Methylation Profiling SOCS2 in Philadelphia-Negative Myeloproliferative Neoplasm

The Janus kinase signal transducer and activator of transcription (JAK / STAT) signalling, which is crucial in Philadelphia-negative myeloproliferative neoplasms’ (MPNs), is negatively regulated by molecules such as SOCS, CISH, and SHP1. SOCS2 methylation has been studied in MPN with conflicting results. Here we examine the methylation status of SOCS2 by specific methylation-specific polymerase chain reaction (MSP) in cell lines and 130 diagnostic prepheral blood samples from Ph-ve MPN. Furthermore, we tried to explain the mismatch of the methylation frequency by assigning the investigated MSP primers to the respective genes. Methylation was detected in normal controls using SOCS2-MSP primers in the 3 'translated exon sequence, but not with primers around the transcription start site in the 5' untranslated regions (5'UTR). SOCS2 was completely unmethylated in primary MPN samples and cell lines. In contrast, SOCS2 methylation when using MSP primers located at the 5'UTR is rare in all studies. In conclusion, SOCS2 methylation is rare in Ph-ve MPN. The appropriate MSP primers are important for an accurate estimate of the methylation frequency. The role of SOCS2 methylation in MPN pathogenesis requires further investigation.

Construction and Validation of a Lung Cancer Diagnostic Model Based on 6-Gene Methylation Frequency in Blood, Clinical Features, and Serum Tumor Markers

Lung cancer has a high mortality rate. Promoting early diagnosis and screening of lung cancer is the most effective way to enhance the survival rate of lung cancer patients. Through computer technology, a comprehensive evaluation of genetic testing results and basic clinical information of lung cancer patients could effectively diagnose early lung cancer and indicate cancer risks. This study retrospectively collected 70 pairs of lung cancer tissue samples and normal human tissue samples. The methylation frequencies of 6 genes (FHIT, p16, MGMT, RASSF1A, APC, DAPK) in lung cancer patients, the basic clinical information, and tumor marker levels of these patients were analyzed. Then, the python package “sklearn” was employed to build a support vector machine (SVM) classifier which performed 10-fold cross-validation to construct diagnostic models that could identify lung cancer risk of suspected cases. Receiver operation characteristic (ROC) curves were drawn, and the performance of the combined diagnostic model based on several factors (clinical information, tumor marker level, and methylation frequency of 6 genes in blood) was shown to be better than that of models with only one pathological feature. The AUC value of the combined model was 0.963, and the sensitivity, specificity, and accuracy were 0.900, 0.971, and 0.936, respectively. The above results revealed that the diagnostic model based on these features was highly reliable, which could screen and diagnose suspected early lung cancer patients, contributing to increasing diagnosis rate and survival rate of lung cancer patients.

