Lyon Hypertensive Rats Research Articles

Body Composition and Metabolic Changes in a Lyon Hypertensive Congenic Rat and Identification of Ercc6l2 as a Positional Candidate Gene.

Central obesity is genetically complex, and its exponential increase in the last decades have made it a critical public health issue. The Lyon Hypertensive (LH) rat is a well-characterized hypertensive model that also exhibits spontaneous and profound differences in body weight and adiposity, relative to its metabolically healthy control, the Lyon Normotensive (LN) rat. The mechanisms underlying the body weight differences between these strains are not well-understood, thus a congenic model (LH17LNa) was developed where a portion of the proximal arm of LN chromosome 17 is introgressed on the LH genomic background to assess the contribution of LN alleles on obesity features. Male and female LH17LNa rats were studied, but male congenics did not significantly differ from LH in this study. Female LH17LNa rats exhibited decreases in total body growth, as well as major alterations to their body composition and adiposity. The LH17LNa female rats also showed decreases in metabolic rate, and a reduction in food intake. The increased adiposity in the female LH17LNa rats was specific to abdominal white adipose tissue, and this phenomenon was further explained by significant hypertrophy in those adipocytes, with no evidence of adipocyte hyperplasia. Sequencing of the parental strains identified a novel frameshift mutation in the candidate gene Ercc6l2, which is involved in transcription-coupled DNA repair, and is implicated in premature aging. The discovery of the significance of Ercc6l2 in the context of female-specific adipocyte biology could represent a novel role of DNA repair failure syndromes in obesity pathogenesis.

Female‐Specific Features of Metabolic Syndrome in an LH Congenic Rat

Central obesity, high blood pressure, dyslipidemia, and insulin resistance are a collection of cardiovascular and metabolic risk factors that form the basis of the Metabolic Syndrome (MetS) and represent a major public health burden worldwide. The phenotypes that define MetS are all highly heritable, but their genetic complexity necessitates the use of animal models to tease apart novel pathways.The Lyon Hypertensive (LH) rat is a well‐characterized model of MetS, exhibiting spontaneous and profound differences in features of MetS compared to its metabolically healthy control, the Lyon Normotensive (LN) rat. To understand the genomic causes of MetS in the LH rat, we developed a congenic rat model, where a portion of LN chromosome 17 is introgressed on the LH genomic background. Our hypothesis was that differences in metabolic features between the LH controls and the congenic rats (LH17LNa) would be due to the replaced region of the genome. Male and female LH17LNa rats and LH controls were phenotyped for a variety of MetS characteristics, including weekly body growth until tissue collection at 15 weeks of age, body composition by nuclear magnetic resonance (NMR) at 8, 11, and 14 weeks of age, metabolic rate (Promethion system) between 10 and 12 weeks of age, and adipose tissue collection and histological examination.We observed a significant decrease in total body growth in female congenics, beginning at 12 weeks of age and persisting throughout the rest of the study. Prior to the divergence in body weight, female congenics showed significant increases in fat mass and significant decreases in fat free mass that remained consistent at all timepoints measured. We also found significant female‐specific decreases in metabolic rate. Tissue collection revealed the source of the increased adiposity in the female LH17LNa rats was specific to abdominal white adipose tissue, and this phenomenon was further explained by significant hypertrophy in those adipocytes, with no evidence of adipocyte hyperplasia.Genome resequencing of the parental strains identified a nonsense mutation in Ercc6l2, implicating this gene as a strong contender underlying the phenotype differences in the congenics. Ercc6l2 is a helicase involved in transcription‐coupled DNA repair, which is specifically mobilized to repair oxidative stress‐induced DNA damage in post‐mitotic cells. Truncating mutations in Ercc6l2 have previously been implicated in genome instability and premature aging in hematopoetic stem cells in patients with bone marrow failure. The discovery of the significance of Ercc6l2 in the context of female‐specific adipocyte biology could represent a novel role of DNA repair syndromes in MetS pathogenesis.

