Violence against Women and Stress-Related Disorders: Seeking for Associated Epigenetic Signatures, a Pilot Study.

BACKGROUND: Violence against women is a relevant health and social problem with negative consequences on women's health. The interaction between genome and environmental factors, such as violence, represents one of the major challenges in molecular medicine. The Epigenetics for WomEn (EpiWE) project is a multidisciplinary pilot study that intends to investigate the epigenetic signatures associated with intimate partner and sexual violence-induced stress-related disorders. MATERIALS AND METHODS: In 2020, 62 women exposed to violence (13 women suffering from sexual violence and 49 from Intimate Partner Violence, IPV) and 50 women with no history of violence were recruited at the Service for Sexual and Domestic Violence. All women aged 18-65 were monitored for their physical and psychological conditions. Blood samples were collected, and DNAs were extracted and underwent the epigenetic analysis of 10 stress-related genes. RESULTS: PTSD prevalence in victims was assessed at 8.1%. Quantitative methylation evaluation of the ten selected trauma/stress-related genes revealed the differential iper-methylation of brain-derived neurotrophic factor, dopamine receptor D2 and insulin-like growth factor 2 genes. These genes are among those related to brain plasticity, learning, and memory pathways. CONCLUSIONS: The association of early detection of posttraumatic distress and epigenetic marker identification could represent a new avenue for addressing women survivors toward resilience. This innovative approach in gender-based violence studies could identify new molecular pathways associated with the long-term effects of violence and implement innovative protocols of precision medicine.

A perspective on diet, epigenetics and complex diseases: where is the field headed next?

Dietary factors can regulate epigenetic processes during life, modulating the intracellular pools of metabolites necessary for epigenetic reactions and regulating the activity of epigenetic enzymes. Their effects are strong during the prenatal life, when epigenetic patterns are written, allowing organogenesis. However, interactions between diet and the epigenome continue throughout life and likely contribute to the onset and progression of various complex diseases. Here, we review the contribution of dietary factors to the epigenetic changes observed in complex diseases and suggest future steps to better address this issue, focusing on neurobehavioral, neuropsychiatric and neurodegenerative disorders, cardiovascular diseases, obesity and Type 2 diabetes, cancer and inflammatory skin diseases.

Correction: Rondinone et al. Extensive Placental Methylation Profiling in Normal Pregnancies. Int. J. Mol. Sci. 2021, 22, 2136.

In the original publication [...].

Cohesin Mutations Induce Chromatin Conformation Perturbation of the H19/IGF2 Imprinted Region and Gene Expression Dysregulation in Cornelia de Lange Syndrome Cell Lines.

Traditionally, Cornelia de Lange Syndrome (CdLS) is considered a cohesinopathy caused by constitutive mutations in cohesin complex genes. Cohesin is a major regulator of chromatin architecture, including the formation of chromatin loops at the imprinted IGF2/H19 domain. We used 3C analysis on lymphoblastoid cells from CdLS patients carrying mutations in NIPBL and SMC1A genes to explore 3D chromatin structure of the IGF2/H19 locus and evaluate the influence of cohesin alterations in chromatin architecture. We also assessed quantitative expression of imprinted loci and WNT pathway genes, together with DMR methylation status of the imprinted genes. A general impairment of chromatin architecture and the emergence of new interactions were found. Moreover, imprinting alterations also involved the expression and methylation levels of imprinted genes, suggesting an association among cohesin genetic defects, chromatin architecture impairment, and imprinting network alteration. The WNT pathway resulted dysregulated: canonical WNT, cell cycle, and WNT signal negative regulation were the most significantly affected subpathways. Among the deregulated pathway nodes, the key node of the frizzled receptors was repressed. Our study provides new evidence that mutations in genes of the cohesin complex have effects on the chromatin architecture and epigenetic stability of genes commonly regulated by high order chromatin structure.

Clinical and Molecular Diagnosis of Beckwith-Wiedemann Syndrome with Single- or Multi-Locus Imprinting Disturbance.

