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Abstract Background The epigenetic status of patients 6-month post-COVID-19 infection remains largely unexplored. The existence of long-COVID, or post-acute sequelae of SARS-CoV-2 infection (PASC), suggests potential long-term changes. Long-COVID includes symptoms like fatigue, neurological issues, and organ-related problems, regardless of initial infection severity. The mechanisms behind long-COVID are unclear, but virus-induced epigenetic changes could play a role. Methods and results Our study explores the lasting epigenetic impacts of SARS-CoV-2 infection. We analyzed genome-wide DNA methylation patterns in an Italian cohort of 96 patients 6 months after COVID-19 exposure, comparing them to 191 healthy controls. We identified 42 CpG sites with significant methylation differences (FDR

Healthcare workers (HCWs) are a high-risk group for hepatitis B virus (HBV) infection. Notably, about 5-10% of the general population does not respond to the HBV vaccination. In this study, we aimed to investigate DNA methylation (DNAm) in order to estimate the biological age of B cells from HCW of both sexes, either responder (R) or non-responder (NR), to HBV vaccination. We used genome-wide DNA methylation data to calculate a set of biomarkers in B cells collected from 41 Rs and 30 NRs between 22 and 62 years old. Unresponsiveness to HBV vaccination was associated with accelerated epigenetic aging (DNAmAge, AltumAge, DunedinPoAm) and was accompanied by epigenetic drift. Female non-responders had higher estimates of telomere length and lower CRP inflammation risk score when compared to responders. Overall, epigenetic differences between responders and non-responders were more evident in females than males. In this study we demonstrated that several methylation DNAm-based clocks and biomarkers are associated with an increased risk of non-response to HBV vaccination, particularly in females. Based on these results, we propose that accelerated epigenetic age could contribute to vaccine unresponsiveness. These insights may help improve the evaluation of the effectiveness of vaccination strategies, especially among HCWs and vulnerable patients.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a strong genetic component in which rare variants contribute significantly to risk. We performed whole genome and/or exome sequencing (WGS and WES) and SNP-array analysis to identify both rare sequence and copy number variants (SNVs and CNVs) in 435 individuals from 116 ASD families. We identified 37 rare potentially damaging de novo SNVs (pdSNVs) in the cases (n = 144). Interestingly, two of them (one stop-gain and one missense variant) occurred in the same gene, BRSK2. Moreover, the identification of 8 severe de novo pdSNVs in genes not previously implicated in ASD (AGPAT3, IRX5, MGAT5B, RAB8B, RAP1A, RASAL2, SLC9A1, YME1L1) highlighted promising candidates. Potentially damaging CNVs (pdCNVs) provided support to the involvement of inherited variants in PHF3, NEGR1, TIAM1 and HOMER1 in neurodevelopmental disorders (NDD), although mostly acting as susceptibility factors with incomplete penetrance. Interpretation of identified pdSNVs/pdCNVs according to the ACMG guidelines led to a molecular diagnosis in 19/144 cases, although this figure represents a lower limit and is expected to increase thanks to further clarification of the role of likely pathogenic variants in ASD/NDD candidate genes not yet established. In conclusion, our study highlights promising ASD candidate genes and contributes to characterize the allelic diversity, mode of inheritance and phenotypic impact of de novo and inherited risk variants in ASD/NDD genes., (© 2024. The Author(s).)

