Mapping of a quantitative trait locus for blood pressure on rat chromosome 2.

A genetic map for rat chromosome 2 that includes five candidate genes for blood pressure regulation was constructed in a region containing a quantitative trait locus (QTL) for blood pressure. Two F2 populations of male rats raised on high salt (8% NaCI) diet from weaning were studied: F2(WKY x S), derived from a cross of Dahl salt-sensitive rats (S) and Wistar-Kyoto rats (WKY); and F2(MNS x S), derived from a cross of S rats and Milan normotensive strain (MNS). In both populations a blood pressure QTL was localized between Na+,K(+)-ATPase alpha 1 isoform and calmodulin-dependent protein kinase II-delta loci. The LOD score for existence of this blood pressure QTL based on the combined populations (n = 330) was 5.66 and accounted for 9.2% of the total variance and 26% of the genetic variance.     

Genetic mapping of two blood pressure quantitative trait loci on rat chromosome 1.

A genetic map for rat chromosome 1 was constructed using 66 microsatellite markers typed on either or both of two populations derived from inbred Dahl salt-sensitive (S) rats: F2(LEW x S) n = 151, and F2(WKY x S) n = 159. These populations had been raised on a high salt (8% NaCl) diet. Systolic blood pressure and heart weight were found to be genetically linked to two separate regions on rat chromosome 1 in the F2(LEW x S) population. One region was centered around the anonymous SA locus and accounted for 24 mmHg of blood pressure. The other region was 55 cM from the SA locus centered around a cluster of cytochromes P450 loci, and accounted for 30 mmHg of blood pressure. Since blood pressure and heart weight were highly correlated these same regions were also linked to heart weight. These results were cross-specific as linkage of these chromosome 1 regions to blood pressure and heart weight was not observed in several other F2 populations derived by crossing S and other normotensive control strains. This is presumably due to different alleles and/or different genetic backgrounds in the various populations. The SA region of chromosome 1 was found to influence body weight in F2(LEW x S) rats. Combining the present data with our previously published data on the F2(LEW x S) population showed that four separate quantitative trait loci with additive effects accounted for 106 mmHg and 38% of the total variance of blood pressure and for 506 mg and 34% of the total variance of heart wt.     

Genetic mapping of two new blood pressure quantitative trait loci in the rat by genotyping endothelin system genes.

The endothelin system, consisting of a series of potent vasoconstrictor peptides and their receptors, is potentially important in the control of blood pressure. We found that the gene coding for endothelin-2 (ET2), also known as vasoctive intestine peptide, cosegregated strongly with systolic blood pressure in a F2 population [F2(S x LEW)] derived from a cross of the Dahl salt-sensitive (S) rat and the Lewis (LEW/NCrlBR) (LEW) rat. The ET2 locus was assigned to rat chromosome 5. The testis-specific histone (HITH) locus also strongly cosegregated with blood pressure in the F2(S x LEW) population and was assigned to rat chromosome 17. Genetic maps of the regions containing the quantitative trait loci (QTL) for blood pressure on chromosomes 5 and 17 were constructed and the QTL were localized using the MAPMAKER/QTL program. The rat genes for endothelin-1, endothelin-3, and endothelin receptor A did not cosegregate with blood pressure in several F2 populations tested and were assigned to rat chromosomes 17, 3, and 19, respectively. Endothelin receptor B cosegregated weakly with blood pressure and was provisionally assigned to rat chromosome 15. We conclude that, in the rat, one new blood pressure QTL is located on chromosome 5 marked by the ET2 locus and another new QTL is located on chromosome 17 near the HITH locus.     

Mapping quantitative trait loci in humans: achievements and limitations

Recent advances in statistical methods and genomic technologies have ushered in a new era in mapping clinically important quantitative traits. However, many refinements and novel statistical approaches are required to enable greater successes in this mapping. The possible impact of recent findings pertaining to the structure of the human genome on efforts to map quantitative traits is yet unclear.     

