Quantitative trait locus mapping of airway responsiveness to chromosomes 6 and 7 in inbred mice

G. T. De Sanctis1, J. B. Singer2, A. Jiao1, C. N. Yandava1, Y. H. Lee1, T. C. Haynes3, E. S. Lander2,4, D. R. Beier5, and J. M. Drazen1

1 Combined Program in Pulmonary and Critical Care Medicine and 5 Genetics Division, Brigham and Women's Hospital and Harvard Medical School, Boston 02115; 2 Whitehead Institute/ Massachusetts Institute of Technology Center for Genome Research, Whitehead Institute for Biomedical Research, Cambridge 02142; 4 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and 3 The Jackson Laboratory, Bar Harbor, Maine 04609


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Quantitative trait locus (QTL) mapping was used to identify chromosomal regions contributing to airway hyperresponsiveness in mice. Airway responsiveness to methacholine was measured in A/J and C3H/HeJ parental strains as well as in progeny derived from crosses between these strains. QTL mapping of backcross [(A/J × C3H/HeJ) × C3H/HeJ] progeny (n = 137-227 informative mice for markers tested) revealed two significant linkages to loci on chromosomes 6 and 7. The QTL on chromosome 6 confirms the previous report by others of a linkage in this region in the same genetic backgrounds; the second QTL, on chromosome 7, represents a novel locus. In addition, we obtained suggestive evidence for linkage (logarithm of odds ratio = 1.7) on chromosome 17, which lies in the same region previously identified in a cross between A/J and C57BL/6J mice. Airway responsiveness in a cross between A/J and C3H/HeJ mice is under the control of at least two major genetic loci, with evidence for a third locus that has been previously implicated in an A/J and C57BL/6J cross; this indicates that multiple genetic factors control the expression of this phenotype.

asthma; methacholine; A/J mice; C3H/HeJ mice


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AIRWAY HYPERRESPONSIVENESS is one of the defining characteristics of asthma (1). Although enhanced reactivity to a variety of bronchoconstrictor agonists is well documented among asthmatic patients, the genetic and molecular mechanisms responsible for this condition are poorly understood. In addition, the biological variability of this complex phenotype (9, 10) reflects the contribution of both genetic and environmental influences to varying degrees on the overall phenotype.

Airway hyperresponsiveness in the absence of administration of stimuli leading to pulmonary inflammation, i.e., intrinsic hyperresponsiveness, is a trait under genetic control (11, 12). Analysis of strain distribution patterns for intrinsic airway responsiveness led to the identification of hyperresponsive and hyporesponsive inbred mouse strains. Examination of these inbred strains reveals that although there is considerable variation in airway responsiveness among strains, the variation found within a strain is smaller, thus demonstrating the heritability of this trait (11-13). Mice with a hyper- or hyporesponsive phenotype have been used as the progenitor strains in genetic mapping experiments to successfully identify quantitative trait loci (QTLs) contributing to airway hyperresponsiveness in inbred strains of mice (4, 8).

In a study by Ewart et al. (8), two different methods of phenotypic analysis were used to quantitate the airflow obstruction induced by a single intravenous dose of the bronchoconstrictor acetylcholine in progeny derived from crosses between C3H/HeJ and A/J mice. The first phenotype involved the peak increase in pulmonary impedance resulting from infusion of a fixed amount of acetylcholine, and the second phenotype involved the airway pressure in phase with airflow to derive the changes in respiratory system resistance resulting from acetylcholine infusion. A single significant linkage to chromosome 6 [logarithm of odds ratio (LOD) = 3.1] was found with the first phenotype; no significant linkages were found for the second.

These results, obtained by Ewart et al. (8) in their cross between C3H/HeJ and A/J mice, differed from findings by De Sanctis et al. (4) in a cross between the A/J and C57BL/6J inbred strains. In that study, they used pulmonary resistance (RL) as the phenotypic outcome measure and identified QTLs on chromosomes 2, 15, and 17. The differences in the two experiments may be due either to differences in the methods of phenotypic assessment, which were clearly shown to affect the identification of loci in the study by Ewart et al. (8), or to differences in the strains studied in each cross.