Abstract 60: Perirenal Adipose Hypertrophy In A Congenic LH Rat: A Role For C17h6orf52

Central obesity, high blood pressure, dyslipidemia, and insulin resistance are a collection of cardiovascular and metabolic risk factors that form the basis of the Metabolic Syndrome (MetS) and represent a major public health burden worldwide. The phenotypes that define MetS are all highly heritable, but their genetic complexity necessitates the use of animal models to tease apart novel pathways. The Lyon Hypertensive (LH) rat is a well-characterized model of MetS, exhibiting profound differences in features of MetS compared to its metabolically healthy control, the Lyon Normotensive (LN) rat. To understand the genomic causes of MetS, we developed a congenic rat model, where a portion of LN chromosome 17 is introgressed on the LH genomic background. Male and female LH congenic (CON) rats and LH controls were phenotyped for a variety of MetS characteristics, including body growth and composition by nuclear magnetic resonance (NMR), metabolic rate (Promethion system), and adipose tissue collection and histological examination. There were significant decreases in body weight in the CON rats of both sexes compared to LH. We also found significant female-specific increases in body fat and decreases in metabolic rate. Tissue collection revealed the source of the increased adiposity in the female CON rats was specific to perirenal white adipose tissue (PWAT) and was further explained by significant hypertrophy in those adipocytes. Genome resequencing of the parental strains identified a gene, C17h6orf52 , as a strong contender underlying the phenotype differences in the congenics, with predicted amino acid changes and the loss of Nr2f2 transcription factor binding sites.

Abstract P117: Blood Pressure Heterosis And Genotype-Specific Regulation Of C17h6orf52

Although blood pressure (BP) is highly heritable, the genetic etiology is complex, and causative genes do not completely explain observed variation. Using the Lyon Hypertensive (LH) rat—a well-characterized polygenic inbred model—we previously identified a novel candidate gene ( C17h6orf52 ) as a master regulator of gene expression regulating cardiometabolic traits. CRISPR-Cas9 gene editing was used to generate a mutation in C17h6orf52 , and blood pressure in homozygous wildtype (WT), heterozygous (HET) and homozygous mutant (MUT) females was characterized via radiotelemetry. Herein, we show evidence of BP heterosis (i.e. hybrid vigor) in females from the C17h6orf52 strain. Systolic (SBP) and diastolic (DBP) pressures (mmHg) were determined in WT, HET, and MUT male and female rats on normal chow (CHOW), and 4% salt (NaCl) diet (to determine salt sensitivity). All measures are presented as mean±SEM, and significant results are indicated according to statistical test. No significant differences were identified in males, but the HET females (n=5) had significantly lower systolic blood pressure than either WT (n=7) or MUT (n=8) at both baseline and salt-stressed conditions: SBP-CHOW: (WT: 144±3.28; HET: 136.0±0.83 * ; MUT: 142.6±2.13)DBP-CHOW: (WT: 98.3±2.29; HET: 94.0±0.68; MUT: 97.4±1.76)SBP-NaCl: (WT: 150.4±3.40; HET: 142.2±1.19 * ; MUT: 150.2±1.83)DBP-NaCl: (WT: 103.3±2.75; HET: 98.2±1.68; MUT: 103.1±1.68) Tissues were collected to assess C17h6orf52 expression relative to WT, displayed below as confidence intervals. While there were no differences between WT and MUT expression, liver C17h6orf52 was significantly overexpressed in heterozygous females. C17h6orf52 2^-ddCT: (WT: 0.91-1.09, n=8; HET: 1.33-1.73 $ , n=5; MUT: 0.85-1.19, n=8) There were no striking differences in any metabolic phenotypes, such as body weight and adiposity, thus these data suggest that C17h6orf52 is protective against increases in blood pressure specifically. Continued study will attempt to tease apart allele-specific regulation and protein functions in heterozygous animals of both sexes. * p<0.05 by Kruskal-Wallis $ p<0.05 by 1-WAY ANOVA

Abstract 053: Gene Editing Yields a Novel Rat Model of Cardiometabolic Disease

Despite strong evidence that the Metabolic Syndrome (MetS) is highly heritable, the genetic etiology is complex and few causative genes are known. Using the Lyon Hypertensive (LH) rat we previously identified a novel candidate MetS gene ( C17h6orf52 ) as a putative master regulator of gene expression with pleiotropic effects on body weight and blood pressure regulation. In this study, CRISPR-Cas9 gene editing was used to generate mutations in C17h6orf52 , and females from the resulting strains, designated M1 and M2, were characterized, with all measures presented as mean±SEM. Lean and fat body mass as a percentage of total body composition was measured via nuclear magnetic resonance (NMR), and resting metabolic rate (RMR) was measured via indirect calorimetry. Blood pressure (BP) measurements were obtained via radiotelemetry. Surprisingly, the M1 and M2 mutations in C17h6orf52 result in different splicing variants that are shown to differentially modulate several cardiometabolic phenotypes. The M1 mutation causes significant changes in BP, body composition and RMR, without affecting body weight. The M2 mutation significantly increased body weight and adiposity without changing BP or RMR. These data suggest that C17h6orf52 is protective against cardiometabolic risk factors, given that the mutation confers a greater burden of MetS features on an already obese and hypertensive rat. The continued study of this rat model of MetS has the potential to functionally validate an uncharacterized regulatory gene and provide novel targets for pharmacological intervention in the treatment of cardiometabolic diseases. * p<0.05 by Students t-test & p<0.05 by ANCOVA $ p<0.05 by 2-WAY ANOVA

