Exposure to aflatoxin B1 in utero is associated with DNA methylation in white blood cells of infants in The Gambia

BACKGROUND: Exposure to environmental toxins during embryonic development may lead to epigenetic changes that influence disease risk in later life. Aflatoxin is a contaminant of staple foods in sub-Saharan Africa, is a known human liver carcinogen and has been associated with stunting in infants.

METHODS: We have measured aflatoxin exposure in 115 pregnant women in The Gambia and examined the DNA methylation status of white blood cells from their infants at 2-8 months old (mean 3.6 ± 0.9). Aflatoxin exposure in women was assessed using an ELISA method to measure aflatoxin albumin (AF-alb) adducts in plasma taken at 1-16 weeks of pregnancy. Genome-wide DNA methylation of infant white blood cells was measured using the Illumina Infinium HumanMethylation450beadchip.

RESULTS: AF-alb levels ranged from 3.9 to 458.4 pg/mg albumin. We found that aflatoxin exposure in the mothers was associated to DNA methylation in their infants for 71 CpG sites (false discovery rate < 0.05), with an average effect size of 1.7% change in methylation. Aflatoxin-associated differential methylation was observed in growth factor genes such as FGF12 and IGF1, and immune-related genes such as CCL28, TLR2 and TGFBI. Moreover, one aflatoxin-associated methylation region (corresponding to the miR-4520b locus) was identified.

CONCLUSIONS: This study shows that maternal exposure to aflatoxin during the early stages of pregnancy is associated with differential DNA methylation patterns of infants, including in genes related to growth and immune function. This reinforces the need for interventions to reduce aflatoxin exposure, especially during critical periods of fetal and infant development.

Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer

BACKGROUND: Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

METHODS: Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens.

RESULTS: The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression.

CONCLUSION: Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.

Gender-specific genomic profiling in metastatic colorectal cancer patients treated with 5-fluorouracil and oxaliplatin

AIMS: Survival and response rates in metastatic colorectal cancer remain poor, despite advances in drug development. There is increasing evidence to suggest that gender-specific differences may contribute to poor clinical outcome. We tested the hypothesis that genomic profiling of metastatic colorectal cancer is dependent on gender.

MATERIALS & METHODS: A total of 152 patients with metastatic colorectal cancer who were treated with oxaliplatin and continuous infusion 5-fluorouracil were genotyped for 21 polymorphisms in 13 cancer-related genes by PCR. Classification and regression tree analysis tested for gender-related association of polymorphisms with overall survival, progression-free survival and tumor response.

RESULTS: Classification and regression tree analysis of all polymorphisms, age and race resulted in gender-specific predictors of overall survival, progression-free survival and tumor response. Polymorphisms in the following genes were associated with gender-specific clinical outcome: estrogen receptor β, EGF receptor, xeroderma pigmentosum group D, voltage-gated sodium channel and phospholipase A2.

CONCLUSION: Genetic profiling to predict the clinical outcome of patients with metastatic colorectal cancer may depend on gender.

A global expression-based analysis of the consequences of the t(4;14) translocation in myeloma

Purpose: Our purpose in this report was to define genes and pathways dysregulated as a consequence of the t(4;14) in myeloma, and to gain insight into the downstream functional effects that may explain the different prognosis of this subgroup.Experimental Design: Fibroblast growth factor receptor 3 (FGFR3) overexpression, the presence of immunoglobulin heavy chain-multiple myeloma SET domain (IgH-MMSET) fusion products and the identification of t(4;14) breakpoints were determined in a series of myeloma cases. Differentially expressed genes were identified between cases with (n = 55) and without (n = 24) a t(4;14) by using global gene expression analysis.Results: Cases with a t(4;14) have a distinct expression pattern compared with other cases of myeloma. A total of 127 genes were identified as being differentially expressed including MMSET and cyclin D2, which have been previously reported as being associated with this translocation. Other important functional classes of genes include cell signaling, apoptosis and related genes, oncogenes, chromatin structure, and DNA repair genes. Interestingly, 25% of myeloma cases lacking evidence of this translocation had up-regulation of the MMSET transcript to the same level as cases with a translocation.Conclusions: t(4;14) cases form a distinct subgroup of myeloma cases with a unique gene signature that may account for their poor prognosis. A number of non-t(4;14) cases also express MMSET consistent with this gene playing a role in myeloma pathogenesis.

Deregulation of genes related to iron and mitochondrial metabolism in refractory anemia with ring sideroblasts

The presence of SF3B1 gene mutations is a hallmark of refractory anemia with ring sideroblasts (RARS). However, the mechanisms responsible for iron accumulation that characterize the Myelodysplastic Syndrome with ring sideroblasts (MDS-RS) are not completely understood. In order to gain insight in the molecular basis of MDS-RS, an integrative study of the expression and mutational status of genes related to iron and mitochondrial metabolism was carried out. A total of 231 low-risk MDS patients and 81 controls were studied. Gene expression analysis revealed that iron metabolism and mitochondrial function had the highest number of genes deregulated in RARS patients compared to controls and the refractory cytopenias with unilineage dysplasia (RCUD). Thus mitochondrial transporters SLC25 (SLC25A37 and SLC25A38) and ALAD genes were over-expressed in RARS. Moreover, significant differences were observed between patients with SF3B1 mutations and patients without the mutations. The deregulation of genes involved in iron and mitochondrial metabolism provides new insights in our knowledge of MDS-RS. New variants that could be involved in the pathogenesis of these diseases have been identified.

