Allelotyping of pooled DNA with 250 K SNP microarrays

BACKGROUND: Genotyping technologies for whole genome association studies are now available. To perform such studies to an affordable price, pooled DNA can be used. Recent studies have shown that GeneChip Human Mapping 10 K and 50 K arrays are suitable for the estimation of the allele frequency in pooled DNA. In the present study, we tested the accuracy of the 250 K Nsp array, which is part of the 500 K array set representing 500,568 SNPs. Furthermore, we compared different algorithms to estimate allele frequencies of pooled DNA. RESULTS: We could confirm that the polynomial based probe specific correction (PPC) was the most accurate method for allele frequency estimation. However, a simple k-correction, using the relative allele signal (RAS) of heterozygous individuals, performed only slightly worse and provided results for more SNPs. Using four replicates of the 250 K array and the k-correction using heterozygous RAS values, we obtained results for 104.141 SNPs. The correlation between estimated and real allele frequency was 0.983 and the average error was 0.046, which was comparable to the results obtained with the 10 K array. Furthermore, we could show how the estimation accuracy depended on the SNP type (average error for A/T SNPs: 0.043 and for G/C SNPs: 0.052). CONCLUSION: The combination of DNA pooling and analysis of single nucleotide polymorphisms (SNPs) on high density microarrays is a promising tool for whole genome association studies

Blood-based DNA methylation as biomarker for breast cancer: a systematic review

Multiple studies have investigated global DNA methylation profiles and gene-specific DNA methylation in blood-based DNA to develop powerful screening markers for cancer. This systematic review summarizes the current evidence on methylation studies that investigated methylation level of blood-derived DNA of breast cancer (BC) patients in comparison to healthy controls by conducting a systematic literature review in PubMed and Web of Science. Essential results, such as methylation levels of BC cases and healthy controls, p values, and odds ratios, were extracted from these studies by two investigators independently. Overall, 45 publications met the inclusion criteria for this review. DNA from whole blood, as well as cell-free DNA (cfDNA) from serum or plasma, was used in these studies. The most common method used for measuring global DNA methylation was the investigation of repetitive elements as surrogates and the application of array-based genome-wide methylation analysis. For measuring gene-specific methylation level, methylation-specific PCR and pyrosequencing were the most frequently used methods. Epigenome-wide blood DNA hypomethylation in BC patients were reported in several studies; however, the evidence is still not conclusive. The most frequently investigated gene in whole blood was BRCA1, which was found more frequently methylated in patients compared to controls. RASSF1A was the most widely investigated gene in cfDNA of serum or plasma, which was also found more frequently methylated in patients compared to controls. Several of the eligible studies reported the associations of global hypomethylation and increased BC risk. Studies investigated associations between gene-specific methylation and BC risk, while got heterogeneous results. But two studies reported that hypermethylation of ATM gene was associated with increased BC risk, which suggest the potential use of this gene for BC risk stratification. Overall, our review suggests the possibility of using blood-based DNA methylation marker as promising marker for BC risk stratification, as several studies found associations between certain methylation level in blood and BC risk. However, so far, the evidence is still quite limited. Optimal markers are yet to be developed and promising results needed to be validated in prospective study cohorts and tested in large screening populations

Exploring the genetics of irritable bowel syndrome: A GWA study in the general population and replication in multinational case-control cohorts

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11\u2005326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31 710(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions

Genetic variants in the vitamin D pathway, 25(OH)D levels, and mortality in a large population-based cohort study

