Esther Martínez-Membrives

Combined sequence-based and genetic mapping analysis of complex traits in outbred rats

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

A resource for the simultaneous high-resolution mapping of multiple quantitative trait loci in rats: The NIH heterogeneous stock

The laboratory rat (Rattus norvegicus) is a key tool for the study of medicine and pharmacology for human health. A large database of phenotypes for integrated fields such as cardiovascular, neuroscience, and exercise physiology exists in the literature. However, the molecular characterization of the genetic loci that give rise to variation in these traits has proven to be difficult. Here we show how one obstacle to progress, the fine-mapping of quantitative trait loci (QTL), can be overcome by using an outbred population of rats. By use of a genetically heterogeneous stock of rats, we map a locus contributing to variation in a fear-related measure (two-way active avoidance in the shuttle box) to a region on chromosome 5 containing nine genes. By establishing a protocol measuring multiple phenotypes including immunology, neuroinflammation, and hematology, as well as cardiovascular, metabolic, and behavioral traits, we establish the rat HS as a new resource for the fine-mapping of QTLs contributing to variation in complex traits of biomedical relevance.

Prepulse inhibition predicts spatial working memory performance in the inbred Roman high- and low-avoidance rats and in genetically heterogeneous NIH-HS rats: relevance for studying pre-attentive and cognitive anomalies in schizophrenia

Animal models of schizophrenia-relevant symptoms are increasingly important for progress in our understanding of the neurobiological basis of the disorder and for discovering novel and more specific treatments. Prepulse inhibition (PPI) and working memory, which are impaired in schizophrenic patients, are among the symptoms/processes modeled in those animal analogs. We have evaluated whether a genetically-selected rat model, the Roman high-avoidance inbred strain (RHA-I), displays PPI deficits as compared with its Roman low-avoidance (RLA-I) counterpart and the genetically heterogeneous NIH-HS rat stock. We have investigated whether PPI deficits predict spatial working memory impairments (in the Morris water maze; MWM) in these three rat types (Experiment 1), as well as in a separate sample of NIH-HS rats stratified according to their extreme (High, Medium, Low) PPI scores (Experiment 2). The results from Experiment 1 show that RHA-I rats display PPI and spatial working memory deficits compared to both RLA-I and NIH-HS rats. Likewise, in Experiment 2, “Low-PPI” NIH-HS rats present significantly impaired working memory with respect to “Medium-PPI” and “High-PPI” NIH-HS subgroups. Further support to these results comes from correlational, factorial, and multiple regression analyses, which reveal that PPI is positively associated with spatial working memory performance. Conversely, cued learning in the MWM was not associated with PPI. Thus, using genetically-selected and genetically heterogeneous rats, the present study shows, for the first time, that PPI is a positive predictor of performance in a spatial working memory task. These results may have translational value for schizophrenia symptom research in humans, as they suggest that either by psychogenetic selection or by focusing on extreme PPI scores from a genetically heterogeneous rat stock, it is possible to detect a useful (perhaps “at risk”) phenotype to study cognitive anomalies linked to schizophrenia.

Heterogeneous stock rat: A unique animal model for mapping genes influencing bone fragility

Previously, we demonstrated that skeletal mass, structure and biomechanical properties vary considerably among 11 different inbred rat strains. Subsequently, we performed quantitative trait loci (QTL) analysis in four inbred rat strains (F344, LEW, COP and DA) for different bone phenotypes and identified several candidate genes influencing various bone traits. The standard approach to narrowing QTL intervals down to a few candidate genes typically employs the generation of congenic lines, which is time consuming and often not successful. A potential alternative approach is to use a highly genetically informative animal model resource capable of delivering very high resolution gene mapping such as Heterogeneous stock (HS) rat. HS rat was derived from eight inbred progenitors: ACI/N, BN/SsN, BUF/N, F344/N, M520/N, MR/N, WKY/N and WN/N. The genetic recombination pattern generated across 50 generations in these rats has been shown to deliver ultra-high even gene-level resolution for complex genetic studies. The purpose of this study is to investigate the usefulness of the HS rat model for fine mapping and identification of genes underlying bone fragility phenotypes. We compared bone geometry, density and strength phenotypes at multiple skeletal sites in HS rats with those obtained from five of the eight progenitor inbred strains. In addition, we estimated the heritability for different bone phenotypes in these rats and employed principal component analysis to explore relationships among bone phenotypes in the HS rats. Our study demonstrates that significant variability exists for different skeletal phenotypes in HS rats compared with their inbred progenitors. In addition, we estimated high heritability for several bone phenotypes and biologically interpretable factors explaining significant overall variability, suggesting that the HS rat model could be a unique genetic resource for rapid and efficient discovery of the genetic determinants of bone fragility.

Neonatal handling decreases unconditioned anxiety, conditioned fear, and improves two-way avoidance acquisition: a study with the inbred Roman high (RHA-I)- and low-avoidance (RLA-I) rats of both sexes.

