Angela Lopomo

Genetic and epigenetic biomarkers for diagnosis, prognosis and treatment of colorectal cancer

Colorectal cancer (CRC) is one of the most common cancer worldwide and results from the accumulation of mutations and epimutations in colonic mucosa cells ultimately leading to cell proliferation and metastasis. Unfortunately, CRC prognosis is still poor and the search of novel diagnostic and prognostic biomarkers is highly desired to prevent CRC-related deaths. The present article aims to summarize the most recent findings concerning the use of either genetic or epigenetic (mainly related to DNA methylation) biomarkers for CRC diagnosis, prognosis, and response to treatment. Recent large-scale DNA methylation studies suggest that CRC can be divided into several subtypes according to the frequency of DNA methylation and those of mutations in key CRC genes, and that this is reflected by different prognostic outcomes. Increasing evidence suggests that the analysis of DNA methylation in blood or fecal specimens could represent a valuable non-invasive diagnostic tool for CRC. Moreover, a broad spectrum of studies indicates that the inter-individual response to chemotherapeutic treatments depends on both epigenetic modifications and genetic mutations occurring in colorectal cancer cells, thereby opening the way for a personalized medicine. Overall, combining genetic and epigenetic data might represent the most promising tool for a proper diagnostic, prognostic and therapeutic approach.

Obesity and diabetes: from genetics to epigenetics.

Obesity is becoming an epidemic health problem. During the last years not only genetic but also, and primarily, environmental factors have been supposed to contribute to the susceptibility to weight gain or to develop complications such as type 2 diabetes. In spite of the intense efforts to identify genetic predisposing variants, progress has been slow and success limited, and the common obesity susceptibility variants identified only explains a small part of the individual variation in risk. Moreover, there is evidence that the current epidemic of obesity and diabetes is environment-driven. Recent studies indicate that normal metabolic regulation during adulthood besides requiring a good balance between energy intake and energy expenditure, can be also affected by pre- and post-natal environments. In fact, maternal nutritional constraint during pregnancy can alter the metabolic phenotype of the offspring by means of epigenetic regulation of specific genes, and this can be passed to the next generations. Studies focused on epigenetic marks in obesity found altered methylation and/or histone acetylation levels in genes involved in specific but also in more general metabolic processes. Recent researches point out the continuous increase of “obesogens”, in the environment and food chains, above all endocrine disruptors, chemicals that interfere with many homeostatic mechanisms. Taken into account the already existing data on the effects of obesogens, and the multiple potential targets with which they might interfere daily, it seems likely that the exposure to obesogens can have an important role in the obesity and diabesity pandemic.

Epigenetics of Obesity.

Obesity is a metabolic disease, which is becoming an epidemic health problem: it has been recently defined in terms of Global Pandemic. Over the years, the approaches through family, twins and adoption studies led to the identification of some causal genes in monogenic forms of obesity but the origins of the pandemic of obesity cannot be considered essentially due to genetic factors, because human genome is not likely to change in just a few years. Epigenetic studies have offered in recent years valuable tools for the understanding of the worldwide spread of the pandemic of obesity. The involvement of epigenetic modifications-DNA methylation, histone tails, and miRNAs modifications-in the development of obesity is more and more evident. In the epigenetic literature, there are evidences that the entire embryo-fetal and perinatal period of development plays a key role in the programming of all human organs and tissues. Therefore, the molecular mechanisms involved in the epigenetic programming require a new and general pathogenic paradigm, the Developmental Origins of Health and Disease theory, to explain the current epidemiological transition, that is, the worldwide increase of chronic, degenerative, and inflammatory diseases such as obesity, diabetes, cardiovascular diseases, neurodegenerative diseases, and cancer. Obesity and its related complications are more and more associated with environmental pollutants (obesogens), gut microbiota modifications and unbalanced food intake, which can induce, through epigenetic mechanisms, weight gain, and altered metabolic consequences.

