High-resolution mapping of quantitative trait loci for emotionality in ...

Short Communications Incorporating Mouse Genome

Mammalian Genome 10, 1098–1101 (1999).

© Springer-Verlag New York Inc. 1999

High-resolution mapping of quantitative trait loci for emotionality in selected strains of mice Maria Grazia Turri,1 Christopher J. Talbot,1 Richard A. Radcliffe,2 Jeanne M. Wehner,2 Jonathan Flint1 1 2

Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, USA

Received: 16 April 1999 / Accepted: 8 July 1999

While a genetic contribution to many behavioral traits is not in doubt, attempts to find the genes themselves have not met with much success. One advance towards this goal has been the demonstration that it is possible to map genes that determine variation in quantitatively measured behavioral traits with crosses between inbred strains (both rodents and insects have been used). Further progress requires high-resolution mapping of quantitative trait loci (QTL). Current detection methods place QTLs within an interval of about half a chromosome, far too large for positional cloning to be a viable option, and a number of approaches have been advocated to increase resolution (Darvasi 1998). Here we report a novel, high-resolution mapping strategy that exploits the effects of artificial selection followed by inbreeding. The strategy requires prior information about the likely position of QTLs, such as would be available from an F2 or backcross genome scan, and the localizations it provides need to be confirmed by further tests of segregation. For the latter purpose, we describe a variant of the recombinant inbred segregation test (RIST; Darvasi 1998), which can be employed for confirmation and further finemapping of QTLs. We have previously shown, using an F2 genome scan, that three QTL (on Chrs 1, 12, and 15) contribute to the genetic variance of a psychological trait in mice termed emotionality. In that experiment, we used two measures of emotionality: total distance the animal traverses in 5 minutes in an open-field arena (open-field activity, OFA) and the number of fecal boli produced in the open field during the 5-min test period (open-field defecation, OFD; Flint et al. 1995). Calvin Hall devised the open-field arena in the 1930s (Hall 1934) as a way of assessing emotionality in rats primarily because strong emotion, especially fear, is known to result in defecation and urination in humans (Stouffer et al. 1949). The open-field arena is a white box or circular arena that is brightly lit and considered to be unpleasant for rats and mice. Animals that are relatively inactive (low OFA scores) and have high defecation scores are regarded as having higher emotionality scores (or being more emotionally reactive) than active animals with low defecation scores. The two inbred strains in which we detected QTLs influencing emotionality are the product of an earlier selection experiment for open-field activity: BALB/cJ and C57BL/6 mice were intercrossed and offspring selected for differences in activity scores for 30 generations. In addition, unselected control lines were bred for the same period. The selection experiment was replicated, yielding six lines in all (DeFries et al. 1978). Following suspension of selection, the lines were randomly mated for a further 18 generations and then full-sib mated for 35 generations (DeFries et al. 1978; DeFries et al. 1970). The following inbred strains were thus established (hereafter referred to as DeFries strains): H1 and H2 are Correspondence to: J. Flint