Confirmation and fine mapping of ethanol sensitivity quantitative trait loci, and candidate gene testing in the LXS recombinant inbred mice

In previous studies, we have mapped quantitative trait loci (QTLs) for hypnotic sensitivity to ethanol using a small recombinant inbred (RI) panel and a large F(2) backcross. Alcohol sensitivity is a major predictor of long-term risk for alcoholism. We remapped hypnotic sensitivity using a new set of 75 RI strains, the LXS, derived from Inbred Long Sleep and Inbred Short Sleep strains. We expected to improve mapping resolution in the QTL regions and to identify novel QTLs for loss of the righting reflex due to ethanol. We used three common mapping algorithms (R/qtl, QTL Cartographer, and WebQTL) to map QTLs in the LXS, and we compared the results. Most mapping studies use only a single algorithm, an approach that may result in failure to identify minor QTLs. We confirmed most of our previously reported QTLs, although one major QTL from earlier work (Lore2) failed to replicate, possibly because it represented multiple linked genes separated by recombination in the RI strains. We also report narrowed confidence intervals, based on mapping with a new genetic resource of more than 4000 polymorphic single-nucleotide polymorphism markers. These narrowed confidence intervals will facilitate candidate gene identification and assessment of overlap with human regions specifying risk for alcoholism. Finally, we present an approach for using these RI strains to assess evidence for candidate genes in the narrowed intervals, and we apply this method to a strong candidate, the serotonin transporter.     

Quantitative trait loci influencing cholesterol and phospholipid phenotypes map to chromosomes that contain genes regulating blood pressure in the spontaneously hypertensive rat.

The frequent coincidence of hypertension and dyslipidemia suggests that related genetic factors might underlie these common risk factors for cardiovascular disease. To investigate whether quantitative trait loci (QTLs) regulating lipid levels map to chromosomes known to contain genes regulating blood pressure, we used a genome scanning approach to map QTLs influencing cholesterol and phospholipid phenotypes in a large set of recombinant inbred strains and in congenic strains derived from the spontaneously hypertensive rat and normotensive Brown-Norway (BN.Lx) rat fed normal and high cholesterol diets. QTLs regulating lipid phenotypes were mapped by scanning the genome with 534 genetic markers. A significant relationship (P < 0.00006) was found between basal HDL2 cholesterol levels and the D19Mit2 marker on chromosome 19. Analysis of congenic strains of spontaneously hypertensive rat indicated that QTLs regulating postdietary lipid phenotypes exist also on chromosomes 8 and 20. Previous studies in the recombinant inbred and congenic strains have demonstrated the presence of blood pressure regulatory genes in corresponding segments of chromosomes 8, 19, and 20. These findings provide support for the hypothesis that blood pressure and certain lipid subfractions can be modulated by linked genes or perhaps even the same genes.     

Identification of quantitative trait loci for haloperidol-induced catalepsy on mouse chromosome 14.

Previous studies have established that neuroleptic-induced catalepsy in mice is a highly heritable trait. The current study focuses on the detection of quantitative trait loci (QTL) for haloperidol-induced catalepsy in a BALB/cJ x LP/J F(2) intercross. One thousand thirty-seven F(2) animals were phenotyped and divided into four categories: very responsive (RR), responsive, nonresponsive, and very nonresponsive (NN). The RR and NN phenotypes comprised approximately 18% each of the total and differed in their haloperidol sensitivity by >10-fold. Sex differed significantly between the NN and RR groups (chi(2) = 14.0; p <.0002); females comprised 58% of the RR individuals but only 38% of the NN individuals. The difference between the extreme phenotypes in the number of piebald animals was highly significant (chi(2) = 30, p <. 00001). Eight percent of the RR individuals were piebald compared with 30% of the NN individuals. A genome wide scan confirmed the presence of a QTL (peak LOD = 6.4) on chromosome 14 near the piebald (Ednrb) and 5-hydroxytryptamine(2A) (Htr2a) loci. Although the parental BALB/cJ and LP/J strains differed significantly in striatal 5-hydroxytryptamine(2A) receptor binding, no marked differences were detected between the phenotypic extremes. A second QTL was detected on chromosome 14 (peak LOD = 6.9), which was located more proximally and included the Chat locus. No QTLs were detected on chromosomes 1 and 9, thus differentiating this cross from previous results obtained for a C57BL/6J x DBA/2J intercross.     

Identification of quantitative trait loci for chemical/inflammatory nociception in mice

Sensitivity to pain is widely variable, and much of this variability is genetic in origin. The specific genes responsible have begun to be identified, but only for thermal nociception. In order to facilitate the identification of polymorphic, pain-related genes with more clinical relevance, we performed quantitative trait locus (QTL) mapping studies of the most common assay of inflammatory nociception, the formalin test. QTL mapping is a technique that exploits naturally occurring variability among inbred strains for the identification of genomic locations containing genes contributing to that variability. An F2 intercross was constructed using inbred A/J and C57BL/6J mice as progenitors, strains previously shown to display resistance and sensitivity, respectively, to formalin-induced nociception. Following phenotypic testing (5% formalin, 25 microl intraplantar injection), mice were genotyped at 90 microsatellite markers spanning the genome. We provide evidence for two statistically significant formalin test QTLs - chromosomal regions whose inheritance is associated with trait variability - on distal mouse chromosomes 9 and 10. Identification of the genes underlying these QTLs may illuminate the basis of individual differences in inflammatory pain, and lead to novel analgesic treatment strategies.     

