[CANCER RESEARCH 62, 3782–3788, July 1, 2002] Universal and Radiation-specific Loci Influence Murine Susceptibility to Radiation-induced Pulmonary Fibrosis 1 Christina K. Haston, 2 Xinhui Zhou, 2 Laura Gumbiner-Russo, Roxanna Irani, Robert Dejournett, Xiangjun Gu, Michael Weil, Christopher I. Amos, and Elizabeth L. Travis 3 Departments of Experimental Radiation Oncology [C. K. H., X. Z., L. G-R., R. I., R. D., M. W., E. L. T.] and Epidemiology [X. G., C. I. A.], University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 ABSTRACT Susceptibility to radiation-induced pulmonary fibrosis is a heritable trait in mice. In a prior study of C57BL/6J (susceptible), C3Hf/Kam (resistant), and F1 and F2 mice derived from these strains, we estimated that 38% of the measured phenotypic variation could be attributed to effects from a few genetic factors. In addition, we identified one genetic factor on chromosome 17 in the MHC region. To identify any additional genetic loci that might influence interstrain variability, we conducted a genome-wide linkage scan using 214 markers and the phenotypically extreme 94 (of 268) F2 mice. In regions exceeding suggestive linkage (LOD  2.8), we followed up with additional markers. This scan revealed evidence for quantitative trait locus (QTL) on chromosomes 17 (LOD  4.2), 1 (LOD  4.5), and 18 (LOD  3.9), which influence susceptibility to radiation-induced pulmonary fibrosis. An additional re- gion containing a QTL on chromosome 6, LOD  4.6, showed linkage in female mice only. The evidence for linkage to chromosome 18 weakened when it was analyzed jointly with other markers. These four loci are estimated to account for 70% of the genetic contribution to this trait with chromosome 17 and 1 accounting for 28 and 24%, respectively. To con- firm and better define the influence of the chromosome 17-linked QTL on radiation sensitivity, we conducted studies on congenic mice in which the linked region on chromosome 17 had been transferred onto a B6.AKR or a C3.SW background. The chromosome 17-linked QTL was confirmed to influence the phenotype as the fibrotic radiation response of B6.AKR-H2 k mice was significantly less than that of B6 mice (P  0.0001). The QTL on chromosome 17 for radiation-induced lung fibrosis is within the same region as QTLs identified for lung damage after other insults, including bleomycin, ozone, and particle exposure, as well as for asthma, suggesting that this region of chromosome 17 may harbor a “universal” lung injury gene. INTRODUCTION Radiotherapy involving the thoracic cavity may result in the normal tissue complication of pulmonary fibrosis (1–3). If patients could be ranked in terms of normal tissue radiosensitivity, Tucker et al. (4) has estimated that radiation doses could be escalated by 20% to resistant patients without exceeding an acceptable incidence of normal tissue complications. Although the proportion of sensitive individuals in a patient population is most likely small, this group of patients defines dose limitations to the whole population. Thus, the ability to prospec- tively screen patients for normal tissue complication susceptibility could allow more patient-individualized treatments to be given, which could enhance the therapeutic benefit. There are currently no assays, either genetic based or otherwise, available to determine individual susceptibility to radiation-induced lung damage. The finding that inbred strains of mice differ in their propensity to develop lung fibrosis after exposure to radiation (5–9) provides a useful model to identify genetic factors influencing this trait. Studies by Sharplin and Franko (8, 9) and by our lab (5) showed that after whole thorax irradiation, various substrains of C3H mice develop a diffuse alveoli- tis, which resolves with no fibrosis, or is lethal, depending on the dose. C57BL/6J mice, in contrast, respond to lung irradiation with a less severe alveolitis and atelectatic regions of fibrosis, which increase with radiation dose. We completed previously an inheritance study in F1 and F2 mice derived from an intercross of C3Hf/Kam (C3H) and C57BL/6J (B6) mice and reported that susceptibility to radiation- induced pulmonary fibrosis has a heritability of 40% and is influ- enced by a few genetic factors (5). Our segregation results were consistent with the inheritance model proposed by Franko et al. (7), suggesting that susceptibility to radiation-induced lung fibrosis is controlled by two autosomal genes. In the prior inheritance study (5), we reported an association of the fibrotic phenotype with a marker on chromosome 17. Mapping in this prior study was limited to investigating loci demonstrated to predict for pulmonary response to bleomycin, 4 motivated by the consistent strain difference of B6 mice as susceptible to fibrosis in response to radiation or bleomycin, and C3H mice as resistant. We estimated that the chromosome 17 association accounted for 17% of the phenotypic variability of this trait in the radiation study. In the present study, a genome-wide scan was undertaken to identify additional loci influ- encing susceptibility to radiation-induced pulmonary fibrosis and to define the linkage region on chromosome 17. Furthermore, the influ- ence of the chromosome 17-linked QTL 5 on this trait was assessed by phenotyping congenic mice after radiation treatment. MATERIALS AND METHODS Phenotype. The phenotypic data are from our prior inheritance study (5), and the methods are reported more extensively there. Briefly, the mice were given whole lung irradiation of either 14 (LD50 of C3H mice) or 16 Gy (LD100 of C3H mice) and were sacrificed when moribund or at 33 weeks, which was the end of the experiment. At sacrifice, the lungs were removed and analyzed histologically. We defined the fibrotic phenotype as the percentage of the left lung with fibrosis by image analysis of a Masson’s Trichrome-stained section (10). In the F2 generation, there was no difference in the extent of fibrosis between mice treated with 14 Gy and mice treated with 16 Gy (P = 0.32), and this result does not change when mice are segregated by sex; therefore, analyses were completed of the F2 data set as a whole. As the F2 lung fibrosis values were not normally distributed, the data were arcsine transformed (11) to stabilize the variance, in compliance with an assumption of the linkage analyses that mean levels are not correlated with the variance. Analyses using either raw or transformed data yielded similar results. Genotype. DNA from F2 animals was prepared, using a DNA extraction kit from liver samples collected at necropsy. We genotyped the F2 intercross progeny by PCRs based on simple sequence repeats using mouse markers defined by Dietrich et al. (12). PCR was performed with one radiolabeled primer (P 32 ) and one unlabelled primer, and we visualized the products by autoradiography of 6% polyacrylamide gels. Received 1/11/02; accepted 5/2/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NCI RO1CA 64193 (to E. L. T.) and RO1HG02275 (to C. I. A.). 2 C. K. H. and X. Z. contributed equally to this manuscript. 3 To whom requests for reprints should be addressed, at the Department of Experi- mental Radiation Oncology, University of Texas M. D. Anderson Cancer Center, Box 066, 1515 Holcombe Boulevard, Houston, TX 77030. 4 E. L. Travis, unpublished data. 5 The abbreviations used are: QTL, quantitative trait locus, Radpf, radiation pulmonary fibrosis; TNF, tumor necrosis factor; MnSOD, manganese superoxide dismutase; PF, pulmonary fibrosis; LRS, likelihood ratio statistic. 3782 on April 10, 2017. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from