the result of the selection for high OFA scores, L1 and L2 selection for low OFA scores, and C1 and C2 are derived from the control lines. The selection experiment demonstrated that open-field behaviors had a genetic basis and confirmed that there was a negative genetic correlation between OFA and OFD. At some level, therefore, OFA and OFD share a genetic basis and, on the grounds that ambulation (motor activity) and defecation (sympathetic nervous activity) are not known to be under the same peripheral nervous system control, the correlation probably represents a central state, termed emotionality. Successful characterization of the molecular basis of rodent emotionality may open new avenues in our understanding of human anxiety disorders: tests of rodent emotionality are widely used in the development of anxiolytic agents, and a large body of data suggests that the same neural structures subserve susceptibility to anxiety in humans and emotionality in rodents (Gray 1982, 1987). We have taken advantage of the way selection alters allele frequencies to fine-map the QTLs found in the F2 intercross of the H1 and L1 DeFries strains. During selection, allele frequencies at loci closely linked to genes influencing the trait tend to diverge, thus providing a method to detect the chromosomal position of such genes. At each new generation of selection, recombination breaks down linkage until only closely linked markers remain in association. The consequence of selection is to recombine regions of the BALB/cJ and C57BL/6J chromosomes that do not determine OFA while driving the region around each QTL to homozygosity. Our F2 intercross analysis indicated that QTLs increasing OFA were carried by the C57BL/6 strain (Flint et al. 1995). Since the selection experiment was carried out four times (twice for high OFA and twice for low OFA) and the mice subsequently inbred, the QTL is likely to be in a region where H1 and H2 DeFries strains (selected for high activity) are C57BL/6 and where L1 and L2 DeFries strains (selected for low activity) are BALB/cJ, while the control line haplotypes will be randomly either C57BL/6 or BALB/cJ. Consequently a simple strategy to refine the region that contains the QTL is to determine haplotypes in the six lines. We were particularly interested in characterizing the loci on Chrs 1 and 15 because they have been identified in more than one QTL detection experiment. Loci influencing OFA have been found on Chrs 1 and 15 with a cross between C57BL/6 and A/J strains (Gershenfeld et al. 1997; Gershenfeld and Paul 1997). Furthermore, two QTL studies of mice, one with a cross between DBA and C57BL/6 (Wehner et al. 1997), the other between C3H and C57BL/6 (Caldarone et al. 1997), detected a locus on Chr 1 influencing fear conditioning that overlaps those found in the openfield studies crosses. Should a pleiotropic locus be found, it is very likely to regulate variation in individual differences in fear and anxiety (LeDoux 1995; Rogan and LeDoux 1996).

M.G. Turri et al.: Fine-mapping of QTL for emotionality

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Fig. 1. Haplotypes of control and selected strains. Speckled bar represents BALB/cJ alleles, black bar C57BL/6 alleles. Dashed lines represent MIT polymorphic markers. Scale at top in centiMorgans (MGD distances). Re-

gions of allele sharing shown with arrows. C1 ⳱ Control line 1, C2 ⳱ control line 2, L1 ⳱ low activity line 1, L2 ⳱ low activity line 2, H1 ⳱ high activity line 1, H2 ⳱ high activity strain 2.

We genotyped all six strains over a 50-centimorgan (cM) region of Chr 1 and 20 cM of Chr 15. Alleles at all loci were identified as either C57BL/6 or BALB/cJ in origin, and markers were ordered using a panel of EUCIB mice (Rhodes et al. 1998). Figure 1 shows the haplotypes that we derived across both regions. Note that the boundary of each haplotype is shown as lying exactly between two markers lying either side of a point of recombination. In both regions there was one segment of chromosome with the expected haplotype distribution among the strains. Between markers D1Mit113 and D1Mit409 on Chr 1, both high strains were C57BL/6 and both low strains were BALB/cJ. The marker with the highest LOD score (D1Mit150) from our F2 intercross mapping (Flint et al. 1995) lies in the center of this region. Availability of markers that distinguish C57BL/6 and BALB/cJ limits our ability to size the region accurately on Chr 1. The distance between D1Mit113, which detects the most distal C57BL/6 allele, and the telomere is 10.1 cM [based on EUCIB map distances (Rhodes et al. 1998)], but we cannot exclude the possibility that a recombinant event occurred just distal to D1Mit452 in the H2, L1, and L2

DeFries strains, giving a distance of over 16.4 cM (EUCIB; Rhodes et al. 1998). On Chr 15 there is a much smaller region with the expected haplotype: a maximum distance of 3.2 cM (between D15Mit185 and D15Mit68) and a minimum distance of 1.4 cM (between D15Mit105 and D15Mit28). Again the marker with the maximum LOD score in the QTL detection experiment (D15Mit28) lies within the region (Flint et al. 1995). In order to confirm the results of the haplotype analysis, we carried out a variant of the recombinant inbred segregation test (Darvasi 1998). In this procedure, after detecting QTLs in an intercross, high-resolution mapping is achieved by crossing parental inbred strains with a set of recombinant inbreds (RI) chosen to have recombinations equally distributed within the interval containing the QTL. F1 populations are separately intercrossed to produce F2 populations, in some of which the QTL will segregate, depending on where the QTL lies with respect to a recombination point. Our experiment diverged from the RIST described above. The

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M.G. Turri et al.: Fine-mapping of QTL for emotionality Table 1. RIST data and ANOVA.