Quantitative trait loci affecting initial sensitivity and acute functional tolerance to ethanol-induced ataxia and brain cAMP signaling in BXD recombinant inbred mice

In previous work, we identified genetic correlations between cAMP accumulation in the cerebellum and sensitivity to the incoordinating effects of ethanol. A genetic correlation suggests that common genes underlie the phenotypes investigated. One method for provisionally identifying genes involved in a given phenotypic measure is quantitative trait locus (QTL) analysis. Using a panel of 30 BXD recombinant inbred strains of mice and the progenitors (DBA/2J and C57BL/6J), and the dowel test for ataxia, we measured the blood ethanol concentrations at the time an animal first fell from the dowel and acute functional tolerance (AFT), and investigated cAMP signaling in the cerebellum. Cyclic AMP accumulation was measured in whole-cell preparations of cerebellar minces from individual mice under basal or stimulated conditions. We conducted a genome-wide QTL analysis of the behavioral and biochemical measures with >2000 genetic markers to identify significant associations. Western blot and comparative sequencing analysis were used to compare cAMP response element binding protein (CREB) levels and protein-coding sequence, respectively. QTL analyses correlating strain means with allelic status at genetic markers identified several significant associations (p < 0.01). Analysis of variance revealed an effect of strain on behavioral and biochemical measures. There was a significant genetic correlation between initial sensitivity and basal cAMP accumulation in the cerebellum. We identified 6 provisional QTLs for initial sensitivity on four chromosomes, 6 provisional QTLs for AFT on four chromosomes, and 11 provisional QTLs for cAMP signaling on nine chromosomes. Two loci were found to overlap for measures of initial sensitivity and for cAMP signaling. Given the genetic correlation between initial sensitivity and basal cAMP accumulation, we investigated candidate genes in a QTL on chromosome 1. Comparative sequence analysis was performed, and protein levels were compared between C57 and DBA mice for Creb1. No significant differences were detected in coding sequence or protein levels for CREB. These results suggest that although ethanol sensitivity and cAMP signaling are determined by multiple genes, they may share certain genetic codetermination.     

Mapping genes that regulate density of dopamine transporters and correlated behaviors in recombinant inbred mice.

Binding of 3beta-(4-iodophenyl) tropane-2beta-carboxylic acid methyl ester ([125I]RTI-55) to the dopamine transporter (DAT) in neostriatum from C57BL/6J, DBA/2J, and 21 BXD recombinant inbred (RI) mouse strains indicated highly significant strain differences in DAT density (Bmax) but no significant differences in affinity (Kd) for this radioligand. Strain mean Bmax values and the known genomic locations of 1390 marker loci were used to carry out a genome-wide search for quantitative trait loci (QTLs), which are chromosomal sites containing genes that influence DAT expression. This search revealed an unusually large effect QTL on chromosome 19 in the region of the proopiomelanocortin pseudogene Pomc-ps1 (8-11 cM), homologous to regions of human chromosomes 9q21 and 11q12-13. This QTL (logarithm of the odds 4.7, df = 1, p = 3 x 10(-6)) by conservative estimates accounts for just over half of the genetic variation in DAT binding site density. The QTL is not the DAT gene itself (Dat1, chromosome 13), but a powerful modulator of DAT expression in neostriatum. Furthermore, DAT expression levels in 20 of the BXD RI strains and the chromosome 19 QTL were correlated with cocaine and methamphetamine-induced locomotor activation and thermic responses (hypo- or hyperthermia), but were not correlated with behaviors related to sensitization, reward, voluntary consumption, stereotypy, or seizures induced by these two psychostimulant drugs. The results suggest that there is a gene(s) on proximal chromosome 19 that strongly influences DAT expression in neostriatum and may influence psychostimulant-induced activity and thermal responses.     

Genetic isolation of a region of chromosome 8 that exerts major effects on blood pressure and cardiac mass in the spontaneously hypertensive rat.

