Yimei Fan

Germline promoter hypermethylation of tumor suppressor genes in gastric cancer.

AIM To explore germline hypermethylation of the tumor suppressor genes MLH1, CDH1 and P16(INK4a) in suspected cases of hereditary gastric cancer (GC). METHODS A group of 140 Chinese GC patients in whom the primary cancer had developed before the age of 60 or who had a familial history of cancer were screened for germline hypermethylation of the MLH1, CDH1 and P16(INK4a) tumor suppressor genes. Genomic DNA was extracted from peripheral blood leukocytes and modified by sodium bisulfite. The treated DNA was then subjected to bisulfite DNA sequencing for a specific region of the MLH1 promoter. The methylation status of CDH1 or P16(INK4a) was assayed using methylation-specific PCR. Clonal bisulfite allelic sequencing in positive samples was performed to obtain a comprehensive analysis of the CpG island methylation status of these promoter regions. RESULTS Methylation of the MLH1 gene promoter was detected in the peripheral blood DNA of only 1/140 (0.7%) of the GC patient group. However, this methylation pattern was mosaic rather than the allelic pattern which has previously been reported for MLH1 in hereditary non-polyposis colorectal cancer (HNPCC) patients. We found that 10% of the MLH1 alleles in the peripheral blood DNA of this patient were methylated, consistent with 20% of cells having one methylated allele. No germline promoter methylation of the CDH1 or P16(INK4a) genes was detected. CONCLUSION Mosaic germline epimutation of the MLH1 gene is present in suspected hereditary GC patients in China but at a very low level. Germline epimutation of the CDH1 or P16(INK4a) gene is not a frequent event.

Novel CDH1 germline mutations identified in Chinese gastric cancer patients

AIM To give a comprehensive report of E-cadherin gene (CDH1) variations in a population at a high risk for gastric cancer (GC). METHODS The samples consisted of 178 men and 58 women with a mean age of 62.3 ± 9.4 years and an age range of 30-84 years. A total of 240 cancer-free controls were recruited (mean age of 61.8 ± 10.1 years, age range of 26-82 years). Samples were screened for CDH1 germline mutations by high-resolution melting analysis or directly sequencing. Luciferase reporter assay, RNA splicing assay and bioinformatic analysis were used to evaluate the effect of mutations. RESULTS Four novel CDH1 sequence alterations were identified in GC patients including a G>T transition 49 bp before the start codon; a three-nucleotide deletion, c.44_46del TGC; one missense mutation, c.604G>A (V202I); and one variation in the intron, c.1320+7A>G. In addition, polymorphism frequencies were observed for CDH1-164delT, -161C>A, -73A>C, c.48+6C>T, c.48+62_48+63delinsCGTGCCCCAGCCC, c.894C>T (A298A), c.1224G>A (A408A), c.1888C>G (L630V), c.2076T>C (A692A), and c.2253C>T (N751N) which is similar to the data reported in http://www.ncbi.nlm.nih.gov/projects/SNP/. RNA splicing analysis suggested that the c.1320+7A>G and c.1224G>A variations did not affect exon splicing ability. Luciferase reporter assay demonstrated that the c.-49T variation might be helpful for E-cadherin transcription, though the increase in transcription activity is limited (only 33%). SIFT score and PolyPhen analysis both demonstrated that the L630V missense mutation probably damages protein function, while the V202I variant does not. CONCLUSION This study reveals novel mutations in sporadic GC patients which had been poorly investigated for susceptibility genes.

Epigenetic regulation of CDH1 exon 8 alternative splicing in gastric cancer.

BackgroundThe tumor suppressor gene CDH1 is critical for intercellular adhesion. In our previous work, we reported a nonfunctional CDH1 transcript that lacks the final 83 base pairs of exon 8 (1054del83). In this work, we probed the role of histone epigenetic modifications as well as DNA methylation in selection of this isoform.MethodsRT-qPCR was used to detect CDH1 RNA expression. Methylation of CDH1 was analyzed by bisulphite sequencing PCR. ChIP assay was performed to show histones level. Cell lines were treated with DNA methyltransferase inhibitor AZA, HDAC inhibitor TSA, or siRNA oligonucleotides to test regulation of CDH1 splicing.ResultsGreater CDH1 1054del83 transcripts were observed in gastric cancer (GC) cell lines than human gastric mucosal epithelial cell line GES-1. All the cell lines showed significant methylation pattern at the CpG sites of CDH1 exon 8. AZA treatment did not influence selection of 1054del83 transcripts. A significant decrease in acetylation for histones H3 and H4K16Ac in an internal region of the CDH1 gene surrounding the alternative exon 8 were detected in GC cell lines. Treatment with TSA preferentially expressed the correctly spliced transcript and not the exon 8 skipped aberrant transcripts, showing that histone acetylation was involved in the splicing regulation. SiRNA-mediated knockdown of SETD2 (The specific methyltransferase of H3K36) decreased exclusion of exon 8, suggesting that the presence of this mark correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. However, CDH1 splicing was not affected by SRSF2 knockdown.ConclusionsH3K36me3 correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. Histone acetylation was involved in the splicing regulation as well.

