Vacuolar Proton-Translocating ATPase May Take Part in the Drug Resistance Phenotype of Glioma Stem Cells.

The vacuolar proton-translocating ATPase (V-ATPase) is a transmembrane multi-protein complex fundamental in maintaining a normal intracellular pH. In the tumoral contest, its role is crucial since the metabolism underlying carcinogenesis is mainly based on anaerobic glycolytic reactions. Moreover, neoplastic cells use the V-ATPase to extrude chemotherapy drugs into the extra-cellular compartment as a drug resistance mechanism. In glioblastoma (GBM), the most malignant and incurable primary brain tumor, the expression of this pump is upregulated, making it a new possible therapeutic target. In this work, the bafilomycin A1-induced inhibition of V-ATPase in patient-derived glioma stem cell (GSC) lines was evaluated together with temozolomide, the first-line therapy against GBM. In contrast with previous published data, the proposed treatment did not overcome resistance to the standard therapy. In addition, our data showed that nanomolar dosages of bafilomycin A1 led to the blockage of the autophagy process and cellular necrosis, making the drug unusable in models which are more complex. Nevertheless, the increased expression of V-ATPase following bafilomycin A1 suggests a critical role of the proton pump in GBM stem components, encouraging the search for novel strategies to limit its activity in order to circumvent resistance to conventional therapy.

Liquid biopsy in non-small cell lung cancer: a meta-analysis of state-of-the-art and future perspectives.

Introduction: To date, tissue biopsy represents the gold standard for characterizing non-small-cell lung cancer (NSCLC), however, the complex architecture of the disease has introduced the need for new investigative approaches, such as liquid biopsy. Indeed, DNA analyzed in liquid biopsy is much more representative of tumour heterogeneity. Materials and methods: We performed a meta-analysis of 17 selected papers, to attest to the diagnostic performance of liquid biopsy in identifying EGFR mutations in NSCLC. Results: In the overall studies, we found a sensitivity of 0.59, specificity of 0.96 and diagnostic odds ratio of 24,69. Since we noticed a high heterogeneity among different papers, we also performed the meta-analysis in separate subsets of papers, divided by 1) stage of disease, 2) experimental design and 3) method of mutation detection. Liquid biopsy has the highest sensitivity/specificity in high-stage tumours, and prospective studies are more reliable than retrospective ones in terms of sensitivity and specificity, both NGS and PCR-based techniques can be used to detect tumour DNA in liquid biopsy. Discussion: Overall, liquid biopsy has the potential to help the management of NSCLC, but at present the non-homogeneous literature data, lack of optimal detection methods, together with relatively high costs make its applicability in routine diagnostics still challenging.

Granular cell tumor of the neurohypophysis presenting as a third ventricle mass.

Granular cell tumors of the neurohypophysis (GCT) are rare benign neoplasms belonging, along with pituicytoma and spindle cell oncocytoma, to the family of TTF1-positive low-grade neoplasms of the posterior pituitary gland. GCT usually present as a solid sellar mass, slowly growing and causing compressive symptoms over time, occasionally with suprasellar extension. They comprise polygonal monomorphous cells with abundant granular cytoplasm, which is ultrastructurally filled with lysosomes. Here we report the case of a GCT presenting as a third ventricle mass, radiologically mimicking chordoid glioma, with aberrant expression of GFAP and Annexin-A, which lends itself as an example of an integrated diagnostic approach to sellar/suprasellar and third ventricle masses.

A Rare Case of Urinary Bladder Hamartoma Clinically Mimicking an Urothelial Carcinoma: A Case Report and Review of the Literature.

Urinary bladder hamartoma is a rare benign proliferation with only 14 cases reported in the literature at present. Urinary bladder hamartoma is composed of a disorderly admixture of normal urinary bladder components, essentially represented by glands lined by transitional epithelium and a variable percentage of fibrous stroma, smooth muscle bundles, and adipose tissue. Urinary bladder hamartomas do not exhibit cytological or architectural abnormalities and show no necrosis or increase in mitotic activity. Clinical manifestations are usually represented by lower urinary tract symptoms, more or less frequently paired with gross hematuria. Several pediatric cases of urinary bladder hamartoma have been reported, sometimes with syndromic associations. Transurethral resection has been curative in all cases reported, with no evidence of recurrence. Here we report an additional rare urinary bladder hamartoma, clinically mimicking urothelial carcinoma, providing a review of the literature regarding this unusual entity.

