Search

Abstract 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.

Abstract 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.