Gustavo F. Bayón

Aberrant DNA methylation patterns of spermatozoa in men with unexplained infertility

STUDY QUESTION Are there DNA methylation alterations in sperm that could explain the reduced biological fertility of male partners from couples with unexplained infertility? SUMMARY ANSWER DNA methylation patterns, not only at specific loci but also at Alu Yb8 repetitive sequences, are altered in infertile individuals compared with fertile controls. WHAT IS KNOWN ALREADY Aberrant DNA methylation of sperm has been associated with human male infertility in patients demonstrating either deficiencies in the process of spermatogenesis or low semen quality. STUDY DESIGN, SIZE, DURATION Case and control prospective study. This study compares 46 sperm samples obtained from 17 normospermic fertile men and 29 normospermic infertile patients. PARTICIPANTS/MATERIALS, SETTING, METHODS Illumina Infinium HD Human Methylation 450K arrays were used to identify genomic regions showing differences in sperm DNA methylation patterns between five fertile and seven infertile individuals. Additionally, global DNA methylation of sperm was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek) in 14 samples, and DNA methylation at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4) measured by bisulfite pyrosequencing in 44 sperm samples. A sperm-specific DNA methylation pattern was obtained by comparing the sperm methylomes with the DNA methylomes of differentiated somatic cells using data obtained from methylation arrays (Illumina 450 K) of blood, neural and glial cells deposited in public databases. MAIN RESULTS AND THE ROLE OF CHANCE In this study we conduct, for the first time, a genome-wide study to identify alterations of sperm DNA methylation in individuals with unexplained infertility that may account for the differences in their biological fertility compared with fertile individuals. We have identified 2752 CpGs showing aberrant DNA methylation patterns, and more importantly, these differentially methylated CpGs were significantly associated with CpG sites which are specifically methylated in sperm when compared with somatic cells. We also found statistically significant (P < 0.001) associations between DNA hypomethylation and regions corresponding to those which, in somatic cells, are enriched in the repressive histone mark H3K9me3, and between DNA hypermethylation and regions enriched in H3K4me1 and CTCF, suggesting that the relationship between chromatin context and aberrant DNA methylation of sperm in infertile men could be locus-dependent. Finally, we also show that DNA methylation patterns, not only at specific loci but also at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4), were lower in sperm than in somatic cells. Interestingly, sperm samples at Alu Yb8 repetitive sequences of infertile patients showed significantly lower DNA methylation levels than controls. LIMITATIONS, REASONS FOR CAUTION Our results are descriptive and further studies would be needed to elucidate the functional effects of aberrant DNA methylation on male fertility. WIDER IMPLICATIONS OF THE FINDINGS Overall, our data suggest that aberrant sperm DNA methylation might contribute to fertility impairment in couples with unexplained infertility and they provide a promising basis for future research. STUDY FUNDING/COMPETING INTERESTS This work has been financially supported by Fundación Cientifica de la AECC (to R.G.U.); IUOPA (to G.F.B.); FICYT (to E.G.T.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F); the Plan Nacional de I+D+I 2008-2011/2013-2016/FEDER (PI11/01728 to AF.F., PI12/01080 to M.F.F. and PI12/00361 to S.L.); the PN de I+D+I 2008-20011 and the Generalitat de Catalunya (2009SGR01490). A.F.F. is sponsored by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (CP11/00131). S.L. is sponsored by the Researchers Stabilization Program from the Spanish National Health System (CES09/020). The IUOPA is supported by the Obra Social Cajastur, Spain.

DNA methylation signatures identify biologically distinct thyroid cancer subtypes.

OBJECTIVE The purpose of this study was to determine the global patterns of aberrant DNA methylation in thyroid cancer. RESEARCH DESIGN AND METHODS We have used DNA methylation arrays to determine, for the first time, the genome-wide promoter methylation status of papillary, follicular, medullary, and anaplastic thyroid tumors. RESULTS We identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in differentiated papillary and follicular tumors, respectively. Interestingly, the other tumor types analyzed displayed more hypomethylated genes (280 in anaplastic and 393 in medullary tumors) than aberrantly hypermethylated genes (86 in anaplastic and 131 in medullary tumors). Among the genes indentified, we show that 4 potential tumor suppressor genes (ADAMTS8, HOXB4, ZIC1, and KISS1R) and 4 potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) are frequently regulated by aberrant methylation in primary thyroid tumors. In addition, we show that aberrant promoter hypomethylation-associated overexpression of MAP17 might promote tumor growth in thyroid cancer. CONCLUSIONS Thyroid cancer subtypes present differential promoter methylation signatures, and nondifferentiated subtypes are characterized by aberrant promoter hypomethylation rather than hypermethylation. Additional studies are needed to determine the potential clinical interest of the tumor subtype-specific DNA methylation signatures described herein and the role of aberrant promoter hypomethylation in nondifferentiated thyroid tumors.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors.

