Hybrid mouse diversity panel: a panel of inbred mouse strains suitable for analysis of complex genetic traits

We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5?% of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.     

Simulating the collaborative cross: power of quantitative trait loci detection and mapping resolution in large sets of recombinant inbred strains of mice

It has been suggested that the collaborative cross, a large set of recombinant inbred strains derived from eight inbred mouse strains, would be a powerful resource for the dissection of complex phenotypes. Here we use simulation to investigate the power of the collaborative cross to detect and map small genetic effects. We show that for a fixed population of 1000 individuals, 500 RI lines bred using a modified version of the collaborative cross design are adequate to map a single additive locus that accounts for 5% of the phenotypic variation to within 0.96 cM. In the presence of strong epistasis more strains can improve detection, but 500 lines still provide sufficient resolution to meet most goals of the collaborative cross. However, even with a very large panel of RILs, mapping resolution may not be sufficient to identify single genes unambiguously. Our results are generally applicable to the design of RILs in other species.     

Meta-Analysis Identifies Gene-by-Environment Interactions as Demonstrated in a Study of 4,965 Mice

Identifying environmentally-specific genetic effects is a key challenge in understanding the structure of complex traits. Model organisms play a crucial role in the identification of such gene-by-environment interactions, as a result of the unique ability to observe genetically similar individuals across multiple distinct environments. Many model organism studies examine the same traits but under varying environmental conditions. For example, knock-out or diet-controlled studies are often used to examine cholesterol in mice. These studies, when examined in aggregate, provide an opportunity to identify genomic loci exhibiting environmentally-dependent effects. However, the straightforward application of traditional methodologies to aggregate separate studies suffers from several problems. First, environmental conditions are often variable and do not fit the standard univariate model for interactions. Additionally, applying a multivariate model results in increased degrees of freedom and low statistical power. In this paper, we jointly analyze multiple studies with varying environmental conditions using a meta-analytic approach based on a random effects model to identify loci involved in gene-by-environment interactions. Our approach is motivated by the observation that methods for discovering gene-by-environment interactions are closely related to random effects models for meta-analysis. We show that interactions can be interpreted as heterogeneity and can be detected without utilizing the traditional uni- or multi-variate approaches for discovery of gene-by-environment interactions. We apply our new method to combine 17 mouse studies containing in aggregate 4,965 distinct animals. We identify 26 significant loci involved in High-density lipoprotein (HDL) cholesterol, many of which are consistent with previous findings. Several of these loci show significant evidence of involvement in gene-by-environment interactions. An additional advantage of our meta-analysis approach is that our combined study has significantly higher power and improved resolution compared to any single study thus explaining the large number of loci discovered in the combined study.     

A Radiation Hybrid Map of Mouse Chromosome 13

A mouse radiation hybrid (RH) panel was used to make a framework map for the entire length of mouse chromosome (Chr) 13. Forty-one loci were typed, and while most used primers flanking simple sequence repeats, some genes were included. The most proximal and distal loci are D13Mit132 and D13Mit35. The estimate of map length for Chr 13 is 1328 cR. The map is compared with the same set of loci from the consensus map for Chr 13, which is 70 cM in length, and also with a recombinational map derived from an intraspecies cross typed for many of the same loci. The mouse RH panel gave good resolution for Chr 13 and at the distal end allowed separation of previously nonrecombinant markers that are present on a single 620-kb YAC clone. Data analysis was performed using the RH option for Map Manager QT. This framework RH map of Chr 13 is the second of a series of RH maps for mouse chromosomes.     

Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis

Significant advances have been made in the discovery of genes affecting bone mineral density (BMD); however, our understanding of its genetic basis remains incomplete. In the current study, genome-wide association (GWA) and co-expression network analysis were used in the recently described Hybrid Mouse Diversity Panel (HMDP) to identify and functionally characterize novel BMD genes. In the HMDP, a GWA of total body, spinal, and femoral BMD revealed four significant associations (-log10P>5.39) affecting at least one BMD trait on chromosomes (Chrs.) 7, 11, 12, and 17. The associations implicated a total of 163 genes with each association harboring between 14 and 112 genes. This list was reduced to 26 functional candidates by identifying those genes that were regulated by local eQTL in bone or harbored potentially functional non-synonymous (NS) SNPs. This analysis revealed that the most significant BMD SNP on Chr. 12 was a NS SNP in the additional sex combs like-2 (Asxl2) gene that was predicted to be functional. The involvement of Asxl2 in the regulation of bone mass was confirmed by the observation that Asxl2 knockout mice had reduced BMD. To begin to unravel the mechanism through which Asxl2 influenced BMD, a gene co-expression network was created using cortical bone gene expression microarray data from the HMDP strains. Asxl2 was identified as a member of a co-expression module enriched for genes involved in the differentiation of myeloid cells. In bone, osteoclasts are bone-resorbing cells of myeloid origin, suggesting that Asxl2 may play a role in osteoclast differentiation. In agreement, the knockdown of Asxl2 in bone marrow macrophages impaired their ability to form osteoclasts. This study identifies a new regulator of BMD and osteoclastogenesis and highlights the power of GWA and systems genetics in the mouse for dissecting complex genetic traits.     

Development of a gene-based radiation hybrid map of chicken Chromosome 7 and comparison to human and mouse

To validate the ChickRH6 whole-genome radiation hybrid (WGRH) panel, we constructed a map of chicken Chromosome 7 based on 19 microsatellite markers from the genetic map and 76 ESTs (expressed sequence tags), whose efficient targeted development was made possible by using the ICCARE software. This high-density radiation hybrid (RH) map of a chicken macrochromosome gives us indications on characteristics of ChickRH6. The potential resolution of the panel is 325 kb and the practical resolution of our framework map is 1.3 Mb. Based on these results, a complete framework map of the chicken genome would comprise 1000 markers. The marker order is in good agreement with the genetic map and comparison with the human and mouse sequence maps revealed a number of internal rearrangements.     

Combining two Meishan F2 crosses improves the detection of QTL on pig chromosomes 2, 4 and 6

Background

In pig, a number of experiments have been set up to identify QTL and a multitude of chromosomal regions harbouring genes influencing traits of interest have been identified. However, the mapping resolution remains limited in most cases and the detected QTL are rather inaccurately located. Mapping accuracy can be improved by increasing the number of phenotyped and genotyped individuals and/or the number of informative markers. An alternative approach to overcome the limited power of individual studies is to combine data from two or more independent designs.

Methods

In the present study we report a combined analysis of two independent design (a French and a Dutch F2 experimental designs), with 2000 F2 individuals. The purpose was to further map QTL for growth and fatness on pig chromosomes 2, 4 and 6. Using QTL-map software, uni- and multiple-QTL detection analyses were applied separately on the two pedigrees and then on the combination of the two pedigrees.

Results

Joint analyses of the combined pedigree provided (1) greater significance of shared QTL, (2) exclusion of false suggestive QTL and (3) greater mapping precision for shared QTL.

Conclusions

Combining two Meishan x European breeds F2 pedigrees improved the mapping of QTL compared to analysing pedigrees separately. Our work was facilitated by the access to raw phenotypic data and DNA of animals from both pedigrees and the combination of the two designs with the addition of new markers allowed us to fine map QTL without phenotyping additional animals.     

Development and applications of a molecular genetic linkage map of the mouse genome

Interspecific mouse backcrosses provide almost limitless genetic variation for gene mapping. We have used interspecific backcrosses to develop the first comprehensive molecular genetic linkage map of the mouse genome. More than 600 loci have been positioned on the map; the current average map resolution is less than 3 cM. Since all loci were mapped using a single backcross panel, gene order can be determined unambiguously. With this level of resolution, it is now possible to position any new locus on the linkage map with virtually 100% certainty. In this article, we review how interspecific linkage maps are constructed, the salient features of our linkage map, and some of the many applications of interspecific linkage maps, in general, for future research.     

