A Simple Method for Combining Genetic Mapping Data from Multiple Crosses and Experimental Designs

Over the past decade many linkage studies have defined chromosomal intervals containing polymorphisms that modulate a variety of traits. Many phenotypes are now associated with enough mapping data that meta-analysis could help refine locations of known QTLs and detect many novel QTLs.We describe a simple approach to combining QTL mapping results for multiple studies and demonstrate its utility using two hippocampus weight loci. Using data taken from two populations, a recombinant inbred strain set and an advanced intercross population we demonstrate considerable improvements in significance and resolution for both loci. 1-LOD support intervals were improved 51% for Hipp1a and 37% for Hipp9a. We first generate locus-wise permuted P-values for association with the phenotype from multiple maps, which can be done using a permutation method appropriate to each population. These results are then assigned to defined physical positions by interpolation between markers with known physical and genetic positions. We then use Fisher's combination test to combine position-by-position probabilities among experiments. Finally, we calculate genome-wide combined P-values by generating locus-specific P-values for each permuted map for each experiment. These permuted maps are then sampled with replacement and combined. The distribution of best locus-specific P-values for each combined map is the null distribution of genome-wide adjusted P-values.Our approach is applicable to a wide variety of segregating and non-segregating mapping populations, facilitates rapid refinement of physical QTL position, is complementary to other QTL fine mapping methods, and provides an appropriate genome-wide criterion of significance for combined mapping results

A Copine family member, Cpne8, is a candidate quantitative trait gene for prion disease incubation time in mouse

Prion disease incubation time in mice is determined by many factors including genetic background. The prion gene itself plays a major role in incubation time; however, other genes are also known to be important. Whilst quantitative trait loci (QTL) studies have identified multiple loci across the genome, these regions are often large, and with the exception of Hectd2 on Mmu19, no quantitative trait genes or nucleotides for prion disease incubation time have been demonstrated. In this study, we use the Northport heterogeneous stock of mice to reduce the size of a previously identified QTL on Mmu15 from approximately 25 to 1.2 cM. We further characterised the genes in this region and identify Cpne8, a member of the copine family, as the most promising candidate gene. We also show that Cpne8 mRNA is upregulated at the terminal stage of disease, supporting a role in prion disease. Applying these techniques to other loci will facilitate the identification of key pathways in prion disease pathogenesis

Dissection of a QTL Hotspot on Mouse Distal Chromosome 1 that Modulates Neurobehavioral Phenotypes and Gene Expression

A remarkably diverse set of traits maps to a region on mouse distal chromosome 1 (Chr 1) that corresponds to human Chr 1q21–q23. This region is highly enriched in quantitative trait loci (QTLs) that control neural and behavioral phenotypes, including motor behavior, escape latency, emotionality, seizure susceptibility (Szs1), and responses to ethanol, caffeine, pentobarbital, and haloperidol. This region also controls the expression of a remarkably large number of genes, including genes that are associated with some of the classical traits that map to distal Chr 1 (e.g., seizure susceptibility). Here, we ask whether this QTL-rich region on Chr 1 (Qrr1) consists of a single master locus or a mixture of linked, but functionally unrelated, QTLs. To answer this question and to evaluate candidate genes, we generated and analyzed several gene expression, haplotype, and sequence datasets. We exploited six complementary mouse crosses, and combed through 18 expression datasets to determine class membership of genes modulated by Qrr1. Qrr1 can be broadly divided into a proximal part (Qrr1p) and a distal part (Qrr1d), each associated with the expression of distinct subsets of genes. Qrr1d controls RNA metabolism and protein synthesis, including the expression of ∼20 aminoacyl-tRNA synthetases. Qrr1d contains a tRNA cluster, and this is a functionally pertinent candidate for the tRNA synthetases. Rgs7 and Fmn2 are other strong candidates in Qrr1d. FMN2 protein has pronounced expression in neurons, including in the dendrites, and deletion of Fmn2 had a strong effect on the expression of few genes modulated by Qrr1d. Our analysis revealed a highly complex gene expression regulatory interval in Qrr1, composed of multiple loci modulating the expression of functionally cognate sets of genes

Markers of serotonergic function in the orbitofrontal cortex and dorsal raphé nucleus predict individual variation in spatial-discrimination serial reversal learning.