CRISPR‐Cas9 Gene Editing Yields a Novel Rat Model of Cardiometabolic Disease

Despite strong evidence that the Metabolic Syndrome (MetS) and its defining features (central obesity, dyslipidemia, hypertension, hyperglycemia) are highly heritable, the genetic etiology is complex and relatively few causative genes are known. For this reason, we employ the Lyon Hypertensive (LH) rat, a well‐characterized polygenic inbred MetS model. Previous genetic and computational approaches identified a novel gene (C17h6orf52) as a putative master regulator of gene expression and oxidative phosphorylation with pleiotropic effects on body weight and blood pressure regulation.In this study, two different frameshift mutations were generated independently by CRISPR‐Cas9 gene editing, producing either a 5 base pair deletion or a 10 base pair insertion in exon 2 of C17h6orf52, and females from the resulting mutant rat strains, designated M1 and M2, respectively, were characterized (Table 1). Herein, mutations in C17h6orf52 are shown to significantly increase blood pressure and serum cholesterol in vivo, despite no significant changes in body composition or body weight in LH‐derived female rats.Either mutation appears to cause modest, but significant or nearly significant, increases in blood pressure on a 0.3% NaCl diet. M1−/− females exhibit significantly increased systolic and diastolic blood pressure, while M2−/− females' systolic and diastolic blood pressure is more moderately elevated. This effect is potentiated when animals are administered a high salt diet. Finally, although neither total nor low‐density lipoprotein (LDL) cholesterol is changed in the M1 female rats with respect to their wildtype control, M2 females develop higher serum cholesterol, as well as significantly higher LDL cholesterol.The differences in phenotype between the M1 and M2 mutations were unexpected. Preliminary data indicates that 5 base pair deletion present in the M1−/− rats results in two distinct splicing variants that may be contributing to the observed phenotype differences. Nevertheless, these data suggest that C17h6orf52 is protective against cardiometabolic risk factors, given that C17h6orf52 mRNA is more highly expressed in MetS‐susceptible LH rats than MetS resistant Lyon Normotensive (LN) rats, and the mutation confers a greater burden of MetS features on an already obese and hypertensive rat. The continued study of this rat model of Metabolic Syndrome has the potential to functionally validate an uncharacterized regulatory gene, and provide novel targets for pharmacological intervention in the treatment of obesity.Support or Funding InformationR01HL089895, R21DK089417, T32GM008629, 14GRNT20410043This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P469: CRISPR-Cas9 Gene Editing Yields Genetic Regulation of Obesity and Metabolism in Female LH-derived Rats

Despite strong evidence that the Metabolic Syndrome (MetS) and its defining features (central obesity, dyslipidemia, hypertension, hyperglycemia) are highly heritable, the genetic etiology is complex and relatively few causative genes are known. For this reason, we employ the Lyon Hypertensive (LH) rat, a well-characterized genetic model of MetS. Previously, we identified a novel gene ( RGD1562963 ) on LH rat chromosome 17 (RNO17) that affects components of MetS, using a combination of genetic mapping and gene network analyses. In this study, frameshift mutations (by CRISPR-Cas9) in exon 2 of RGD1562963 is shown to change resting metabolic rate (RMR) and body composition in LH-derived female rats, measured at 8 and 11 weeks of age, despite no differences in either total body weight or food consumption. RGD1562963 -/- ( RGD -/- ) females have significantly more body fat mass than their wild-type control at 8 weeks of age, measured by nuclear magnetic resonance (NMR) as a percentage of total body weight ( # 8 weeks: WT = 5.08±0.099, n=5; RGD -/- = 6.52±0.260, n=8; *p<.001), and this difference increases by 11 weeks of age (WT = 5.39±0.140, n=5; RGD -/- = 7.81±0.293, n=8, *p<.0001). RMR measurements at 8 weeks showed that both genotypes had identical resting energy expenditure (WT: 0.721±0.033 kcal/hr, n=5; RGD -/- : 0.721±0.021 kcal/hr, n=6) and body weights (WT = 160.5g±6.884, n=5; RGD -/- = 160.7g±4.246, n=8). Though no body weight differences appear by 11 weeks (WT: 196.5g±7.226, n=5; RGD -/- : 196.1g±3.765, n=8), the wild-type females have an unexpected decrease in RMR, while the RMR of RGD -/- females remains high (WT: 0.654±0.005 kcal/hr, n=4; RGD -/- : 0.759±0.014 kcal/hr, n=7, *p<.01). Our studies suggest RGD1562963 is an obesity susceptibility gene. Furthermore, inhibition of this gene at the whole body level is tolerated, and unexpectedly induced increased resting metabolic rate despite increased adiposity, possibly as a protective mechanism. The continued study of this rat model of Metabolic Syndrome has the potential to functionally validate an uncharacterized regulatory gene, and provide novel targets for pharmacological intervention in the treatment of obesity. # all measures are presented as mean±SEM, * RM 2-WAY ANOVA, Sidak’s correction