During the last decades, our knowledge about the genetic architecture of sporadic amyotrophic lateral sclerosis (sALS) has significantly increased. However, besides the recognized genetic risk factors, also the environment is supposed to have a role in disease pathogenesis. Epigenetic modifications reflect the results of the interaction between environmental factors and genes and may play a role in the development and progression of ALS. A recent epigenome-wide association study (EWAS) in blood identified differentially methylated positions mapping to 42 genes involved in cholesterol biosynthesis and immune-related pathways. Here we performed a genome-wide DNA methylation analysis in the blood of an Italian cohort of 61 sALS patients and 61 healthy controls. Initially, a conventional genome-wide association analysis was performed, and results were subsequently integrated with the findings from the previous EWAS using a meta-analytical approach. To delve deeper into the significant outcomes, over-representation analysis (ORA) was employed. Moreover, the epigenetic signature obtained from the meta-analysis was examined to determine potential associations with chemical compounds, utilizing the Toxicogenomic Database. Expanding the scope of the epigenetic analysis, we explored both epigenetic drift and rare epivariations. Notably, we observed an elevated epigenetic drift in sALS patients compared to controls, both at a global and single gene level. Interestingly, epigenetic drift at a single gene level revealed an enrichment of genes related to the neurotrophin signaling pathway. Moreover, for the first time, we identified rare epivariations exclusively enriched in sALS cases associated with 153 genes, 88 of whom with a strong expression in cerebral areas. Overall, our study reinforces the evidence that epigenetics may contribute to the pathogenesis of ALS and that epigenetic drift may be a useful diagnostic marker. Moreover, this study suggests the potential role of epivariations in ALS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Brusati, Peverelli, Calzari, Tiloca, Casiraghi, Sorce, Invernizzi, Carbone, Cavagnola, Verde, Silani, Ticozzi, Ratti and Gentilini.)

Background and Objectives: Epigenetic estimators based on DNA methylation levels have emerged as promising biomarkers of human aging. These estimators exhibit natural variations across human groups, but data about indigenous populations remain underrepresented in research. This study aims to investigate differences in epigenetic estimators between two distinct human populations, both residing in the Gran Chaco region of Argentina, the Native-American Wichí, and admixed Criollos who are descendants of intermarriages between Native Americans and the first European colonizers, using a population genetic approach., Methodology: We analyzed 24 Wichí (mean age: 39.2 ± 12.9 yo) and 24 Criollos (mean age: 41.1 ± 14.0 yo) for DNA methylation levels using the Infinium MethylationEPIC (Illumina) to calculate 16 epigenetic estimators. Additionally, we examined genome-wide genetic variation using the HumanOmniExpress BeadChip (Illumina) to gain insights into the genetic history of these populations., Results: Our results indicate that Native-American Wichí are epigenetically older compared to Criollos according to five epigenetic estimators. Analyses within the Criollos population reveal that global ancestry does not influence the differences observed, while local (chromosomal) ancestry shows positive associations between specific SNPs located in genomic regions over-represented by Native-American ancestry and measures of epigenetic age acceleration (AgeAccelHannum). Furthermore, we demonstrate that differences in population ecologies also contribute to observed epigenetic differences., Conclusions and Implications: Overall, our study suggests that while the genomic history may partially account for the observed epigenetic differences, non-genetic factors, such as lifestyle and ecological factors, play a substantial role in the variability of epigenetic estimators, thereby contributing to variations in human epigenetic aging., Competing Interests: The authors declare no conflict of interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health.)

Oestrogen receptor expression in breast cancer (BC) cells is a marker of high cellular differentiation and allows the identification of two BC groups (ER-positive and ER-negative) that, although not completely homogeneous, differ in biological characteristics, clinical behaviour, and therapeutic options. The study, based on three publicly available EWAS (Epigenetic Wide Association Study) datasets, focuses on the comparison between these two groups of breast cancer using an epimutation score. The score is calculated not only based on the presence of the epimutation, but also on the deviation amplitude of the methylation outlier value. For each dataset, we performed a functional analysis based first on the functional gene region of each annotated gene (we aggregated the data per gene region TSS1500, TSS200, first-exon, and body-gene identified by the information from the Illumina Data Sheet), and then, we performed a pathway enrichment analysis through the REACTOME database based on the genes with the highest epimutation score. Thus, we blended our results and found common pathways for all three datasets. We found that a higher and significant epimutation score due to hypermethylation in ER-positive BC is present in the promoter region of the genes belonging to the SUMOylation pathway, the Notch pathway, the IFN-γ signalling pathway, and the deubiquitination protease pathway, while a higher and significant level of epimutation due to hypomethylation in ER-positive BC is present in the promoter region of the genes belonging to the ESR-mediated pathway. The presence of this state of promoter hypomethylation in the ESR-mediated signalling genes is consistent and coherent with an active signalling pathway mediated by oestrogen function in the group of ER-positive BC. The SUMOylation and Notch pathways are associated with BC pathogenesis and have been found to play distinct roles in the two BC subgroups. We speculated that the altered methylation profile may play a role in regulating signalling pathways with specific functions in the two subgroups of ER BC.

