miR-486-5p expression is regulated by DNA methylation in osteosarcoma.

BACKGROUND: Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. RESULTS: To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2'-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. CONCLUSIONS: miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.

Diagnostic mRNA splicing assay for variants in BRCA1 and BRCA2 identified two novel pathogenic splicing aberrations.

BACKGROUND: Pathogenic variants in BRCA1 and BRCA2 cause hereditary breast and ovarian cancer. Screening of these genes has become easily accessible in diagnostic laboratories. Sequencing and copy number analyses are used to detect pathogenic variants, but also lead to identification of variants of unknown clinical significance (VUS). If the effect of a VUS can be clarified, it has direct consequence for the clinical management of the patient and family members. A splicing assay is one of several tools that might help in the classification of VUS. We therefore established mRNA analyses for BRCA1 and BRCA2 in the diagnostic laboratory in 2015. We hereby report the results of mRNA analysis variants in BRCA1 and BRCA2 after three years. METHODS: Variants predicted to alter splicing and variants within the canonical splice sites were selected for splicing analyses. Splicing assays were performed by reverse transcription-PCR of patient RNA. A biallalic expression analysis was carried out whenever possible. RESULTS: Twenty-five variants in BRCA1 and BRCA2 were analyzed by splicing assays; nine showed altered transcripts and 16 showed normal splicing patterns. The two novel pathogenic variants in BRCA1 c.4484 + 3 A > C and c.5407-10G > A were characterized. CONCLUSIONS: We conclude that mRNA analyses are useful in characterization of variants that may affect splicing. The results can guide classification of variants from unknown clinical significance to pathogenic or benign in a diagnostic laboratory, and thus be of direct clinical importance.

A tissue-based comparative effectiveness analysis of biomarkers for early detection of colorectal tumors.

OBJECTIVES: We recently identified a six-gene methylation-based biomarker panel suitable for early detection of colorectal cancer (CRC). In this study, we compared the performance of this novel epi-panel with that of previously identified DNA methylation markers in the same clinical tissue sample sets. METHODS: Quantitative methylation-specific PCR was used to analyze the promoter region of SEPT9 and VIM in a total of 485 tissue samples, divided into test and validation sets. ITGA4, NTRK2, OSMR, and TUBG2 were also included in the analyses. Receiver operating characteristic (ROC) curves were used to compare the performances of the individual biomarkers with that of the novel epi-panel. RESULTS: SEPT9 and VIM were methylated in 82 and 67% of CRCs (n=169) and in 88 and 54% of the adenomas (n=104). Only 3% of the normal mucosa samples (n=107) were methylated for these genes, confirming that the methylation was highly cancer-specific. Areas under the ROC curve (AUC), distinguishing CRCs from normal mucosa, were 0.94 for SEPT9 and 0.81 for VIM. AUC values for separating adenomas from normal mucosa samples were 0.96 and 0.81 for the same genes. In comparison, the novel epi-panel achieved an AUC of 0.98 (CRC) and 0.97 (adenomas).ITGA4, OSMR, NTRK2, and TUBG2 were methylated in 90, 78, 7, and 1% of the CRCs, and in 76, 77, 3, and 0% of the adenomas. Between 0 and 2% of the normal mucosa samples were methylated for the same genes. ITGA4 and OSMR achieved an AUC of 0.96 and 0.92 (CRC vs. normal mucosa), and 0.93 and 0.92 (adenomas vs. normal mucosa). CONCLUSIONS: We have confirmed the high performance of some of the previously identified DNA methylation markers. Furthermore, we showed that a recently reported epi-panel performed better than the individual DNA methylation biomarkers when analyzed in the same tissue samples. This observation was also true for VIM and SEPT9, which are included in commercially available noninvasive tests for CRC. These results further underscore the value of combining a manageable number of individual markers into a panel, which in addition to having a higher sensitivity and specificity might provide a more profound robustness to a noninvasive test compared with single markers.

Quantitative validation of GJC1 promoter hypermethylation in benign and malignant colorectal tumors.

We have previously shown that the gap junction protein γ 1 (GJC1) gene, encoding the connexin-45 protein, is inactivated by promoter hypermethylation in colorectal cancer. This was confirmed in a recent Endocrine-Related Cancer publication analyzing a limited number of samples. The aim of this study was to analyze GJC1 in a larger clinical cohort (n=485) and to assess whether or not the promoter hypermethylation was associated with clinical or pathological features. The methylation of GJC1 was confirmed to be tumor specific and was observed in 33% of colorectal cancers and 12% of adenomas. The methylation was strongly associated with BRAF mutations (P=5.64×10(-13)) as well as with proximal tumor location (P=1.42×10(-3)), features compatible with a CpG island methylator phenotype.

DNA methylation analyses of the connexin gene family reveal silencing of GJC1 (Connexin45) by promoter hypermethylation in colorectal cancer.

Gap junctions are specialized plasma membrane domains consisting of channels formed by members of the connexin protein family. Gap junctional intercellular communication is often lost in cancers due to aberrant localization or downregulation of connexins, and connexins are therefore suggested to act as tumor suppressor genes in various tissues. The aim of this study was to investigate the expression pattern and DNA promoter methylation status of connexins in colorectal cancer. Expression of six (GJA1, GJA9, GJB1, GJB2, GJC1 and GJD3) connexin genes was detected in normal colonic tissue samples. GJC1 expression was reduced in colorectal carcinomas compared to normal tissue samples. All analyzed connexins were hypermethylated in colon cancer cell lines, although at various frequencies. GJA4, GJB6 and GJD2 were hypermethylated in 60% (29/48), 25% (12/48) and 96% (46/48) of primary colorectal carcinomas, respectively. However, the methylation status was not associated with gene expression. GJC1 has two alternative promoters, which were methylated in 42% (32/76) and 38% (25/65) of colorectal tumors, and in none of the normal mucosa samples. Expression of GJC1 was significantly lower in methylated compared with unmethylated samples (p < 0.01) and was restored in cell lines treated with the demethylating drug 5-aza-2'deoxycytidine. Taken together, DNA hypermethylation of the promoter region of GJC1, encoding connexin45, is an important mechanism in silencing gene expression in colorectal cancer.