Experiment
The authors previously reported the mapping of QTL, Stmm3 (skin tumor modifier of MSM 3), in a cross between the resistant Japanese wild-derived inbred strain MSM/Ms and the wild derived inbred, susceptible strain FVB/NJcl to a region on mouse chromosome 4 flanked by markers D4Mit174 and D4Mit15. [J:193231, PMID:22843548].
In the present study resistant FVB/NJcl x (MSM/Ms x FVB/NJcl) F1 backcrossed mice were selected for further backcrossing to FVB/NJcL over at least 10 generations ultimately leading to two congenic lines covering the extended region of Stmm3:
4a - FBB.MSM-(D4Mit26-D4Mit254); and
4b - FVB.MSM-(D4Mit17-D4Mit157) containing the MSM/Ms allele of Stmm3 on the FVB/N background. [Fig 1A]. These mice were subjected to the same DMBA/TPA-induced skin tumor induction protocol as previously reported. Papilloma numbers were counted every week until 20 weeks post initiation.
Heterozygous M/F mice of congenic line 4a, which were susceptible to papilloma, developed an average of 34.7 (+/- 8.1) papillomas at 20 weeks after initiation.
Heterozygous M/F mice of congenic line 4b showed a strong suppressive effect on papilloma development; an average of 18.1 (+/-14.6) papillomas at 20 weeks after initiation, p=0.0114. By excluding the region of the negative congenic line (4a) from the region of the positive congenic line (4b) the location of Stmm3 was narrowed to an interval of about 34 Mb, mapping between D4Mit17 (31.3 cM) and D4Mit26 (48.5 cM) of congenic line 4b, FVB.MSM-(D4Mit17-D4Mit157).
In the original analysis strong linkage to the number of papillomas >6mm in diameter was revealed. However, the linkage peak completely disappeared when the analysis was confined to the number of papillomas less than or equal to 6mm in diameter. The conclusion was that Stmm3 genes function only at the late stage of papillomas and were not involved in early stage development.
To confirm this stage-specific effect papillomas were classified into two categories based on size. The number of M/F heterozygous mice that developed papillomas more than 6mm in diameter in line 4a was nearly the same as those developed in the F/F homozygous mice.
Whereas, M/F heterozygous mice from line 4b developed almost no papilloma more than 6mm in diameter compared with the F/F homozygous mice. Furthermore, M/F heterozygous mice of line 4b developed fewer papillomas less than or equal to 6 mm in diameter than either F/F homozygous or M/F heterozygous mice in line 4a. Taken together the results suggest Stmm3 conferred resistance to larger papillomas and showed a weaker suppressive effect on smaller papillomas, confirming the original analysis.
Histological analysis was also performed. The results suggested that Stmm3 reduced proliferative cells in papillomas, rather than development.
Twelve informative microsatellite markers were added to investigate allelic imbalance on Chromosome 4 to determine whether somatic change would allow more specific localization of Stmm3. Maximal allele-specific imbalance was detected using marker D4Mit1003 and D4Mit26 at approximately 81.0 Mb and 88.7 Mb, respectively. Somatic mapping data allowed the refinement of the interval containing Stmm3 to a shorter region, revealing two allelic imbalance peaks. The proximal peak was the region from D4Mit17 (63.0 Mb), D4Mit1003 (81.3 Mb) and D4Mit328 (83.6 Mb), the physical size of about 20.6 Mb. The distal peak was from D4Mit327 (86.1 Mb), D4Mit26 (88.7 Mb) and D4Mit165 (90.7 Mb), the physical size of about 4.6 Mb. When these two regions were combined, the total physical size of the candidate region was about 25 Mb. Allelic imbalance analysis revealed Cdkn2a is localized in the peak region (app 88 MB) showing loss of MSM allele. Authors suggest this could be a candidate gene for late stage papilloma on Chr 4 in a future study.
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