Generation and characterization of a 12,000-rad radiation hybrid panel for fine mapping in pig

We have constructed a 12,000-rad porcine whole-genome radiation hybrid panel to complement the first generation 7,000-rad panel (IMpRH) and allow higher resolution mapping studies both in specific areas of interest and on the whole genome. We analyzed 243 hybrid clones on the basis of their marker retention frequency to produce a final panel of 90 hybrid clones with an average retention frequency of 35.4%. The resolution of this 12,000-rad panel (IMNpRH2) was compared to the resolution of the 7,000-rad panel (IMpRH) by constructing framework maps in the 2.4-Mb region of porcine chromosome 15 containing the acid meat RN gene. In this region, two-point analysis was used to estimate RH distances and demonstrates their reliability with the estimation of physical distances. This study demonstrates that the 12,000-rad panel constitutes a powerful tool for constructing high-resolution maps. Indeed, the resolution of IMNpRH2 (12-14 kb/cR(12,000)) is two to three times more than that of IMpRH (35-37 kb/cR(7,000)). As expected, the increase in the radiation dose allows an increase of the mapping resolution in terms of kb/cR with the same suppleness of use for mapping experiments. In addition the RH map constructed in the region investigated proved to be more homogeneous on IMNpRH2 than on IMpRH.     

Efficient control of population structure in model organism association mapping

Genomewide association mapping in model organisms such as inbred mouse strains is a promising approach for the identification of risk factors related to human diseases. However, genetic association studies in inbred model organisms are confronted by the problem of complex population structure among strains. This induces inflated false positive rates, which cannot be corrected using standard approaches applied in human association studies such as genomic control or structured association. Recent studies demonstrated that mixed models successfully correct for the genetic relatedness in association mapping in maize and Arabidopsis panel data sets. However, the currently available mixed-model methods suffer from computational inefficiency. In this article, we propose a new method, efficient mixed-model association (EMMA), which corrects for population structure and genetic relatedness in model organism association mapping. Our method takes advantage of the specific nature of the optimization problem in applying mixed models for association mapping, which allows us to substantially increase the computational speed and reliability of the results. We applied EMMA to in silico whole-genome association mapping of inbred mouse strains involving hundreds of thousands of SNPs, in addition to Arabidopsis and maize data sets. We also performed extensive simulation studies to estimate the statistical power of EMMA under various SNP effects, varying degrees of population structure, and differing numbers of multiple measurements per strain. Despite the limited power of inbred mouse association mapping due to the limited number of available inbred strains, we are able to identify significantly associated SNPs, which fall into known QTL or genes identified through previous studies while avoiding an inflation of false positives. An R package implementation and webserver of our EMMA method are publicly available.     

High-resolution cytogenetic-based physical map of human chromosome 16

A panel of 54 mouse/human somatic cell hybrids, each possessing various portions of chromosome 16, was constructed; 46 were constructed from naturally occurring rearrangements of this chromosome, which were ascertained in clinical cytogenetics laboratories, and a further 8 from rearrangements spontaneously arising during tissue culture. By mapping 235 DNA markers to this panel of hybrids, and in relation to four fragile sites and the centromere, a cytogenetic-based physical map of chromosome 16 with an average resolution of 1.6 Mb was generated. Included are 66 DNA markers that have been typed in the CEPH pedigrees, and these will allow the construction of a detailed correlation of the cytogenetic-based physical map and the genetic map of this chromosome. Cosmids from chromosome 16 that have been assembled into contigs by use of repetitive sequence fingerprinting have been mapped to the hybrid panel. Approximately 11% of the euchromatin is now both represented in such contigs and located on the cytogenetic-based physical map. This high-resolution cytogenetic-based physical map of chromosome 16 will provide the basis for the cloning of genetically mapped disease genes, genes disrupted in cytogenetic rearrangements that have produced abnormal phenotypes, and cancer breakpoints.     