To address these issues, we now studied a cross between A/J and C3H/HeJ strains and used the change in RL after the infusion of methacholine as our outcome indicator. Our data demonstrate a polygenic mode of inheritance for airway hyperresponsiveness in the A/J and C3H/HeJ cross. We confirm the previously reported evidence of significant linkage on chromosome 6 (8) and report a novel linkage on chromosome 7 and a suggestive linkage on chromosome 17.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male viral antibody-free (VAF) C3H/HeJ (n = 34), A/J (n = 49), (C3H/HeJ × A/J) F1 hybrid (n = 40), (A/J × C3H/HeJ) F1 hybrid (n = 40), (A/J × C3H/HeJ) F2 intercross (n = 104), and [(A/J × C3H/HeJ) × C3H/HeJ] backcross (n = 369) mice 5-6 wk of age were purchased from Jackson Laboratory (Bar Harbor, ME). To minimize environmental effects, the mice were housed in isolation cages under VAF conditions. The mice were acclimatized for 10-14 days after transport and allowed free access to commercial pelleted mouse food and water, which were autoclaved to ensure sterility. VAF status was routinely confirmed by testing blood samples taken from sentinel animals. Because age (6, 21), gender (11), and airway infections (18) can alter airway responsiveness, only male, 8-wk-old mice housed in a barrier facility were studied.

Phenotype analysis. The methodology for measuring airway responsiveness was as originally described by Martin et al. (14) and modified by De Sanctis and co-workers (3, 4). Dose-response curves to methacholine were obtained by administering sequentially increasing doses of methacholine (acetyl-beta -methylcholine chloride; 33-3,300 µg/kg iv; Sigma, St. Louis, MO) with a 0.1-ml Hamilton (Reno, NV) microsyringe. The volume of fluid injected with each dose produced no measurable physiological effects. RL was determined with the use of signals derived from transpulmonary pressure and lung volume (14). Each animal's dose-response curve was log transformed and then subjected to regression analysis to calculate the dose required for a 1.50-fold increase in RL (log ED150RL). Because the doses of methacholine are given in geometrically increasing amounts, it is common to log transform this index. QTL analysis was also carried out with the individual RL values (as a percentage of baseline) for the 330 and 1,000 µg/kg doses from 369 backcross progeny.

DNA preparation. Animals were removed from the plethysmograph after the dose-response curves were obtained and killed by exsanguination under surgical anesthesia. Both kidneys were carefully removed, snap-frozen in liquid nitrogen, and subsequently stored in a -80°F freezer. Purified genomic DNA was obtained from one of the kidneys with a DNA extraction kit (Stratagene, La Jolla, CA). Spectrophotometric readings of the DNA samples were taken to verify their purity (ratio of absorbance at 260 nm to that at 280 nm) and to quantitate (absorbance at 260 nm) the DNA concentration.

Genotype analysis. Genotype analysis was performed with mouse simple sequence length polymorphic (SSLP) markers as previously described (5). Initially, a panel of between 81 and 97 SSLP markers, distributed at ~15-centimorgan (cM) intervals throughout the genome, was used to genotype 232 of the phenotypically most extreme backcross progeny. Because we identified a linkage on chromosome 6 in an area previously reported by others (8), four markers around the peak of the QTL on chromosome 6 were used to type all 369 backcross progeny. Primers were purchased from Research Genetics. PCR amplification was performed in 10 mM Tris, pH 8.3, 45 mM KCl, 1.5 mM MgCl2, and 0.2 mM each dATP, dCTP, dTTP, and dGTP. PCR was performed for 35 cycles at 94°C for 15 s, 58°C for 45 s, and 72°C for 45 s. Product size was analyzed by electrophoresis with 3.5% MetaPhor agarose and ethidium bromide staining.

Statistical and linkage analysis. Computations were performed with the JMP 3.1.5 (SAS Institute, Cary, NC) statistical package. A Tukey-Kramer honestly significant difference test was used to assess differences between the parental strains and individual crosses. For nonparametric data, differences between groups were analyzed by the Wilcoxon rank sum test. Where appropriate, a Shapiro-Wilk W-test was used to assess normality. Standard ANOVA, including cross-terms for two-way interactions, was used to evaluate possible interactions between candidate QTLs (chromosomes 6 and 7) and the mouse airway responsiveness phenotypes (330 and 1,000 µg/kg and log ED150RL). QTL analysis was carried out with the Map Manager QT computer package from the genotype data and the following airway responsiveness phenotypes: 1) the response of RL to individual methacholine doses of 100, 330, and 1,000 µg/kg and 2) the log ED150RL values computed from the dose-response curves. The likelihood ratio statistic that Map Manager QT calculates for QTL mapping is a LOD score based on a natural logarithm. Conventional base 10 LOD scores were calculated from the likelihood ratio statistic values by multiplication with a constant (0.217). Results, expressed as means ± SD unless otherwise stated, were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Airway responsiveness was measured in 40 A/J, 41 C3H/HeJ, 40 (C3H/HeJ × A/J) F1, 40 (A/J × C3H/HeJ) F1, 104 (A/J × C3H/HeJ) F2 intercross, and 369 [(A/J × C3H/HeJ)F1 × C3H/HeJ] backcross mice; the values for RL, expressed as a percentage of baseline for the 330 and 1,000 µg/kg doses, are shown in Table 1 along with the log ED150RL values. A scatterplot of RL values for the 330 µg/kg dose (expressed as a percentage of baseline) from individual parental, F1, F2, and backcross mice is presented in Fig. 1 and as the log ED150RL values in Fig. 2. Analysis of the distribution of airway responsiveness phenotypes in the A/J and C3H/HeJ parental strains confirmed the original observations that A/J mice are more responsive to challenge with cholinergic bronchoconstrictors than C3H/HeJ mice (8, 11).