The majority of patients with Follicular Lymphoma (FL) experience subsequent phases of remission and relapse, making the disease "virtually" incurable. To predict the outcome of FL patients at diagnosis, various clinical-based prognostic scores have been proposed; nonetheless, they continue to fail for a subset of patients. Gene expression profiling has highlighted the pivotal role of the tumor microenvironment (TME) in the FL prognosis; nevertheless, there is still a need to standardize the assessment of immune-infiltrating cells for the prognostic classification of patients with early or late progressing disease. We studied a retrospective cohort of 49 FL lymph node biopsies at the time of the initial diagnosis using pathologist-guided analysis on whole slide images, and we characterized the immune repertoire for both quantity and distribution (intrafollicular, IF and extrafollicular, EF) of cell subsets in relation to clinical outcome. We looked for the natural killer (CD56), T lymphocyte (CD8, CD4, PD1) and macrophage (CD68, CD163, MA4A4A)-associated markers. High CD163/CD8 EF ratios and high CD56/MS4A4A EF ratios, according to Kaplan-Meier estimates were linked with shorter EFS (event-free survival), with the former being the only one associated with POD24. In contrast to IF CD68+ cells, which represent a more homogeneous population, higher in non-progressing patients, EF CD68+ macrophages did not stratify according to survival. We also identify distinctive MS4A4A+CD163-macrophage populations with different prognostic weights. Enlarging the macrophage characterization and combining it with a lymphoid marker in the rituximab era, in our opinion, may enable prognostic stratification for low-/high-grade FL patients beyond POD24. These findings warrant validation across larger FL cohorts.

The aim of the present study was to provide a comprehensive characterization of whole genome DNA methylation patterns in replicative and ionizing irradiation- or doxorubicin-induced premature senescence, exhaustively exploring epigenetic modifications in three different human cell types: in somatic diploid skin fibroblasts and in bone marrow- and adipose-derived mesenchymal stem cells. With CpG-wise differential analysis, three epigenetic signatures were identified: (a) cell type- and treatment-specific signature; (b) cell type-specific senescence-related signature; and (c) cell type-transversal replicative senescence-related signature. Cluster analysis revealed that only replicative senescent cells created a distinct group reflecting notable alterations in the DNA methylation patterns accompanying this cellular state. Replicative senescence-associated epigenetic changes seemed to be of such an extent that they surpassed interpersonal dissimilarities. Enrichment in pathways linked to the nervous system and involved in the neurological functions was shown after pathway analysis of genes involved in the cell type-transversal replicative senescence-related signature. Although DNA methylation clock analysis provided no statistically significant evidence on epigenetic age acceleration related to senescence, a persistent trend of increased biological age in replicative senescent cultures of all three cell types was observed. Overall, this work indicates the heterogeneity of senescent cells depending on the tissue of origin and the type of senescence inducer that could be putatively translated to a distinct impact on tissue homeostasis.

