Genetic analysis of metabolic defects in the spontaneously hypertensive rat

Abnormalities in carbohydrate and lipid metabolism are common in patients with essential hypertension and in the spontaneously hypertensive rat (SHR). To identify chromosome regions contributing to this clustering of cardiovascular risk factors in the SHR, we searched for quantitative trait loci (QTL) associated with insulin resistance, glucose intolerance, and dyslipidemia by using the HXB/BXH recombinant inbred (RI) strains. Analysis of variance in RI strains suggested significant effects of genetic factors. A genome screening of the RI strains with more than 700 markers revealed QTL significantly associated with insulin resistance on Chromosomes (Chrs) 3 and 19. The Chr 19 QTL was confirmed by testing a previously derived SHR-19 congenic strain: transfer of a Chr 19 segment delineated by markers D19Rat57 and D19Mit7 from the Brown Norway (BN/Cr) strain onto the genetic background of the SHR/Ola was associated with decreased insulin and glucose concentrations and ameliorated insulin resistance at the tissue level. These findings suggest that closely linked genes on Chr 19, or perhaps even a single gene with pleiotropic effects, influence the clustering of metabolic disturbances in the SHR-BN model.     

Closely linked non-additive blood pressure quantitative trait loci

There is enough evidence through linkage and substitution mapping to indicate that rat chromosome 1 harbors multiple blood pressure (BP) quantitative trait loci (QTLs). Of these, BP QTL1b was previously reported from our laboratory using congenic strains derived by introgressing normotensive alleles from the LEW rat onto the genetic background of the hypertensive Dahl salt-sensitive (S) rat. The region spanned by QTL1b is quite large (20.92 Mb), thus requiring further mapping with improved resolution so as to facilitate systematic identification of the underlying genetic determinant(s). Using congenic strains containing the LEW rat chromosomal segments on the Dahl salt-sensitive (S) rat background, further iterations of congenic substrains were constructed and characterized. Collective data obtained from this new iteration of congenic substrains provided evidence for further fragmentation of QTL1b with improved resolution. At least two separate genetic determinants of blood pressure underlie QTL1b. These are within 7.40 Mb and 7.31 Mb and are known as the QTL1b1 region and the QTL1b2 region, respectively. A genetic interaction was detected between the two BP QTLs. Interestingly, five of the previously reported differentially expressed genes located within the newly mapped QTL1b1 region remained differentially expressed. The congenic strain S.LEW(D1Mco36-D1Mco101), which harbors the QTL1b1 region alone but not the QTL1b2 region, serves as a genetic tool for further dissection of the QTL1b1 region and validation of Nr2f2 as a positional candidate gene. Overall, this study represents an intermediary yet obligatory progression towards the identification of genetic elements controlling BP. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. E. J. Toland and Y. Saad contributed equally to this work.     

Genetic isolation of quantitative trait loci for blood pressure development and renal mass on chromosome 5 in the spontaneously hypertensive rat

Total genome scans of genetically segregating populations derived from spontaneously hypertensive rats (SHR) and other rat models of essential hypertension suggested a presence of quantitative trait loci (QTL) regulating blood pressure on multiple chromosomes, including chromosome 5. The objective of the current study was to test directly a hypothesis that chromosome 5 of the SHR carries a blood pressure regulatory QTL. A new congenic strain was derived by replacing a segment of chromosome 5 in the SHR/Ola between the D5Wox20 and D5Rat63 markers with the corresponding chromosome segment from the normotensive Brown Norway (BN/Crl) rat. Arterial pressures were directly monitored in conscious, unrestrained rats by radiotelemetry. The transfer of a segment of chromosome 5 from the BN strain onto the SHR genetic background was associated with a significant decrease of systolic blood pressure, that was accompanied by amelioration of renal hypertrophy. The heart rates were not significantly different in the SHR compared to SHR chromosome 5 congenic strain. The findings of the current study demonstrate that gene(s) with major effects on blood pressure and renal mass exist in the differential segment of chromosome 5 trapped within the new SHR.BN congenic strain.     