Aberrant Methylation and Decreased Expression of NRIP1 in IGHV-Unmutated CLL

Aberrant DNA methylation patterning occurs in many cancers, including chronic lymphocytic leukemia (CLL). Methylation programming changes as B cells differentiate from immature progenitors to mature B cells. CLL clones originate from a continuum of B cell maturation states, which differ in their "methylome." Emerging evidence suggests that aberrant methylation programming occurs at numerous genes that are differentially expressed in IGHV-defined CLL subtypes. Nuclear receptor interacting protein 1 (NRIP1) reproducibly shows significant gene expression differences in IGHV unmutated (U-CLL) and mutated CLL (M-CLL), with lower transcript levels observed in U-CLL cells. Low expression of NRIP1 is associated with poorer overall survival and time-to-treatment. NRIP1 encodes a cofactor that interacts with various nuclear receptors and other proteins to regulate transcription of genes involved in cellular proliferation, survival, metabolism, inflammation, and other processes. Published data reveal several CpG dinucleotides near the NRIP1 promoter that undergo increasing methylation during the maturation and differentiation of normal peripheral blood B cells (NBC), concordant with a stepwise decrease in transcript levels of NRIP1 over the course of maturation. Interestingly, our analysis of publically available expression data revealed significantly lower NRIP1 expression in U-CLL cells (the less mature CLL subtype) than NBC, while M-CLL cells (the more mature CLL subtype) express levels comparable to NBC. Given these observations, our objective was to interrogate the promoter and regulatory regions at the NRIP1 locus for differences in CpG methylation in genomic DNA isolated from U-CLL, M-CLL, and NBC. We hypothesized that the NRIP1 locus is more highly methylated in U-CLL than in M-CLL and NBC, and that this increased methylation represses NRIP1 gene transcription, contributing to its aberrantly low expression in U-CLL compared to M-CLL and NBC. We bisulfite-converted genomic DNA from negatively-selected CD19+ treatment-naïve CLL samples (n=74 U-CLL, n=37 M-CLL) and NBC from healthy donors (n=5). We PCR-amplified regions in or near the CpG island of NRIP1 and used Agena Bioscience's EpiTyper kit to prepare the samples for analysis on a MassARRAY spectrometer. EpiTyper software was used to detect mass differences that indicate either non-methylated or methylated CpG-containing fragments, and to calculate their relative frequency in each sample. For each CpG locus that was assayed, we fit a beta-regression model to detect differential methylation between M-CLL and U-CLL. We also computed Pearson correlation coefficients comparing methylation levels to gene expression levels. We assessed 47 CpGs mapping to the promoter and nearby regulatory elements of NRIP1 for methylation frequency and correlation with NRIP1 expression. From our comparison of methylation frequency in U-CLL and M-CLL, 3 adjacent CpG loci were significant at false discovery rate (FDR) =1% (CpG 58, p=0.00016; CpG 144, p=0.00062; and CpG 155, p=0.00287). The three most significant correlation coefficients arose from the same three CpG loci (CpG 58, R=0.677, p=2.2e-16; CpG 144, R=0.539, p=7.16e-10; CpG 155, R=0.651, p=5.77e-15). Unexpectedly, all 3 loci were methylated at higher levels in M-CLL and at lower levels in U-CLL (on average), and expression was positively correlated with methylation levels. These CpGs map near a documented binding site for the repressive transcription factor RUNX3, prompting us to hypothesize that differential methylation in U-CLL versus M-CLL may affect the binding affinity of RUNX3 and contribute to the robust NRIP1 expression differences in these CLL subtypes. Future work will test this hypothesis using gel-shift and luciferase reporter assays followed by chromatin immunoprecipitation experiments. Few studies have focused on aberrant methylation patterning in CLL and the consequences of deregulated gene expression. We are the first to look at NRIP1 for differences in DNA methylation across IGHV subtypes. Future studies aimed at understanding how methylation at this locus affects NRIP1 transcription, as well as how levels of NRIP1 affect cell survival pathways, will enhance our understanding of the role of this gene in CLL, and the contributions of deregulated methylation to disease pathophysiology. Disclosures No relevant conflicts of interest to declare.

CpG Island Methylation of the MLH1, MGMT, DAPK, and CASP8 Genes in Cancerous and Adjacent Noncancerous Stomach Tissues

Background and Objective. Many factors are involved in the development of gastric adenocarcinoma. The CpG island methylation of apoptosis and mismatch repair genes by the loss of their function is important in gastric adenocarcinoma. The aim of this study was to determine the methylation frequency of MLH1, MGMT, CASP8, and DAPK in cancerous and adjacent noncancerous stomach tissues, to determine possible associations with the selected clinicopathological characteristics, and to identify possible correlation between the methylation of individual genes. Material and Methods. The methylation status of MLH1, MGMT, DAPK, and CASP8 was investigated in 69 patients with gastric adenocarcinoma by using methylation-specific polymerase chain reaction. The associations between patients’ clinical characteristics and methylation status were assessed. Results. The methylation frequency of the MLH1, DAPK, MGMT, and CASP8 gene promoters in cancerous and adjacent noncancerous tissues was 31.9% and 27.5%; 47.8% and 46.4%; 36.2% and 44.9%; and 5.8% and 5.8%, respectively, but the differences were not significant. There was no significant association between the methylation status of the mentioned genes and clinicopathological characteristics, such as age, sex, tumor type by the Lauren classification, degree of differentiation G, and TNM staging. An inverse correlation between the methylation of the DAPK and MLH1 gene promoters in cancerous and surrounding noncancerous tissues was found. Conclusions. The methylation of the MLH1, MGMT, DAPK, and CASP8 genes was found to occur both in cancerous and noncancerous stomach tissues. These findings provide additional insights into gene methylation patterns in gastric adenocarcinoma.