CONTEXT: Low 25-hydroxyvitamin D [25(OH)D] concentrations have been consistently associated with excess mortality in epidemiological studies, but this association could be due to confounding by health impairments going along with low 25(OH)D levels. An association of vitamin D related genetic variants with all-cause mortality could strengthen the claims of causality, because this association is assumed to be unaffected by confounding. OBJECTIVE: To assess the associations of low 25(OH)D with mortality in the presence or absence of genetic variants in the vitamin D pathway. DESIGN, SETTING AND PARTICIPANTS: A population-based cohort of German men and women. Data from 8,417 participants with complete follow-up for mortality. Six genetic variants were genotyped. MAIN OUTCOME MEASURES: All-cause mortality. RESULTS: Two SNPs, rs3755967 (GC) and rs11603330 (DHCR7), were associated with higher risk of low vitamin D status (OR per minor allele, 95% CI: 1.27, 1.18-1.36 and 1.16, 1.08-1.25, respectively). Low 25(OH)D (below the season-specific 33rd percentile) was associated with increased all-cause, cardiovascular and cancer mortality. However, none of the SNPs were associated with increased mortality. Furthermore, the increase in mortality for those with low 25(OH)D was mostly smaller in the presence of the risk alleles for low 25(OH)D ("genetically low 25(OH)D") than in the absence of those risk alleles ("otherwise low 25(OH)D"). CONCLUSIONS: Although we may have been limited by a low statistical power to detect small associations, our study showed that the strong relationship between low 25(OH)D and increased mortality may be at least partly due to other factors related with low 25(OH)D levels

The associations of DNA methylation alterations in oxidative stress-related genes with cancer incidence and mortality outcomes: a population-based cohort study

Background: Reactive oxygen species may be involved in epigenetic gene activation or silencing. We aimed to identify CpG sites, at which DNA methylation is related to urinary 8-isoprostane levels (biomarker of lipid peroxidation) and cancer or mortality outcomes. This investigation was based on a German, population-based cohort with linkage to cancer and mortality registry data (2000–2016). Results: Blood DNA methylation in promoter regions of 519 genes, known to be involved in pathways from oxidative stress (OS) to cancer, was obtained at the cohort's baseline examination. Inverse associations of DNA methylation at cg25365794 (ALOXE3) and cg08862778 (MTOR) with 8-isoprostane levels were observed in a derivation set (n = 1000) and validated in two independent subsets of the cohort (n = 548 and n = 741). Multivariate regression models were used to evaluate the associations of DNA methylation at the two CpG sites with lung, colorectal, prostate, breast, and overall cancer incidence as well as CVD, cancer, and all-cause mortality. DNA methylation at cg25365794 (ALOXE3) was inversely associated with lung and prostate cancer incidence. DNA methylation at cg08862778 (MTOR) was associated with a 43% lower breast cancer incidence in the top vs. bottom tertile. Conclusion: The finding for ALOXE3 may not be causal. As ALOXE3 is mainly expressed in skin tissue, the observed association might reflect the fact that both DNA methylation at the ALOXE3 gene and urinary 8-isoprostane concentrations depend on the level of OS in tissues. Contrarily, the finding for the MTOR gene and breast cancer is biologically plausible because the MTOR protein plays an important role in PI3K/Akt signaling, which is a pathway related to cancer development and cell senescence

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer.

Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM -/- patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

Characterization of extracellular circulating microRNA

MicroRNAs (miRNAs), a class of post-transcriptional gene expression regulators, have recently been detected in human body fluids, including peripheral blood plasma as extracellular nuclease resistant entities. However, the origin and function of extracellular circulating miRNA remain essentially unknown. Here, we confirmed that circulating mature miRNA in contrast to mRNA or snRNA is strikingly stable in blood plasma and cell culture media. Furthermore, we found that most miRNA in plasma and cell culture media completely passed through 0.22 µm filters but remained in the supernatant after ultracentrifugation at 110 000g indicating the non-vesicular origin of the extracellular miRNA. Furthermore, western blot immunoassay revealed that extracellular miRNA ultrafiltrated together with the 96 kDa Ago2 protein, a part of RNA-induced silencing complex. Moreover, miRNAs in both blood plasma and cell culture media co-immunoprecipited with anti-Ago2 antibody in a detergent free environment. This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins. We hypothesize that extracellular miRNAs are in the most part by-products of dead cells that remain in extracellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex. Nevertheless, our data does not reject the possibility that some miRNAs can be associated with exosomes