Background: COVID-19 has a wide spectrum of clinical manifestations and given its impact on morbidity and mortality, there is an unmet medical need to discover endogenous cellular and molecular biomarkers that predict the expected clinical course of the disease. Recently, epigenetics and especially DNA methylation have been pointed out as a promising tool for outcome prediction in several diseases., Methods and Results: Using the Illumina Infinium Methylation EPIC BeadChip850K, we investigated genome-wide differences in DNA methylation in an Italian Cohort of patients with comorbidities and compared severe (n = 64) and mild (123) prognosis. Results showed that the epigenetic signature, already present at the time of Hospital admission, can significantly predict risk of severe outcomes. Further analyses provided evidence of an association between age acceleration and a severe prognosis after COVID-19 infection. The burden of Stochastic Epigenetic Mutation (SEMs) has been significantly increased in patients with poor prognosis. Results have been replicated in silico considering COVID-19 negative subjects and available previously published datasets., Conclusions: Using original methylation data and taking advantage of already published datasets, we confirmed in the blood that epigenetics is actively involved in immune response after COVID-19 infection, allowing the identification of a specific signature able to discriminate the disease evolution. Furthermore, the study showed that epigenetic drift and age acceleration are associated with severe prognosis. All these findings prove that host epigenetics undergoes notable and specific rearrangements to respond to COVID-19 infection which can be used for a personalized, timely, and targeted management of COVID-19 patients during the first stages of hospitalization., (© 2023. The Author(s).)

Background: Epigenetics of Autism Spectrum Disorders (ASD) is still an understudied field. The majority of the studies on the topic used an approach based on mere classification of cases and controls., Objective: The present study aimed at providing a multi-level approach in which different types of epigenetic analysis (epigenetic drift, age acceleration) are combined., Methods: We used publicly available datasets from blood (n = 3) and brain tissues (n = 3), separately. Firstly, we evaluated for each dataset and meta-analyzed the differential methylation profile between cases and controls. Secondly, we analyzed age acceleration, epigenetic drift and rare epigenetic variations., Results: We observed a significant epi-signature of ASD in blood but not in brain specimens. We did not observe significant age acceleration in ASD, while epigenetic drift was significantly higher compared to controls. We reported the presence of significant rare epigenetic variations in 41 genes, 35 of which were never associated with ASD. Almost all genes were involved in pathways linked to ASD etiopathogenesis (i.e., neuronal development, mitochondrial metabolism, lipid biosynthesis and antigen presentation)., Conclusion: Our data support the hypothesis of the use of blood epi-signature as a potential tool for diagnosis and prognosis of ASD. The presence of an enhanced epigenetic drift, especially in brain, which is linked to cellular replication, may suggest that alteration in epigenetics may occur at a very early developmental stage (i.e., fetal) when neuronal replication is still high., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Beckwith-Wiedemann syndrome (BWS, OMIM # 130650) is an imprinting disorder, associated with overgrowth and increased risk of embryonal tumors. Patients carrying hypomethylation in the KCNQ1OT1:TSS DMR (11p15.5) show MLID (Multilocus Imprinting Disturbance) upon epimutations at other imprinted regions. Few cases of BWS MLID's mothers with biallelic pathogenetic variants in maternal effect genes, mainly components of the subcortical maternal complex, are reported. We describe two families, one with a history of conception difficulties with a novel homozygous nonsense NLRP2 variant and another experiencing 8 miscarriages with a compound heterozygous PADI6 variant., (© 2022. The Author(s).)