Dysfunction of the orbitofrontal cortex (OFC) impairs the ability of individuals to flexibly adapt behavior to changing stimulus-reward (S-R) contingencies. Impaired flexibility also results from interventions that alter serotonin (5-HT) and dopamine (DA) transmission in the OFC and dorsomedial striatum (DMS). However, it is unclear whether similar mechanisms underpin naturally occurring variations in behavioral flexibility. In the present study, we used a spatial-discrimination serial reversal procedure to investigate interindividual variability in behavioral flexibility in rats. We show that flexibility on this task is improved following systemic administration of the 5-HT reuptake inhibitor citalopram and by low doses of the DA reuptake inhibitor GBR12909. Rats in the upper quintile of the distribution of perseverative responses during repeated S-R reversals showed significantly reduced levels of the 5-HT metabolite, 5-hydroxy-indoleacetic acid, in the OFC. Additionally, 5-HT2A receptor binding in the OFC of mid- and high-quintile rats was significantly reduced compared with rats in the low-quintile group. These perturbations were accompanied by an increase in the expression of monoamine oxidase-A (MAO-A) and MAO-B in the lateral OFC and by a decrease in the expression of MAO-A, MAO-B, and tryptophan hydroxylase in the dorsal raphé nucleus of highly perseverative rats. We found no evidence of significant differences in markers of DA and 5-HT function in the DMS or MAO expression in the ventral tegmental area of low- vs high-perseverative rats. These findings indicate that diminished serotonergic tone in the OFC may be an endophenotype that predisposes to behavioral inflexibility and other forms of compulsive behavior.This work was supported by Medical Research Council Grants (G0701500; G0802729), a 503 Wellcome Trust Programme Grant (grant number 089589/Z/09/Z), and by a Core Award 504 from the Medical Research Council and the Wellcome Trust to the Behavioural and Clinical 505 21 Neuroscience Institute (MRC Ref G1000183; WT Ref 093875/Z/10/Z). RLB was supported 506 by a studentship from the Medical Research Council. JA was supported by a Fellowship from 507 the Swedish Research Council (350-2012-230). BJ was supported by Fellowships from the 508 AXA Research Fund and the National Health and Medical Research Council of Australia. 509 Financial support from the Fredrik and Ingrid Thuring Foundation is also acknowledged.This is the accepted manuscript. The final version is available from Nature Publishing at http://www.nature.com/npp/journal/vaop/ncurrent/full/npp2014335a.html

HECTD2 Is Associated with Susceptibility to Mouse and Human Prion Disease

Prion diseases are fatal transmissible neurodegenerative disorders, which include Scrapie, Bovine Spongiform Encephalopathy (BSE), Creutzfeldt-Jakob Disease (CJD), and kuru. They are characterised by a prolonged clinically silent incubation period, variation in which is determined by many factors, including genetic background. We have used a heterogeneous stock of mice to identify Hectd2, an E3 ubiquitin ligase, as a quantitative trait gene for prion disease incubation time in mice. Further, we report an association between HECTD2 haplotypes and susceptibility to the acquired human prion diseases, vCJD and kuru. We report a genotype-associated differential expression of Hectd2 mRNA in mouse brains and human lymphocytes and a significant up-regulation of transcript in mice at the terminal stage of prion disease. Although the substrate of HECTD2 is unknown, these data highlight the importance of proteosome-directed protein degradation in neurodegeneration. This is the first demonstration of a mouse quantitative trait gene that also influences susceptibility to human prion diseases. Characterisation of such genes is key to understanding human risk and the molecular basis of incubation periods

Treatment- and Population-Dependent Activity Patterns of Behavioral and Expression QTLs