Exome sequencing coupled with mRNA analysis identifies NDUFAF6 as a Leigh gene

We report here the case of a young male who started to show verbal fluency disturbance, clumsiness and gait anomalies at the age of 3.5years and presented bilateral striatal necrosis. Clinically, the diagnosis was compatible with Leigh syndrome but the underlying molecular defect remained elusive even after exome analysis using autosomal/X-linked recessive or de novo models. Dosage of respiratory chain activity on fibroblasts, but not in muscle, underlined a deficit in complex I. Re-analysis of heterozygous probably pathogenic variants, inherited from one healthy parent, identified the p.Ala178Pro in NDUFAF6, a complex I assembly factor. RNA analysis showed an almost mono-allelic expression of the mutated allele in blood and fibroblasts and puromycin treatment on cultured fibroblasts did not lead to the rescue of the maternal allele expression, not supporting the involvement of nonsense-mediated RNA decay mechanism. Complementation assay underlined a recovery of complex I activity after transduction of the wild-type gene. Since the second mutation was not detected and promoter methylation analysis resulted normal, we hypothesized a non-exonic event in the maternal allele affecting a regulatory element that, in conjunction with the paternal mutation, leads to the autosomal recessive disorder and the different allele expression in various tissues. This paper confirms NDUFAF6 as a genuine morbid gene and proposes the coupling of exome sequencing with mRNA analysis as a method useful for enhancing the exome sequencing detection rate when the simple application of classical inheritance models fails.

Application of artificial neural networks to link genetic and environmental factors to DNA methylation in colorectal cancer.

AIMSnWe applied artificial neural networks (ANNs) to understand the connections among polymorphisms of genes involved in folate metabolism, clinico-pathological features and promoter methylation levels of MLH1, APC, CDKN2A(INK4A), MGMT and RASSF1A in 83 sporadic colorectal cancer (CRC) tissues, and to link dietary and lifestyle factors with gene promoter methylation.nnnMATERIALS & METHODSnPromoter methylation was assessed by means of methylation-sensitive high-resolution melting and genotyping by PCR-RFLP technique. Data were analyzed with the Auto Contractive Map, a special kind of ANN able to define the strength of the association of each variable with all the others and to visually show the map of the main connections.nnnRESULTSnWe observed a strong connection between the low methylation levels of the five CRC genes and the MTR 2756AA genotype. Several other connections were revealed, including those between dietary and lifestyle factors and the methylation levels of CRC genes.nnnCONCLUSIONnANNs revealed the complexity of the interconnections among factors linked to DNA methylation in CRC.

Autoimmune Thyroiditis and Myasthenia Gravis

Autoimmune diseases (AIDs) are the result of specific immune responses directed against structures of the self. In normal conditions, the molecules recognized as “self” are tolerated by immune system, but when the self-tolerance is lost, the immune system could react against molecules from the body, causing the loss of self-tolerance, and subsequently the onset of AID that differs for organ target and etiology. Autoimmune thyroid disease (ATD) is caused by the development of autoimmunity against thyroid antigens and comprises Hashimoto’s thyroiditis and Graves disease. They are frequently associated with other organ or non-organ specific AIDs, such as myasthenia gravis (MG). In fact, ATD seems to be the most associated pathology to MG. The etiology of both diseases is multifactorial and it is due to genetic and environmental factors, and each of them has specific characteristics. The two pathologies show many commonalities, such as the organ-specificity with a clear pathogenic effect of antibodies, the pathological mechanisms, such as deregulation of the immune system and the implication of the genetic predisposition. They also show some differences, such as the mode of action of the antibodies and therapies. In this review that focuses on ATD and MG, the common features and the differences between the two diseases are discussed.

Pharmacoepigenetics and pharmacoepigenomics of gastrointestinal cancers

ABSTRACT Introduction: Our understanding of the epigenetic changes occurring in gastrointestinal cancers has gained tremendous advancements in recent years, and some epigenetic biomarkers are already translated into the clinics for cancer diagnostics. In parallel, pharmacoepigenetics and pharmacoepigenomics of solid tumors are relevant novel, but emerging and promising fields. Areas covered: A comprehensive review of the literature to summarize and update the emerging field of pharmacoepigenetics and pharmacoepigenomics of gastrointestinal cancers. Expert commentary: Several epigenetic modifications have been proposed to account for interindividual variations in drug response in gastrointestinal cancers. Similarly, single-agent or combined strategies with high doses of drugs that target epigenetic modifications (epi-drugs) were scarcely tolerated by the patients, and current research has moved to their combination with standard therapies to achieve chemosensitization, radiosensitization, and immune modulation of cancerous cells. In parallel, recent genome-wide technologies are revealing the pathways that are epigenetically deregulated during cancer-acquired resistance, including those targeted by non-coding RNAs. Indeed, novel, less toxic, and more specific molecules are under investigation to specifically target those pathways. The field is rapidly expanding and gathering together information coming from these investigations has the potential to lead to clinical applications in the coming new years.

