1. Volume 131, Issue 5, Pages 1418-1430 (November 2006)
Aristaless-like Homeobox-4 Gene Methylation Is a Potential Marker for Colorectal Adenocarcinomas 
Matthias P.A. Ebert, Fabian Model, Suzanne Mooney, Kari Hale, Joe Lograsso, Lori Tonnes–Priddy, Juliane Hoffmann, Antal Csepregi, Christoph Röcken, Bela Molnar, Hans–Ulrich Schulz, Peter Malfertheiner, Catherine Lofton–Day 
Gastroenterology 
Volume 131, Issue 5, Pages (November 2006)
DOI: /j.gastro
Copyright © 2006 AGA Institute Terms and Conditions

2. Figure 1
Results of the APPCR analysis of pooled DNA from normal and adenoma tissues. A hypermethylated gene (ALX4) was detected in the adenoma DNA (box, arrow). R, RsaI; H, RsaI/HpaII; M, RsaI/MspI.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

3. Figure 2
Degree of methylation of the ALX4 gene as assessed by MethyLight assay as outlined in Materials and Methods. (A) ALX4 gene methylation in normal colon mucosa and matched colon cancer. (B) ALX4 gene methylation in matched cases from 11 patients with primary colorectal cancers and liver metastasis. (C) ALX4 gene methylation in primary colorectal cancers and metastasis. (D) ALX4 methylation in colorectal cancer cell lines.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

4. Figure 3
Bisulfite sequencing. Four cases of cancers with a high degree of methylation and their matched normal nonmalignant colon mucosa tissues were selected (inset). ALX4 gene methylation was confirmed by sequencing of bisulfite-treated genomic DNA of samples 1–4. N, normal mucosa; T, tumor. Numbers 1–4 correspond to cases 1–4 in the MethyLight assay (inset); MethyLight assay indicates the CpG sites that were covered by both the MethyLight assay and sequencing of the respective DNA fragments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

5. Figure 4
ALX4 gene expression in colon cancer cell lines. Four colon cancer cell lines were analyzed for ALX4 gene expression by reverse-transcription PCR. In these 4 cell lines, ALX4 messenger RNA levels were below the level of detection; however, after treatment with the methylation inhibitor 5-aza-2′-deoxycytidine, Lovo cells readily exhibited ALX4 messenger RNA. β2M, β2-microglobulin; A, incubation with 5′-azadeocxycytidine; D, incubation with DMSO; −, no DNA; M, DNA ladder.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

6. Figure 5
Gene methylation in colorectal cancer. The methylation status of 6 genes in 47 colorectal cancers (TPEF/HPP1, APC, TIMP3, DAPK, Caveolin-2, p16) has been reported by our group recently.18 Now ALX4 gene methylation was assessed by MethyLight analysis in the same set of patients. While ALX4 and TPEF/HPP1 gene methylation occurred in the majority of colorectal cancers, the other 6 genes were less frequently methylated. Each box indicates a sample from a patient with colorectal cancer (n = 47). ■, methylated sample, PMR ≥4%; □, unmethylated sample, PMR <4%.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

7. Figure 6
ALX4 gene methylation in gastrointestinal and other neoplasias. Methylation of the ALX4 gene was analyzed using MethyLight analysis. Each box indicates a sample from a patient with the respective preneoplastic or neoplastic condition. ■, methylated sample, PMR ≥4%; □, unmethylated sample, PMR <4%; N, normal; T, tumor; D, dysplasia; L, liver; M, metastasis; IEN, intraepithelial neoplasia; SCC, squamous cell cancer; HCC, hepatocellular cancer; CCC, adenocarcinomas of the biliary tree including gallbladder cancer and cholangiocellular carcinomas.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

8. Figure 7
Serum ALX4 methylation levels in patients with colorectal cancer and adenomas. (A) The serum levels in 30 patients with colorectal cancer were significantly increased compared with noncancer controls (P < .0001). Mean ± SEM. (B) Receiver operating characteristic curve. The area under the curve generated from serum ALX4 gene methylation was (95% confidence interval, 0.721–0.921) for all 30 patients with colorectal cancer. (C) Overall, there was no significant difference in the mean ALX4 methylation levels of patients with hyperplastic or adenomatous polyps compared with patients without polyps (normal). Mean ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions