Two-Locus Linkage Analysis Using Recombinant Inbred Strains and Bayes'' Theorem

Recombinant inbred (RI) strains are useful in linkage analysis and gene mapping. The currently available statistical tests of linkage using data derived from the study of RI strains, including a previous Bayesian analysis, have not been stringent enough guides for conclusions about linkage. In this paper, the probability of linkage was estimated using Bayes' theorem. Tables are presented that give the probability of linkage in sets of up to 30 RI strains and the critical values of i (the number of recombinants) in sets of up to 100 RI strains. Several means of increasing the power of RI strains in linkage analysis are discussed.     

Genetics of body weight in the LXS recombinant inbred mouse strains

This is the first phenotypic analysis of 75 new recombinant inbred (RI) strains derived from ILS and ISS progenitors. We analyzed body weight in two independent cohorts of female mice at various ages and in males at 60 days. Body weight is a complex trait which has been mapped in numerous crosses in rodents. The LXS RI strains displayed a large range of weights, transgressing those of the inbred progenitors, supporting the utility of this large panel for mapping traits not selected in the progenitors. Numerous QTLs for body weight mapped in single- and multilocus scans. We assessed replication between these and previously reported QTLs based on overlapping confidence intervals of published QTLs for body weight at 60 days and used meta-analyses to determine combined p values for three QTL regions located on Chromosomes 4, 5, and 11. Strain distribution patterns of microsatellite marker genotypes, weight, and other phenotypes are available on WebQTL () and allow genetic mapping of any heritable quantitative phenotype measured in these strains. We report one such analysis, correlating brain and body weights. Large reference panels of RI strains, such as the LXS, are invaluable for identifying genetic correlations, GXE (Gene X Environment) interactions, and replicating previously identified QTLs. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Mapping of the gene coding for the muscle protein nebulin (Neb) to the proximal region of mouse chromosome 2.

Using five different series of recombinant inbred (RI) strains, we have mapped the gene encoding nebulin, a muscle-specific protein. The strain distribution pattern of Neb-specific RFLPs segregating in the RI strains showed significant concordance with those of Pmv-7 and Hc, two loci previously located to proximal mouse Chromosome 2. The gene order, intergene distances, and confidence intervals were determined according to standard maximum likelihood estimates as: centromere-Hc-5.4 cM (1.4 cM-15.4 cM; 95% confidence interval)-Neb-3.6 cM (1.5-6.7 cM)-Pmv-7.     

Genetic characterization of a new set of recombinant inbred lines (LGXSM) formed from the intercross of SM/J and LG/J inbred mouse strains

A new set of LGXSM recombinant inbred (RI) strains is presented. The RI strain panel consists of 18 remaining strains of the original 55 founding strains. Strain characterization is based on 506 polymorphic microsatellites and 4289 single nucleotide polymorphisms (SNPs) distributed across the genome. Average microsatellite intermarker distance is 4.80 ± 4.84 Mb or 2.91 ± 3.21 F₂ cM. SNPs are more densely spaced at 0.57 ± 1.27 Mb. Ninety-five percent of all microsatellite intermarker intervals are separated by less than 15.00 Mb or 8.50 F₂ cM, while 95% of the SNPs are less than 0.95 Mb apart. Strains show expected low levels of nonsyntenic association among loci and complete genomic independence. During inbreeding, the RI strains went through strong natural selection on the agouti locus on Chromosome 2, especially when the epistatically interacting tyrosinase locus on Chromosome 7 carried the wild-type allele. The LG/J and SM/J strains differ in a large number of biomedically important traits, and they and their intercross progeny have been used in multiple mapping studies. The LG×SM RI strain panel provides a powerful new resource for mapping the genetic bases of complex traits and should prove to be of great biomedical utility in modeling complex human diseases such as obesity and diabetes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Use of AFLP Markers for Gene Mapping and QTL Detection in the Rat

The AFLP technique is a new DNA marker technology based on the selective amplification of restriction fragments. Multiple polymorphic markers are simultaneously produced and can be tested in one PCR. No prior information on genomic DNA sequences is needed. In the current study, we contribute 18 AFLP markers to the linkage map of the rat. Seven AFLP markers were assigned to specific chromosomes by analysis of a (BN × ACI)F1 × ACI backcross progeny. Another 11 AFLP markers were mapped by using a panel of the H × B/B × H recombinant inbred (RI) strains. Genotypes of these AFLP markers were also tested for correlations with some blood pressure phenotypes in the RI strains. Suggestive correlation was found between the mean arterial pressure and two closely linked AFLP markers located on chromosome 20. The current study illustrates the value of AFLP markers for the construction of linkage maps and the detection of quantitative trait loci.     