The spontaneously hypertensive rat (SHR) is the most widely studied animal model of essential hypertension. Despite > 30 yr of research, the primary genetic lesions responsible for hypertension in the SHR remain undefined. In this report, we describe the construction and hemodynamic characterization of a congenic strain of SHR (SHR-Lx) that carries a defined segment of chromosome 8 from a normotensive strain of Brown-Norway rats (BN-Lx strain). Transfer of this segment of chromosome 8 from the BN-Lx strain onto the SHR background resulted in substantial reductions in systolic and diastolic blood pressure and cardiac mass. Linkage and comparative mapping studies indicate that the transferred chromosome segment contains a number of candidate genes for hypertension, including genes encoding a brain dopamine receptor and a renal epithelial potassium channel. These findings demonstrate that BP regulatory gene(s) exist within the differential chromosome segment trapped in the SHR-Lx congenic strain and that this region of chromosome 8 plays a major role in the hypertension of SHR vs. BN-Lx rats.     

Rheumatoid factors in 129XB recombinant inbred strains. Igh-1-linked control of allotypic and isotypic specificities

To examine the role of autologous IgG in the induction of murine rheumatoid factors (RF) we have analyzed the allotypic specificity of anti-IgG2a RF in recombinant inbred strains derived from 129/Sv (Igh- 1a) and C57BL/6 (Igh-1b) mice. In five of six Igh-1a strains, anti- IgG2a RF reacted with IgG2aa but failed to react with IgG2ab. In contrast, isotype-specific RF, which reacted equally well with a and b allotypes of IgG2a, represented the major RF species in one Igh-1a and all five Igh-1b strains tested. An additional form of RF specific for IgG2ab and not reactive with IgG2aa was detected in one Igh-1b strain. RF specific for a give allotype was thus only found in the presence of that allotype, which strongly suggests the involvement of autologous IgG in the induction of mouse RF synthesis. The specificity of RF was apparently further controlled by genes linked to but different from the Igh-C locus, as indicated by the absence of IgG2aa-specific RF in one of the 6 Igh-1a strains tested. Because this strain, 129XBG, has been shown to express idiotypic markers characteristic of Igh-1b mice, it is likely that the genes, which in the presence of a given allotype induce the production of isotype rather than allotype-specific RF, are identical to those that control the expression of idiotypes. Evidence was also obtained to indicate that Igh-1-linked genes influence the isotypic specificity and the isotype of RF itself: IgA anti-IgG2a predominated in Igh-1a strains and IgM anti-IgG1 in Igh-1b strains. Interestingly enough, total IgA and IgG2a levels also were higher in Igh-1a than in Igh-1b strains.     

Cocaine-induced seizure thresholds: quantitative trait loci detection and mapping in two populations derived from the C57BL/6 and DBA/2 mouse strains

Seizures are a well known consequence of human cocaine abuse, and in rodent models, sensitivity to cocaine seizures has been shown to be strongly influenced by genotype. For example, several studies have reported significant differences between the C57BL/6 (B6) and DBA/2 (D2) inbred mouse strains in their sensitivity to cocaine-induced seizures. This prompted our use of the BXD recombinant inbred (RI) strain set and an F(2) population derived from the B6 and D2 progenitor strains for further genetic analyses and for gene mapping efforts in this study. Cocaine was infused into the lateral tail vein, and the doses needed to induce a running bouncing clonic seizure and a tonic hindlimb extensor seizure were recorded for each mouse. In the BXD RI set, a genome-wide search was carried out for QTLs (quantitative trait loci), which are sites on a chromosome containing genes that influence seizure susceptibility. An F(2) population (B6D2F2, n = 408) was subsequently used as a second, confirmation step. Based on both RI and F(2) results, three QTLs emerged as significant (P <.00005): one for clonic seizures on chromosome 9 (distal), and two for tonic seizures on chromosomes 14 (proximal to mid) and 15 (distal). Two additional QTLs emerged as suggestive (P <.0015), both associated with clonic seizures on chromosomes 9 (proximal) and 15 (distal). Both QTLs on chromosome 9 were sex-specific, with much larger effects on the phenotype seen in females than in males.     