Contribution of independent and pleiotropic genetic effects in the metabolic syndrome in a hypertensive rat.

Hypertension is a major risk factor for cardiovascular disease, Type 2 diabetes, and end organ failure, and is often found concomitant with disorders characteristic of the Metabolic Syndrome (MetS), including obesity, dyslipidemia, and insulin resistance. While the associated features often occur together, the pathway(s) or mechanism(s) linking hypertension in MetS are not well understood. Previous work determined that genetic variation on rat chromosome 17 (RNO17) contributes to several MetS-defining traits (including hypertension, obesity, and dyslipidemia) in the Lyon Hypertensive (LH) rat, a genetically determined MetS model. We hypothesized that at least some of the traits on RNO17 are controlled by a single gene with pleiotropic effects. To address this hypothesis, consomic and congenic strains were developed, whereby a defined fragment of RNO17 from the LH rat was substituted with the control Lyon Normotensive (LN) rat, and MetS phenotypes were measured in the resultant progeny. Compared to LH rats, LH-17LN consomic rats have significantly reduced body weight, blood pressure, and lipid profiles. A congenic strain (LH-17LNc), with a substituted fragment at the distal end of RNO17 (17q12.3; 74–97 Mb; rn4 assembly), showed differences from the LH rat in blood pressure and serum total cholesterol and triglycerides. Interestingly, there was no difference in body weight between the LH-17LNc and the parental LH rat. These data indicate that blood pressure and serum lipids are regulated by a gene(s) in the distal congenic interval, and could be due to pleiotropy. The data also indicate that body weight is not determined by the same gene(s) at this locus. Interestingly, only two small haplotypes spanning a total of approximately 0.5 Mb differ between the LH and LN genomes in the congenic interval. Genes in these haplotypes are strong candidate genes for causing dyslipidemia in the LH rat. Overall, MetS, even in a simplified genetic model such as the LH-17LN rat, is likely due to both independent and pleiotropic gene effects.

Independent and Pleiotropic Gene Effects on Hypertension and Metabolic Syndrome in Lyon Hypertensive Rats

The Lyon Hypertensive (LH) rat is an inbred selection model of spontaneous hypertension. However through the selection and inbreeding of the LH strain for high blood pressure, genes causing salt‐sensitivity and several features of the Metabolic Syndrome (MetS) including obesity and dyslipidemia were also fixed; thus the LH rat is a genetic model of MetS associated hypertension. Conversely, the Lyon Normotensive (LN) control strain is normotensive, salt‐resistant and displays no features of MetS. Both strains were selectively bred from the same outbred Sprague Dawley founders, based on divergent blood pressures, yet their genomes are >99.98% identical. Our overall goal is to identify novel genes and pathways linking disparate phenotypes in MetS. We performed comprehensive systems genetic studies in LH rats and determined that rat chromosome (RNO) 17 contributes not only hypertension but also obesity and dyslipidemia. Genetic mapping studies identified overlapping quantitative trait loci (QTL) for blood pressure, body weight, and plasma lipids on RNO17 in the LH rat. Whole genome sequencing of LH and LN strains revealed large blocks of RNO17 are of shared ancestry; haplotype fine‐mapping refined a list of 26 candidate causal genes. Consomic and congenic studies determined that the whole chromosome substitution of LH RNO17 with that of the LN was sufficient to reduce blood pressure, lipid levels, and body weight. Interestingly, a congenic strain substituting only the distal end of LH RNO17 with the LN allele reduces blood pressure and serum lipids independent of body weight, suggesting both independent and pleiotropic gene effects in LH hypertension. We used RNA‐seq to identify transcriptome differences between LH and LN strains, and to map genetic regulation of gene expression (eQTL). Proximal RNO17 was found to regulate the expression of over 60 genes located elsewhere in the genome (a trans‐eQTL hotspot). A single candidate gene in this locus (RGD1562963), is cis‐regulated, has sequence variation between LH and LN rats, is differentially expressed in multiple tissues between LH and LN, and controls the expression of downstream genes mapping to the trans‐eQTL hotspot that are associated with hypertension and MetS. Our integrative studies suggest that genetic variation in LH RNO17 has independent and pleiotropic effects on hypertension and features of MetS.Support or Funding InformationThis work is supported by NIH grants R01HL089895 and R21DK089417, AHA grant 14GRNT20410043, and the Fraternal Order of Eagles Diabetes Research Center at the University of Iowa.