                              
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Table 1.   RL and log ED150RL values in A/J, C3H/HeJ, and hybrid mice derived from simple crosses



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Fig. 1.   Scatterplot of pulmonary resistance (RL) values (expressed as a percentage of baseline) from individual parental and hybrid mice. ACF1, A/J female mated with CeH/HeJ male F1 hybrid; CAF1, C3H/HeJ female mated with A/J male F1 hybrid; backcross, [(A/J × C3H/HeJ)F1 × C3H/HeJ]; n, no. of mice. * Significantly different from C3H/HeJ mice, P < 0.05.



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Fig. 2.   Scatterplot of RL values for log-transformed dose required for 1.50-fold increase in RL (log ED150RL) for parental and hybrid mice. Distribution of airway responsiveness phenotypes and therefore genetically informative progeny in F2 intercross group is narrower than range of responses to 330 µg/kg dose (expressed as a percentage of baseline). n, No. of mice.

Analysis of the distribution of individual animals demonstrates that the mode of inheritance does not follow a simple Mendelian or complex genetic pattern. The RL values for the 330 µg/kg dose, expressed as a percentage of baseline, are presented in Fig. 1. Analysis of F1 mice derived from reciprocal crosses [A/J females mated with C3H/HeJ males (ACF1) and C3H/HeJ females with A/J males (CAF1)] revealed significant differences in airway responsiveness. Both F1 hybrids had a phenotype that was intermediate between the A/J and C3H/HeJ strains but was skewed more toward the A/J strain (Table 1). However, significantly greater responses were observed in the ACF1 hybrid strain than in the CAF1 strain (P < 0.0047; Table 1, Fig. 1). Airway responses were broadly distributed to encompass the range of values observed in the C3H/HeJ, F1, and A/J groups (Fig. 1) in F2 mice derived from ACF1 mice. A similar analysis of the log ED150RL values for the parental and hybrid crosses revealed a narrower distribution of airway responses (Fig. 2) than that in response to 330 µg/kg of methacholine alone. Airway responsiveness in F2 mice, ascertained with the RL response to 330 or 1,000 µg/kg of methacholine or log ED150RL values, was not normally distributed (P < 0.0001 by Shapiro-Wilk W-test for normality). Analysis of airway responses for the 330 µg/kg dose in the F2 intercross and CAF1 mice indicated that 48% of the total variance in the intercross was genetic. The CAF1 group was used to calculate the genetic component of the variance because the variability in airway responses in this F1 group was significantly less compared with that in the ACF1 cross.

Because airway hyperresponsiveness appeared to be intermediate in the ACF1 mice but skewed toward the A/J phenotype, they were backcrossed to the hyporesponsive C3H/HeJ parental strain [(A/J × C3H/HeJ) F1 × C3H/HeJ]. Airway responsiveness in 369 backcross progeny (Fig. 1) was not normally distributed (P < 0.0001), a finding similar to that for the F2 progeny (Fig. 3).


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Fig. 3.   Estimated distribution of methacholine response and normal quantile plot of backcross (A) and F2 intercross (B) mice studied in terms of RL values for 330 µg/kg dose. n, No. of mice. Neither F2 intercross progeny nor backcross progeny were normally distributed.