Background: Imprinting disorders are a group of congenital diseases which are characterized by molecular alterations affecting differentially methylated regions (DMRs). To date, at least twelve imprinting disorders have been defined with overlapping but variable clinical features including growth and metabolic disturbances, cognitive dysfunction, abdominal wall defects and asymmetry. In general, a single specific DMR is affected in an individual with a given imprinting disorder, but there are a growing number of reports on individuals with so-called multilocus imprinting disturbances (MLID), where aberrant imprinting marks (most commonly loss of methylation) occur at multiple DMRs. However, as the literature is fragmented, we reviewed the molecular and clinical data of 55 previously reported or newly identified MLID families with putative pathogenic variants in maternal effect genes (NLRP2, NLRP5, NLRP7, KHDC3L, OOEP, PADI6) and in other candidate genes (ZFP57, ARID4A, ZAR1, UHRF1, ZNF445)., Results: In 55 families, a total of 68 different candidate pathogenic variants were identified (7 in NLRP2, 16 in NLRP5, 7 in NLRP7, 17 in PADI6, 15 in ZFP57, and a single variant in each of the genes ARID4A, ZAR1, OOEP, UHRF1, KHDC3L and ZNF445). Clinical diagnoses of affected offspring included Beckwith-Wiedemann syndrome spectrum, Silver-Russell syndrome spectrum, transient neonatal diabetes mellitus, or they were suspected for an imprinting disorder (undiagnosed). Some families had recurrent pregnancy loss., Conclusions: Genomic maternal effect and foetal variants causing MLID allow insights into the mechanisms behind the imprinting cycle of life, and the spatial and temporal function of the different factors involved in oocyte maturation and early development. Further basic research together with identification of new MLID families will enable a better understanding of the link between the different reproductive issues such as recurrent miscarriages and preeclampsia in maternal effect variant carriers/families and aneuploidy and the MLID observed in the offsprings. The current knowledge can already be employed in reproductive and genetic counselling in specific situations., (© 2022. The Author(s).)

Taking advantage of the fast-growing knowledge of RNA-binding proteins (RBPs) we review the signature of downregulated genes for RBPs in the transcriptome of induced pluripotent stem cell neurons (iNeurons) modelling the neurodevelopmental Rubinstein Taybi Syndrome (RSTS) caused by mutations in the genes encoding CBP/p300 acetyltransferases. We discuss top and functionally connected downregulated genes sorted to "RNA processing" and "Ribonucleoprotein complex biogenesis" Gene Ontology clusters. The first set of downregulated RBPs includes members of hnRNHP (A1, A2B1, D, G, H2-H1, MAGOHB, PAPBC), core subunits of U small nuclear ribonucleoproteins and Serine-Arginine splicing regulators families, acting in precursor messenger RNA alternative splicing and processing. Consistent with literature findings on reduced transcript levels of serine/arginine repetitive matrix 4 (SRRM4) protein, the main regulator of the neural-specific microexons splicing program upon depletion of Ep300 and Crebbp in mouse neurons, RSTS iNeurons show downregulated genes for proteins impacting this network. We link downregulated genes to neurological disorders including the new HNRNPH1-related intellectual disability syndrome with clinical overlap to RSTS. The set of downregulated genes for Ribosome biogenesis includes several components of ribosomal subunits and nucleolar proteins, such NOP58 and fibrillarin that form complexes with snoRNAs with a central role in guiding post-transcriptional modifications needed for rRNA maturation. These nucleolar proteins are "dual" players as fibrillarin is also required for epigenetic regulation of ribosomal genes and conversely NOP58-associated snoRNA levels are under the control of NOP58 interactor BMAL1, a transcriptional regulator of the circadian rhythm. Additional downregulated genes for "dual specificity" RBPs such as RUVBL1 and METTL1 highlight the links between chromatin and the RBP-ome and the contribution of perturbations in their cross-talk to RSTS. We underline the hub position of CBP/p300 in chromatin regulation, the impact of its defect on neurons' post-transcriptional regulation of gene expression and the potential use of epidrugs in therapeutics of RBP-caused neurodevelopmental disorders., Competing Interests: None

