Precise Epigenetic Analysis Using Targeted Bisulfite Genomic Sequencing Distinguishes FSHD1, FSHD2, and Healthy Subjects

The true prevalence of facioscapulohumeral muscular dystrophy (FSHD) is unknown due to difficulties with accurate clinical evaluation and the complexities of current genetic diagnostics. Interestingly, all forms of FSHD are linked to epigenetic changes in the chromosome 4q35 D4Z4 macrosatellite, suggesting that epigenetic analysis could provide an avenue for sequence-based FSHD diagnostics. However, studies assessing DNA methylation at the FSHD locus have produced conflicting results; thus, the utility of this technique as an FSHD diagnostic remains controversial. Here, we critically compared two protocols for epigenetic analysis of the FSHD region using bisulfite genomic sequencing: Jones et al., that contends to be individually diagnostic for FSHD1 and FSHD2, and Gaillard et al., that can identify some changes in DNA methylation levels between groups of clinically affected FSHD and healthy subjects, but is not individually diagnostic for any form of FSHD. We performed both sets of assays on the same genetically confirmed samples and showed that this discrepancy was due strictly to differences in amplicon specificity. We propose that the epigenetic status of the FSHD-associated D4Z4 arrays, when accurately assessed, is a diagnostic for genetic FSHD and can readily distinguish between healthy, FSHD1 and FSHD2. Thus, epigenetic diagnosis of FSHD, which can be performed on saliva DNA, will greatly increase accessibility to FSHD diagnostics for populations around the world.

A proof-of-concept study for the pathogenetic role of enhancer hypomethylation of MYBPHL in multiple myeloma

Enhancer DNA methylation and expression of MYBPHL was studied in multiple myeloma (MM). By bisulfite genomic sequencing, among the three CpGs inside the MYBPHL enhancer, CpG1 was significantly hypomethylated in MM cell lines (6.7–50.0%) than normal plasma cells (37.5–75.0%) (P = 0.007), which was negatively correlated with qPCR-measured MYBPHL expression. In RPMI-8226 and WL-2 cells, bearing the highest CpG1 methylation, 5-azadC caused enhancer demethylation and expression of MYBPHL. In primary samples, higher CpG1 methylation was associated with lower MYBPHL expression. By luciferase assay, luciferase activity was enhanced by MYBPHL enhancer compared with empty vector control, but reduced by site-directed mutagenesis of each CpG. RNA-seq data of newly diagnosed MM patients showed that MYBPHL expression was associated with t(11;14). MOLP-8 cells carrying t(11;14) express the highest levels of MYBPHL, and its knockdown reduced cellular proliferation and increased cell death. Herein, as a proof-of-concept, our data demonstrated that the MYBPHL enhancer, particularly CpG1, was hypomethylated and associated with increased MYBPHL expression in MM, which was implicated in myelomagenesis.

Transcriptome profiling unveils GAP43 regulates ABC transporters and EIF2 signaling in colorectal cancer cells

Background The growth- and plasticity-associated protein-43 (GAP43) is biasedly expressed in indigestive system and nervous system. Recent study has shown that GAP43 is responsible for the development of neuronal growth and axonal regeneration in normal nervous tissue, while serves as a specific biomarker of relapsed or refractory neuroblastoma. However, its expression pattern and function in digestive system cancer remains to be clarified. Methods In this study, we examined the GAP43 status with qRT-PCR and bisulfite genomic sequencing in colorectal cancer (CRC). We investigated the effect of overexpressed GAP43 in CRC cells with RNA-seq. The RNA-seq data was analyzed with DAVID and IPA. Results GAP43 was downregulated in CRC compared to the adjacent tissues. DNA methylase inhibitor 5-Aza-CdR treatment could significantly induce GAP43, indicated that the silencing of GAP43 gene in CRC is closely related to DNA methylation. Bisulfite genomic sequencing confirmed the promoter methylation of GAP43 in CRC. To explore the transcriptional alterations by overexpressed GAP43 in CRC, we performed RNA-seq and found that upregulated genes were significantly enriched in the signaling pathways of ABC transporters and ECM-receptor interaction, while downregulated genes were significantly enriched in Ribosome signaling pathway. Further Ingenuity Pathway Analysis (IPA) showed that EIF2 signaling pathway was significantly repressed by overexpression of GAP43. Conclusion Our findings provide a novel mechanistic insight of GAP43 in CRC. Transcriptome profiling of overexpressed GAP43 in CRC uncovered the functional roles of GAP43 in the development of human CRC.