Animals, adenoma induction and Scoring
A recombinant inbred line (Line I) of ApcMin/+ mice on a C57BL/6 background (B6Min/+) that showed limited intraline variation in adenoma numbers was established and maintained as detailed previously (23). Female BALB/cByJ (BALB) mice were crossed with male B6Min/+ to yield F1 (N1) progeny. N1 male ApcMin/+ were then backcrossed to female BALB to produce N2 offspring that carry at least one copy of the BALB-derived modifier of Min (Mom1) dominant resistance allele (Mom1R). Irradiations and sham-irradiations were performed as given previously on N2 ApcMin/+ mice (8). Animals were housed in conventional cages with water, and standard maintenance diet, provided ad libitum. Mice were killed by CO2 asphyxiation when quality of life was compromised. Intestinal tracts were prepared for adenoma counting as detailed earlier (8,23). The small intestine was divided into two equal portions with the upper segment containing all of the duodenum and jejunum. The number of adenomas arising in the upper (USI) and lower small intestine (LSI) was determined at time of sacrifice. Given that numbers are unchanged All procedures involving mice were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and with guidance from local ethics committees on animal experimentation.
Genotyping of ApcMin/+ was performed as given earlier (24). Genome-wide microsatellite analysis was conducted using standard methods and involved the following markers: chromosome (chr) 1, D1Mit430, 169, 132, 495, 17 and 292; chr2, D2Mit327, 395, 411, 229, 148, and 230; chr3, D3Mit178, 51, 320 and 352; chr 4, D4Mit227, 18, 193, 348, 308 and 256; chr5, D5Mit387, 352, 201, 157, 188, 168 and 409; chr6, D6Mit1, 83, 123, 284, 287, 194, 15 and 198; chr7, D7Mit178, 117, 228, 276, 323, 101 and 362; chr8, D8Mit155, 124, 289, 292, 45, 211, 213 and 49; chr9, D9Mit250, 90, 285, 336, 355, 347, 201 and 151; chr10, D10Mit189, 106, 194, 31, 42, 95, 233 and 103; chr11, D11Mit216, 339, 177 and 179 ; chr 12, D12Mit182, 91, 143 and 17; chr13, D13Mit115, 179, 142, 107, 260 and 78; chr14, D14Mit126, 127, 174, 60, 102 and 107; chr15, D15Mit100, 70 and 159; chr16, D16Mit182, 4, 5, 189 and 106; chr17, D17Mit143, 51, 180, 20 and 142; chr18, D18Mit222, 208, 49 and 4; chr19, D19Mit28, 106, 90, 103 and 6. Prkdc was genotyped as given in (19). All available irradiated and un-irradiated mice were genotyped.
The MapManager QTX program (25) was used to perform interval mapping (IM) and permutation testing (PT). Mapping of likeliest position of a QTL on a chromosome and the association with a given trait is determined by the statistic value described as the Likelihood Ratio Statistic (LRS) or the Log of Odds (LOD). The observed LRS was tested against the null hypothesis that the QTL did not occur by chance in the genome. Empirical significance thresholds (suggestive, significant and highly significant) were computed for each individual trait (USI and LSI) and empirical p values calculated for each observed LRS. IM analysis was also performed using Cartographer QTL (9). Composite interval mapping (CIM; (11,13,14) to investigate multiple significant peaks observed from IM was conducted in Cartographer.
Selection and interrogation of target genes
Following the identification of the physical positions of each QTL, a comprehensive list of genes mapping to a region ±15 cM from the most likely position of the QTLs was collated using the Ensemble databases. These lists were checked for known tumour susceptibility loci, and searched for coding non-synonymous single nucleotide polymorphisms (CnSNP) between BALB and C57BL/6J using MPD database (26). All selected polymorphisms were verified by re-sequencing DNA taken from the mice used in the backcross. Sequencing was carried out on a 3100 ABI prism sequence analyser; primers and protocols are available on request.
Bioinformatic tools used to predict functional influence of non-synonymous polymorphisms
The functional influences of CnSNPs were studied using bioinformatics software, including Polyphen (27) and SIFT (28). Sequence alignment and conservation of the SNPs were analysed by EMBL-EBI and Polyphen. A Search Tool for the Retrieval of Interacting Genes or Proteins (STRING) was used to identify whether the CnSNPs belonged to known or predicted functional domains (29). These domains were also confirmed using scientific literature searches.