Genetic control of gene expression and higher-order phenotypes is almost invariably dependent on environment and experimental conditions. We use two families of recombinant inbred strains of mice (LXS and BXD) to study treatment- and genotype-dependent control of hippocampal gene expression and behavioral phenotypes. We analyzed responses to all combinations of two experimental perturbations, ethanol and restraint stress, in both families, allowing for comparisons across 8 combinations of treatment and population. We introduce the concept of QTL activity patterns to characterize how associations between genomic loci and traits vary across treatments. We identified several significant behavioral QTLs and many expression QTLs (eQTLs). The behavioral QTLs are highly dependent on treatment and population. We classified eQTLs into three groups: cis-eQTLs (expression variation that maps to within 5 Mb of the cognate gene), syntenic trans-eQTLs (the gene and the QTL are on the same chromosome but not within 5 Mb), and non-syntenic trans-eQTLs (the gene and the QTL are on different chromosomes). We found that most non-syntenic trans-eQTLs were treatment-specific whereas both classes of syntenic eQTLs were more conserved across treatments. We also found there was a correlation between regions along the genome enriched for eQTLs and SNPs that were conserved across the LXS and BXD families. Genes with eQTLs that co-localized with the behavioral QTLs and displayed similar QTL activity patterns were identified as potential candidate genes associated with the phenotypes, yielding identification of novel genes as well as genes that have been previously associated with responses to ethanol

Quantitative trait loci for sensitivity to ethanol intoxication in a C57BL/6J × 129S1/SvImJ inbred mouse cross

Individual variation in sensitivity to acute ethanol (EtOH) challenge is associated with alcohol drinking and is a predictor of alcohol abuse. Previous studies have shown that the C57BL/6J (B6) and 129S1/SvImJ (S1) inbred mouse strains differ in responses on certain measures of acute EtOH intoxication. To gain insight into genetic factors contributing to these differences, we performed quantitative trait locus (QTL) analysis of measures of EtOH-induced ataxia (accelerating rotarod), hypothermia, and loss of righting reflex (LORR) duration in a B6 × S1 F2 population. We confirmed that S1 showed greater EtOH-induced hypothermia (specifically at a high dose) and longer LORR compared to B6. QTL analysis revealed several additive and interacting loci for various phenotypes, as well as examples of genotype interactions with sex. QTLs for different EtOH phenotypes were largely non-overlapping, suggesting separable genetic influences on these behaviors. The most compelling main-effect QTLs were for hypothermia on chromosome 16 and for LORR on chromosomes 4 and 6. Several QTLs overlapped with loci repeatedly linked to EtOH drinking in previous mouse studies. The architecture of the traits we examined was complex but clearly amenable to dissection in future studies. Using integrative genomics strategies, plausible functional and positional candidates may be found. Uncovering candidate genes associated with variation in these phenotypes in this population could ultimately shed light on genetic factors underlying sensitivity to EtOH intoxication and risk for alcoholism in humans

Genetic Control of a Central Pattern Generator: Rhythmic Oromotor Movement in Mice Is Controlled by a Major Locus near Atp1a2

Fluid licking in mice is a rhythmic behavior that is controlled by a central pattern generator (CPG) located in a complex of brainstem nuclei. C57BL/6J (B6) and DBA/2J (D2) strains differ significantly in water-restricted licking, with a highly heritable difference in rates (h2≥0.62) and a corresponding 20% difference in interlick interval (mean ± SEM = 116.3±1 vs 95.4±1.1 ms). We systematically quantified motor output in these strains, their F1 hybrids, and a set of 64 BXD progeny strains. The mean primary interlick interval (MPI) varied continuously among progeny strains. We detected a significant quantitative trait locus (QTL) for a CPG controlling lick rate on Chr 1 (Lick1), and a suggestive locus on Chr 10 (Lick10). Linkage was verified by testing of B6.D2-1D congenic stock in which a segment of Chr 1 of the D2 strain was introgressed onto the B6 parent. The Lick1 interval on distal Chr 1 contains several strong candidate genes. One of these is a sodium/potassium pump subunit (Atp1a2) with widespread expression in astrocytes, as well as in a restricted population of neurons. Both this subunit and the entire Na+/K+-ATPase molecule have been implicated in rhythmogenesis for respiration and locomotion. Sequence variants in or near Apt1a2 strongly modulate expression of the cognate mRNA in multiple brain regions. This gene region has recently been sequenced exhaustively and we have cataloged over 300 non-coding and synonymous mutations segregating among BXD strains, one or more of which is likely to contribute to differences in central pattern generator tempo