains were first identified by the analysis of the distribution of observed strain differences (OSD) calculated from the result of genome resequencing. Divergent haplotype regions totaling less than 16% of the rat genome that contain more than 95% of the identified SNPs in each of the three pairwise comparisons between the Lyon strains have been identified; in particular, there are 14 divergent haplotype blocks between LH and LN spanning 7.7% of RNO17 that harbor more than 97% of SNPs identified on RNO17. Twenty-five genes in these regions were thus identified as potential genetic determinants for MetS.
Phenotypic QTLs (pQTL) and expression QTLs (eQTL) mapping from a cohort of male LH × LN F2 rats were performed by putting the cohort on a 15-week phenotyping protocol and genome-wide genotyping. Total liver RNA from 36 individuals from the cohort were sequenced to provide expression data for eQTL mapping. We have mapped 22 pQTLs that are statistically linked to 15 traits, with RNO17 linked to 15 traits associated with blood pressure, leptin and body weight. We have also identified 1,200 eQTLs from this cohort, including 11 eQTLs with cis-linkage with one or more genes. On RNO17, we have identified two SNPs between 29-39 Mb which are significantly linked to the expression of 85 genes; the only gene with cis-linkage with these SNPs, RGD1562963, was hence identified as a putative master regulator.
Transcriptome analyses were then performed on the Lyon parental animals; the total liver and kidney of RNA from 6 each of LH, LL and LN strains that were subjected to the same 15-week phenotyping protocol were sequenced for differential expression analysis, gene coexpression network analysis and quantitative trait transcript analysis. Differential expression analysis identified 4 genes on RNO17's divergent haplotype regions: Cul2 and the aforementioned RGD1562963 for liver, Amph and Bambi for kidney. Quantitative trait transcript analyses have shown significant correlations between the expressions of these four genes with one or more of the traits of the animals treated, validating their status as potential genetic determinants for MetS. However, out of the 84 genes that RGD1562963 potentially regulates, only two other genes (Cul2 and Supt4h1) have significant correlations with one or more traits. Gene coexpression network analyses have shown a relationship between genes on the TGF-β pathway and the differentially expressed genes in the kidney, supporting our speculation on the hyperactivity of the TGF-β system in the etiology of the LH phenotypes.
An LH-17LN consomic strain was also generated by introgressing an LN copy of RNO17 onto the LH genomic background to validate in vivo the role of RNO17 in the etiology of MetS symptoms in LH. We have observed that the consomic strain has significantly decreased body weight, adiposity, blood pressure, and inter-week blood pressure differences that may be a surrogate for salt sensitivity. Thus, the role of RNO17 on the LH genotype is validated.
In summary, we have been able to identify, by in vivo and in silico methods, that RNO17 is related to the MetS traits in LH; that 4 genes, Amph, Bambi, Cul2, and RGD1562963, are potential genetic contributors to RNO17's effects; and that their effects may include, but are not limited to, the activation of TGF-Β signals.