The present study evaluated the long-lasting effects of neonatal handling (NH; administered during the first 21 days of life) on unlearned and learned anxiety-related responses in inbred Roman High- (RHA-I) and Low-avoidance (RLA-I) rats. To this aim, untreated and neonatally-handled RHA-I and RLA-I rats of both sexes were tested in the following tests/tasks: a novel object exploration (NOE) test, the elevated zero maze (ZM) test, a “baseline acoustic startle” (BAS) test, a “context-conditioned fear” (CCF) test and the acquisition of two-way active—shuttle box—avoidance (SHAV). RLA-I rats showed higher unconditioned (novel object exploration test -“NOE”-, elevated zero maze test -“ZM”-, BAS), and conditioned (CCF, SHAV) anxiety. NH increased exploration of the novel object in the NOE test as well as exploration of the open sections of the ZM test in both rat strains and sexes, although the effects were relatively more marked in the (high anxious) RLA-I strain and in females. NH did not affect BAS, but reduced CCF in both strains and sexes, and improved shuttle box avoidance acquisition especially in RLA-I (and particularly in females) and in female RHA-I rats. These are completely novel findings, which indicate that even some genetically-based anxiety/fear-related phenotypes can be significantly modulated by previous environmental experiences such as the NH manipulation.

High-resolution genome screen for bone mineral density in heterogeneous stock rat

We previously demonstrated that skeletal mass, structure, and biomechanical properties vary considerably in heterogeneous stock (HS) rat strains. In addition, we observed strong heritability for several of these skeletal phenotypes in the HS rat model, suggesting that it represents a unique genetic resource for dissecting the complex genetics underlying bone fragility. The purpose of this study was to identify and localize genes associated with bone mineral density in HS rats. We measured bone phenotypes from 1524 adult male and female HS rats between 17 and 20 weeks of age. Phenotypes included dual‐energy X‐ray absorptiometry (DXA) measurements for bone mineral content and areal bone mineral density (aBMD) for femur and lumbar spine (L3–L5), and volumetric BMD measurements by CT for the midshaft and distal femur, femur neck, and fifth lumbar vertebra (L5). A total of 70,000 polymorphic single‐nucleotide polymorphisms (SNPs) distributed throughout the genome were selected from genotypes obtained from the Affymetrix rat custom SNPs array for the HS rat population. These SNPs spanned the HS rat genome with a mean linkage disequilibrium coefficient between neighboring SNPs of 0.95. Haplotypes were estimated across the entire genome for each rat using a multipoint haplotype reconstruction method, which calculates the probability of descent for each genotyped locus from each of the eight founder HS strains. The haplotypes were tested for association with each bone density phenotype via a mixed model with covariate adjustment. We identified quantitative trait loci (QTLs) for BMD phenotypes on chromosomes 2, 9, 10, and 13 meeting a conservative genomewide empiric significance threshold (false discovery rate [FDR] = 5%; p < 3 × 10−6). Importantly, most QTLs were localized to very small genomic regions (1–3 megabases [Mb]), allowing us to identify a narrow set of potential candidate genes including both novel genes and genes previously shown to have roles in skeletal development and homeostasis.

Neonatal handling enduringly decreases anxiety and stress responses and reduces hippocampus and amygdala volume in a genetic model of differential anxiety: Behavioral-volumetric associations in the Roman rat strains

The hippocampus and amygdala have been proposed as key neural structures related to anxiety. A more active hippocampus/amygdala system has been related to greater anxious responses in situations involving conflict/novelty. The Roman Low- (RLA) and High-avoidance (RHA) rat lines/strains constitute a genetic model of differential anxiety. Relative to RHA rats, RLA rats exhibit enhanced anxiety/fearfulness, augmented hippocampal/amygdala c-Fos expression following exposure to novelty/conflict, increased hippocampal neuronal density and higher endocrine responses to stress. Neonatal handling (NH) is an environmental treatment with long-lasting anxiety/stress-reducing effects in rodents. Since hippocampus and amygdala volume are supposed to be related to anxiety/fear, we hypothesized a greater volume of both areas in RLA than in RHA rats, as well as that NH treatment would reduce anxiety and the volume of both structures, in particular in the RLA strain. Adult untreated and NH-treated RHA and RLA rats were tested for anxiety, sensorimotor gating (PPI), stress-induced corticosterone and prolactin responses, two-way active avoidance acquisition and in vivo 7 T 1H-Magnetic resonance image. As expected, untreated RLA rats showed higher anxiety and post-stress hormone responses, as well as greater hippocampus and amygdala volumes than untreated RHA rats. NH decreased anxiety/stress responses, especially in RLA rats, and significantly reduced hippocampus and amygdala volumes in this strain. Dorsal striatum volume was not different between the strains nor it was affected by NH. Finally, there were positive associations (as shown by correlations, factor analysis and multiple regression) between anxiety and PPI and hippocampus/amygdala volumes.