Locus D1Mit150 Genotype:

D15Mit187 Genotype:

Genotype BALBc/J C57BL/6 C57BL/6

BALBc/J BALBc/J C57BL/6

BALBc/J C57BL/6 C57BL/6

BALBc/J BALBc/J C57BL/6

ANOVA P-Value

EMO Mean

OFA Mean

OFD Mean

0.0006

0.76 0.22 −0.12

185.94 210.24 235.22

7.76 5.64 4.72

0.31 0.08 −0.44

190.45 225.69 264.74

5.41 5.5 4.03

0.0038

Animals from an F2 intercross between H2 and C1 DeFries strains have been classified by genotype at two loci (D1Mit150 and D15Mit187). Alleles are given the name of the inbred strain from which they derive (either BALBc/J or C57BL/6). The means of three phenotypes (OFA, OFD and EMO) associated with each genotype are shown. ANOVA P-values are given for the EMO phenotype only (at both loci the F statistic degrees of freedom are 2, 71).

Fig. 2. Outcome of an F2 cross between the H2 and C1 strains, which determines whether the QTL lies in region a or b, either side of the markers D1Mit150 and D1MIt361. The figure shows non-recombinant chromosomes over the terminal 20 cM of Chr 1. The increasing QTL allele (associated with C57BL/6) is shown in bold, and the decreasing QTL allele (associated with BALB/cJ) is shown in italics. It can be seen that if the QTL lies in region b, then there will be no phenotypic differences segregating with D1MIT150; in contrast, if the QTL lies in region a, D1Mit150 will detect an effect.

strains we used to detect QTLs are in fact themselves RI strains (derived from C57BL/6 and BALB/cJ); crossing these strains back onto C57BL/6 and BALB/cJ and looking for segregation of the parental haplotype with the trait would confirm the position of the QTLs, but would not further increase resolution. However, by crossing RI strains with each other, we can test whether QTLs segregate either side of a recombination, and hence both confirm and refine our localization at the same time. None of the available C57BL/6 and BALB/cJ RI strains were suitable for further delimiting the loci we had identified, but a cross between the controls strain C1 and H2 would determine whether the QTL on Chr 1 lies distal to D1Mit150. The expected results of a cross between C1 and H2 for the two possible QTL locations are shown in Fig. 2. This cross would also confirm that the QTL on Chr 15 is carried within the small region indicated by the haplotype analysis. An F2 was generated from a cross between C1 and H2, and 71 animals were tested in an open-field arena as described above (Flint et al. 1995). We obtain an increase in power by mapping a joint trait derived from OFA and OFD: both scores are standardized to a mean of 0 and variance of 1, the sign of OFA is reversed (to take into account the known negative genetic correlation of the measures), and the mean of the derived scores used to define a new phenotype, EMO (Talbot et al. 1999). In order to test for segregation of the QTL on Chr 1, we used the marker D1Mit150. We test for a difference between genotypes by an analysis of variance (ANOVA) in which the phenotypic mean and variance associated with each genotype is calculated and a variance ratio used to test for the equality of the variances. The ANOVA P-value was significant [P ⳱ 0.0006 (see table)]. This result indicates that the QTL must be located in the proximal interval (a) indicated in Fig. 2, lying proximal to D1Mit361 (87.0 cM) and distal to D1Mit452 (73.9 cM), within a minimum interval bounded by D1Mit150 (84.3 cM) and D1Mit113 (80.3 cM). On Chr 15, the marker D15Mit187 gave an ANOVA P-value of 0.0038, confirming that a QTL is segregating in this F2 at that location (see Table 1). This experiment does not rule out the possibility that the QTL lies within the C57BL/6 haplotype proximal to D15Mit68 in the control strain, but the results of our initial F2 intercross used a cross between H1 and L1 in which there is no