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.     

Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.     

Genetic loci for diet-induced atherosclerotic lesions and plasma lipids in mice

Genetic factors independent of those affecting plasma lipid levels are a major contributor to risk for atherosclerosis in humans, yet the basis for these is poorly understood. This study examined plasma lipids and diet-induced atherosclerosis in 16-month-old female mice of strains C56BL/6J and DBA/2J. Mice of the parental strains, from recombinant inbred strains derived from these (BXD RI), and F₂ progeny were fed an atherogenic diet for 16 weeks, beginning at 1 year of age. This induced atherosclerotic lesion formation in both parental strains, accompanied by increased plasma LDL levels. However, individual BXD RI strains and the BXD F₂ mice demonstrated a range of atherosclerotic lesion formation that was not or at best weakly correlated with plasma lipid levels. Quantitative trait locus (QTL) analysis of the BXD F₂ mice identified a locus with significant linkage (lod 4.5) for aortic lesion size on Chromosome (Chr) 10 that was independent of plasma lipids. Other loci with suggestive or significant linkage for various plasma lipid measures were identified on Chr 2, 3, 4, 5, 6, 7, 11, and 17. In this intercross, the genes primarily influencing atherosclerosis are distinct from those controlling plasma lipid levels.     

Genetic control of neuroadapted sindbis virus replication in female mice maps to chromosome 2 and associates with paralysis and mortality

Neuroadapted Sindbis virus (NSV) infection of mice causes hindlimb paralysis and 100% mortality in the C57BL/6 mouse strain, while adults of the BALB/cBy mouse strain are resistant to fatal encephalomyelitis. Levels of viral RNA are higher in the brains of infected C57BL/6 mice than in BALB/cBy mice (D. C. Thach et al., J. Virol. 74:6156-6161, 2000). These phenotypic differences between the two strains allowed us to map genetic loci involved in mouse susceptibility to NSV and to find relationships between mortality, paralysis, and viral RNA levels. Analysis of percent mortality in H2-congenic and F(1) mice suggested that the H2 locus, sex linkage, and imprinting were not involved in determining susceptibility and that resistance was partially dominant over susceptibility. Segregation analysis using CXB recombinant inbred (RI) mice indicated that the percent mortality was multigenic. Interval mapping detected a suggestive quantitative trait locus (QTL) on chromosome 2 near marker D2Mit447. Analysis of paralysis in the RI mice detected the same suggestive QTL. Viral RNA level in F(1) mice was intermediate. Interval mapping using viral RNA levels in RI mice detected a significant QTL near marker D2Mit447 that explained 69% of the genetic variance. This QTL was confirmed in F2 mice and was designated as Nsv1. Viral RNA level, percent paralyzed, and percent mortality were linearly correlated (r = 0.8 to 0.9). These results indicate that mortality, paralysis, and viral RNA levels are related complex traits and that Nsv1 controls early viral load and determines the likelihood of paralysis and death.     

Use of recombinant inbred strains to map genes of aging

Recombinant inbred strains have been used in a number of organisms for segregation and linkage analysis of quantitative traits. One major advantage of the recombinant inbred (RI) methodology is that the genetic identity of individuals within a strain permits replicate measures of the same recombinant genotype. Such replicability is important for traits such as aging inDrosophila, where phenotypic expression is highly influenced by different environmental conditions. RI strain methodology has an added advantage for DNA marker-based linkage analysis of traits measured over the lifespan of the organism. The DNA can be extracted from individuals of the same genotype as those measured in a longevity study. In this paper an argument is presented for the use of a set of recombinant inbred strains to map the quantitative trait loci involved in the aging process inDrosophila. A unique use of a set of stable, transposable moleular markers to trace the quantitative trait loci involved is suggested.     

RI manager,a microcomputer program for analysis of data from recombinant inbred strains

RI Manager is a microcomputer program for storage and analysis of genetic mapping data from recombinant inbred strains. The program rapidly identifies statistically significant linkage between known loci and a newly described locus, and it facilitates rapid evaluation of alternative map orders for linked loci.     

Chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality.