Abstract P638: Investigating Gene Pleiotropy in the Metabolic Syndrome in Lyon Hypertensive Rats

The metabolic syndrome (MetS) - hypertension, obesity, dyslipidemia, and insulin resistance - is a major risk factor for cardiovascular disease and stroke. Our overall goal is to identify novel genes and pathways causing MetS. Our previous work determined that rat chromosome 17 (RNO17) contributes to several MetS-defining traits (including high blood pressure, obesity, and dyslipidemia) in the Lyon Hypertensive (LH) rat, a genetically determined MetS rat model. We hypothesized that at least some of the traits on RNO17 are controlled by a single gene with pleiotropic effects. To address this hypothesis, we generated congenic strains where a defined fragment of RNO17 from the LH rat was substituted by that of the control Lyon Normotensive (LN) rat, and measured MetS phenotypes. One congenic (LH-17 LN a), with the proximal 30 Mb of RNO17 from the LH genome substituted with that of the LN genome, did not show significant differences from the LH parental strain. However, another congenic strain (LH-17 LN c), with a substituted fragment at the distal end of RNO17 (84-97 Mb), showed significant differences from the LH rat in serum total cholesterol (3.15 ± 0.15 vs. 4.29 ± 0.17 mMol; p<0.01) and triglycerides (0.47 ± 0.06 vs. 1.27± 0.13 mMol; p<0.001), and a trend for reduced blood pressure (SBP 150.8 ± 3.4 vs. 157.1 ± 1.7 mmHg; p=0.1). Interestingly, there was no difference in body weight between the LH-17 LN c and the parental LH rat (440 + 7.2 vs. 435 + 9.1 g). These data indicate that serum cholesterol and triglycerides, and possibly blood pressure are regulated by a gene(s) in the distal congenic interval, and could be due to pleiotropy. The data also indicate body weight is not determined by the same gene(s). Interestingly, only two small haplotypes spanning a total of 1 Mb differ between the LH and LN genomes in the congenic interval. Genes in these haplotypes are being studied as candidate genes for causing dyslipidemia in the LH rat. Overall MetS, even in a simplified genetic model such as the LH-17 LN rat, is likely due to both independent and pleiotropic gene effects.

Systems biology with high-throughput sequencing reveals genetic mechanisms underlying the metabolic syndrome in the Lyon hypertensive rat.

The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including obesity, hypertension, dyslipidemia, and insulin resistance. The Lyon hypertensive and Lyon normotensive rats are models of MetS sensitivity and resistance, respectively. To identify genetic determinants and mechanisms underlying MetS, an F2 intercross between Lyon hypertensive and Lyon normotensive was comprehensively studied. Multidimensional data were obtained including genotypes of 1536 single-nucleotide polymorphisms, 23 physiological traits, and >150 billion nucleotides of RNA-seq reads from the livers of F2 intercross offspring and parental rats. Phenotypic and expression quantitative trait loci (eQTL) were mapped. Application of systems biology methods identified 17 candidate MetS genes. Several putative causal cis-eQTL were identified corresponding with phenotypic QTL loci. We found an eQTL hotspot on rat chromosome 17 that is causally associated with multiple MetS-related traits and found RGD1562963, a gene regulated in cis by this eQTL hotspot, as the most likely eQTL driver gene directly affected by genetic variation between Lyon hypertensive and Lyon normotensive rats. Our study sheds light on the intricate pathogenesis of MetS and demonstrates that systems biology with high-throughput sequencing is a powerful method to study the pathogenesis of complex genetic diseases.

