From QTL to causative gene.

<p>Three phases of QTL gene identification are depicted, with the length of time for each phase shown for phenotypes, such as cancer endpoints, that may take several months to a year to assess. a) Linkage analysis. Two inbred mouse or rat strains that differ in cancer risk are intercrossed. Chromosomal segments from the resistant strain (R) are shown in white and those from the susceptible strain (S) are shown in red. After phenotyping 50–200 progeny and genotyping them at approximately 100 markers, linkage analysis reveals two QTLs (<i>QTL1</i> and <i>QTL2</i>) on chromosomes 2 and 4. b) Fine mapping. A congenic line carrying the <i>QTL1</i> chromosomal region from the sensitive strain on the genetic background of the resistant strain is constructed by repeated backcrossing to the resistant strain, with selection for markers from the sensitive strain in the desired interval. The heterozygous congenic is backcrossed to the resistant strain to produce recombinant lines (X1 to X10) that carry various segments of the region from the sensitive strain (shown as red lines). A smaller chromosomal region (Minimal Interval) containing <i>QTL1</i> is inferred from phenotypic analysis of the lines. c) Candidate prioritization and testing. Candidate genes within the minimal interval may be prioritized by several complementary approaches. Comparison of SNP haplotypes for the resistant and sensitive strains (R and S) to those for other strains, such as S2, another sensitive strain with a QTL mapping to the same region as for S, may allow definition of a smaller interval for <i>QTL1</i>. Direct sequence analysis of alleles in the resistant (R) and sensitive (S) strains may identify polymorphisms with potential biological consequences. Differential expression of candidate genes in tumors or appropriate normal tissues from sensitive and resistant recombinant lines may be assessed by transcriptome analysis. Mutations or altered expression of the orthologue of a candidate may be observed in human tumors. Ultimately, a candidate gene may be tested directly for its role in cancer development by modifying the genome of the resistant or sensitive strain through transgenesis or homologous recombination.