Four genetic loci were tested for linkage with loci that control genetic resistance to lethal ectromelia virus infection in mice. Three of the loci were selected because of concordance with genotypes assigned to recombinant inbred (RI) strains of mice derived from resistant C57BL/6 and susceptible DBA/2 (BXD) mice on the basis of their responses to challenge infection. Thirty-six of 167 male (C57BL/6 x DBA/2)F1 x DBA/2 backcross (BC) mice died (22%), of which 27 (75%) were homozygous for DBA/2 alleles at Hc and H-2D. Twenty-eight percent of sham-castrated and 6% of sham-ovariectomized BC mice were susceptible to lethal mousepox, whereas 50% of gonadectomized mice were susceptible. There was no linkage evident between Hc or H-2D and loci that controlled resistance to lethal ectromelia virus infection in 44 castrated BC mice. Mortality among female mice of BXD RI strains with susceptible or intermediate male phenotypes was strongly correlated (r = 0.834) with male mortality. Gonadectomized C57BL/6 mice were as resistant as intact mice to lethal ectromelia virus infection. These results indicate that two gonad-dependent genes on chromosomes 2 and 17 and one gonad-independent gene control resistance to mousepox virus infection, that males and females share gonad-dependent genes, and that the gonad-independent gene is fully protective.     

Analysis of oocyte quality in recombinant inbred mouse strains developed from KE and CBA strains that differ in fertilization efficiency

Recombinant inbred (RI) mouse strains were developed from reciprocal crosses between two inbred strains differing in the proportion of fertilized ova (CBA, 100%; KE, 77%), to analyse the underlying factors. A correlation (r = 0.83, P < 0.01) between fertilization efficiency within 22 RI strains and after mating RI females with KE males proved that oocyte quality was involved. The following oocyte parameters were analysed in RI and progenitor strains: time of meiotic maturation, rapidity of enzymatic removal of egg investments, and proportion of fertilized ova with supplementary spermatozoa in the perivitelline space. Among the RI strains, high incidence of supplementary spermatozoa was correlated with lower efficiency of fertilization (r = -0.58, P < 0.05) and with slow meiotic maturation (r = -64, P < 0.01), suggesting that delayed maturation may affect oocyte ability of being fertilized by the first penetrating spermatozoon. However, significant correlations were also found between characters which coexist within the progenitor strains, but are not likely to be physiologically related; this suggests that RI strains have inherited large blocks of progenitor genomes, not disrupted by recombination. The strain distribution pattern (SDP) of the analysed traits revealed CBA-like, KE-like, and intermediate phenotypes, indicating that they are polygenically determined. No linkages were found between the studied traits and 12 enzymatic markers. However, the SDP for fertilization efficiency showed a preponderance of non-matching strains (15/19) in relation to agouti locus; the known instability of this chromosome region makes it possible that a putative linkage was disrupted by recombination when RI strains were created.     

Construction of a confidence set of markers for the location of a disease gene using affected sib pair data

We have previously proposed a confidence set approach for finding tightly linked genomic regions under the setting of parametric linkage analysis. In this article, we extend the confidence set approach to nonparametric linkage analysis of affected sib pair (ASP) data based on their identity-by-descent (IBD) information. Two well-known statistics in nonparametric linkage analysis, the Two-IBD test (proportion of ASPs sharing two alleles IBD), and the Mean test (average number of alleles shared IBD in the ASPs), are used for constructing confidence sets. Some numerical analyses as well as a simulation study were carried out to demonstrate the utility of the methods. Our results show that the fundamental advantages of the confidence set approach in parametric linkage analysis are retained when the method is generalized to nonparametric analysis. Our study on the accuracy of confidence sets, in terms of choice of tests, underlying disease incidence data, and amount of data available, leads us to conclude, among other things, that the Mean test outperforms the Two-IBD test in most situations, with the reverse being true only for traits with small additive variance. Although we describe how to construct confidence sets based on only two familiar tests, one can construct confidence sets similarly using other allele sharing statistics.     

Genetic counseling in rare syndromes: a resampling method for determining an approximate confidence interval for gene location with linkage data from a single pedigree.

Multipoint linkage analysis is a powerful method for mapping a rare disease gene on the human gene map despite limited genotype and pedigree data. However, there is no standard procedure for determining a confidence interval for gene location by using multipoint linkage analysis. A genetic counselor needs to know the confidence interval for gene location in order to determine the uncertainty of risk estimates provided to a consultant on the basis of DNA studies. We describe a resampling, or "bootstrap," method for deriving an approximate confidence interval for gene location on the basis of data from a single pedigree. This method was used to define an approximate confidence interval for the location of a gene causing nonsyndromal X-linked mental retardation in a single pedigree. The approach seemed robust in that similar confidence intervals were derived by using different resampling protocols. Quantitative bounds for the confidence interval were dependent on the genetic map chosen. Once an approximate confidence interval for gene location was determined for this pedigree, it was possible to use multipoint risk analysis to estimate risk intervals for women of unknown carrier status. Despite the limited genotype data, the combination of the resampling method and multipoint risk analysis had a dramatic impact on the genetic advice available to consultants.     