Abstract 617: The Consomic LH-17 LN Rat Is A Single Chromosome Model Of Metabolic Syndrome

The Lyon Hypertensive (LH) rat is an inbred model of Metabolic Syndrome, exhibiting spontaneous hypertension, high body weight, high plasma lipids, altered insulin:glucose ratio. The Lyon Normotensive (LN) control strain, selectively bred for normal blood pressure from the same Sprague Dawley (SD) colony, is genetically quite similar to the LH, but displays no features of MetS. Genetic mapping in an LHxLN cross determined that all of the MetS phenotypes mapped to rat chromosome (RNO 17). In order to identify the gene(s) causing MetS on RNO17 we generated a consomic strain (LH-17 LN ) where the LH RNO17 was replaced with that of the LN strain. Substitution of the single chromosome was sufficient to significantly lower blood pressure lower systolic blood pressure (145 + 2 vs. 161 + 2 mmHg; p<0.05), body weight (408 + 6 vs 453 + 5 g; p<0.05), and plasma cholesterol (2.5 ± 0.1 vs 2.9 ± 0.08; p<0.05) compared to the LH parental strain. To identify putative causal genes for MetS, we fine-mapped RNO17 in silico utilizing the genome sequence of the LH and LN rat strains. The genomes of the LH and LN strains are highly similar, differing by <0.02% at the sequence level (in comparison, humans typically differ by about 1%). Moreover, we have shown that the variants cluster into <500 divergent haplotype blocks with an average length of 880 kb, which are presumed to contain the genetic variation causing the phenotypic variation between the LH and LN strains. On RNO17 there are just 14 divergent haplotypes, containing 11 genes with coding sequence differences in the LH. The combined in vivo and in silico approaches allowed for the identification of candidate causal variants for the phenotypes on RNO17 and will be studied further to determine their role(s) in MetS.

Genomic structure of nucleotide diversity among Lyon rat models of metabolic syndrome

BackgroundThe metabolic syndrome (MetS), a complex disorder involving hypertension, obesity, dyslipidemia and insulin resistance, is a major risk factor for heart disease, stroke, and diabetes. The Lyon Hypertensive (LH), Lyon Normotensive (LN) and Lyon Low-pressure (LL) rats are inbred strains simultaneously derived from a common outbred Sprague Dawley colony by selection for high, normal, and low blood pressure, respectively. Further studies found that LH is a MetS susceptible strain, while LN is resistant and LL has an intermediate phenotype. Whole genome sequencing determined that, while the strains are phenotypically divergent, they are nearly 98% similar at the nucleotide level. Using the sequence of the three strains, we applied an approach that harnesses the distribution of Observed Strain Differences (OSD), or nucleotide diversity, to distinguish genomic regions of identity-by-descent (IBD) from those with divergent ancestry between the three strains. This information was then used to fine-map QTL identified in a cross between LH and LN rats in order to identify candidate genes causing the phenotypes.ResultsWe identified haplotypes that, in total, contain at least 95% of the identifiable polymorphisms between the Lyon strains that are likely of differing ancestral origin. By intersecting the identified haplotype blocks with Quantitative Trait Loci (QTL) previously identified in a cross between LH and LN strains, the candidate QTL regions have been narrowed by 78%. Because the genome sequence has been determined, we were further able to identify putative functional variants in genes that are candidates for causing the QTL.ConclusionsWhole genome sequence analysis between the LH, LN, and LL strains identified the haplotype structure of these three strains and identified candidate genes with sequence variants predicted to affect gene function. This approach, merged with additional integrative genetics approaches, will likely lead to novel mechanisms underlying complex disease and provide new drug targets and therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-197) contains supplementary material, which is available to authorized users.

Abstract 18436: Systems Biology Identifies a Gene Driving Expression of Metabolic Syndrome in Lyon Rats

The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including obesity, hypertension, dyslipidemia, and insulin resistance. The Lyon Hypertensive (LH) and Lyon Normotensive (LN) rats are models of MetS sensitivity and resistance, respectively. To identify genetic loci influencing phenotypes underlying MetS, an F2 intercross between LH and LN was studied to identify physiological quantitative trait loci (pQTL) contributing to plasma lipid and leptin levels, blood pressure, and body weight/adiposity. We performed mRNA-seq in F2 rats to identify the loci regulating gene expression, or expression (e)QTL, followed by gene expression network analyses. Systems biology methods were applied to identify candidate MetS genes, including genes previously shown to be MetS-related. We found an eQTL hotspot on RNO17, which was also located within pQTLs for MetS-related traits. The genes regulated by this eQTL hotspot were mainly in two co-expression network modules (a mitochondria related module and a gene regulation related module) and causally affect many MetS-related traits. Multiple evidences strongly and consistently support RGD1562963, a gene regulated in cis by this eQTL hotspot as the eQTL driver gene directly affected by genetic variation between LH and LN rats. Our study sheds light on the intricate pathogenesis of MetS and that systems biology with high-throughput sequencing is a powerful method to study the etiology of complex multifactorial diseases.

