DIAPH3 predicts survival of patients with MGMT-methylated glioblastoma.

Background: Glioblastoma is one of the most aggressive primary brain tumors, with a poor outcome despite multimodal treatment. Methylation of the MGMT promoter, which predicts the response to temozolomide, is a well-established prognostic marker for glioblastoma. However, a difference in survival can still be detected within the MGMT methylated group, with some patients exhibiting a shorter survival than others, emphasizing the need for additional predictive factors. Methods: We analyzed DIAPH3 expression in glioblastoma samples from the cancer genome atlas (TCGA). We also retrospectively analyzed one hundred seventeen histological glioblastomas from patients operated on at Saint-Luc University Hospital between May 2013 and August 2019. We analyzed the DIAPH3 expression, explored the relationship between mRNA levels and Patient's survival after the surgical resection. Finally, we assessed the methylation pattern of the DIAPH3 promoter using a targeted deep bisulfite sequencing approach. Results: We found that 36% and 1% of the TCGA glioblastoma samples exhibit copy number alterations and mutations in DIAPH3, respectively. We scrutinized the expression of DIAPH3 at single cell level and detected an overlap with MKI67 expression in glioblastoma proliferating cells, including neural progenitor-like, oligodendrocyte progenitor-like and astrocyte-like states. We quantitatively analyzed DIAPH3 expression in our cohort and uncovered a positive correlation between DIAPH3 mRNA level and patient's survival. The effect of DIAPH3 was prominent in MGMT-methylated glioblastoma. Finally, we report that the expression of DIAPH3 is at least partially regulated by the methylation of three CpG sites in the promoter region. Conclusion: We propose that combining the DIAPH3 expression with MGMT methylation could offer a better prediction of survival and more adapted postsurgical treatment for patients with MGMT-methylated glioblastoma.

Exploring Differentially Methylated Genes in Vulvar Squamous Cell Carcinoma.

DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC (n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies (n = 6). Using a false-discovery rate of 0.05, beta-difference (Δß) of ±0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

Prenatal parental tobacco smoking, gene specific DNA methylation, and newborns size: the Generation R study.

BACKGROUND: Deleterious effects of prenatal tobacco smoking on fetal growth and newborn weight are well-established. One of the proposed mechanisms underlying this relationship is alterations in epigenetic programming. We selected 506 newborns from a population-based prospective birth cohort in the Netherlands. Prenatal parental tobacco smoking was assessed using self-reporting questionnaires. Information on birth outcomes was obtained from medical records. The deoxyribonucleic acid (DNA) methylation of the growth genes IGF2DMR and H19 was measured in newborn umbilical cord white blood cells. Associations were assessed between parental tobacco smoking and DNA methylation using linear mixed models and adjusted for potential confounders. RESULTS: The DNA methylation levels of IGF2DMR and H19 in the non-smoking group were median (90 % range), 54.0 % (44.6-62.0), and 30.0 % (25.5-34.0), in the first trimester only smoking group 52.2 % (44.5-61.1) and 30.8 % (27.1-34.1), and in the continued smoking group 51.6 % (43.9-61.3) and 30.2 % (23.7-34.8), respectively. Continued prenatal maternal smoking was inversely associated with IGF2DMR methylation (ß = -1.03, 95 % CI -1.76; -0.30) in a dose-dependent manner (P-trend = 0.030). This association seemed to be slightly more profound among newborn girls (ß = -1.38, 95 % CI -2.63; -0.14) than boys (ß = -0.72, 95 % CI -1.68; 0.24). H19 methylation was also inversely associated continued smoking <5 cigarettes/day (ß = -0.96, 95 % CI -1.78; -0.14). Moreover, the association between maternal smoking and newborns small for gestational age seems to be partially explained by IGF2DMR methylation (ß = -0.095, 95 % CI -0.249; -0.018). Among non-smoking mothers, paternal tobacco smoking was not associated with IGF2DMR or H19 methylation. CONCLUSIONS: Maternal smoking is inversely associated with IGF2DMR methylation in newborns, which can be one of the underlying mechanisms through which smoking affects fetal growth.

Inhibin alpha-subunit (INHA) expression in adrenocortical cancer is linked to genetic and epigenetic INHA promoter variation.

Adrenocortical carcinoma (ACC) is a rare, but highly malignant tumor of unknown origin. Inhibin α-subunit (Inha) knockout mice develop ACCs following gonadectomy. In man, INHA expression varies widely within ACC tissues and its circulating peptide inhibin pro-αC has been described as a novel tumor marker for ACC. We investigated whether genetic and epigenetic changes of the INHA gene in human ACC cause loss or variation of INHA expression. To this end, analyses of INHA sequence, promoter methylation and mRNA expression were performed in human adrenocortical tissues. Serum inhibin pro-αC levels were also measured in ACC patients. INHA genetic analysis in 37 unique ACCs revealed 10 novel, heterozygous rare variants. Of the 3 coding bases affected, one variant was synonymous and two were missense variants: S72F and S184F. The minor allele of rs11893842 at -124 bp was observed at a low frequency (24%) in ACC samples and was associated with decreased INHA mRNA levels: 4.7±1.9 arbitrary units for AA, compared to 26±11 for AG/GG genotypes (P = 0.034). The methylation of four proximal INHA promoter CpGs was aberrantly increased in five ACCs (47.7±3.9%), compared to normal adrenals (18.4±0.6%, P = 0.0052), whereas the other 14 ACCs studied showed diminished promoter methylation (9.8±1.1%, P = 0.020). CpG methylation was inversely correlated to INHA mRNA levels in ACCs (r = -0.701, p = 0.0036), but not associated with serum inhibin pro-αC levels. In conclusion, aberrant methylation and common genetic variation in the INHA promoter occur in human ACCs and are associated with decreased INHA expression.