Spatial learning in the genetically heterogeneous NIH-HS rat stock and RLA-I/RHA-I rats: Revisiting the relationship with unconditioned and conditioned anxiety

To characterize learning/memory profiles for the first time in the genetically heterogeneous NIH-HS rat stock, and to examine whether these are associated with anxiety, we evaluated NIH-HS rats for spatial learning/memory in the Morris water maze (MWM) and in the following anxiety/fear tests: the elevated zero-maze (ZM; unconditioned anxiety), a context-conditioned fear test and the acquisition of two-way active avoidance (conditioned anxiety). NIH-HS rats were compared with the Roman High- (RHA-I) and Low-Avoidance (RLA-I) rat strains, given the well-known differences between the Roman strains/lines in anxiety-related behavior and in spatial learning/memory. The results show that: (i) As expected, RLA-I rats were more anxious in the ZM test, displayed more frequent context-conditioned freezing episodes and fewer avoidances than RHA-I rats. (ii) Scores of NIH-HS rats in these tests/tasks mostly fell in between those of the Roman rat strains, and were usually closer to the values of the RLA-I strain. (iii) Pigmented NIH-HS (only a small part of NIH-HS rats were albino) rats were the best spatial learners and displayed better spatial memory than the other three (RHA-I, RLA-I and NIH-HS albino) groups. (iv) Albino NIH-HS and RLA-I rats also showed better learning/memory than the RHA-I strain. (v) Within the NIH-HS stock, the most anxious rats in the ZM test presented the best learning and/or memory efficiency (regardless of pigmentation). In summary, NIH-HS rats display a high performance in spatial learning/memory tasks and a passive coping strategy when facing conditioned conflict situations. In addition, unconditioned anxiety in NIH-HS rats predicts better spatial learning/memory.

Fine mapping of bone structure and strength QTLs in heterogeneous stock rat

We previously demonstrated that skeletal structure and strength phenotypes vary considerably in heterogeneous stock (HS) rats. These phenotypes were found to be strongly heritable, suggesting that the HS rat model represents a unique genetic resource for dissecting the complex genetic etiology underlying bone fragility. The purpose of this study was to identify and localize genes associated with bone structure and strength phenotypes using 1524 adult male and female HS rats between 17 to 20 weeks of age. Structure measures included femur length, neck width, head width; femur and lumbar spine (L3-5) areas obtained by DXA; and cross-sectional areas (CSA) at the midshaft, distal femur and femoral neck, and the 5th lumbar vertebra measured by CT. In addition, measures of strength of the whole femur and femoral neck were obtained. Approximately 70,000 polymorphic SNPs distributed throughout the rat genome were selected for genotyping, with a mean linkage disequilibrium coefficient between neighboring SNPs of 0.95. Haplotypes were estimated across the entire genome for each rat using a multipoint haplotype reconstruction method, which calculates the probability of descent at each locus from each of the 8 HS founder strains. The haplotypes were then tested for association with each structure and strength phenotype via a mixed model with covariate adjustment. We identified quantitative trait loci (QTLs) for structure phenotypes on chromosomes 3, 8, 10, 12, 17 and 20, and QTLs for strength phenotypes on chromosomes 5, 10 and 11 that met a conservative genome-wide empiric significance threshold (FDR=5%; P<3×10(-6)). Importantly, most QTLs were localized to very narrow genomic regions (as small as 0.3 Mb and up to 3 Mb), each harboring a small set of candidate genes, both novel and previously shown to have roles in skeletal development and homeostasis.

Coping-Style Behavior Identified by a Survey of Parent-of-Origin Effects in the Rat

In this study we investigate the effects of parent of origin on complex traits in the laboratory rat, with a focus on coping style behavior in stressful situations. We develop theory, based on earlier work, to partition heritability into a component due to a combination of parent of origin, maternal, paternal and shared environment, and another component that estimates classical additive genetic variance. We use this theory to investigate the effects on heritability of the parental origin of alleles in 798 outbred heterogeneous stock rats across 199 complex traits. Parent-of-origin-like heritability was on average 2.7fold larger than classical additive heritability. Among the phenotypes with the most enhanced parent-of-origin heritability were 10 coping style behaviors, with average 3.2 fold heritability enrichment. To confirm these findings on coping behavior, and to eliminate the possibility that the parent of origin effects are due to confounding with shared environment, we performed a reciprocal F1 cross between the behaviorally divergent RHA and RLA rat strains. We observed parent-of-origin effects on F1 rat anxiety/coping-related behavior in the Elevated Zero Maze test. Our study is the first to assess genetic parent-of-origin effects in rats, and confirm earlier findings in mice that such effects influence coping and impulsive behavior, and suggest these effects might be significant in other mammals, including humans.