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.

Mental health and the effects on methylation of stress-related genes in front-line versus other health care professionals during the second wave of COVID-19 pandemic: an Italian pilot study.

Healthcare workers experienced high degree of stress during COVID-19. Purpose of the present article is to compare mental health (depressive and Post-Traumatic-Stress-Disorders-PTSD-symptoms) and epigenetics aspects (degree of methylation of stress-related genes) in front-line healthcare professionals versus healthcare working in non-COVID-19 wards. Sixty-eight healthcare workers were included in the study: 39 were working in COVID-19 wards (cases) and 29 in non-COVID wards (controls). From all participants, demographic and clinical information were collected by an ad-hoc questionnaire. Depressive and PTSD symptoms were evaluated by the Patient Health Questionnaire-9 (PHQ-9) and the Impact of Event Scale-Revised (IES-R), respectively. Methylation analyses of 9 promoter/regulatory regions of genes known to be implicated in depression/PTSD (ADCYAP1, BDNF, CRHR1, DRD2, IGF2, LSD1/KDM1A, NR3C1, OXTR, SLC6A4) were performed on DNA from blood samples by the MassARRAY EpiTYPER platform, with MassCleave settings. Controls showed more frequent lifetime history of anxiety/depression with respect to cases (χ2 = 5.72, p = 0.03). On the contrary, cases versus controls presented higher PHQ-9 (t = 2.13, p = 0.04), PHQ-9 sleep item (t = 2.26, p = 0.03), IES-R total (t = 2.17, p = 0.03), IES-R intrusion (t = 2.46, p = 0.02), IES-R avoidance (t = 1.99, p = 0.05) mean total scores. Methylation levels at CRHR1, DRD2 and LSD1 genes was significantly higher in cases with respect to controls (p < 0.01, p = 0.03 and p = 0.03, respectively). Frontline health professionals experienced more negative effects on mental health during COVID-19 pandemic than non-frontline healthcare workers. Methylation levels were increased in genes regulating HPA axis (CRHR1) and dopamine neurotransmission (DRD2 and LSD1), thus supporting the involvement of these biological processes in depression/PTSD and indicating that methylation of these genes can be modulated by stress conditions, such as working as healthcare front-line during COVID-19 pandemic.

3D bioprinting of multi-layered segments of a vessel-like structure with ECM and novel derived bioink.

3D-Bioprinting leads to the realization of tridimensional customized constructs to reproduce the biological structural complexity. The new technological challenge focuses on obtaining a 3D structure with several distinct layers to replicate the hierarchical organization of natural tissues. This work aims to reproduce large blood vessel substitutes compliant with the original tissue, combining the advantages of the 3D bioprinting, decellularization, and accounting for the presence of different cells. The decellularization process was performed on porcine aortas. Various decellularization protocols were tested and evaluated through DNA extraction, quantification, and amplification by PCR to define the adequate one. The decellularized extracellular matrix (dECM), lyophilized and solubilized, was combined with gelatin, alginate, and cells to obtain a novel bioink. Several solutions were tested, tuning the percentage of the components to obtain the adequate structural properties. The geometrical model of the large blood vessel constructs was designed with SolidWorks, and the construct slicing was done using the HeartWare software, which allowed generating the G-Code. The final constructs were 3D bioprinted with the Inkredible + using dual print heads. The composition of the bioink was tuned so that it could withstand the printing of a segment of a tubular construct up to 10 mm and reproduce the multicellular complexity. Among the several compositions tested, the suspension resulting from 8% w/v gelatin, 7% w/v alginate, and 3% w/v dECM, and cells successfully produced the designed structures. With this bioink, it was possible to print structures made up of 20 layers. The dimensions of the printed structures were consistent with the designed ones. We were able to avoid the double bioink overlap in the thickness, despite the increase in the number of layers during the printing process. The optimization of the parameters allowed the production of structures with a height of 20 layers corresponding to 9 mm. Theoretical and real structures were very close. The differences were 14% in height, 20% internal diameter, and 9% thickness. By tailoring the printing parameters and the amount of dECM, adequate mechanical properties could be met. In this study, we developed an innovative printable bioink able to finely reproduce the native complex structure of the large blood vessel.

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

In the original publication [...].

Preferential X Chromosome Inactivation as a Mechanism to Explain Female Preponderance in Myasthenia Gravis.

Myasthenia gravis (MG) is a neuromuscular autoimmune disease characterized by prevalence in young women (3:1). Several mechanisms proposed as explanations for gender bias, including skewed X chromosome inactivation (XCI) and dosage or sex hormones, are often involved in the development of autoimmunity. The skewed XCI pattern can lead to an unbalanced expression of some X-linked genes, as observed in several autoimmune disorders characterized by female predominance. No data are yet available regarding XCI and MG. We hypothesize that the preferential XCI pattern may contribute to the female bias observed in the onset of MG, especially among younger women. XCI analysis was performed on blood samples of 284 women between the ages of 20 and 82. XCI was tested using the Human Androgen Receptor Assay (HUMARA). XCI patterns were classified as random (XCI < 75%) and preferential (XCI ≥ 75%). In 121 informative patients, the frequency of skewed XCI patterns was 47%, significantly higher than in healthy controls (17%; p ≤ 0.00001). Interestingly, the phenomenon was observed mainly in younger patients (<45 years; p ≤ 0.00001). Furthermore, considering the XCI pattern and the other clinical characteristics of patients, no significant differences were found. In conclusion, we observed preferential XCI in MG female patients, suggesting its potential role in the aetiology of MG, as observed in other autoimmune diseases in women.

MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines.

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness.

Assessment of pregnancy dietary intake and association with maternal and neonatal outcomes.

BACKGROUND: Maternal dietary habits are contributors of maternal and fetal health; however, available data are heterogeneous and not conclusive. METHODS: Nutrient intake during pregnancy was assessed in 503 women with uncomplicated pregnancies, using the validated Food Frequency Questionnaire developed by the European Prospective Investigation into Cancer and Nutrition (EPIC-FFQ). RESULTS: In all, 68% of women had a normal body mass index at the beginning of pregnancy, and 83% of newborns had an appropriate weight for gestational age. Maternal pre-pregnancy body mass index (BMI), gestational weight gain (GWG), and placental weight were independently correlated with birth weight. GWG was not related to the pre-pregnancy BMI. EPIC-FFQ evaluation showed that 30% of women adhered to the European Food Safety Authority (EFSA) ranges for macronutrient intake. In most pregnant women (98.1%), consumption of water was below recommendations. Comparing women with intakes within EFSA ranges for macronutrients with those who did not, no differences were found in BMI, GWG, and neonatal or placental weight. Neither maternal nor neonatal parameters were associated with the maternal dietary profiles. CONCLUSIONS: In our population, maternal pre-pregnancy BMI, GWG, and placental weight are determinants of birth weight percentile, while no association was found with maternal nutrition. Future studies should explore associations through all infancy. IMPACT: Maternal anthropometrics and nutrition status may affect offspring birth weight. In 503 healthy women, maternal pre-pregnancy body mass index (BMI), gestational weight gain (GWG), and placental weight were independently correlated to neonatal birth weight. GWG was not related to the pre-pregnancy BMI. In all, 30% of women respected the EFSA ranges for macronutrients. Neither maternal nor neonatal parameters were associated with maternal dietary profiles considered in this study. Maternal pre-pregnancy BMI, GWG, and placental weight are determinants of neonatal birth weight percentile, while a connection with maternal nutrition profiles was not found.

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.

The Classification of Myeloproliferative Neoplasms: Rationale, Historical Background and Future Perspectives with Focus on Unclassifiable Cases.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by increased proliferation of one or more myeloid lineages in the bone marrow. The classification and diagnostic criteria of MPNs have undergone relevant changes over the years, reflecting the increased awareness on these conditions and a better understanding of their biological and clinical-pathological features. The current World Health Organization (WHO) Classification acknowledges four main sub-groups of MPNs: (i) Chronic Myeloid Leukemia; (ii) classical Philadelphia-negative MPNs (Polycythemia Vera; Essential Thrombocythemia; Primary Myelofibrosis); (iii) non-classical Philadelphia-negative MPNs (Chronic Neutrophilic Leukemia; Chronic Eosinophilic Leukemia); and (iv) MPNs, unclassifiable (MPN-U). The latter are currently defined as MPNs with clinical-pathological findings not fulfilling the diagnostic criteria for any other entity. The MPN-U spectrum traditionally encompasses early phase MPNs, terminal (i.e., advanced fibrotic) MPNs, and cases associated with inflammatory or neoplastic disorders that obscure the clinical-histological picture. Several lines of evidence and clinical practice suggest the existence of additional myeloid neoplasms that may expand the spectrum of MPN-U. To gain insight into such disorders, this review addresses the history of MPN classification, the evolution of their diagnostic criteria and the complex clinical-pathological and biological features of MPN-U.

Triple-Negative Essential Thrombocythemia: Clinical-Pathological and Molecular Features. A Single-Center Cohort Study.

Lack of demonstrable mutations affecting JAK2, CALR, or MPL driver genes within the spectrum of BCR-ABL1-negative myeloproliferative neoplasms (MPNs) is currently referred to as a triple-negative genotype, which is found in about 10% of patients with essential thrombocythemia (ET) and 5-10% of those with primary myelofibrosis (PMF). Very few papers are presently available on triple-negative ET, which is basically described as an indolent disease, differently from triple-negative PMF, which is an aggressive myeloid neoplasm, with a significantly higher risk of leukemic evolution. The aim of the present study was to evaluate the bone marrow morphology and the clinical-laboratory parameters of triple-negative ET patients, as well as to determine their molecular profile using next-generation sequencing (NGS) to identify any potential clonal biomarkers. We evaluated a single-center series of 40 triple-negative ET patients, diagnosed according to the 2017 WHO classification criteria and regularly followed up at the Hematology Unit of our Institution, between January 1983 and January 2019. In all patients, NGS was performed using the Illumina Ampliseq Myeloid Panel; morphological and immunohistochemical features of the bone marrow trephine biopsies were also thoroughly reviewed. Nucleotide variants were detected in 35 out of 40 patients. In detail, 29 subjects harbored one or two variants and six cases showed three or more concomitant nucleotide changes. The most frequent sequence variants involved the TET2 gene (55.0%), followed by KIT (27.5%). Histologically, most of the cases displayed a classical ET morphology. Interestingly, prevalent megakaryocytes morphology was more frequently polymorphic with a mixture of giant megakaryocytes with hyperlobulated nuclei, normal and small sized maturing elements, and naked nuclei. Finally, in five cases a mild degree of reticulin fibrosis (MF-1) was evident together with an increase in the micro-vessel density. By means of NGS we were able to identify nucleotide variants in most cases, thus we suggest that a sizeable proportion of triple-negative ET patients do have a clonal disease. In analogy with driver genes-mutated MPNs, these observations may prevent issues arising concerning triple-negative ET treatment, especially when a cytoreductive therapy may be warranted.

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.

(Epi)genetic profiling of extraembryonic and postnatal tissues from female monozygotic twins discordant for Beckwith-Wiedemann syndrome.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder caused by defects at the 11p15.5 imprinted region. Many cases of female monozygotic (MZ) twins discordant for BWS have been reported, but no definitive conclusions have been drawn regarding the link between epigenetic defects, twinning process, and gender. Here, we report a comprehensive characterization and follow-up of female MZ twins discordant for BWS. METHODS: Methylation pattern at 11p15.5 and multilocus methylation disturbance (MLID) profiling were performed by pyrosequencing and MassARRAY in placental/umbilical cord samples and postnatal tissues. Whole-exome sequencing was carried out to identify MLID causative mutations. X-chromosome inactivation (XCI) was determined by HUMARA test. RESULTS: Both twins share KCNQ1OT1:TSS-DMR loss of methylation (LOM) and MLID in blood and the epigenetic defect remained stable in the healthy twin over time. KCNQ1OT1:TSS-DMRLOM was nonhomogeneously distributed in placental samples and the twins showed the same severely skewed XCI pattern. No MLID-causative mutations were identified. CONCLUSION: This is the first report on BWS-discordant twins with methylation analyses extended to extraembryonic tissues. The results suggest that caution is required when attempting prenatal diagnosis in similar cases. Although the causative mechanism underlying LOM remains undiscovered, the XCI pattern and mosaic LOM suggest that both twinning and LOM/MLID occurred after XCI commitment.

Profound alterations of the chromatin architecture at chromosome 11p15.5 in cells from Beckwith-Wiedemann and Silver-Russell syndromes patients.

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are imprinting-related disorders associated with genetic/epigenetic alterations of the 11p15.5 region, which harbours two clusters of imprinted genes (IGs). 11p15.5 IGs are regulated by the methylation status of imprinting control regions ICR1 and ICR2. 3D chromatin structure is thought to play a pivotal role in gene expression control; however, chromatin architecture models are still poorly defined in most cases, particularly for IGs. Our study aimed at elucidating 11p15.5 3D structure, via 3C and 3D FISH analyses of cell lines derived from healthy, BWS or SRS children. We found that, in healthy cells, IGF2/H19 and CDKN1C/KCNQ1OT1 domains fold in complex chromatin conformations, that facilitate the control of IGs mediated by distant enhancers. In patient-derived cell lines, we observed a profound impairment of such a chromatin architecture. Specifically, we identified a cross-talk between IGF2/H19 and CDKN1C/KCNQ1OT1 domains, consisting in in cis, monoallelic interactions, that are present in healthy cells but lost in patient cell lines: an inter-domain association that sees ICR2 move close to IGF2 on one allele, and to H19 on the other. Moreover, an intra-domain association within the CDKN1C/KCNQ1OT1 locus seems to be crucial for maintaining the 3D organization of the region.

DNA Methylation in the Diagnosis of Monogenic Diseases.

DNA methylation in the human genome is largely programmed and shaped by transcription factor binding and interaction between DNA methyltransferases and histone marks during gamete and embryo development. Normal methylation profiles can be modified at single or multiple loci, more frequently as consequences of genetic variants acting in cis or in trans, or in some cases stochastically or through interaction with environmental factors. For many developmental disorders, specific methylation patterns or signatures can be detected in blood DNA. The recent use of high-throughput assays investigating the whole genome has largely increased the number of diseases for which DNA methylation analysis provides information for their diagnosis. Here, we review the methylation abnormalities that have been associated with mono/oligogenic diseases, their relationship with genotype and phenotype and relevance for diagnosis, as well as the limitations in their use and interpretation of results.

Analysis of BRCA1 and RAD51C Promoter Methylation in Italian Families at High-Risk of Breast and Ovarian Cancer.

Previous studies on breast and ovarian carcinoma (BC and OC) revealed constitutional BRCA1 and RAD51C promoter hypermethylation as epigenetic alterations leading to tumor predisposition. Nevertheless, the impact of epimutations at these genes is still debated. One hundred and eight women affected by BC, OC, or both and considered at very high risk of carrying BRCA1 germline mutations were studied. All samples were negative for pathogenic variants or variants of uncertain significance at BRCA testing. Quantitative BRCA1 and RAD51C promoter methylation analyses were performed by Epityper mass spectrometry on peripheral blood samples and results were compared with those in controls. All the 108 analyzed cases showed methylation levels at the BRCA1/RAD51C promoter comparable with controls. Mean methylation levels (± stdev) at the BRCA1 promoter were 4.3% (± 1.4%) and 4.4% (± 1.4%) in controls and patients, respectively (p > 0.05; t-test); mean methylation levels (± stdev) at the RAD51C promoter were 4.3% (± 0.9%) and 3.7% (± 0.9%) in controls and patients, respectively (p > 0.05; t-test). Based on these observations; the analysis of constitutional methylation at promoters of these genes does not seem to substantially improve the definition of cancer risks in patients. These data support the idea that epimutations represent a very rare event in high-risk BC/OC populations.