Identification of QTLs Involved in the Development of Amygdala Kindling in the Rat

Amygdala kindling is useful for modeling human epilepsy development. It has been known that genetic factors are involved in the development of amygdala kindling. The purpose of this study was to identify the loci that are responsible for the development of amygdala kindling. To achieve this, rat strains from a LEXF/FXLE recombinant inbred (RI) strain panel were used. The phenotypes of amygdala kindling-related parameters for seven RI strains and parental LE/Stm and F344/Stm strains were determined. They included the afterdischarge threshold (ADT), the afterdischarge duration (ADD), and the kindling rate, an incidence of development of kindling. Quantitative trait loci (QTL) analysis was performed to identify linkage relationships between these phenotypes and 1,033 SNP markers. Although no significant differences in pre-kindling ADT and ADD were observed, a significant difference in the kindling rate was found for the LEXF/FXLE RI strain. Two QTLs for the amygdala kindling rate (Agkr1 and Agkr2) were identified on rat chromosome 2. These findings clearly prove the existence of genetic influences that are involved in kindling development and suggest that substantial genetic components contribute to the progression of partial seizures into generalized seizures.     

Estimation of Linkage and Association from Allele Transmission Data

The TDT provides a hypothesis test for the presence of linkage or association (linkage disequilibrium). However, since the TDT is a single test statistic, it cannot be used to separate association and linkage. The importance of this difficulty, following a significant TDT result, has been recently emphasized by Whittaker , Denham and Morris (2000), who alert the community to the possibility that a significant TDT may result from loose linkage and strong association, or from tight linkage and weak association. To attack this problem we start with the parametric model for family‐based allele transmission data of Sham and Curtis (1995) (or Sham (1998)) and find that the parameters in the model are not always identifiable. So we introduce a reparameterization that resolves the identifiability issues and leads to a valid likelihood ratio (LR) test for linkage. Since the linkage and association parameters are both of interest, we next introduce and apply an integrated likelihood (IL) approach to provide separate point estimates and confidence intervals for these parameters. The estimates are shown to have generally small bias and mean square error, while the confidence intervals have good average length and coverage probabilities. We compare the power of the IL approach for testing linkage and, separately association, with the TDT and LR.     

An empirical Bayes method for updating inferences in analysis of quantitative trait loci using information from related genome scans

Individual genome scans for quantitative trait loci (QTL) mapping often suffer from low statistical power and imprecise estimates of QTL location and effect. This lack of precision yields large confidence intervals for QTL location, which are problematic for subsequent fine mapping and positional cloning. In prioritizing areas for follow-up after an initial genome scan and in evaluating the credibility of apparent linkage signals, investigators typically examine the results of other genome scans of the same phenotype and informally update their beliefs about which linkage signals in their scan most merit confidence and follow-up via a subjective-intuitive integration approach. A method that acknowledges the wisdom of this general paradigm but formally borrows information from other scans to increase confidence in objectivity would be a benefit. We developed an empirical Bayes analytic method to integrate information from multiple genome scans. The linkage statistic obtained from a single genome scan study is updated by incorporating statistics from other genome scans as prior information. This technique does not require that all studies have an identical marker map or a common estimated QTL effect. The updated linkage statistic can then be used for the estimation of QTL location and effect. We evaluate the performance of our method by using extensive simulations based on actual marker spacing and allele frequencies from available data. Results indicate that the empirical Bayes method can account for between-study heterogeneity, estimate the QTL location and effect more precisely, and provide narrower confidence intervals than results from any single individual study. We also compared the empirical Bayes method with a method originally developed for meta-analysis (a closely related but distinct purpose). In the face of marked heterogeneity among studies, the empirical Bayes method outperforms the comparator.     

Typing recombinant inbred mouse strains for microsatellite markers on Chromosomes 10, 16, 18, 19, and X

A total of 57 different microsatellite variants have been typed in one or more of five different sets of recombinant inbred (RI) mouse strains. The present report concentrates on markers for Chromosomes (Chrs) 10, 16, 18, 19 and X. These markers extend the regions swept in these RI strains, provide reference markers for integrating RI and conventional maps, and provide additional estimates of genetic distances. Multilocus maps, based on maximum likelihood analysis of present and previously published RI SDPs on five chromosomes, are presented. Unexpectedly, three microsatellite markers, previously assigned to Chr 10, detected polymorphic fragments mapping to other chromosomes.