Methylation of migraine-related genes in different tissues of the rat.

17ß-Estradiol, an epigenetic modulator, is involved in the increased prevalence of migraine in women. Together with the prophylactic efficacy of valproate, which influences DNA methylation and histone modification, this points to the involvement of epigenetic mechanisms. Epigenetic studies are often performed on leukocytes, but it is unclear to what extent methylation is similar in other tissues. Therefore, we investigated methylation of migraine-related genes that might be epigenetically regulated (CGRP-ergic pathway, estrogen receptors, endothelial NOS, as well as MTHFR) in different migraine-related tissues and compared this to methylation in rat as well as human leukocytes. Further, we studied whether 17ß-estradiol has a prominent role in methylation of these genes. Female rats (n = 35) were ovariectomized or sham-operated and treated with 17ß-estradiol or placebo. DNA was isolated and methylation was assessed through bisulphite treatment and mass spectrometry. Human methylation data were obtained using the Illumina 450k genome-wide methylation array in 395 female subjects from a population-based cohort study. We showed that methylation of the Crcp, Calcrl, Esr1 and Nos3 genes is tissue-specific and that methylation in leukocytes was not correlated to that in other tissues. Interestingly, the interindividual variation in methylation differed considerably between genes and tissues. Furthermore we showed that methylation in human leukocytes was similar to that in rat leukocytes in our genes of interest, suggesting that rat may be a good model to study human DNA methylation in tissues that are difficult to obtain. In none of the genes a significant effect of estradiol treatment was observed.

Epigenetic profiles in children with a neural tube defect; a case-control study in two populations.

Folate deficiency is implicated in the causation of neural tube defects (NTDs). The preventive effect of periconceptional folic acid supplement use is partially explained by the treatment of a deranged folate-dependent one carbon metabolism, which provides methyl groups for DNA-methylation as an epigenetic mechanism. Here, we hypothesize that variations in DNA-methylation of genes implicated in the development of NTDs and embryonic growth are part of the underlying mechanism. In 48 children with a neural tube defect and 62 controls from a Dutch case-control study and 34 children with a neural tube defect and 78 controls from a Texan case-control study, we measured the DNA-methylation levels of imprinted candidate genes (IGF2-DMR, H19, KCNQ1OT1) and non-imprinted genes (the LEKR/CCNL gene region associated with birth weight, and MTHFR and VANGL1 associated with NTD). We used the MassARRAY EpiTYPER assay from Sequenom for the assessment of DNA-methylation. Linear mixed model analysis was used to estimate associations between DNA-methylation levels of the genes and a neural tube defect. In the Dutch study group, but not in the Texan study group we found a significant association between the risk of having an NTD and DNA methylation levels of MTHFR (absolute decrease in methylation of -0.33% in cases, P-value = 0.001), and LEKR/CCNL (absolute increase in methylation: 1.36% in cases, P-value = 0.048), and a borderline significant association for VANGL (absolute increase in methylation: 0.17% in cases, P-value = 0.063). Only the association between MTHFR and NTD-risk remained significant after multiple testing correction. The associations in the Dutch study were not replicated in the Texan study. We conclude that the associations between NTDs and the methylation of the MTHFR gene, and maybe VANGL and LEKKR/CNNL, are in line with previous studies showing polymorphisms in the same genes in association with NTDs and embryonic development, respectively.

Loci at chromosomes 13, 19 and 20 influence age at natural menopause.

We conducted a genome-wide association study for age at natural menopause in 2,979 European women and identified six SNPs in three loci associated with age at natural menopause: chromosome 19q13.4 (rs1172822; -0.4 year per T allele (39%); P = 6.3 × 10(-11)), chromosome 20p12.3 (rs236114; +0.5 year per A allele (21%); P = 9.7 × 10(-11)) and chromosome 13q34 (rs7333181; +0.5 year per A allele (12%); P = 2.5 × 10(-8)). These common genetic variants regulate timing of ovarian aging, an important risk factor for breast cancer, osteoporosis and cardiovascular disease.