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Silver Russell Syndrome (SRS, MIM #180860) is a rare growth retardation disorder in which clinical diagnosis is based on six features: pre- and postnatal growth failure, relative macrocephaly, prominent forehead, body asymmetry, and feeding difficulties (Netchine-Harbison clinical scoring system (NH-CSS)). The molecular mechanisms consist in (epi)genetic deregulations at multiple loci: the loss of methylation (LOM) at the paternal H19/IGF2 :IG-DMR (chr11p15.5) (50%) and the maternal uniparental disomy of chromosome 7 (UPD(7)mat) (10%) are the most frequent causes. Thus far, about 40% of SRS remains undiagnosed, pointing to the need to define the rare mechanisms in such a consistent fraction of unsolved patients. Within a cohort of 176 SRS with an NH-CSS ≥ 3, a molecular diagnosis was disclosed in about 45%. Among the remaining patients, we identified in 3 probands (1.7%) with UPD(20)mat (Mulchandani-Bhoj-Conlin syndrome, OMIM #617352), a molecular mechanism deregulating the GNAS locus and described in 21 cases, characterized by severe feeding difficulties associated with failure to thrive, preterm birth, and intrauterine/postnatal growth retardation. Our patients share prominent forehead, feeding difficulties, postnatal growth delay, and advanced maternal age. Their clinical assessment and molecular diagnostic flowchart contribute to better define the characteristics of this rare imprinting disorder and to rank UPD(20)mat as the fourth most common pathogenic molecular defect causative of SRS.

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.

Oculo-auriculo-vertebral-spectrum (OAVS; OMIM 164210) is a rare disorder originating from abnormal development of the first and second branchial arch. The clinical phenotype is extremely heterogeneous with ear anomalies, hemifacial microsomia, ocular defects, and vertebral malformations being the main features. MYT1 , AMIGO2 , and ZYG11B gene variants were reported in a few OAVS patients, but the etiology remains largely unknown. A multifactorial origin has been proposed, including the involvement of environmental and epigenetic mechanisms. To identify the epigenetic mechanisms contributing to OAVS, we evaluated the DNA-methylation profiles of 41 OAVS unrelated affected individuals by using a genome-wide microarray-based methylation approach. The analysis was first carried out comparing OAVS patients with controls at the group level. It revealed a moderate epigenetic variation in a large number of genes implicated in basic chromatin dynamics such as DNA packaging and protein-DNA organization. The alternative analysis in individual profiles based on the searching for Stochastic Epigenetic Variants (SEV) identified an increased number of SEVs in OAVS patients compared to controls. Although no recurrent deregulated enriched regions were found, isolated patients harboring suggestive epigenetic deregulations were identified. The recognition of a different DNA methylation pattern in the OAVS cohort and the identification of isolated patients with suggestive epigenetic variations provide consistent evidence for the contribution of epigenetic mechanisms to the etiology of this complex and heterogeneous disorder.

Ring chromosome 20 [r(20)] syndrome is a rare condition characterized by a non-supernumerary ring chromosome 20 replacing a normal chromosome 20. It is commonly seen in a mosaic state and is diagnosed by means of karyotyping. r(20) syndrome is characterized by a recognizable epileptic phenotype with typical EEG pattern, intellectual disability manifesting after seizure onset in otherwise normally developing children, and behavioral changes. Despite the distinctive phenotype, many patients still lack a diagnosis-especially in the genomic era-and the pathomechanisms of ring formation are poorly understood. In this review we address the genetic and clinical aspects of r(20) syndrome, and discuss differential diagnoses and overlapping phenotypes, providing the reader with useful tools for clinical and laboratory practice. We also discuss the current issues in understanding the mechanisms through which ring 20 chromosome causes the typical manifestations, and present unpublished data about methylation studies. Ultimately, we explore future perspectives of r(20) research. Our intended audience is clinical and laboratory geneticists, child and adult neurologists, and genetic counselors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Peron, Catusi, Recalcati, Calzari, Larizza, Vignoli and Canevini.)