The MLH1 2101C>A (Q701K) variant increases the risk of gastric cancer in Chinese males

BackgroundGastric cancer is one of the most common cancers affecting East Asians, and MLH1 could play a critical role during tumorigenesis in this condition.MethodsSamples from 236 Chinese patients suffering from gastric cancer were screened for MLH1 germline mutations. Carrier frequencies of the mutations were compared between gastric cancer patients and 240 cancer-free controls. Bioinformatic analysis was used to predict the effect of these mutations on protein function and mRNA splicing.ResultsSix MLH1 sequence alterations were identified in gastric cancer patients including two promoter region substitutions, -93G>A and -28A>G, and four missense mutations 649C>T (R217C), 655A>G (I219V), 1151T>A (V384D) and 2101C>A (Q701K). Compared with the MLH1 2101CC genotype, the 2101CA genotype was associated with a risk of gastric cancer (OR = 8.42, 95% CI = 1.04-68.06) in males. Furthermore, the MLH1 2101C>A mutant was predicted by in silico analysis to affect exon splicing ability. Immunohistochemistry of one index patient carrying the MLH1 2101C>A mutation demonstrated a loss of MLH1 protein and normal expression of MSH2 and E-cadherin. No significant differences were demonstrated between cases and controls for the other five MLH1 variants but the data indicated an ethnic difference in the frequency of these variations between Eastern Asians and Western populations.ConclusionsAn ethnic-specific MLH1 mutation spectrum occurred in Chinese gastric cancer patients. The MLH1 2101C>A mutation could be a marker for susceptibility to gastric cancer, particularly in males.

Influence of eight unclassified missense variants of the MLH1 gene on Lynch syndrome susceptibility.

Missense mutations in MLH1 have frequently been detected in patients with Lynch syndrome, but their genetic significance has not been extensively assessed. In this study, we attempt to evaluate the etiological role of eight MLH1 missense variants. The variants were analyzed for their ability to affect MLH1 protein interaction with its partner PMS2 in vivo employing a yeast two-hybrid system. In addition, a SIFT (sorting intolerant from tolerant) algorithm was adopted to predict the effects of amino acid substitutions. Finally, scanning of mutations in a normal Chinese population and assay of the clinical characteristics have all been taken into account. Our results demonstrated that the MLH1 variants D485E and L653R cause functional alterations of the human MutLα complex significantly. The R265C, D304V, A586P, and R755S variants affect partial interaction. The remaining two variants, N38D and L559R, could be nonfunctional polymorphisms or might affect the mismatch repair system through other mechanisms.

Effect of histone modifications on hMLH1 alternative splicing in gastric cancer

hMLH1 is one of the mismatch genes closely related to the occurrence of gastric cancer. Epigenetic regulation may play more important roles than gene mutations in DNA damage repair genes to drive carcinogenesis. In this article, we discuss the role of epigenetic changes, especially histone modifications in the regulation of hMLH1 alternative splicing. Our results showed that hMLH1 delEx10, delEx11, delEx10–11, delEx16 and delEx17 transcripts were ubiquitous in sporadic Chinese gastric cancer patients and gastric cancer cell lines. Lower level of H4K16ac and H3ac was detected in hMLH1 exon 10–11 region in gastric cancer cell lines when compared with human gastric mucosal epithelial cell line GES-1. A significant decrease of hMLH1 delEx11 and delEx10–11 was observed in gastric cancer cell lines after trichostatin A treatment. H3K36me3 and H3K4me2 levels were lower in hMLH1 exon 10–11 and exon 16–17 regions in gastric cancer lines when compared with GES-1. Aberrant transcripts such as hMLH1 delEx11 and delEx10–11 were significantly higher in gastric cancer cell lines after small interfering RNA–mediated knockdown of SETD2 (the specific methyltransferase of H3K36). The hMLH1 delEx10 and delEx10–11 transcripts were increased after interference of SRSF2. Taken together, our study demonstrates that lower level of histone acetylation and specific histone methylation such as H3K36me3 correlate with aberrant transcripts in hMLH1 exon 10–11 region. SRSF2 may be involved in these specific exons skipping as well.

Identification the potential of stool-based SNCA methylation as a candidate biomarker for early colorectal cancer detection

Background: Stool-based DNA methylation are emerging as promising biomarkers for colorectal cancer (CRC), but their capability for detecting curable stages (adenomas) remains unclear. This study aims to explore the potential of selected candidate biomarkers (SNCA, SPG20 and FBN1) in stool samples for identifying patients with adenomas or CRC. Methods: Thirty-one patients with CRC, 49 patients with adenomas, and 64 normal controls were selected. Stool samples were processed by the stool intestinal mucosal cells collector and analyzed by quantitative methylation-specific polymerase chain reaction (qMSP). Results: Promoter methylation status of genes was denoted as percentage of methylated reference (PMR) values. The SNCA PMR values were significantly higher in CRC and adenomas groups than normal controls. The area under the ROC curve (AUC) for SNCA was 0.836 and 0.772 for detection of CRC and adenomas, respectively. The sensitivity of stool-based SNCA methylation was 83.9% for CRC and 75.5% for adenomas, with the specificity of 75% for both. The odds ratio (OR) for CRC and adenomas with stool SNCA hypermethylation was 11.291 and 9.234, respectively, after adjustment for patients’ age and sex. Though there was a trend of increase of SNCA promoter methylation with disease progression, the difference was not significant. No significant difference in stool-based SNCA methylation was found between proximal and distal lesions. SNCA methylation significantly reduced after tumor resection compared to the initial status. The SPG20 and FBN1 methylation status was not significantly different among groups. Conclusions: The results validated the capability of SNCA methylation in stool samples for the identification of patients with adenomas or CRC.