Equilibrium and kinetic studies of substrate binding to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, an intermediate in the pathway for folic acid biosynthesis. The enzyme has been identified as a potential target for antimicrobial drugs. Equilibrium binding studies showed that Escherichia coli HPPK-bound ATP or the nonhydrolyzable ATP analogue alpha, beta-methyleneadenosine triphosphate (AMPCPP) with high affinity. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate (MANT-ATP) exhibited a substantial fluorescence enhancement upon binding to HPPK, with an equilibrium dissociation constant comparable with that for ATP (10.4 and 4.5 micrometer, respectively). The apoenzyme did not bind the second substrate HMDP, however, unless AMPCPP was present, suggesting that the enzyme binds ATP first, followed by HMDP. Equilibrium titration of HPPK into HMDP and AMPCPP showed an enhancement of fluorescence from the pterin ring of the substrate, and a dissociation constant of 36 nm was deduced for HMDP binding to the HPPK.AMPCPP binary complex. Stopped flow fluorimetry measurements showed that the rate constants for the binding of MANT-ATP and AMPCPP to HPPK were relatively slow (3.9 x 10(5) and 1.05 x 10(5) m(-1) s(-1), respectively) compared with the on rate for binding of HMDP to the HPPK.AMPCPP binary complex. The significance of these results with respect to the crystal structures of HPPK is discussed.     

Recalculation of 23 mouse HDL QTL datasets improves accuracy and allows for better candidate gene analysis

In the past 15 years, the quantitative trait locus (QTL) mapping approach has been applied to crosses between different inbred mouse strains to identify genetic loci associated with plasma HDL cholesterol levels. Although successful, a disadvantage of this method is low mapping resolution, as often several hundred candidate genes fall within the confidence interval for each locus. Methods have been developed to narrow these loci by combining the data from the different crosses, but they rely on the accurate mapping of the QTL and the treatment of the data in a consistent manner. We collected 23 raw datasets used for the mapping of previously published HDL QTL and reanalyzed the data from each cross using a consistent method and the latest mouse genetic map. By utilizing this approach, we identified novel QTL and QTL that were mapped to the wrong part of chromosomes. Our new HDL QTL map allows for reliable combining of QTL data and candidate gene analysis, which we demonstrate by identifying Grin3a and Etv6, as candidate genes for QTL on chromosomes 4 and 6, respectively. In addition, we were able to narrow a QTL on Chr 19 to five candidates.     

Extension of the physical map in the region of the mouse X chromosome homologous to human Xq28 and identification of an exception to conserved linkage.

We have extended our pulsed-field gel map of the region of the mouse X chromosome homologous to human Xq28 to include the loci Gdx (DXS254Eh), P3 (DXS253Eh), G6pd, Cf-8, and F8a. Gdx, P3, and G6pd are demonstrated to be physically linked to the X-linked visual pigment locus (Rsvp) within a maximal distance of 340 kb, while G6pd and Cf-8 are approximately 900 kb apart. These studies favor a gene order of cen-Rsvp-Gdx-P3-G6pd-(Cf-8)-tel and extend the physical map of this region to 5 million bp. In conjunction with previous physical mapping studies in both mouse and human, the results suggest conserved linkage for loci in this region of the mouse X chromosome and human Xq28. However, employing pulsed-field gel electrophoresis and genetic pedigree analysis of interspecific backcross progeny, we have found close linkage of a clone encoding a mouse homolog for human factor VIII-associated gene A (F8A) to DXPas8, thus revealing the first exception to conserved gene order between murine and human loci in the region.     