Selective genotyping of 232 of the phenotypically extreme backcross progeny was carried out with a panel of between 81 and 97 SSLP markers, distributed at ~15-cM intervals throughout the mouse genome. The phenotypically extreme progeny were initially selected because they are the genetically most informative mice and provide the most linkage information (9). After QTL analysis revealed regions of linkage, the complete set of 369 progeny was genotyped with a set of four markers on chromosome 6 selected in a region showing evidence of linkage in our cross as well as in the study by Ewart et al. (8) using the same parental strains but with a different method of phenotyping airway responsiveness. Linkage analysis of the backcross progeny was carried out with the Map Manager QT computer package with the RL achieved after infusion of a methacholine dose of 330 or 1,000 µg/kg or the log ED150RL value as an outcome indicator. The LOD scores for the phenotypic outcome parameters are presented in Table 2 along with the genetic markers.

                              
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Table 2.   Peak LOD scores in segregating backcross progeny with airway responsiveness phenotypes

Two significant QTLs were identified with the RL after infusion of 330 µg/kg of methacholine but not with the other outcome indicators. The first QTL identified on chromosome 6 (peak LOD score = 3.32; Fig. 4) maps within the region of the linkage previously reported by Ewart et al. (8) on chromosome 6 in the same genetic background, i.e., A/J and C3H/HeJ. The region in which the maximum LOD score was identified on chromosome 6 was contiguous with a region (~27 cM) of recombination suppression noted by us and also previously noted by Ewart et al. The lack of recombinant events was observed in 96 (A/J × C3H/HeJ) F2 intercross progeny genotyped at these loci and encompassed the following markers: D6Mit243, D6Mit101, D6Mit108, and D6Mit366.


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Fig. 4.   Logarithm of odds ratio (LOD) score from genotypes of murine simple sequence length polymorphic markers for 128-361 informative backcross progeny on chromosome 6. cM, centimorgan.

Besides the significant linkage found on chromosome 6, linkage was also detected on chromosome 7 (LOD = 3.8; Fig. 5); the peak LOD score was observed between D7Mit21 and D7Mit249. Significant linkage was demonstrable when the response to either the 330 or 1,000 µg/kg dose of methacholine was used as the phenotypic index. We tested for genetic interactions between the loci using standard ANOVA, including cross-terms for two-way interactions. Although each of the two loci had a significant effect on airway hyperreactivity when present by itself, there was no evidence of synergistic or antagonistic interactions affecting airway responsiveness between the QTLs on chromosomes 6 and 7 when both loci were present in the backcross progeny.


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Fig. 5.   LOD scores from genotypes of murine simple sequence length polymorphic markers for 137-224 informative backcross progeny on chromosome 7.

In addition to the QTLs identified on chromosomes 6 and 7, we found suggestive evidence for a third locus on chromosome 17 (LOD score = 1.7; only with 100 µg/kg dose). This result is interesting because we had previously found evidence for a QTL controlling airway hyperresponsiveness in the same region of chromosome 17 in a cross between A/J and C57BL/6J inbred strains (4). The results of the QTL analysis for the present study are presented in Table 3 along with the previous QTLs identified in the A/J and C57BL/6J genetic background (4). This region was the only one of the three regions showing linkage in the (A/J × C57BL/6J) cross in which any evidence for linkage was obtained in this (A/J × C3H/HeJ) cross; the other regions in which we had previously identified linkage in the (A/J × C57BL/6J) cross were on chromosome 2 (LOD = 3.0) and chromosome 15 (LOD = 3.7).

                              
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Table 3.   Chromosomal peak LOD scores in [(A/J × C3H/HeJ)F1 × C3H/HeJ] and [(C57BL/6J × A/J)F1 × C57BL/6J] backcross progeny


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intrinsic or native airway responsiveness, i.e., the state of airway responsiveness that exists in the absence of any external inflammatory stimuli, is an important feature of human asthma. People with high levels of airway responsiveness have an accelerated loss of lung function (15, 19) and a persistently high level of airway responsiveness, a marker for asthma severity (20). Data from studies (4, 8, 16, 17, 22) in both humans and animals are consistent with the intrinsic level of airway responsiveness as a heritable trait. Our data confirm the findings of Ewart et al. (8) by identifying linkage in the same region of chromosome 6 and extend these findings by demonstrating the presence of an additional linkage on chromosome 7. Each of these QTLs exhibits significant effects on its own, and together they illustrate the complexity of the heritability of airway hyperresponsiveness.

We studied reciprocal F1 crosses to examine the role of zygotic genotype on airway responsiveness. We found a small but significant difference between the CAF1 and ACF1 progeny. These results are in agreement with those reported previously by Levitt and Mitzner (11) in which ACF1 mice were significantly more responsive than CAF1 mice; the mechanistic basis for this effect remains unexplained.