Age-Related Macular Degeneration-Associated Genes in Alzheimer Disease

Objectives: Given the clinical and pathological similarities between age-relatedmacular degeneration (AMD) and Alzheimer disease (AD), to assess whether AMDassociatedsingle nucleotide polymorphisms (SNPs), including those from complementrelatedgenes, are associated with AD. 
Design: A case-control association study-typedesign. 
Setting: A UK tertiary care dementia clinic. 
Participants: 322 cognitivelynormal participants and 258 cases with a clinical diagnosis of AD.
Measurements:Polymorphisms in the following genes were studied: CFH, ARMS2, C2/CFB, C3, CFI/PLA2G12a, SERPING1, TLR3, TLR4, CRP, APOE, and TOMM40. Haplotypes were analysedfor CFH, TOMM40, and APOE. Univariate analysis was performed for each geneticchange and case-comparator status, and then correction for multiple testing performed. 
Results: The presence of an ε4 APOE allele was significantly associated with AD. Noassociation was evident between CFH SNPs or haplotypes, or other AMD-associated SNPstested, and AD. The exceptions were TOMM40 SNPs, which were associated with AD evenafter correction for multiple comparisons. The associations disappeared, however, whenentered into a regression model including APOE genotypes. 
Conclusions: The resultsfor most SNPs tested, as well as CFH haplotypes, are novel. The functional effects ofabnormal complement activity in AD’s pathogenesis may be contradictory, butmethodological reasons may underlie the lack of association—for example, geneticchanges other than SNPs being involved.

Inhibitors of alpha-synuclein oligomerization and toxicity: a future therapeutic strategy for Parkinson's disease and related disorders.

An abundance of genetic, histopathological, and biochemical evidence has implicated the neuronal protein, alpha-synuclein (alpha-syn) as a key player in the development of several neurodegenerative diseases, the so-called synucleinopathies, of which Parkinson's disease (PD) is the most prevalent. Development of disease appears to be linked to events that increase the intracellular concentration of alpha-syn or cause its chemical modification, either of which can accelerate the rate at which it forms aggregates. Examples of such events include increased copy number of genes, decreased rate of degradation via the proteasome or other proteases, or altered forms of alpha-syn, such as truncations, missense mutations, or chemical modifications by oxidative reactions. Aggregated forms of the protein, especially newly formed soluble aggregates, are toxic to cells, so that one therapeutic strategy would be to reduce the rate at which such oligomerization occurs. We have therefore designed several peptides and also identified small molecules that can inhibit alpha-syn oligomerization and toxicity in vitro. These compounds could serve as lead compounds for the design of new drugs for the treatment of PD and related disorders in the future.

A novel diagnostic test detects a low frequency of the hemicentin Gln5345Arg variant among Northern Irish age related macular degeneration patients.

Purpose: Age related macular degeneration (AMD) is a common cause of severe vision loss. Identification of genes involved in AMD will facilitate early detection and ultimately help to identify pathways for treatment for this disorder. The A16,263G mutation in the HEMICENTIN-1 gene produces a non-conservative substitution of arginine for glutamine at codon 5345 which has been implicated in familial AMD. The aim of this study is to develop a rapid diagnostic assay for the detection of this mutation and to evaluate its frequency in a sample of AMD patients. Methods: A primer probe set was designed from exon 104 of the HEMICENTIN-1 gene to differentiate between mutant and wild type alleles. A region spanning the mutation was amplified by PCR using a LightCycler (Roche Diagnostic). The mutation was then detected by melt curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. The frequency of the mutation within the Northern Ireland population was evaluated by assaying 508 affected AMD patients, 25 possibly affected and 163 controls. Results: This assay clearly discriminates between the A16,263G mutant and wild type HEMICENTIN-1 alleles. The wild type sequence has a single base mismatch with the probe which decreases the stability of the hybrid, resulting in a lower TM (TM=51.27 °C) than that observed for the perfectly matched mutant allele (TM=59.9 °C). The mutant allele was detected in only one of the 696 subjects, an affected AMD patient. Conclusions: We describe a rapid assay for the genotyping of the Gln5345Arg mutation using real-time fluorescence PCR to facilitate rapid processing of samples through combined amplification and detection steps. These characteristics are suitable for a clinical setting where high throughput diagnostic procedures are required. The frequency of this mutation within the Northern Ireland population has been estimated at 0.2%, concurring with previous findings that this mutation is a rare variant associated with AMD. A rapid diagnostic assay will facilitate a reliable and convenient evaluation of the frequency of the Gln5345Arg mutation and its association with AMD within other populations.