DNA methylation patterns in newborns exposed to tobacco in utero

BackgroundMaternal smoking during pregnancy is a major risk factor for adverse health outcomes. The main objective of the study was to assess the impact of in utero tobacco exposure on DNA methylation in children born at term with appropriate weight at birth.MethodsTwenty mother-newborn dyads, after uncomplicated pregnancies, in the absence of perinatal illness were included. All mothers were healthy with no cardiovascular risk factors, except for the associated risks among those mothers who smoked. Umbilical cord blood and maternal peripheral venous blood were collected and an epigenome-wide association study was performed using a 450 K epigenome-wide scan (Illumina Infinium HumanMethylation 450BeadChip) with adjustment to normalize the DNA methylation for data cell variability in whole blood.ResultsThe maternal plasmatic cotinine levels ranged from 10.70-115.40 ng/ml in the exposed group to 0-0.59 ng/ml in the non-exposed group. After adjusting for multiple comparisons in 427102 probes, statistically significant differences for 31 CpG sites, associated to 25 genes were observed. There was a greater than expected proportion of statistically-significant loci located in CpG islands (Fisher’s exact test, p = 0.029) and of those CpG islands, 90.3% exhibit higher methylation levels in the exposed group. The most striking and significant CpG site, cg05727225, is located in the chromosome 11p15.4, within the adrenomedullin gene.ConclusionsIn utero tobacco exposure, even in the absence of fetal growth restriction, may alter the epigenome, contributing to global DNA hypomethylation. Therefore, DNA status can be used as a biomarker of prenatal insults. Considering the possibility to reverse epigenetic modifications, a window of opportunity exists to change the programmed chronic disease.

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

Summary Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

DNA methylation changes in human lung epithelia cells exposed to multi-walled carbon nanotubes

Abstract Humans are increasingly exposed to nanoparticles and, although many of their physiological effects have been described, the molecular mechanisms underlying them are still largely unknown. The present study aimed to determine the possible role of certain epigenetic mechanisms in the cellular response of human lung epithelial cells that are triggered by long-term exposure to titanium dioxide nanoparticles (TiO2NPs) and multi-walled carbon nanotubes (MWCNTs). The results showed that exposure to TiO2NPs had only minor effects on genome-wide DNA methylation. However, we identified 755 CpG sites showing consistent DNA hypomethylation in cells exposed to MWCNTs. These sites were mainly located at low density CpG regions and enhancers, and very frequently on the X chromosome. Our results thus suggest that long-term MWCNT exposure may have important effects on the epigenome.

Cumulative/dynamic ROC curve estimation

ABSTRACT Receiver operating-characteristic (ROC) curve is a popular graphical method frequently used in order to study the diagnostic capacity of continuous (bio)markers. When the considered outcome is a time-dependent variable, the direct generalization is known as cumulative/dynamic ROC curve. For a fixed point of time, t, one subject is allocated into the positive group if the event happens before t and into the negative group if the event is not happened at t. The presence of censored subject, which can not be directly assigned into a group, is the main handicap of this approach. The proposed cumulative/dynamic ROC curve estimator assigns a probability to belong to the negative (positive) group to the subjects censored previously to t. The performance of the resulting estimator is studied from Monte Carlo simulations. Some real-world applications are reported. Results suggest that the new estimators provide a good approximation to the real cumulative/dynamic ROC curve.

Epigenetic dysregulation of TET2 in human glioblastoma

Ten-eleven translocation (TET) enzymes are frequently deregulated in cancer, but the underlying molecular mechanisms are still poorly understood. Here we report that TET2 shows frequent epigenetic alterations in human glioblastoma including DNA hypermethylation and hypo-hydroxymethylation, as well as loss of histone acetylation. Ectopic overexpression of TET2 regulated neural differentiation in glioblastoma cell lines and impaired tumor growth. Our results suggest that epigenetic dysregulation of TET2 plays a role in human glioblastoma.

Bioinformatics Tools in Epigenomics Studies

One of the most common problems in Epigenomics studies comes from the average size of the data set generated by modern sequencing and microarray platforms. Traditional analyses of data become unfeasible when dealing with huge sets of reads or big matrices, thus forcing the analyst not only to keep the biologic question in mind but also to master several, unrelated software tools. This book chapter aims to provide a gentle introduction to the vast universe of available software tools for the analysis of Epigenomics data. A general introduction to the most common epigenetic alterations is followed by a classification of available analysis tools. Although there are tools that are not mentioned in this chapter, those presented here will be a good starting point for anyone interested in Epigenomics Data Analysis.

SDHC Promoter Methylation, a Novel Pathogenic Mechanism in Parasympathetic Paragangliomas

Context Germline mutations in the succinate dehydrogenase A, B, C, and D genes (collectively, SDHx) predispose to the development of paragangliomas (PGLs) arising at the parasympathetic or sympathetic neuroendocrine systems. SDHx mutations cause absence of tumoral immunostaining for SDHB. However, negative SDHB immunostaining has also been found in a subset of PGLs that lack SDHx mutations. Settings Here, we report the comprehensive molecular characterization of one such a tumor of parasympathetic origin compared with healthy paraganglia and other PGLs with or without SDHx mutations. Results Integration of multiplatform data revealed somatic SDHC methylation and loss of the 1q23.3 region containing the SDHC gene. This correlated with decreased SDHC messenger RNA (mRNA) and protein levels. Furthermore, another genetic event found affected the VHL gene, which showed a decreased DNA copy number, associated with low VHL mRNA levels, and an absence of VHL protein detected by immunohistochemistry. In addition, the tumor displayed a pseudohypoxic phenotype consisting in overexpression of the hypoxia-inducible factor (HIF)-1α and miR-210, as well as downregulation of the iron-sulfur cluster assembly enzyme (ISCU) involved in SDHB maturation. This profile resembles that of SDHx- or VHL-mutated PGLs but not of PGLs with decreased VHL copy number, pointing to SDHC rather than VHL as the pathogenic driver. Conclusions Collectively, these findings demonstrate the potential importance of both the SDHC epigenomic event and the activation of the HIF-1α/miR-210/ISCU axis in the pathogenesis of SDHx wild-type/SDHB-negative PGLs. To our knowledge, this is the first case of a sporadic parasympathetic PGL that carries silencing of SDHC, fulfilling the two-hit Knudsons model for tumorigenesis.