We have shown that the RL phenotype used in our study to measure methacholine-induced airway obstruction is a sensitive and reproducible method of phenotypic assessment. Interestingly, we observed different peak LOD scores for linkage when different aspects of the airway responsiveness phenotype, as measured by RL, were assessed (Table 2). It is likely that these different outcomes reflect the varying ability of each index to discriminate between the two progenitor populations and gradations of airway responsiveness between them. Thus there may not be a single best outcome indicator to use, but rather the choice of index to use may depend on the specific spread of phenotypes among the progeny in a cross. Our linkage results are similar to the results found when the airway pressure-time index method was used by Ewart et al. (8) to identify the previously reported QTL on chromosome 6 (LOD score = 3.11). Because the airway pressure-time index method measures total pulmonary impedance to inflation, whereas the measurement of RL depends on the component of transpulmonary pressure in phase with flow, these data suggest that the murine pulmonary response to infused methacholine is due predominantly to airway narrowing rather than to changes in the mechanical properties of the lung parenchyma.

Our linkage on chromosome 6 (LOD score = 3.32) is in close proximity to that reported by Ewart et al. (8) and is likely to represent the same locus; the finding is strengthened by the fact that linkage is seen with multiple techniques of phenotypic assessment. In both studies, recombination suppression was observed in a large number of segregating progeny in a region that has been measured to be ~20-25 cM in other crosses (8). The mechanism of this recombination suppression is not known, but its presence precludes finer mapping of this QTL in this cross.

In addition to the linkage found on chromosome 6 (LOD = 3.32), another linkage was also detected on chromosome 7 (LOD = 3.8). This identification was unexpected given that it was not detected in the previous study by Ewart et al. (8) in the same genetic background. Our ability to demonstrate this linkage may be due to the greater number of segregating progeny we studied or because our method of phenotypic assessment is better able to detect subtle physiological alterations; our data do not allow us to distinguish these possibilities. Nevertheless the evidence for linkage is strong, and we assert the presence of a fifth locus associated with intrinsic airway hyperresponsiveness. Based on their prior QTL analysis, De Sanctis et al. (4) previously reported the presence of Bhr1, Bhr2, and Bhr3 on murine chromosomes 2, 15, and 17, respectively. We propose the name Bhr4 for the locus on chromosome 7 and Bhr5 for the linkage on chromosome 6, reported previously by Ewart et al. (8) and confirmed by us.

Bhr4 maps close to an interesting candidate gene, kallikrein (Klk), on chromosome 7. Kallikrein has been implicated in the pathobiology of human asthma. For example, bronchial tissue kallikrein activity is highly correlated with the appearance of immunoreactive histamine and kinin after endobronchial challenge with an aeroallergen in mildly asthmatic patients (2). In addition, there is a close association between immediate-type hypersensitivity events and the appearance of active kallikrein in the bronchoalveolar lavage in mildly asthmatic patients. The involvement of tissue kallikrein in the pathobiology of asthma has also been demonstrated in another recent investigation (7) in which a tissue kallikrein inhibitor was shown to reduce eosinophilia in a model of allergic inflammation.

In our previous QTL analysis of an (A/J × C57BL/6J) cross (4), we obtained tentative evidence for a locus on chromosome 17 (LOD = 2.83). Here, we identified suggestive evidence (maximum LOD = 1.7) in an (A/J × C3H/HeJ) cross. Although the evidence for linkage is not strong, the fact that suggestive linkage is seen in two crosses probably indicates the presence of A/J alleles in this region, affecting airway hyperresponsiveness phenotypes in multiple genetic backgrounds.

In conclusion, we have demonstrated that the genetic control of airway hyperresponsiveness, a defining characteristic of human asthma and a marker for asthmalike conditions in mice, is complex and controlled by at least two quantitative trait loci on chromosomes 6 and 7 in the A/J and C3H/HeJ strains. There may be an additional A/J locus on chromosome 17 that contributes to this phenotype in more than one cross. Thus airway responsiveness in mice is inherited in a complex fashion, with a number of distinct and identifiable genetic loci contributing to this phenotype.


    ACKNOWLEDGEMENTS

This work was supported by the American Lung Association (G. T. De Sanctis); National Heart, Lung, and Blood Institute Grant HL-36110 (to J. M. Drazen); and a grant from the National Center for Human Genome Research (to E. S. Lander).


    FOOTNOTES

G. T. De Sanctis is a recipient of a Partners Nesson (Boston, MA) Research Award.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. M. Drazen, Divisions of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (E-mail: jdrazen{at}rics.bwh.harvard.edu).

Received 1 March 1999; accepted in final form 23 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(6):L1118-L1123
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society