Isolation of a Genomic Region Affecting Most Components of Metabolic Syndrome in a Chromosome-16 Congenic Rat Model

Metabolic syndrome is a highly prevalent human disease with substantial genomic and environmental components. Previous studies indicate the presence of significant genetic determinants of several features of metabolic syndrome on rat chromosome 16 (RNO16) and the syntenic regions of human genome. We derived the SHR.BN16 congenic strain by introgression of a limited RNO16 region from the Brown Norway congenic strain (BN-Lx) into the genomic background of the spontaneously hypertensive rat (SHR) strain. We compared the morphometric, metabolic, and hemodynamic profiles of adult male SHR and SHR.BN16 rats. We also compared in silico the DNA sequences for the differential segment in the BN-Lx and SHR parental strains. SHR.BN16 congenic rats had significantly lower weight, decreased concentrations of total triglycerides and cholesterol, and improved glucose tolerance compared with SHR rats. The concentrations of insulin, free fatty acids, and adiponectin were comparable between the two strains. SHR.BN16 rats had significantly lower systolic (18–28 mmHg difference) and diastolic (10–15 mmHg difference) blood pressure throughout the experiment (repeated-measures ANOVA, P < 0.001). The differential segment spans approximately 22 Mb of the telomeric part of the short arm of RNO16. The in silico analyses revealed over 1200 DNA variants between the BN-Lx and SHR genomes in the SHR.BN16 differential segment, 44 of which lead to missense mutations, and only eight of which (in Asb14, Il17rd, Itih1, Syt15, Ercc6, RGD1564958, Tmem161a, and Gatad2a genes) are predicted to be damaging to the protein product. Furthermore, a number of genes within the RNO16 differential segment associated with metabolic syndrome components in human studies showed polymorphisms between SHR and BN-Lx (including Lpl, Nrg3, Pbx4, Cilp2, and Stab1). Our novel congenic rat model demonstrates that a limited genomic region on RNO16 in the SHR significantly affects many of the features of metabolic syndrome.     

Mapping of quantitative trait loci for seminal vesicle mass and litter size to rat chromosome 8

The spontaneously hypertensive rat (SHR) and the Brown Norway (BN) rat differ significantly in litter size (7.6 versus 4.5 pups). In the HXB and BXH sets of recombinant inbred (RI) strains derived from SHR and BN rats, heritability of litter size and of selected male reproductive parameters such as sperm production, sperm count, sperm morphology and motility, and the mass of the testis, epididymides, and seminal vesicles were estimated and a search was undertaken for quantitative trait loci (QTL) associated with these phenotypes. The mass of seminal vesicles was significantly associated with a QTL near the D8Cebr204S21 marker on chromosome 8 (LOD score = 4.1, P = 0.00001); this QTL was responsible for 46% of the genetic variability of the trait. The same gene marker on chromosome 8 also showed a suggestive association with the litter size. Litter size was significantly correlated with the mass of seminal vesicles (r = 0.58, P = 0.003). These findings indicate that the variability in litter size among RI strains may be due in part to differences in the mass of seminal vesicles and it is possible that both mass of seminal vesicles and litter size are determined by a pleiotropic effect of the same QTL on rat chromosome 8.     

Congenic Mice Provide Evidence for a Genetic Locus That Modulates Spontaneous Arthritis Caused by Deficiency of IL-1RA

To understand the role of genetic factors involved in the development of spontaneous arthritis in mice deficient in IL-1 receptor antagonist protein (IL_1RA), we have identified a genomic region containing a major quantitative trait locus (QTL) for this disease. The QTL is on chromosome 1 and appears to be the strongest genetic region regulating arthritis. To confirm the importance of the QTL and to identify potential candidate genes within it, we conducted speed congenic breeding to transfer the QTL region from DBA/1 mice that are resistant to spontaneous arthritis into BALB/c^−/− which are susceptible. Genetic markers along every chromosome were used to assist in the selection of progeny in each generation to backcross to BALB/c^−/−. By the 6th generation we determined that all of the chromosomes in the progeny were of BALB/c origin with the exception of portions of chromosome 1. At this stage we intercrossed selected mice to produce homozygous strains containing the genomic background of BALB/c^−/− except for the QTL region on chromosome 1, which was from DBA/1. We were able to establish two congenic strains with overlapping DBA/1 DNA segments. These strains were observed for the development of spontaneous arthritis. Both congenic strains were relatively resistant to spontaneous arthritis and had delayed onset and reduced severity of disease. The gene/s that regulates this major QTL would appear to be located in the region of the QTL that is shared by both strains. The common transferred region is between D1Mit110 and D1Mit209 on chromosome 1. We evaluated this region for candidate genes and have identified a limited number of candidates. Confirmation of the identity and precise role of the candidates will require additional study.     