Abstract 518: Systems Biology Approach to Identify Genetic Mechanisms Underlying the Metabolic Syndrome in the LH Rat

The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including hypertension, obesity, dyslipidemia, and insulin resistance. The Lyon Hypertensive (LH) and Lyon Normotensive (LN) rats are models of MetS sensitivity and resistance, respectively. To identify genetic determinants and mechanisms underlying MetS, rats from an F2 intercross between LH and LN were studied. Multi-dimensional data were obtained including SNP genotypes, physiological traits including blood pressure, plasma lipid and leptin levels, and body weight/adiposity, and more than 150 billion nucleotides of RNA-Seq reads from the livers of F2 (n=36), LH (n=6), and LN (n=6) individuals. We identified 17 pQTLs (physiological quantitative trait loci) and 1200 eQTLs (gene expression quantitative trait loci) at genome-wide p<0.05. Systems biology methods were applied to prioritize candidate MetS genes, including genes previously shown to be MetS-related. We identified an eQTL peak on rat chromosome 17 (RNO17) shared by large number of genes; RNO17 also has pQTL for MetS-related traits. Network analysis in these genes identified two major co-regulation modules involving mitochondria and gene regulation; genes in the two modules causally affect multiple MetS traits. A gene, RGD1562963, closest to the eQTL peak was identified as the eQTL driver gene. It is directly affected by genetic variation between LH and LN rats, affects downstream genes in the eQTL cluster, and is correlated with MetS phenotypes. Our study sheds light on the intricate pathogenesis of MetS and proved that systems biology with high-throughput sequencing is a powerful method to study the etiology of complicated diseases.

Abstract 651: Genetic Mapping Of Traits And Transcriptome In The Lyon Hypertensive Rat

The metabolic syndrome (MetS) is characterized by obesity, dyslipidemia, hypertension, and insulin resistance and is a major risk factor for cardiovascular disease. Using the Lyon Hypertensive and Lyon Normotensive rats as models of the metabolic syndrome, we aim to determine both physiological and transcriptional regulation underlying the syndrome. To identify genetic loci influencing phenotypes underlying MetS, an F2 intercross between LH and LN with 150 offspring was studied to identify physiological quantitative trait loci (pQTL) contributing to plasma lipid levels, blood pressure, and body weight/adiposity. We performed RNA-seq in livers from 36 selected rats from the intercross to identify the mapping of gene expression, or expression (e)QTL. Linkage analyses identified pQTL and eQTL with significant logarithm of odds (lod) scores, whose genome-wide significance was determined by permutation testing. We identified 17 pQTL on 5 rat chromosomes and 75 on 16 chromosomes. It has been determined that LH chromosome 17 contributes to multiple features of MetS; furthermore, an eQTL on rat chromosome 17 was identified that regulates the expression of multiple genes in trans, meaning the regulated gene falls outside the QTL interval or on another chromosome. We hypothesize these genes share a common transcriptional regulator which is affected by genetic variation on chromosome 17. eQTLs are likely to influence precursor pathways and cofactors of the metabolic syndrome. Identifying these in the LH rat could lead to translational studies to determine their role in the human MetS.

Durable Improvement of Renal Function After Perindopril Withdrawal in Lyon Hypertensive Rats