Gene-Specific Methylation Analysis in Thymomas of Patients with Myasthenia Gravis

Thymomas are uncommon neoplasms that arise from epithelial cells of the thymus and are often associated with myasthenia gravis (MG), an autoimmune disease characterized by autoantibodies directed to different targets at the neuromuscular junction. Little is known, however, concerning epigenetic changes occurring in thymomas from MG individuals. To further address this issue, we analyzed DNA methylation levels of genes involved in one-carbon metabolism (MTHFR) and DNA methylation (DNMT1, DNMT3A, and DNMT3B) in blood, tumor tissue, and healthy thymic epithelial cells from MG patients that underwent a surgical resection of a thymic neoplasm. For the analyses we applied the methylation-sensitive high-resolution melting technique. Both MTHFR and DNMT3A promoters showed significantly higher methylation in tumor tissue with respect to blood, and MTHFR also showed significantly higher methylation levels in tumor tissue respect to healthy adjacent thymic epithelial cells. Both DNMT1 and DNMT3B promoter regions were mostly hypomethylated in all the investigated tissues. The present study suggests that MTHFR methylation is increased in thymomas obtained from MG patients; furthermore, some degrees of methylation of the DNMT3A gene were observed in thymic tissue with respect to blood.

The thymidylate synthase enhancer region (TSER) polymorphism increases the risk of thymic lymphoid hyperplasia in patients with Myasthenia Gravis

BACKGROUNDnMyasthenia Gravis (MG) is caused, in approximately 80% of the patients, by autoantibodies against the nicotinic acetylcholine receptor (AChR). The disease is often associated with pathological changes of the thymus: thymic epithelial tumours are present in about 10-20% of the patients, while up to 80% of the patients with early disease onset have thymic hyperplasia. Folate metabolism is required for the production of DNA precursors and for proper DNA methylation reactions, and impaired folate metabolism has been often associated with cellular growth and cancer.nnnMETHODSnWe investigated if major polymorphisms of folate-related genes, namely MTHFR c.677C>T, MTR c.2756A>G, MTRR c.66A>G and TYMS TSER (a 28-bp tandem repeat in the 5 promoter enhancer region of TYMS) increase the risk of pathological changes of the thymus in AChR+ MG patients. A total of 526 AChR+ MG patients, including 132 patients with normal (involuted) thymus, 146 patients with thymic hyperplasia, and 248 patients with a thymoma were included in the study. Allele and genotype comparisons were performed among the three study groups, after correcting for multiple testing.nnnRESULTSnThe frequency of the TYMS TSER 3R allele was significantly higher in MG patients with thymic hyperplasia (P=0.004), and the TYMS TSER 3R3R genotype was significantly associated with increased risk of thymic hyperplasia [OR 2.71 (95% CI: 1.34-5.47)].nnnCONCLUSIONSnThe 3R allele in the thymidylate synthase promoter enhancer region results in increased protein production, required for the synthesis of DNA precursors. The present study suggests that the TYMS TSER 3R allele increases the risk of thymic lymphoid hyperplasia in AChR+ MG patients.

Epigenetic Signatures in the Diagnosis and Prognosis of Cancer

Epigenetic marks, such as DNA methylation and histone-tail modifications, as well as noncoding RNAs, are gathering increasing interest as possible diagnostic or prognostic biomarkers of cancer. In this regard, the possibility to detect DNA methylation changes in circulating free DNA or in other specimens, such as urines, fecal material, or sputum samples from patients suffering from various forms of cancer, has already led to the development of noninvasive diagnostic tools or has been suggested as a potential screening approach for several human cancers. Similarly, many epigenetic changes occurring in cancerous tissues are increasingly considered to be predictors of disease aggressiveness or valid biomarkers for the choice of the most proper treatment. This chapter provides some examples of epigenetic biomarkers that have the potential for use in clinical practice of the major human cancers.