A large QTL for fear and anxiety mapped using an F2 cross can be dissected into multiple smaller QTLs

Using chromosome substitution strains (CSS), we previously identified a large quantitative trait locus (QTL) for conditioned fear (CF) on mouse chromosome 10. Here, we used an F₂ cross between CSS‐10 and C57BL/6J (B6) to localize that QTL to distal chromosome 10. That QTL accounted for all the difference between CSS‐10 and B6. We then produced congenic strains to fine‐map that interval. We identified two congenic strains that captured some or all the QTL. The larger congenic strain (Line 1: 122.387121–129.068 Mb; build 37) appeared to account for all the difference between CSS‐10 and B6. The smaller congenic strain (Line 2: 127.277–129.068 Mb) was intermediate between CSS‐10 and B6. We used haplotype mapping followed by quantitative polymerase chain reaction to identify one gene that was differentially expressed in both lines relative to B6 (Rnf41) and one that was differentially expressed between only Line 1 and B6 (Shmt2). These cis‐eQTLs may cause the behavioral QTLs; however, further studies are required to validate these candidate genes. More generally, our observation that a large QTL mapped using CSS and F₂ crosses can be dissected into multiple smaller QTLs shows a weaknesses of two‐stage approaches that seek to use coarse mapping to identify large regions followed by fine‐mapping. Indeed, additional dissection of these congenic strains might result in further subdivision of these QTL regions. Despite these limitations, we have successfully fine‐mapped two QTLs to small regions and identified putative candidate genes, showing that the congenic approach can be effective for fine‐mapping QTLs .     

Derivation of SHR-chromosome 4 congenic sublines for fine genetic mapping of quantitative trait loci with major effects on insulin resistance and blood pressure

Recently, we have found that transfer of a segment of chromosome 4 between I16 and Npy markers from the Brown Norway (BN) rat into the spontaneously hypertensive rat (SHR) significantly attenuated both hypertension (measured by telemetry) and insulin resistance (measured as plasma insulin/glucose ratios before and after a high fructose diet) in the SHR progenitor strain. To map the putative quantitative trait loci (QTL) more precisely, we derived an (SHR×SHR.BN-chr.4)F2 population to search for recombinants that will enable us to produce congenic sublines. The F2 animals were genotyped in markers equally distributed along the interval of the chromosome 4 differential segment. Altogether, five new congenic sublines with overlapping segments of the differential chromosome 4 are being produced. New congenic sublines will enable us to test the hypothesis that insulin resistance and hypertension can be influenced by closely linked genes or perhaps even the same gene(s) on chromosome 4.     

A new framework marker-based linkage map and SDPs for the Rat HXB/BXH strain set

A new contiguous genetic linkage map of the HXB/BXH set of rat recombinant inbred (RI) strains was constructed to enhance QTL mapping power and precision, and thereby make the RI strain set a better genomics resource. The HXB/BXH rat RI strains were developed from a cross between the hypertensive SHR/OlaIpcv and normotensive BN-Lx/Cub rat strains and have been shown useful for identifying quantitative trait loci (QTL) for a variety of cardiovascular, metabolic, and behavioral phenotypes. In the current analysis, the DNAs from 31 existing strains, 1 substrain, and 4 extinct strains were genotyped for a selection of polymorphic microsatellite marker loci, predominantly polymorphic framework markers from high-density integrated rat genome maps. The resulting linkage map consists of 245 microsatellite markers spanning a total length of 1789 cM with an average inter-marker distance of ~8.0 cM. This map covers the rat genome contiguously and completely with the exception of two locations on Chromosomes (Chrs) 11 and 16. The new genotypic information obtained also permitted further genetic characterization of the RI strain set including strain independence, genetic similarity among the individual strains, and non-syntenic associations between loci.