Regional deletion and amplification on chromosome 6 in a uveal melanoma case without abnormalities on chromosomes 1p, 3 and 8.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Loss of the long arm and gain of the short arm of chromosome 6 are frequently observed chromosomal aberrations in UM, together with loss of chromosome 1p36, loss of chromosome 3 and gain of chromosome 8. This suggests the presence of one or more oncogenes on 6p and tumor suppressor genes at 6q that are involved in UM development. Both regions, however, have not been well defined yet. Furthermore in other neoplasms gain of 6p and loss of 6q are frequently occurring events. In this case report, we describe the delineation of a partial gain on chromosome 6p and a partial deletion on 6q in a UM with the objective to pinpoint smaller candidate regions on chromosome 6 involved in UM development. Conventional cytogenetics, comparative genomic hybridization (CGH) and fluorescence in-situ hybridization (FISH) were used to delineate regions of loss and gain on chromosome 6 in this UM patient. With conventional cytogenetics a deleted region was found on chromosome 6q that was further delineated to a region ranging from 6q16.1 to 6q22 using CGH and FISH. A region of gain from 6pter to 6p21.2 was also demarcated with CGH and FISH. No other deletions or amplifications on recurrently involved chromosomes were found in this patient. This study indicates the presence of one or more tumor suppressor genes on chromosomal region 6q16.1-6q22 and the presence of one or more oncogenes on chromosomal region 6pter-6p21.2, which are likely to be important in UM and other tumors.

Expression of APITD1 is not related to copy number changes of chromosomal region 1p36 or the prognosis of uveal melanoma.

PURPOSE: Concurrent loss of chromosome arm 1p, region 36, and chromosome 3 leads to decreased disease-free survival in patients with uveal melanoma. A candidate tumor-suppressor gene APITD1 is located on the critical region on chromosome arm 1p, and it was therefore hypothesized that lower expression levels of this gene could lead to decreased survival in patients, with concurrent loss of a region on chromosome arm 1p and chromosome 3. Using neuroblastoma cells, which, like uveal melanoma, originate from neural crest cells, a former study showed that APITD1 has cell growth and/or cell death properties. In this study, APITD1 expression was analyzed to determine whether it corresponds with the DNA copy number and is related to survival in uveal melanoma. METHODS: To detect whether loss in the copy number of APITD1 results in lowered mRNA expression of the gene, FISH analysis was combined with real-time PCR. In addition, the effect of APITD1 expression on survival was studied by using Kaplan-Meier survival analysis. RESULTS: Expression of APITD1 mRNA was not related to DNA copy number (P = 0.956) or chromosome 3 status (P = 0.958). Kaplan-Meier survival analysis showed very similar survival curves for tumors with high and low APITD1 expression, with a log-rank significance value of P = 0.9682. CONCLUSIONS: These results indicate that APITD1 is not the tumor suppressor gene on 1p36 responsible for the negative prognostic effect in uveal melanoma with concurrent loss of chromosome arm 1p, region 36, and chromosome 3.

Concurrent loss of chromosome arm 1p and chromosome 3 predicts a decreased disease-free survival in uveal melanoma patients.

PURPOSE: Uveal melanoma is a highly malignant disease with a mortality rate of 50% at 10 to 15 years. Previous studies have shown that chromosomal changes are associated with decreased survival of the patient. However, in these studies the small number of tumors analyzed did not allow robust statistical analysis. In the present study, the independent numerical changes in chromosomes 1, 3, 6, and 8 on disease-free survival (DFS) was assessed in a large series of patients with uveal melanoma. METHODS: One hundred twenty tumors from patients with uveal melanoma were analyzed for numerical changes in chromosomes 1, 3, 6, and 8, with cytogenetic analysis, fluorescent in situ hybridization, and/or comparative genomic hybridization. Data were correlated with disease outcome in univariate and multivariate analyses, by Kaplan-Meier and Cox regression analyses. RESULTS: At a mean follow-up time of 45 months, 42 patients had died or had metastatic disease. In the univariate analysis, loss of chromosome 3, gain of 8q, largest tumor diameter, or the presence of epithelioid cells was associated with a decreased DFS. In the multivariate analysis, the effect of monosomy 3 on survival was largely modified by changes in 1p36. Regarding all chromosomal changes, only the concurrent loss of the short arm of chromosome 1 and all of chromosome 3 was an independent prognostic parameter for disease-free survival (P < 0.001). CONCLUSIONS: In uveal melanoma, concurrent loss of the short arm of chromosome 1 and all of chromosome 3 is an independent predictor of decreased DFS.

The RAS-BRAF kinase pathway is not involved in uveal melanoma.

An activating mutation has been recently observed in cutaneous melanoma in a downstream component of RAS-BRAF. The most common mutation, occurring in 80% of cutaneous melanoma samples, is a T-to-A transition resulting in a single amino acid substitution (V599E). Since cutaneous and uveal melanoma (UM) have a common origin, we aimed to establish whether activation of the BRAF proto-oncogene is also an important factor in the development of UM. Exons 11 through 18 of the BRAF gene were screened from 33 primary UMs and 11 UM cell lines. Genomic polymerase chain reaction products were evaluated using single-strand conformation polymorphism analysis, followed by sequencing of aberrant products. The most common mutation, T1796A in the kinase domain of BRAF, was not observed in any of the primary UM samples. This mutation was also absent in 10 of the 11 UM cell lines. In one of the UM cell lines, OCM1, the T1796A mutation was present. We conclude that, in contrast to cutaneous melanoma, BRAF does not appear to be involved in the pathogenesis of UM.