Beckwith-Wiedemann syndrome (BWS) is a clinically and genetically heterogeneous overgrowth disease. BWS is caused by (epi)genetic defects at the 11p15 chromosomal region, which harbors two clusters of imprinted genes, IGF2/H19 and CDKN1C/KCNQ1OT1, regulated by differential methylation of imprinting control regions, H19/IGF2:IG DMR and KCNQ1OT1:TSS DMR, respectively. A subset of BWS patients show multi-locus imprinting disturbances (MLID), with methylation defects extended to other imprinted genes in addition to the disease-specific locus. Specific (epi)genotype-phenotype correlations have been defined in order to help clinicians in the classification of patients and referring them to a timely diagnosis and a tailored follow-up. However, specific phenotypic correlations have not been identified among MLID patients, thus causing a debate on the usefulness of multi-locus testing in clinical diagnosis. Finally, the high incidence of BWS monozygotic twins with discordant phenotypes, the high frequency of BWS among babies conceived by assisted reproductive technologies, and the female prevalence among BWS-MLID cases provide new insights into the timing of imprint establishment during embryo development. In this review, we provide an overview on the clinical and molecular diagnosis of single- and multi-locus BWS in pre- and post-natal settings, and a comprehensive analysis of the literature in order to define possible (epi)genotype-phenotype correlations in MLID patients.

Extensive Placental Methylation Profiling in Normal Pregnancies.

The placental methylation pattern is crucial for the regulation of genes involved in trophoblast invasion and placental development, both key events for fetal growth. We investigated LINE-1 methylation and methylome profiling using a methylation EPIC array and the targeted methylation sequencing of 154 normal, full-term pregnancies, stratified by birth weight percentiles. LINE-1 methylation showed evidence of a more pronounced hypomethylation in small neonates compared with normal and large for gestational age. Genome-wide methylation, performed in two subsets of pregnancies, showed very similar methylation profiles among cord blood samples while placentae from different pregnancies appeared very variable. A unique methylation profile emerged in each placenta, which could represent the sum of adjustments that the placenta made during the pregnancy to preserve the epigenetic homeostasis of the fetus. Investigations into the 1000 most variable sites between cord blood and the placenta showed that promoters and gene bodies that are hypermethylated in the placenta are associated with blood-specific functions, whereas those that are hypomethylated belong mainly to pathways involved in cancer. These features support the functional analogies between a placenta and cancer. Our results, which provide a comprehensive analysis of DNA methylation profiling in the human placenta, suggest that its peculiar dynamicity can be relevant for understanding placental plasticity in response to the environment.

Constitutive BRCA1 Promoter Hypermethylation Can Be a Predisposing Event in Isolated Early-Onset Breast Cancer.

Early age at onset of breast cancer (eoBC) is suggestive of an increased genetic risk. Although genetic testing is offered to all eoBC-affected women, in isolated cases the detection rate of pathogenic variants is <10%. This study aimed at assessing the role of constitutive promoter methylation at BC-associated loci as an underlying predisposing event in women with eoBC and negative family history. Promoter methylation at 12 loci was assessed by the MassARRAY technology in blood from 154 BRCA1/2 negative patients with eoBC and negative family history, and 60 healthy controls. Hypermethylation was determined, within each promoter, by comparing the patient's mean methylation value with thresholds based on one-sided 95% bootstrap confidence interval of the controls' mean. Three patients had hypermethylated results, two at BRCA1 and one at RAD51C. Analyses on tumor tissue from the patient exceeding the highest threshold at BRCA1 revealed a mean methylation >60% and loss of heterozygosity at chromosome 17q. The patient hypermethylated at RAD51C showed low methylation in the tumor sample, ruling out a role for methylation-induced silencing in tumor development. In isolated eoBC patients, BRCA1 constitutive promoter methylation may be a predisposing event. Further studies are required to define the impact of methylation changes occurring at BC-predisposing genes and their role in tumorigenesis.

A novel splice site variant in ITPR1 gene underlying recessive Gillespie syndrome.

Gillespie syndrome (GLSP) is a rare congenital disorder characterized by partial aniridia, hypotonia, progressive cerebellar hypoplasia, nonprogressive ataxia, and intellectual disability. All causative variants to date affect the central or the 3'-terminal domains of ITPR1 gene and exhibit autosomal recessive or dominant inheritance pattern. We investigated by exome sequencing the molecular cause of GLSP in a family composed by consanguineous healthy parents, two affected siblings and one healthy son. We found the novel splice site variant c.278_279 + 2delACGT located at the 5'-end of ITPR1. The affected siblings were homozygotes, their parents heterozygous carriers and the variant was absent in the healthy son, indicating a recessive inheritance pattern. The deletion abolished the splice-donor site at exon 5/intron 5 junction, causing the skipping of exon 5 and the generation of a premature STOP codon. The mutation is predicted to result in the synthesis of a 64-amino acids nonfunctional protein. The mutant transcript comprised >96% of ITPR1 mRNA in the affected siblings, indicating that a small amount of wild-type transcript was still present. The novel autosomal recessive mutation here reported is the first variant affecting the ITPR1 N-terminal suppressor domain, thus extending the spectrum of the pathogenetic variants in GLSP and the range of the associated clinical manifestations.

Molecular profiling of lung cancer specimens and liquid biopsies using MALDI-TOF mass spectrometry.

BACKGROUND: Identification of predictive molecular alterations in lung adenocarcinoma is essential for accurate therapeutic decisions. Although several molecular approaches are available, a number of issues, including tumor heterogeneity, frequent material scarcity, and the large number of loci to be investigated, must be taken into account in selecting the most appropriate technique. MALDI-TOF mass spectrometry (MS), which allows multiplexed genotyping, has been adopted in routine diagnostics as a sensitive, reliable, fast, and cost-effective method. Our aim was to test the reliability of this approach in detecting targetable mutations in non-small cell lung cancer (NSCLC). In addition, we also analyzed low-quality samples, such as cytologic specimens, that often, are the unique source of starting material in lung cancer cases, to test the sensitivity of the system. METHODS: We designed a MS-based assay for testing 158 mutations in the EGFR, KRAS, BRAF, ALK, PIK3CA, ERBB2, DDR2, AKT, and MEK1 genes and applied it to 92 NSCLC specimens and 13 liquid biopsies from another subset of NSCLC patients. We also tested the sensitivity of the method to distinguish low represented mutations using serial dilutions of mutated DNA. RESULTS: Our panel is able to detect the most common NSCLC mutations and the frequency of the mutations observed in our cohort was comparable to literature data. The assay identifies mutated alleles at frequencies of 2.5-10%. In addition, we found that the amount of DNA template was irrelevant to efficiently uncover mutated alleles present at high frequency. However, when using less than 10 ng of DNA, the assay can detect mutations present in at least 10% of the alleles. Finally, using MS and a commercial kit for RT-PCR we tested liquid biopsy from 13 patients with identified mutations in cancers and detected the mutations in 4 (MS) and in 5 samples (RT-PCR). CONCLUSIONS: MS is a powerful method for the routine predictive tests of lung cancer also using low quality and scant tissues. Finally, after appropriate validation and improvement, MS could represent a promising and cost-effective strategy for monitoring the presence and percentage of the mutations also in non-invasive sampling.

Epigenetic alterations in cancer and personalized cancer treatment.

Based on the pivotal importance of epigenetics for transcription regulation, it is not surprising that cancer is characterized by several epigenetic abnormalities. Conversely to genetic alterations, epigenetic changes are not permanent, thus represent opportunities for therapeutic strategies designed to reverse transcriptional abnormalities, and cancer is the first disease in which epigenetic therapies with chromatin remodeling agents were introduced. The role of miRNAs in gene regulation supports their potential as innovative therapeutic strategy. Recent evidences have proven that the environment can profoundly influence the epigenome: diet, smoking and alcohol consumption can negatively impact the expression profile. Given the plasticity of epigenetic marks, it is challenging the idea that the epigenetic alterations are 'druggable' sites using specific food components.

PDGFB hypomethylation is a favourable prognostic biomarker in primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterised by the clonal proliferation of the haematopoietic precursors together with the progressive development of bone marrow fibrosis. This stromal alteration is an important clinical issue and specific prognostic markers are not currently available. In bone marrow biopsies from 58 PMF patients, we explored the methylation pattern of genes encoding cytokines involved in the stromal reaction, namely platelet-derived growth factor-beta (PDGFB), transforming growth factor-beta (TGFB) and basic fibroblast growth factor (FGF2). We also evaluated the methylation profile of the Long Interspersed Nucleotide Element 1 (LINE-1). PDGFB, FGF2 and LINE-1, but not TGFB, were significantly differently methylated in PMF compared to controls. Significantly, PDGFB hypomethylation (<16%) was correlated with a favourable PMF prognosis (grade of marrow fibrosis, p=0.03; International Prognostic Scoring Systems p=0.01 and Dynamic International Prognostic Scoring Systems, p=0.02). Although the basis of the association of PDGFB hypomethylation with favourable prognosis remains to be clarified, we speculate that hypomethylation in PMF could represent the effect of acquired somatic mutations in genes involved in epigenetic regulation of the genome.

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) plays a role in cell cycle progression and cytoskeletal dynamics.

MARK4 is a serine-threonine kinase that phosphorylates MAP proteins, increasing microtubule dynamics. MARK4 differs from the other members of the MARK family for encoding two isoforms (MARK4L and MARK4S), differentially expressed in the nervous system, and for the peculiar localisation at the centrosome and the midbody. By cytofluorimetric analysis we showed that MARK4 is expressed throughout the cell cycle and preferentially activated during mitosis. Depletion of MARK4S affected the morphology and proliferation of fibroblasts and glioma cells, as the percentages of cells in S and G2/M phases were reduced and the percentage of cells in G1 was increased. In MARK4S silenced cells, centrosomes were duplicated and positioned apically to the nucleus, indicating that the centrosome cycle was altered and the cells arrested in G1 phase. Overexpression of MARK4L or MARK4S reduced the density of the microtubule network, confirming microtubules as the main target of MARK4, and revealed a novel co-localisation of MARK4 and vimentin. Taken together, our data confirm that MARK4 is a key component in the regulation of microtubule dynamics and highlight its major role in cell cycle progression, particularly at the G1/S transition. The co-localisation of vimentin and MARK4L suggests that MARK4 has a wide-ranging influence on cytoskeleton.

Genome-wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with changes in transcriptional profiles.

Primary plasma cell leukemia (pPCL) is a rare, yet aggressive form of de novo plasma cell tumor, distinct from secondary PCL (sPCL) which represents a leukemic transformation of pre-existing multiple myeloma (MM). Herein, we performed a comprehensive molecular analysis of a prospective series of pPCLs by means of FISH, single nucleotide polymorphism (SNP) array and gene expression profiling (GEP). IGH@ translocations were identified in 87% of pPCL cases, with prevalence of t(11;14) (40%) and t(14;16) (30.5%), whereas the most frequent numerical alterations involved 1p (38%), 1q (48%), 6q (29%), 8p (42%), 13q (74%), 14q (71%), 16q (53%), and 17p (35%). We identified a minimal biallelic deletion (1.5 Mb) in 8p21.2 encompassing the PPP2R2A gene, belonging to a family of putative tumor suppressors and found to be significantly down-regulated in deleted cases. Mutations of TP53 were identified in four cases, all but one associated with a monoallelic deletion of the gene, whereas activating mutations of the BRAF oncogene occurred in one case and were absent in N- and K-RAS. To evaluate the influence of allelic imbalances in transcriptional expression we performed an integrated genomic analysis with GEP data, showing a significant dosage effect of genes involved in transcription, translation, methyltransferase activity, apoptosis as well as Wnt and NF-kB signaling pathways. Overall, we provide a compendium of genomic alterations in a prospective series of pPCLs which may contribute to improve our understanding of the pathogenesis of this aggressive form of plasma cell dyscrasia and the mechanisms of tumor progression in MM.

Significance of clustered tumor suppressor genes in cancer.

The two-hit model is a well-known mechanism for the inactivation of tumor suppressor genes in cancer and it has been assumed that chromosomal deletions are the second inactivating event. Large deletions are frequently found in cancer and can lead to the haploinsufficiency of the loci mapped to the deleted region. The study by Xue et al. demonstrated that hemizygous 8p deletions can attenuate the activity of multiple genes that control growth and promote tumorigenesis, and showed that the effect of large 8p deletions on tumor phenotype goes beyond the effects of the individual genes as the characteristics of a tumor are also influenced by the additive and/or combined effect of the haploinsufficiency of multiple genes. These convincing findings, demonstrating that the hemizygosity of a cluster of genes negatively regulates proliferation and promotes tumor growth, have opened up new study perspectives aimed at characterizing the genomic organization of this new class of tumor suppressor genes and their role in tumorigenesis.

Juxtaposition of heterochromatic and euchromatic regions by chromosomal translocation mediates a heterochromatic long-range position effect associated with a severe neurological phenotype.

BACKGROUND: The term "position effect" is used when the expression of a gene is deleteriously affected by an alteration in its chromosomal environment even though the integrity of the protein coding sequences is maintained. We describe a patient affected by epilepsy and severe neurodevelopment delay carrying a balanced translocation t(15;16)(p11.2;q12.1)dn that we assume caused a position effect as a result of the accidental juxtaposition of heterochromatin in the euchromatic region. RESULTS: FISH mapped the translocation breakpoints (bkps) to 15p11.2 within satellite III and the 16q12.1 euchromatic band within the ITFG1 gene. The expression of the genes located on both sides of the translocation were tested by means of real-time PCR and three, all located on der(16), were found to be variously perturbed: the euchromatic gene NETO2/BTCL2 was silenced, whereas VPS35 and SHCBP1, located within the major heterochromatic block of chromosome 16q11.2, were over-expressed. Pyrosequencing and chromatin immunoprecipitation of NETO2/BTCL2 and VPS35 confirmed the expression findings. Interphase FISH analysis showed that der(16) localised to regions occupied by the beta satellite heterochromatic blocks more frequently than der(15). CONCLUSIONS: To the best of our knowledge, this is the first report of a heterochromatic position effect in humans caused by the juxtaposition of euchromatin/heterochromatin as a result of chromosomal rearrangement. The overall results are fully in keeping with the observations in Drosophila and suggest the occurrence of a human heterochromatin position effect associated with the nuclear repositioning of the der(16) and its causative role in the patient's syndromic phenotype.

The methylation of the TSC2 promoter underlies the abnormal growth of TSC2 angiomyolipoma-derived smooth muscle cells.

Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that is caused by mutations in either the TSC1 or TSC2 gene. Smooth muscle-like cells (ASMs) were isolated from an angiomyolipoma of a patient with TSC. These cells lacked tuberin, were labeled by both HMB45 and CD44v6 antibodies, and had constitutive S6 phosphorylation. The cells bear a germline TSC2 intron 8-exon 9 junction mutation, but DNA analysis and polymerase chain reaction amplification failed to demonstrate loss of heterozygosity. Testing for an epigenetic alteration, we detected methylation of the TSC2 promoter. Its biological relevance was confirmed by tuberin expression and a reduction in HMB45 labeling and S6 constitutive phosphorylation after exposure to the chromatin-remodeling agents, trichostatin A and 5-azacytidine. These cells were named TSC2(-/meth) ASMs. Their proliferation required epidermal growth factor in the medium as previously described for TSC2(-/-) ASMs. Blockade of epidermal growth factor with monoclonal antibodies caused the death of TSC2(-/meth) ASMs. In addition, rapamycin effectively blocked the proliferation of these cells. Our data show for the first time that methylation of the TSC2 promoter might cause a complete loss of tuberin in TSC2 cells, and that the pathogenesis of angiomyolipomas might also originate from epigenetic defects in smooth muscle cells. Additionally, the effect of chromatin-remodeling agents in these cells suggests a further avenue for the treatment of TSC as well as lymphangioleiomyomatosis.

Loss of heterozygosity on chromosome 4q32-35 in sporadic basal cell carcinomas: evidence for the involvement of p33ING2/ING1L and SAP30 genes.

BACKGROUND: Studies on basal cell carcinoma (BCC) have demonstrated that patched gene and p53 gene located at 9q22.3 and 17p13 are the main genes responsible for the onset of this tumor. In order to identify a possible involvement of other tumor suppressor genes, we screened 19 cases of BCCs for loss of heterozygosity (LOH). METHODS: The analysis was performed on tumoral tissue and on corresponding normal tissue by using a panel of 37 polymorphic markers spanning 26 chromosomal regions. RESULTS: We observed that only four chromosomal regions, 4q32 (30.00%), 4q35 (27.27%), 9q21-22 (28.57%), and 9q22-qter (35.71%), demonstrated a significative LOH (>20%), even if others show a borderline percentage (15-20%) of imbalance (1q32, 3p24, 10p22.1, and 17q21.3). CONCLUSIONS: Our findings suggest that a new chromosomal region mapping in the long arm of chromosome 4 could be involved in sporadic BCC carcinogenesis. Further analyses indicate that the minimal deleted region in 4q32-35 includes p33ING2/ING1L and SAP30, whose deletion could impair the G1-phase checkpoint control and favor gene silencing, respectively.