644 ORIGINAL ARTICLE J Med Genet: first published as 10.1136/jmg.39.9.644 on 1 September 2002. Downloaded from Identification of candidate lung cancer susceptibility genes in mouse using oligonucleotide arrays W J Lemon*, H Bernert*, H Sun, Y Wang, M You ............................................................................................................................. See end of article for J Med Genet 2002;39:644–655 authors’ affiliations *These two authors contributed equally to this work. ....................... We applied microarray gene expression profiling to lungs from mouse strains having variable suscep- Correspondence to: Dr M You, Manuel tibility to lung tumour development as a means to identify, within known quantitative trait loci (QTLs), Tzagournis Medical candidate genes responsible for susceptibility or resistance to lung cancer. At least eight chromosomal Research Facility, Room regions of mice have been mapped and verified to be linked with lung tumour susceptibility or resist- 530, 420W West 12th ance. In this study, high density oligonucleotide arrays were used to measure the relative expression Avenue, Columbus, Ohio levels of >36 000 genes and ESTs in lung tissues of A/J, BALB/cJ, SM/J, C3H/HeJ, and C57BL/6J 43210, USA; [email protected] mice. A number of differentially expressed genes were found in each of the lung cancer susceptibility QTLs. Bioinformatic analysis of the differentially expressed genes located within QTLs produced 28 Revised version received susceptibility candidates and 22 resistance candidates. These candidates may be extremely helpful in 31 May 2002 Accepted for publication the ultimate identification of the precise genes responsible for lung tumour susceptibility or resistance in 14 June 2002 mice and, through follow up, humans. Complete data sets are available at http://thinker.med.ohio- ....................... state.edu. umerous chromosomal regions genetically linked with homologous to human chromosome 6p21; potential candi- susceptibility or resistance to pulmonary adenomas dates at this location are the genes for tumour necrosis factor Nhave been described in mice using inbred strains α and β. Similarly, linkages to lung tumour susceptibility were showing widely different susceptibilities to formation of both also seen at markers on chromosome 19 (Pas3), accounting for spontaneous and chemical induced lung tumours.1–3 Suscepti- 3% of the phenotypic variation in a study on (A/J × C57BL/6J) bility is intrinsic to the lung itself as shown by the classical × C57BL/6J mice, and 2% of the explained phenotypic × experiments involving lung explants from sensitive and variation when (A/J C57BL/6J) F2 mice were used. In this 45 resistant mice. After carcinogen administration to F1 mice latter study, suggestive linkage to a locus on chromosome 9 previously made host to these explants, only the lungs from (Pas4) was determined to explain 4% of the total phenotypic the sensitive mouse strain developed tumours.45 Matings of variance.89Mouse-human synteny for all loci can be examined http://jmg.bmj.com/ sensitive A/J and resistant C57BL/6J mice produce F1 and F2 in detail using the Homology browser at the NCBI offspring, which are of intermediate sensitivity to tumour (www.ncbi.nlm.nih.gov/Homology). induction, thus implicating more than one gene and illustrat- At present, four Par QTLs have been mapped using F2 or ing that tumour size and number are multigenic quantitative backcross populations of mice, including Parl (chromosome traits.6 Production of recombinant inbred (RI) lines of A/J (A) 11), Par2 (chromosome 18), Par3 (chromosome 12), and Par4 and C57BL/6J (B6) mice and subsequent analysis of their (chromosome 4).12 Par1 is a lung tumour resistance locus that tumour sensitivities suggested that three genes, one major was mapped in (A/J × M spretus) × C57BL/6J mice to the and two minor genes, were involved in determining the sensi- retinoic acid receptor-α (Rara) gene locus on chromosome on October 2, 2021 by guest. Protected copyright. tivity to mouse lung tumour development.6 Subsequent 11.13 14 Contributed by the M spretus allele, Par1 gave a linkage studies have identified pulmonary adenoma suscepti- maximum lod score of 5.3 accounting for 23% of phenotypic bility (Pas) and pulmonary adenoma resistance (Par) loci. We variance when coexpressed with the highly penetrant Pas1 thus adopt the definition of quantitative trait locus (QTL) as a allele of the A/J strain. In mice carrying the M spretus instead known chromosomal region in which one or more genes are of the A/J allele of the Pas1 gene, the resistant effect of Par1 on likely to underlie the linkage. tumour incidence, multiplicity, and volume was lessened by Listed in table 1 are the selected QTLs that have been about a half. Par1 behaves like a modulator of Pas1, to some mapped in various mouse