To determine whether the persistent antihypertensive effect observed after withdrawal of angiotensin-converting enzyme inhibition was specific and whether it was associated with durable improvement of renal function. Lyon hypertensive (LH) rats were treated from 3 and 12 weeks of age with perindopril at the dose of 0.4 (P0.4) or 1.5 (P1.5) mg·kg(-1)·d(-1), with double (hydralazine 75 mg·kg(-1)·d(-1) and hydrochlorothiazide 15 mg·kg(-1)·d(-1)), or triple antihypertensive therapy (reserpine 0.75 mg·kg(-1)·d(-1) was added). Blood pressure (BP) was recorded by telemetry, and renal functions were evaluated in freely moving rats. Despite similar BP reduction after perindopril withdrawal in P0.4 and P1.5 groups, greater decrease in proteinuria was observed in P1.5 group. Double or triple therapy prevented the hypertension of LH rats, but without any persistent antihypertensive or antiproteinuric effect after its withdrawal. Salt load elicited an increase in BP that was similar in untreated LH and both perindopril-pretreated groups (+30 mm Hg), but more pronounced in double therapy–pretreated or triple therapy–pretreated groups (+50 mm Hg). The pressure–natriuresis curve shifted to lower BP levels in P0.4 and P1.5 groups, whereas it was blunted in double and triple therapy groups. Angiotensin-converting enzyme inhibition has a durable renoprotection after treatment withdrawal that is independent of BP lowing.

Comments on Point:Counterpoint: The dominant contributor to systemic hypertension: Chronic activation of the sympathetic nervous system vs. Activation of the intrarenal renin-angiotensin system

Comments on Point:Counterpoint: The dominant contributor to systemic hypertension: Chronic activation of the sympathetic nervous system vs. Activation of the intrarenal renin-angiotensin system

Visfatin is associated with lipid metabolic abnormalities in Lyon hypertensive rats

1. Visfatin (also known as nicotinamide phosphoribosyltransferase or Nampt) is a novel adipokine associated with metabolic abnormalities in obesity and diabetes. The aim of the present study was to determine whether visfatin levels in the circulation and visfatin expression in fat tissues are altered in Lyon hypertensive (LH) rats, a hypertensive strain with numerous metabolic abnormalities, and, if so, to explore the possible correlations between blood visfatin levels and the metabolic abnormalities in LH rats. 2. Male 18-20-week-old LH rats and their control, Lyon normotensive (LN) rats, were used. Blood pressure was recorded in conscious, unrestrained rats. Fat tissue weight was determined. Serum visfatin concentrations were determined by enzyme immunoassay, whereas visfatin expression in fat tissues was determined by western blotting. Relationships between two parameters were evaluated by linear regression analysis. 3. The LH rats had significantly higher bodyweight and fat tissue weight compared with LN rats. Serum lipid levels of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) were all significantly higher in LH rats than in LN rats. Moreover, serum visfatin levels were higher in LH rats than in LN rats and were positively correlated with bodyweight, fat tissue weight and serum lipid levels (i.e. TC, HDL-C, LDL-C, TG and the TG:HDL-C ratio). There were no significant differences in serum glucose and insulin concentrations, or in the whole-body insulin resistance index, between LN and LH rats, and serum visfatin levels were not correlated with any of these parameters. Visfatin expression in visceral fat tissue, but not in subcutaneous fat tissue, was markedly upregulated in LH compared with LN rats. 4. In conclusion, the results of the present study demonstrate that serum visfatin levels in LH rats are elevated and are associated with lipid metabolic abnormalities.

Meta-Analysis of Genome-Wide Gene Expression Differences in Onset and Maintenance Phases of Genetic Hypertension

Gene expression differences accompany both the onset and established phases of hypertension. By an integrated genome-transcriptome approach we performed a meta-analysis of data from 74 microarray experiments available on public databases to identify genes with altered expression in the kidney, adrenal, heart, and artery of spontaneously hypertensive and Lyon hypertensive rats. To identify genes responsible for the onset of hypertension we used a statistical approach that sought to eliminate expression differences that occur during maturation unrelated to hypertension. Based on this adjusted fold-difference statistic, we found 36 genes for which the expression differed between the prehypertensive phase and established hypertension. Genes having possible relevance to hypertension onset included Actn2, Ankrd1, ApoE, Cd36, Csrp3, Me1, Myl3, Nppa, Nppb, Pln, Postn, Spp1, Slc21a4, Slc22a2, Thbs4, and Tnni3. In established hypertension 102 genes exhibited altered expression after Bonferroni correction (P<0.05). These included Atp5o, Ech1, Fabp3, Gnb3, Ldhb, Myh6, Lpl, Pkkaca, Vegfb, Vcam1, and reduced nicotinamide-adenine dinucleotide dehydrogenases. Among the genes identified, there was an overrepresentation of gene ontology terms involved in energy production, fatty acid and lipid metabolism, oxidation, and transport. These could contribute to increases in reactive oxygen species. Our meta-analysis has revealed many new genes for which the expression is altered in hypertension, so pointing to novel potential causative, maintenance, and responsive mechanisms and pathways.