Aberrant methylation of the eyes absent 4 gene in ulcerative colitis-associated dysplasia.
OBJECTIVE
This study explored the eyes absent 4 (EYA4) gene promoter methylation in noncolitic colorectal tissues and assessed its discrimination for neoplasia in chronic ulcerative colitis (CUC).


METHODS
The methylation status of noncolitic specimens was confirmed by direct bisulfite sequencing. Methylation-specific polymerase chain reaction (MSP) primers were designed to evaluate colorectal tissues, including 50 noncolitic patients comprising 24 normal epithelia, 14 polyps, and 12 cancers. The assay was tested on tissues from 67 CUC patients including 31 surveillance neoplasia-positive patients and nonneoplastic controls including 22 CUC surveillance-negative and 14 CUC short-disease duration. Remote colonic tissue was included from each of 27 of the 31 CUC neoplasia cases. The expression of EYA4 was quantified in cell lines by use of reverse-transcription polymerase chain reaction.


RESULTS
Within noncolitic tissues, bisulfite sequencing showed EYA4 promoter hypermethylation in 80% (8 of 10) of colorectal cancers but in none (0 of 9) of the normal tissues. MSP was positive in 81% (21 of 26) of cancers and polyps and in only 4% (1 of 14) of normal mucosa. In CUC, MSP was positive in 81% (25 of 31) of neoplastic cases but in none (0 of 36) of the nonneoplastic controls. RNA expression was decreased in methylated compared with unmethylated cell lines (P < .001). Treatment with 5-Aza-2'-deoxycytidine (DAC)/Trichostatin (TSA) increased the overall messenger RNA expression (P = .005).


CONCLUSIONS
The EYA4 gene promoter is hypermethylated commonly in sporadic and colitic neoplasia and may be associated with gene silencing. EYA4 methylation represents a candidate marker for CUC surveillance.
Osborn
Neal K
NK
Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Zou
Hongzhi
H
Molina
Julian R
JR
Lesche
Ralf
R
Lewin
Joern
J
Lofton-Day
Cathy
C
Klatt
Kristie K
KK
Harrington
Jonathan J
JJ
Burgart
Lawrence J
LJ
Ahlquist
David A
DA
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015415:Biomarkers / Q000032:analysis; D002460:Cell Line; D002908:Chronic Disease; D003093:Colitis, Ulcerative / Q000150:complications / Q000235:genetics / Q000473:pathology*; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology; D003111:Colonic Polyps / Q000235:genetics; D000077209:Decitabine; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008745:Methylation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics*
D015415:Biomarkers; C117736:EYA4 protein, human; D006877:Hydroxamic Acids; D012333:RNA, Messenger; D015534:Trans-Activators; C012589:trichostatin A; D000077209:Decitabine; D001374:Azacitidine