Volume 129, Issue 5, Pages 1392-1399 (November 2005) MLH1 Germline Epimutations as a Factor in Hereditary Nonpolyposis Colorectal Cancer  Megan Hitchins, Rachel Williams, Kayfong Cheong, Nimita Halani, Vita A.P. Lin, Deborah Packham, Sue Ku, Andrew Buckle, Nicholas Hawkins, John Burn, Steven Gallinger, Jack Goldblatt, Judy Kirk, Ian Tomlinson, Rodney Scott, Allan Spigelman, Catherine Suter, David Martin, Graeme Suthers, Robyn Ward  Gastroenterology  Volume 129, Issue 5, Pages 1392-1399 (November 2005) DOI: 10.1053/j.gastro.2005.09.003 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Methylation analysis of MLH1 by COBRA. (A) Map of the MLH1 and antisense EPM2AIP1 promoter regions showing positions of the PCR fragments and restriction sites used in COBRA for the detection of methylated CpG sites. Gray rectangles represent gene exons, arrows denote transcriptional direction from transcription initiation sites, and horizontal bars indicate fragments amplified by bisulfite-specific PCR of genomic DNA. The scale is in base pairs with +1 positioned at the transcription start site of MLH1. (B) COBRA of the A and C regions of the MLH1 promoter in PBL DNA from subject ST and his nuclear family. c+, positive control, DNA from RKO colorectal cancer cell line in which MLH1 is biallelically methylated; c−, negative control, DNA from normal PBLs; m, pUC19/MspI DNA marker. PCR products of methylated DNA digest with each of the restriction enzymes, whereas amplicons from unmethylated templates are resistant. Proband ST was positive for methylation at all restriction sites tested, but his parents and unaffected siblings were unmethylated. Gastroenterology 2005 129, 1392-1399DOI: (10.1053/j.gastro.2005.09.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Immunohistochemical analysis of MLH1 expression in the colorectal adenocarcinoma from the subject. Paraffin sections (4 μm) were stained with murine anti-MLH1 antibody (1:200; PharMingen, San Diego, CA), biotinylated horse anti-mouse immunoglobulin (1:100; Dako, Dakopatts, Denmark), horseradish peroxidase–labeled streptavidin (1:200; Dako), and diaminobenzidine substrate. Carcinoma cells show absence of nuclear staining for MLH1, while residual trapped normal colonic crypts and inflammatory cells (upper right) show normal expression. Hematoxylin counterstain. Bar = 100 μm. Gastroenterology 2005 129, 1392-1399DOI: (10.1053/j.gastro.2005.09.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Bisulfite sequencing of the MLH1 and EPM2AIP1 promoters in somatic tissues from individual ST. HF, hair follicles; BM, buccal mucosa. Each horizontal row represents a single allele and each circle a CpG dinucleotide. Methylated CpG sites are black, and unmethylated CpGs are white. The subject showed approximately equal proportions of methylated and unmethylated alleles in each cell type derived from all 3 embryonic germ cell layers, suggestive of soma-wide monoallelic epimutation. Gastroenterology 2005 129, 1392-1399DOI: (10.1053/j.gastro.2005.09.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Monoallelic expression of the paternal MLH1 allele in individual ST. Sequence electropherograms across the delCTT polymorphic site at position 2363–2365 (indicated by gray arrows) within the 3′ untranslated region of MLH1 are shown for both genomic DNA and RNA from the subject and the genomic DNA from his parents. Both genomic DNA and complementary DNA templates were derived from PBLs. Proband ST is heterozygous for the delCTT polymorphism, causing a frameshift in the sequence readout from genomic DNA. Only the delCTT allele was detected in the RNA, indicating that MLH1 is expressed monoallelically. The paternal DNA was also heterozygous for the delCTT polymorphism, whereas the maternal DNA was homozygous for the wild-type allele, indicating the delCTT allele transcriptionally active in the subject was paternally inherited. Gastroenterology 2005 129, 1392-1399DOI: (10.1053/j.gastro.2005.09.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 MLH1 intron 4 SNP pedigree and loss of heterozygosity analyses. (A) TaiI digest of intron 4 SNP PCR products from PBL DNA of subject ST and family members. The 222–base pair PCR products are cleaved to fragments of 120 and 102 base pairs on the G allele, whereas the A allele remains undigested. The familial inheritance pattern indicates obligate maternal transmission of the A allele to all offspring and paternal transmission of the G allele. m, pUC19/MspI DNA ladder. (B) Fluorescent loss of heterozygosity analysis of tumor in proband ST. (Upper panel) DNA from normal colonic mucosa. (Lower panel) DNA from tumor. Boxed numbers represent fragment size in base pairs (top) and peak area in fluorescent units (bottom). The carboxyfluorescein label is attached to the reverse PCR primer, so only the 102–base pair digested fragment on the G allele is detected. Ratio of alleles G/A is 1:5.5 ([2998/1505]/[1038/2871]) in tumor versus normal, indicating somatic loss of the paternal G allele in the tumor. Gastroenterology 2005 129, 1392-1399DOI: (10.1053/j.gastro.2005.09.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions