Distinct promoter methylation patterns of LKB1 in the hamartomatous polyps of Peutz-Jeghers syndrome and its potential in gastrointestinal malignancy prediction

Abstract

Background: Peutz-Jeghers Syndrome (PJS) is known as a rare inherited polyposis due to the malfunction of serine/threonine kinase gene LKB1. However, not all of PJS patients carry LKB1 germline mutation. Previous researches have observed the elevated DNA methylation level in PJS polyps. Nevertheless, the mechanism of such abnormal and its impact on PJS patients remains to be fully described.

Results: The results proved a significant increase on the methylation level of LKB1 promoter in PJS polyps compared with normal colon biopsies through bisulfite PCR followed by Sanger sequencing. Moreover, the methylation pattern in PJS polyps could be further categorized as three different scenarios: hypermethylated, hemimethylated and hypomethylated pattern. Furthermore, immunohistochemistry of DNMT1/3a/3b suggested the up-regulation of DNMT1 and 3a might participate the epigenetic alternation of LKB1 in PJS polyps. Logistic regression suggested hypomethylated LKB1 promoter in PJS polyps as a risk factor for gastrointestinal malignancies in PJS patients.

Conclusions: The promoter methylation level of LKB1 gene in PJS polyps is generally elevated compared with normal colon mucosa. Yet not all of PJS polyps carry hypermethylated LKB1 promoter. Hypomethylation in this region has linked to malignant tumors in PJS patients. Given the rarity of PJS, this work together with previous researches, have proved the importance of LKB1 promoter methylation in PJS development and prognosis.

Keywords: Colorectal Cancer; DNA methylation; Hamartomatous polyp; Liver kinase B1; Peutz-Jeghers syndrome; Prognosis.

Fig. 1

Bisulfite PCR-Sanger sequencing revealed elevated methylation level in the hamartomatous polyps of PJS patients compared with normal mucosa. a, b Histology of PJS polyp samples used in this study, magnification = 100x, n = 50 c, d Histology of normal colon mucosa used in this study, magnification = 100x, n = 50 e Bisulfite PCR Primer design from LKB1 promoter by MethPrimer. f Representative of gel image after bisufite PCR amplifications. The PCR product is 259 bp, n = 100 (g) Average methylation level for LKB1 promoter region, comparison between 50 PJS polyps and 50 normal mucosa samples revealed the gap between two groups. Means ± SEM, *P < 0.05. h The methylation analysis per each CpG site indicated that instead of randomly distributed, DNA methylation was evenly distributed across the whole region. Data presented as means. All bars = 100 μm

Fig. 2

Characterization of DNMTs’ expression in normal colon mucosa, PJS polyps, and colorectal cancer of PJS patients. a-c DNMT1 expression in the above samples. The expression of DNMT1 is increased in PJS polyps and colorectal cancer in PJS patients compared with normal mucosa. n = 15 (d-f) DNMT3a expression in the above samples. The expression of DNMT3a is increased in PJS polyps and colorectal cancer in PJS patients compared with normal mucosa. n = 15 (g-i) DNMT3b immunochemistry results shows negative staining in all the above samples. n = 15. Bar = 100 μm

Fig. 3

Distinct promoter methylation patterns of LKB1 in hamartomatous polyps of PJS patients. a-c Representative of three methylation patterns of LKB1 promoter region presented by lollipop graph. a: hyper-methylated pattern; b: hemi-methylated pattern; c: hypo-methylated pattern. d Linear plot for the average methylation level of LKB1 promoter region for all the above patterns. e Diagram of three possible scenarios for different methylation patterns. M = maternal allele, P = paternal allele