Influence of thiamine deficiency on the response to ethanol in two inbred rat strains

We investigated whether thiamine deficiency (TD), a frequent concomitant of chronic alcoholism, differentially modifies the response to ethanol in two inbred rat strains with highly different genetic susceptibilities to development of TD encephalopathy. Ethanol-induced (3 g/kg i.p.) behavioral impairment and hypothermia were studied after 2, 5 and 7 weeks of TD and after 6 weeks of repletion on normal diet. Controls of the M520/N (TD-sensitive) strain metabolized ethanol more rapidly, had a greater liver to body weight ratio, greater total body water, earlier and lower peak blood ethanol concentrations (BEC), diminished area under the BEC curve and lesser behavioral impairment and hypothermia (even at equivalent BEC values) than those of the F344/N (TD-resistant) strain. In both strains, TD resulted in reduced ethanol metabolic rate and liver to body weight ratio and equivalent ethanol-induced hypothermia and behavioral impairment at lower BEC. Lower and delayed peak BEC and unchanged area under the BEC curve suggest an increased volume of ethanol distribution during TD. Recovery appeared complete after 6 weeks of normal diet. Both strains lost an equivalent proportion of body weight during TD but M520/N rats had lesser decrements in ethanol metabolic rate, had greater reductions in liver weight, peak BEC and baseline body temperature and developed overt encephalopathy whereas F344/N rats did not. Therefore, in the chronic alcoholic, TD may modify ethanol's effects via pharmacokinetic and pharmacodynamic mechanisms. Relatively high ethanol tolerance of the strain with a genetic predisposition to TD encephalopathy is consistent with the hypothesized role of this avitaminosis in the pharmacogenetics of alcoholism.     

Diagnosis of viral myocarditis by cardiac magnetic resonance and viral genome detection in peripheral blood

In patients with acute myocarditis, viral genome can be detected in plasma and peripheral leukocytes. Its relationship with active myocardial inflammation, however, is not well understood. Myocardial edema as a feature of inflammation and myocardial necrosis or fibrosis can be frequently observed in patients with acute myocarditis by cardiovascular magnetic resonance (CMR). We assessed the association of viral genome presence in peripheral blood samples with myocardial edema and irreversible injury. We examined consecutive patients with clinically suspected myocarditis after an episode of viral illness. State-of-the-art methods were used for detecting myocardial edema and irreversible injury using CMR and viral genome applying reverse transcribed, nested polymerase chain reaction in peripheral blood samples. The specificity of viral amplification products was confirmed by automatic DNA sequencing. Of a total of 55 patients (53.5 ± 15.6 years), 21 were positive for viral genome in peripheral leukocytes. Interestingly, 18 (86 %) of these patients also showed global myocardial edema, as compared to only 7/34 (21 %) without PCR evidence for viral genome. The overall agreement between CMR criteria for edema and viral PCR was 84 %. In contrast, there was no significant relationship of viral genome presence with myocardial necrosis or scars. In patients with clinically suspected myocarditis, myocardial edema but not irreversible myocardial injury is associated with the presence of viral genome in peripheral blood.     

Changes in blood pressure and cardiac output during maximal isokinetic exercise.

This study was undertaken to determine the blood pressure (BP) and cardiac output (Qc) responses to maximal isokinetic exercise. The subjects (n = 5) performed unilateral knee extension/flexion exercise (knee exercise) and unilateral elbow extension/flexion exercise (elbow exercise) at 0.52, 1.57, and 2.62 rads.sec-1. The BP was monitored using a cannula placed in the radial artery. Heart rate (HR), stroke volume (SV), and Qc were measured by impedance cardiography. In response to isokinetic exercise, HR and Qc increased significantly (p less than .01), while the SV did not. The BP response was characterized by significant increases in systolic, diastolic, and mean arterial pressure (MAP) (p less than .01). The Qc and MAP, responses were not influenced by the exercise velocity. The adjustments in HR, MAP, and rate pressure product (RPP) to the elbow exercise were qualitatively similar to those seen during the knee exercise, but the absolute values achieved were smaller (p less than .05). Compared with maximal dynamic exercise, the HR and SV responses to the knee exercises were lower. The MAP response to isokinetic exercise equaled the highest value achieved during dynamic exercise. Findings from the present study suggest that the cardiovascular stress (the increase in HR, MAP, and RPP) associated with isokinetic exercise is independent of the velocity of movement and is proportional to the active muscle mass.     

Determination of the bactericidal activity of blood serum by recombinant luminescence strains of Escherichia coli

It has been experimentally proven as possible to use recombinant strains of Escherichia coli carrying the genes of luminescence of natural fluorescent bacteria in evaluating the bactericidal activity of blood serum (BABS). The shaping fluorescence inhibition was shown to be in proportion to the developing bactericidal effect in respect to the luminescent strains of Escherichia coli, which depended on a used concentration of blood serum and time of contact. The list of E. coli recombinant strains, which fit in most of all with the evaluation of BABS, was drawn. The values of the bactericidal activity of blood serum determined by the routine bacteriological and nephelometric methods were demonstrated to coincide with those determined by the worked-out bio-luminescent method.