crosses. A major susceptibility locus degree subduing the dominant effect of Pas1 on lung tumori- × × was mapped in (A/J C3H/HeJ) F2 mice to distal chromosome genesis. Par2 was mapped by linkage studies on (A/J BALB/ × × 6 and was termed the Pas1 locus. This locus produced a maxi- cByJ) A/J and (A/JO1aHsd BALB/cO1aHsd) F2 mice to mum logarithm of the likelihood ratio (lod) score of 9 and chromosome 18 at microsatellite marker D18MIT103. A lod accounted for approximately 45% of the observed phenotypic score of 12.2 was reported at this locus, with a phenotypic variance.7 A lod score of 3 or greater is considered significant variance of 38% for resistance to tumour induction.15 This for linkage. Consistent results were obtained in comprehen- locus was termed Par2. In our own analysis of (A/JO1aHsd × × sive linkage studies using (A/J C57BL/6J) F2 (60% of BALB/cO1aHsd) F2 mice, Par2 had a significant linkage to lung variance), (A/J × C57BL/6J) × C57BL/6J (16% of variance), tumour resistance and produced a maximum lod score of (A/J × M spretus) × C57BL/6J (34% of variance), and A × B & B 11.16 The greatest linkage occurred at the site of the Dcc tumour × A RI mice (51% of variance).8–11 Three other loci were repressor gene.16 Par3 was mapped to chromosome 12 with a mapped to chromosomes 17, 19, and 9.89Linkage to a locus on lod score of 6.47, using backcross population between chromosome 17, the site of the putative Pas2 locus, was SMXA24 RI mice and A/J mice.14 Par3 seems to have a stronger × observed in (A/J C57BL/6J) F2, accounting for 8% of the total resistance to lung tumour induction when coexpressed with variance in phenotype. The location of the Pas2 locus is the A/J allele of the Par2.17 Finally, Par4 or Papg1 was mapped www.jmedgenet.com Mouse lung cancer susceptibility candidates Table 1 Summary of Pas and Par QTLs. Pas QTLs have been largely studied using various genetic crosses of A/J and C57BL/6J; Par 1 and 3 using A/J and SM/J; Par 2 and 4 using A/J and BALB/cJ. Markers flanking the QTLs were taken larger than what some recent fine mapping studies have produced in order to allow identification of multiple genes Start marker End marker Explained Locus Strains Breeding variance Name Genetic Celera Name Genetic Celera Celera Transcripts Affymetrix Mu74K Pas 1 A × B6 F2 60% D6Mit54 48.2 109, 249, 790 D6Mit304 75 142, 276, 365 640 250 Pas 2 A × B6 B6 × A RI 8 D17Mit23 17 22, 057, 276 D17Mit50 23.2 39, 136, 371 468 119 Pas 3 A × B6 F2 2 D19Mit42 2 6, 828, 495 D19Mit19 25 39, 976, 948 295 61 Pas 4 A × B6 F2 4 D9Mit11 42 82, 499, 293 D9Mit282 72 114, 926, 441 932 213 Par 1 (SMXA × A) × ABC ∼10 D11Mit4 37 69, 772, 571 D11Mit14 59 101, 055, 855 1043 345 Par 2 A × B/c F2 ∼50 D18Mit124 32 57, 893, 422 D18Mit4 57 83, 071, 801 373 92 Par 3 (SMXA × A) × ABC ∼10 D12Mit36 13 52, 195, 168 D12Mit6 44 81, 478, 562 457 79 Par 4 A × B/c F2 ∼10 D4Mit39 10.6 32, 493, 011 D4Mit77 42.5 83, 848, 148 611 111 nucleotide arrayent (Santa aliquots were Clara, hybridised to CA): each Affymetrix Mu74Av2 mouse oligo- (A array), from 30 microarray). Singletonturers’ cRNA preparations protocolsour were microarray (www.cancergenetics.med.ohio-state.edu/ produced core facilityRNA samples according were further to purified, standard labelled,Microarray and manufac- analysis processed by mined by reading absorbance atmaldehyde 260/280 nm. agarose gel,Rnase free and water. The the qualitythe of RNA concentration RNA was pellet confirmed was was on30 washed minutes. deter- a After in for- centrifugation at 75% 14 ethanolwith 000 an rpm and equal for dissolved 15 volume minutes, of in minutes; isopropanol, and the incubated on aqueous ice phase for minutes. was The transferred sample tochloroform, was a centrifuged vigorous fresh at mixing, tube 14 and 000 incubation rpm on for ice 20 for 15 mouse strains. for lung cancer susceptibilityregions or resistance for in the several identificationdifferential relevant gene of expression candidate in genes lungwe cancer responsible susceptibility used QTL microarray profiling high to identify densityCelera mouse candidate genome genes. sequence oligonucleotide Specifically, and lungsignificant cancer challenge. genetics arrays with In thepolymorphism to present study, of we detect such combinedEvaluation the a of large differentialresponsible, number fine gene mapping of may expression provefew genes to genes and be can more separated be nucleotide challenging.most by of a more the effect thantypically attributed a assume to that few one the or QTL. centimorgans verybe If few are localised genes several are to genes responsible a or for ficult. 20-30 a One cM obstacle QTL is region. thefor fact Fine the that mapping lung several cancer studies hundred susceptibility genes QTLs can proves to be rather dif- minutes at room temperature,then followed by homogenised addition in of 1nati, 200 ml OH).