Volume 125, Issue 2, Pages 501-509 (August 2003) Pathogenicity of the hereditary colorectal cancer mutation hMLH1 del616 linked to shortage of the functional protein  Tiina E Raevaara, Carlos Vaccaro, Wael M Abdel-Rahman, Esteban Mocetti, Shashi Bala, Karin E Lönnqvist, Reetta Kariola, Henry T Lynch, Päivi Peltomäki, Minna Nyström-Lahti  Gastroenterology  Volume 125, Issue 2, Pages 501-509 (August 2003) DOI: 10.1016/S0016-5085(03)00905-3

Figure 1 Pedigree of family 4401, with relevant clinical and molecular data. Filled symbols (squares for males; circles for females) denote individuals affected with HNPCC-type cancer, whereas open symbols indicate unaffected individuals. Cancer site and age (years) at diagnosis are shown below each symbol. Cancer sites are as follows: Co, colon; Ce, cecum; Tr, transverse colon; Sig, sigmoid colon; Asc, ascending colon; SB, small bowel; Pan, pancreas; ESOF, esophagus; End, endometrium; Mel, melanoma. Markers used for haplotype analysis and their relative distances in centimorgans (cM) are given in a separate key box. The haplotypes for the affected individuals are shown with allele sizes in base pairs and the shared portion boxed. The carrier status for the hMLH1 del616 mutation is given (+, mutation carrier; −, noncarrier; for the youngest generation, the carrier status has been omitted for confidentiality reasons), and results from MSI and hMLH1 immunohistochemical (IHC) studies are shown. For MSI, the number of unstable markers is given as a fraction of all markers successfully analyzed. Gastroenterology 2003 125, 501-509DOI: (10.1016/S0016-5085(03)00905-3)

Figure 2 Serial sections from an ascending colon tumor from individual III:3, stained with (A ) anti-hMLH1, (B) anti-hMSH2, and (C ) anti-hMSH6 antibodies. The anti-hMLH1 antibody shows positive (brown) staining of only a few scattered tumor nuclei and most tumor nuclei are negative. Note the positive staining of a lymphoid follicle (bottom) that serves as an internal control. The hMSH2 and hMSH6 antibodies show homogenous positive staining of most tumor nuclei. Gastroenterology 2003 125, 501-509DOI: (10.1016/S0016-5085(03)00905-3)

Figure 3 (Top) Expression of hMutLα WT and hMutLα del616 in Sf9 and 293T cells, detected by Western blot analysis. The amount of the hMLH1 del616 protein is low compared with the amount of hMLH1 WT in both expression systems, but especially in 293T cells, whereas the amount of naturally expressed β-tubulin protein was equal in all 293T cell clones. (Bottom) Combined coimmunoprecipitation and Western blot analysis shows similar interactions of hMLH1 WT and hMLH1 del616 with hPMS2 WT. Gastroenterology 2003 125, 501-509DOI: (10.1016/S0016-5085(03)00905-3)

Figure 4 MMR activity of MMR-deficient HCT116 nuclear extract (NE) complemented with hMutLα variants. MOCK contains heteroduplex DNA with no protein, TK6 is MMR-proficient NE used as a positive control, and HCT116 is MMR-deficient NE used as a negative control. The top fragment (3193 base pairs [bp]) in each lane shows the migration of the unrepaired linearized G · T mismatch-containing plasmid DNA, and the two lower fragments (1833 and 1360 bp) indicate repaired and digested plasmids. The repair efficiencies are marked in terms of percentages (digested DNA of the total DNA added to the reaction) below the panel. Both protein variants, hMutLα WT (purified protein and total protein extract) and hMutLα del616 (total protein extract), complement HCT116 NE. Gastroenterology 2003 125, 501-509DOI: (10.1016/S0016-5085(03)00905-3)

Figure 5 MMR activity of MMR-deficient HCT116 nuclear extract complemented with different amounts of hMutLα variants. The controls were used and repair efficiencies were counted as in Figure 4. The original amounts, 2.3 and 5.8 μg of hMutLα WT and hMutLα del616, respectively, were reduced by factors of 5, 10, and 20. The values are an average of 2 independent experiments, and the data are presented as the mean ± SD. The repair percentages of hMutLα WT were 33% ± 2.9% (1), 38.5% ± 2.1% (1:5), 37% ± 1.4% (1:10), and 29% ± 3.1% (1:20) and of hMutLα del616 were 31% ± 3.7% (1), 30.5% ± 2.1% (1:5), 20.5% ± 6.4% (1:10), and 7% ± 3.1% (1:20). Gastroenterology 2003 125, 501-509DOI: (10.1016/S0016-5085(03)00905-3)