C57BL/6 haplotype in this region (see Fig. 1). It is much more likely that the QTL lies in the small region of haplotype sharing between D15Mit105 and D15Mit68. We estimated the effect attributable to each marker, using regression analysis. The multiple regression procedure involved partitioning allelic effects into additive and dominance components. For the additive component, a separate predictor variable is created for each allele, with the variable taking the values of 0, 1, or 2 for the number of alleles of the given type present. For such a model to be identified, the effect of one of the alleles must be equated to the negative of the sum of the other allelic effects, with the intercept estimating the overall mean; the effect of the largest allele was fixed at each locus. For the dominance component, a separate predictor variable, coded 0 or 1, was created for each possible heterozygote type. We used each marker locus separately in predicting our individual phenotypes of interest. The regression analysis allowed us to calculate the contribution to the phenotypic variance from each locus: the QTL on Chr 1 contributes 16 percent of the phenotypic variance, and the QTL on Chr 15 contributes 8 percent. Our results confirm that loci on chromosomes 1 and 15 contribute to the genetic variance of emotionality and reduce the intervals that contain those QTLs to 4–13 cM on Chr 1 and 1.4–3.2 cM on Chr 15. We have recently reported evidence that there is a QTL influencing emotionality on Chr 1 segregating in an outbred stock of mice (heterogeneous stock; Talbot et al. 1999) within 1 cM of D1Mit264 (position 64.76 on the EUCIB map). Taken together, the mapping data indicate that the QTLs on Chr 1 in the DeFries strains and in the heterogeneous stock are different loci. We designate the QTL on Chr 1 in the DeFries strains as Emo1, the QTL on Chr 15 as Emo2, and the Chr 1 QTL in the heterogeneous stock as Emo3. The strategy we used for fine mapping QTLs exploits an effect of selection on the trait of interest: tight linkage between markers associated with the QTL will be maintained while recombination will break up association with more distant markers. This effect of selection has been used previously to detect QTLs in the mouse (Keightley et al. 1996), but the work reported here is the first demonstration that inbred strains derived from a selection experiment can be used to fine-map QTLs. The strategy is quick: mapping the regions of haplotype sharing is simple, and confirming the location of QTLs with a suitable cross requires only a small number of animals. In our study, we used 71 F2 animals and were able to confirm the presence of QTLs with relatively small effect sizes. However it does require bi-directionally selected strains in which QTLs have already been detected. It should be emphasized that the strategy we have used cannot be used as a first-line strategy for QTL detection: it requires prior knowledge of QTL location. Moreover, it has limited power. In our case we had four selection lines, but this is unusual. More commonly only two bi-directionally selected strains will be available.

M.G. Turri et al.: Fine-mapping of QTL for emotionality

Finally, it is important to confirm the fine-mapping results by the use of, for example, a segregation test such as RIST. The interval on Chr 15 is now small enough to consider possible candidates. Although the EST map in the mouse is still relatively sparse, the syntenic region is 22q13, for which almost complete sequence is now available (http://www.sanger.ac.uk). The region on Chr 1 is much larger and will require alternative approaches to reduce the interval further, for instance, the approach we have developed using outbred mice (Talbot et al. 1999). With the rapid growth in gene and high-resolution maps, and the development of new breeding strategies capable of fine-mapping QTLs down to regions of well under a centimorgan, molecular identification of the QTLs influencing behavior is likely to be achieved soon. This should soon lead to rapid advances in our understanding of the biology of normal behavior and of psychopathology. Acknowledgments. This work was supported by the Wellcome Trust (M. Turri, C.J. Talbot, and J. Flint), NIMH grant awards MH-53668 (J.M. Wehner and R.A. Radcliffe). RCA to J.M. Wehner (AA-00141), and a postdoctoral traineeship to R.A. Radcliffe (HD-07189). M. Turri is a Scatcherd European scholar, supported by a fellowship from the Universita degli Studi di Roma “Tor Vergata”.

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