Background: PADI6 is a component of the subcortical maternal complex, a group of proteins that is abundantly expressed in the oocyte cytoplasm, but is required for the correct development of early embryo. Maternal-effect variants of the subcortical maternal complex proteins are associated with heterogeneous diseases, including female infertility, hydatidiform mole, and imprinting disorders with multi-locus imprinting disturbance. While the involvement of PADI6 in infertility is well demonstrated, its role in imprinting disorders is less well established., Results: We have identified by whole-exome sequencing analysis four cases of Beckwith-Wiedemann syndrome with multi-locus imprinting disturbance whose mothers are carriers of PADI6 variants. In silico analysis indicates that these variants result in loss of function, and segregation analysis suggests they act as either recessive or dominant-negative maternal-effect mutations. Genome-wide methylation analysis revealed heterogeneous and extensively altered methylation profiles of imprinted loci in the patients, including two affected sisters, but not in their healthy siblings., Conclusion: Our results firmly establish the role of PADI6 in imprinting disorders. We report loss-of-function maternal-effect variants of PADI6 that are associated with heterogeneous multi-locus imprinting disturbances in the progeny. The rare finding of two siblings affected by Beckwith-Wiedemann syndrome suggests that in some cases, familial recurrence risk of these variants may be high. However, the heterogeneous phenotypes of the other pedigrees suggest that altered oocyte PADI6 function results in stochastic maintenance of methylation imprinting with unpredictable consequences on early embryo health.

Preterm birth (PTB) can be defined as the endpoint of a complex process that could be influenced by maternal and environmental factors. Epigenetics recently emerged as an interesting field of investigation since it represents an important mechanism of regulation. This study evaluates epigenetic impact of preterm birth on DNA methylation. Genome-wide DNAm was measured using the Illumina 450K array in cord blood samples obtained from 72 full term and 18 preterm newborns. Lymphocyte composition was calculated based on specific epigenetic markers that are present on the 450k array. Differential methylation analysis was performed both at site and region level; moreover, stochastic epigenetic mutations (SEMs) were also evaluated. The study showed significant differences in blood cell composition between the two groups. Moreover, after multiple testing correction, statistically significant differences in DNA methylation levels emerged between the two groups both at site and region levels. Results obtained were compared to those reported by previous EWAS, leading to a list of more consistent genes associated with PTB. Finally, the SEMs analysis revealed that the burden of SEMs resulted significantly higher in the preterm group. In conclusion, PTB resulted associated to specific epigenetic signatures that involve immune system. Moreover, SEMs analysis revealed an increased epigenetic drift at birth in the preterm group.

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.

Background: Maternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss. Enigmatically, they are also associated with DNA methylation abnormalities at imprinted genes in conceptuses: in the devastating gestational abnormality biparental complete hydatidiform mole (BiCHM) or in multi-locus imprinting disease (MLID). However, the developmental timing, genomic extent and mechanistic basis of these imprinting defects are unknown. The rarity of these disorders and the possibility that methylation defects originate in oocytes have made these questions very challenging to address., Methods: Single-cell bisulphite sequencing (scBS-seq) was used to assess methylation in oocytes from a patient with BiCHM identified to be homozygous for an inactivating mutation in the human SCMC component KHDC3L. Genome-wide methylation analysis of a preimplantation embryo and molar tissue from the same patient was also performed., Results: High-coverage scBS-seq libraries were obtained from five KHDC3L c.1A>G oocytes, which revealed a genome-wide deficit of DNA methylation compared with normal human oocytes. Importantly, germline differentially methylated regions (gDMRs) of imprinted genes were affected similarly to other sequence features that normally become methylated in oocytes, indicating no selectivity towards imprinted genes. A range of methylation losses was observed across genomic features, including gDMRs, indicating variable sensitivity to defects in the SCMC. Genome-wide analysis of a pre-implantation embryo and molar tissue from the same patient showed that following fertilisation methylation defects at imprinted genes persist, while most non-imprinted regions of the genome recover near-normal methylation post-implantation., Conclusions: We show for the first time that the integrity of the SCMC is essential for de novo methylation in the female germline. These findings have important implications for understanding the role of the SCMC in DNA methylation and for the origin of imprinting defects, for counselling affected families, and will help inform future therapeutic approaches.

