Volume 121, Issue 6, Pages 1300-1309 (December 2001) Extensive methylation of hMLH1 promoter region predominates in proximal colon cancer with microsatellite instability  Yasuyuki Miyakura, Kokichi Sugano, Fumio Konishi, Akira Ichikawa, Masato Maekawa, Kazuhisa Shitoh, Seiji Igarashi, Kenjiro Kotake, Yasuo Koyama, Hideo Nagai  Gastroenterology  Volume 121, Issue 6, Pages 1300-1309 (December 2001) DOI: 10.1053/gast.2001.29616 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Map of the 5' CpG islands of the hMLH1 gene and design of the PCR primers. (Top) CpG sites are indicated by vertical lines. (Middle) The expected PCR products for regions A, B, C, D, and E are shown. Their positions relative to the adenine residue at the start codon and the sizes of the amplified DNA fragments are indicated. Numbers in parentheses indicate the numbers of CpG sites in each region. (Bottom) Sequences of each primer set are indicated. I in the primer sequences indicates inosine residues substituted for guanine (methylated) or adenine (unmethylated). Gastroenterology 2001 121, 1300-1309DOI: (10.1053/gast.2001.29616) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 BiPS analysis of the hMLH1 promoter region is shown in the left panel. Case Nos. are the same as those indicated in Table 2. U indicates control unmethylated normal DNA, and M indicates control methylated DNA. Cancer tissue of case J433 (433T) displayed methylated band in all regions, exhibiting full methylation pattern. Cancer tissue of case J262 (262T) displayed methylated band only in regions A and C, exhibiting partial methylation. Normal tissue displayed a faint methylated band in region A. After SSCP analysis, the extra bands were cut from the electrophoresed gels, reamplified by PCR, and sequenced by dideoxy sequencing procedure. Results of the direct sequencing were shown in the right panel. Sequences (J433T) with fully methylated cytosines of CpG dinucleotides in all regions indicate full methylation. Gastroenterology 2001 121, 1300-1309DOI: (10.1053/gast.2001.29616) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunohistochemical staining for hMLH1 protein in colorectal cancer. Case J312 showed positive nuclear staining for hMLH1, both in (A) normal colonic mucosa and (B) cancer tissue, of which the promoter region of hMLH1 was not methylated. In case I376, hMLH1 protein expression was positive in (C) normal mucosa, but decreased in (D) cancer tissue, of which the promoter region of hMLH1 was hypermethylated. In case J449 showed heterogenous staining, (E) most of cancer showed positive nuclear staining, but (F) a part of cancer showed negative nuclear staining. This case exhibited partial methylation of hMLH1 promoter region. Gastroenterology 2001 121, 1300-1309DOI: (10.1053/gast.2001.29616) Copyright © 2001 American Gastroenterological Association Terms and Conditions