Genetic locus on mouse chromosome 7 controls elevated heart rate

Elevated heart rate (HR) is a risk factor for cardiovascular diseases. The goal of the study was to map HR trait in mice using quantitative trait locus (QTL) analysis followed by genome-wide association (GWA) analysis. The first approach provides mapping power and the second increases genome resolution. QTL analyses were performed in a C3HeB×SJL backcross. HR and systolic blood pressure (SBP) were measured by the tail-cuff plethysmography. HR was ～80 beats/min higher in SJL compared with C3HeB. There was a wide distribution of the HR (536-763 beats/min) in N2 mice. We discovered a highly significant QTL (logarithm of odds = 6.7, P < 0.001) on chromosome 7 (41 cM) for HR in the C3HeB×SJL backcross. In the Hybrid Mouse Diversity Panel (58 strains, n = 5-6/strain) we found that HR (beats/min) ranged from 546 ± 12 in C58/J to 717 ± 7 in MA/MyJ mice. SBP (mmHg) ranged from 99 ± 6 in strain I/LnJ to 151 ± 4 in strain BXA4/PgnJ. GWA analyses were done using the HMDP, which revealed a locus (64.2-65.1 Mb) on chromosome 7 that colocalized with the QTL for elevated HR found in the C3HeB×SJL backcross. The peak association was observed for 17 SNPs that are localized within three GABA(A) receptor genes. In summary, we used a combined genetic approach to fine map a novel elevated HR locus on mouse chromosome 7.     

Linkage mapping of 1454 new maize candidate gene Loci

Bioinformatic analyses of maize EST sequences have highlighted large numbers of candidate genes putatively involved in agriculturally important traits. To contribute to ongoing efforts toward mapping of these genes, we used two populations of intermated recombinant inbred lines (IRILs), which allow a higher map resolution than nonintermated RILs. The first panel (IBM), derived from B73 x Mo17, is publicly available from the Maize Genetics Cooperation Stock Center. The second panel (LHRF) was developed from F2 x F252 to map loci monomorphic on IBM. We built framework maps of 237 loci from the IBM panel and 271 loci from the LHRF panel. Both maps were used to place 1454 loci (1056 on map IBM_Gnp2004 and 398 on map LHRF_Gnp2004) that corresponded to 954 cDNA probes previously unmapped. RFLP was mostly used, but PCR-based methods were also performed for some cDNAs to map SNPs. Unlike in usual IRIL-based maps published so far, corrected meiotic centimorgan distances were calculated, taking into account the number of intermating generations undergone by the IRILs. The corrected sizes of our framework maps were 1825 cM for IBM_Gnp2004 and 1862 cM for LHRF_Gnp2004. All loci mapped on LHRF_Gnp2004 were also projected on a consensus map IBMconsensus_Gnp2004. cDNA loci formed clusters near the centromeres except for chromosomes 1 and 8.     

A high-resolution radiation hybrid map of the proximal region of rat Chromosome 4

Radiation hybrid (RH) mapping has been used to produce genome maps in the human and mouse, but as yet the technique has been applied little to other species. We describe the use of RH mapping in the rat, using a newly available rat/hamster RH panel, to construct an RH map of the proximal part of rat Chromosome (Chr) 4. This region is of interest because quantitative trait loci (QTLs) for defective insulin and catecholamine action, hypertension, and dyslipidemia map to this region. The RH map includes 23 rat genes or microsatellites previously mapped to this part of Chr 4, one rat gene not previously mapped in the rat, and markers for four new genes, homologs of which map to the syntenic region of the mouse genome. The RH map integrates genetic markers previously mapped on several rat crosses, increases the resolution of existing maps, and may provide a suitable basis for physical map construction and gene identification in this chromosomal region. Our results demonstrate the utility of RH mapping in the rat genome and show that RH mapping can be used to localize, in the rat genome, the homologs of genes from other species such as the mouse. This will facilitate identification of candidate genes underlying QTLs on this chromosomal segment. Received: 4 December 1998 / Accepted: 19 January 1999