Haplotype-associated lung tumor modifier (<i>Halt</i>) loci identified by haplotype analysis, using the 140K BROAD SNP panel.

a<p>Position of the central SNP, in mega bases (Mb) based on NCBI m37 mouse assembly.</p>b<p>Global -log P is the minus logarithm of the p-value for the haplotype sliding window (window size: 3-SNPs).</p>c<p>UI, urethane-induced lung tumor incidence, UM, urethane-induced lung tumor multiplicity.</p>d<p>At each <i>Halt</i> locus, the intercrosses whose parental strains carry different alleles, and that have herein been analyzed, are indicated. AHF2, (A/J×C3H/He)F2; CHF2, (BALB/c×C3H/He)F2; CWF2, (BALB/c×SWR/J)F2.</p

Threshold probabilities (expressed as −log P) corresponding to experiment-wide type I risk errors α = 0.05 and α = 0.10.

<p>Threshold probabilities (expressed as −log P) corresponding to experiment-wide type I risk errors α = 0.05 and α = 0.10.</p

Spontaneous lung tumor incidence correlates with both urethane-induced lung tumor multiplicity (green) and incidence (red) in mouse inbred strains.

<p>Incidence is given as mean percentages, whereas multiplicity is mean number of tumors/mouse. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen-1000331-t001" target="_blank">Table 1</a> for phenotype values.</p

Putative lung tumor modifier loci identified by previous genome-wide association studies or by the present study using the WTCHG SNP panel.

a<p><i>SLT1</i> to <i>SLT5</i> loci, reported to affect spontaneous lung tumor incidence in 13 strains <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen.1000331-Wang1" target="_blank">[7]</a>; <i>Clas1</i> to <i>Clas4</i> loci, reported to affect lung tumor multiplicity in 21 strains <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen.1000331-Liu1" target="_blank">[8]</a>. Additional <i>SLT</i> and <i>Clas</i> loci derive from the present study; for each locus region (1 Mb size), the SNP showing the best statistical association is shown.</p>b<p>Selected SNPs mapping in the reported <i>SLT1</i> to <i>SLT5</i> or <i>Clas1</i> to <i>Clas4</i> regions were genotyped in all strains for which phenotypes were available (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen-1000331-t001" target="_blank">Table 1</a>); the SNPs showing the highest association with the phenotype are shown.</p>c<p>Position (in Mb) based on Ensembl release 49.</p>d<p>The association between each SNP and lung tumor phenotypes (expressed as log+1 of phenotype value) was tested by t-test. Minus log P values reported in bold type indicate the associations above the statistical threshold obtained by permutation (at α = 0.10 significance level).</p

Lung tumor phenotypes (spontaneous and urethane-induced incidence and urethane-induced tumor multiplicity) of mouse inbred strains used in this study.

1<p>Mean incidence of spontaneous lung tumors; data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen.1000331-Manenti3" target="_blank">[5]</a>, except for CAST/Ei, NZW/LacJ, SM/J, and SPRET/EiJ, which derived from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen.1000331-Wang1" target="_blank">[7]</a>.</p>2<p>Mean incidence of urethane-induced lung tumors; data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen.1000331-Manenti3" target="_blank">[5]</a> or <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen.1000331-Malkinson1" target="_blank">[22]</a>.</p>3<p>Mean lung tumor multiplicity after urethane treatment; data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen.1000331-Manenti3" target="_blank">[5]</a>, except for SPRET/EiJ, which derived from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen.1000331-To1" target="_blank">[23]</a>.</p>4<p>W, WTCHG; B, BROAD.</p

Absence of <i>Lasc1</i> gene expression in mouse normal lung and lung tumors of (A/J×C57BL/6J)F1 mice.

<p>The ethidium bromide-stained gel shows the RT-PCR results: lanes 1–3, normal lung derived from adult mice; lanes 4–6, lung tumors derived from urethane-treated mice; lane M, DNA size marker; lane DNA, genomic DNA. Only genomic DNA (top panel) and the <i>Itpr2</i> housekeeping gene (bottom panel) were amplified.</p

Genome-wide genetic linkage analysis of loci affecting urethane-induced lung tumor multiplicity.

<p>(A) (BALB/c×C3H/He)F2 cross detects the <i>Pas1</i> locus at LOD score = 18.4. (B) (A/J×C3H/He)F2 cross detected the <i>Pas1</i> locus at LOD score = 18.7. Red curves indicate the results of the composite interval mapping, whereas black curves indicate the results of genome scan using the <i>Kras</i> genotype as covariate (conditioning on the <i>Pas1</i> alleles). Horizontal lines indicate the threshold values (α = 0.05) of the LOD score. The <i>Clas2</i> locus (Chromosome 4) showed no significant linkage, despite the presence of the claimed functional polymorphism (D102E) in both crosses. No other locus detected by whole-genome strain survey showed significant linkage.</p

Differential allelic expression of <i>Kras</i>-4A isoform indicates the existence of functional polymorphisms in <i>Kras</i> gene.

<p>Allelic ratios were determined by pyrosequencing for rs30022167 and rs29968550, which map in a region of the <i>Kras</i> 3′-UTR common to both isoforms, on genomic DNA and cDNA from normal lung tissue (n = 20) and lung tumor specimens (n = 15) from ABF4 heterozygous mice. Values are mean and SE; *** P<0.001, two-sided Welch's t test (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004307#pgen-1004307-t001" target="_blank"><b>Table 1</b></a> for complete data).</p

<i>Kras</i>-4A levels in lung tumors from 80 urethane-treated ABF4 mice are controlled by <i>Pas1</i> locus.

<p>(a) Expression QTL analysis of <i>Kras</i> transcripts showed that square-root-transformed levels of <i>Kras</i>-4A linked to the <i>Pas1</i> locus (LOD = 4.5). No significant linkage was observed for the <i>Kras</i>-4B mRNA isoform. Tick marks show the position of the genotyped markers in a recombinational map. Horizontal line at LOD = 2.55 marks the 95% threshold for significance. (b) Relative expression levels of the <i>Kras</i>-4A isoform according to rs6265387 genotype. Mice homozygous for the A/J-derived susceptible allele (GG, n = 37) had higher levels than either heterozygous animals (AG, n = 34) or mice homozygous for the C57BL/6 resistant allele (AA, n = 9). ***<i>P</i><0.001, **<i>P</i><0.01 vs. GG mice, ANOVA followed by Tukey's test for multiple comparisons. Values are means and SE.</p

Lung tumor multiplicity in 183 urethane-treated male ABF4 mice is controlled by the <i>Pa</i>s<i>1</i> locus.

<p>(a) LOD score plot for chromosome 6 on which a quantitative trait locus (QTL) for lung tumor multiplicity (square root transformed values) mapped to the telomeric region. The QTL peak (LOD score = 48, phenotypic variance explained = 69%) overlapped with the <i>Pas1</i> locus. Tick marks show the position of 37 genotyped markers, including rs6265387 at the QTL peak. Horizontal line indicates the 95% LOD threshold. (b) Number of lung tumors per animal, grouped according to genotype at rs6265387. Mice homozygous for the A/J-derived allele (GG; n = 59) had more tumors than either heterozygous animals (n = 82) or mice homozygous for the C57BL/6-derived allele (AA; n = 42). ***<i>P</i><1.0×10⁻⁶ versus the A/J-derived allele, ANOVA followed by Tukey's test for multiple comparisons. The line within each box represents the median; upper and lower edges of each box are 75^th and 25^th percentiles, respectively; upper and lower bars indicate the highest and lowest values less than one interquartile range from the extremes of the box.</p