Background: A subset of individuals affected by imprinting disorders displays multi-locus imprinting disturbances (MLID). MLID has been associated with maternal-effect variants that alter the maintenance of methylation at germline-derived differentially methylated regions (gDMRs) in early embryogenesis. Pedigrees of individuals with MLID also include siblings with healthy phenotype. However, it is unknown if these healthy individuals have MLID themselves or if their methylation patterns differ from those associated with imprinting disorders, and in general, if MLID affects the clinical phenotype., Methods: We have investigated gDMR methylation by locus-specific and whole-genome analyses in a family with multiple pregnancy losses, a child with Beckwith-Wiedemann syndrome (BWS) and a further child with no clinical diagnosis of imprinting disorder or other pathologies., Results: We detected MLID with different methylation profiles in the BWS-affected and healthy siblings. Whole-exome sequencing demonstrated the presence of novel loss-of-function variants of NLRP5 in compound heterozygosity in the mother. The methylation profiles of the two siblings were compared with those of other cases with MLID and control groups by principal component analysis and unsupervised hierarchical clustering, but while their patterns were clearly separated from those of controls, we were unable to cluster those associated with specific clinical phenotypes among the MLID cases., Conclusion: The identification of two novel maternal-effect variants of NLRP5 associated with poly-abortivity and MLID adds further evidence to the role of this gene in the maintenance of genomic imprinting in early embryos. Furthermore, our results demonstrate that within these pedigrees, MLID can also be present in the progeny with healthy phenotype, indicating that some sort of compensation occurs between altered imprinted loci in these individuals. The analysis of larger cohorts of patients with MLID is needed to formulate more accurate epigenotype-phenotype correlations.

Introduction: Silver-Russell syndrome (SRS) is an imprinting disorder primarily caused by genetic and epigenetic aberrations on chromosomes 11 and 7. SRS is a rare growth retardation disorder often misdiagnosed due to its heterogeneous and non-specific clinical features. The Netchine-Harbison clinical scoring system (NH-CSS) is the recommended tool for differentiating patients into clinical SRS or unlikely SRS. However, the clinical diagnosis is molecularly confirmed only in about 60% of patients, leaving the remaining substantial proportion of SRS patients with unknown genetic etiology. Materials and Methods: A cohort of 34 Italian patients with SRS or SRS-like features scored according to the NH-CSS and without any SRS-associated (epi)genetic alterations was analyzed by high-resolution array-based comparative genomic hybridization (CGH) in order to identify potentially pathogenic copy number variants (CNVs). Results and Discussion: In seven patients, making up 21% of the initial cohort, five pathogenic and two potentially pathogenic CNVs were found involving distinct genomic regions either previously associated with growth delay conditions (1q24.3-q25.3, 17p13.3, 17q22, and 22q11.2-q11.22) and with SRS spectrum (7p12.1 and 7p15.3-p14.3) or outlined for the first time (19q13.42), providing a better definition of reported and as yet unreported SRS overlapping syndromes. All the variants involve genes with a defined role in growth pathways, and for two genes mapping at 7p, IGF2BP3 and GRB10 , the association with SRS turns out to be reinforced. The deleterious effect of the two potentially pathogenic variants, comprising GRB10 and ZNF331 genes, was explored by targeted approaches, though further studies are needed to validate their pathogenic role in the SRS etiology. In conclusion, we reconfirm the utility of performing a genome-wide scan to achieve a differential diagnosis in patients with SRS or similar features and to highlight novel chromosome alterations associated with SRS and growth retardation disorders., (Copyright © 2019 Crippa, Bonati, Calzari, Picinelli, Gervasini, Sironi, Bestetti, Guzzetti, Bellone, Selicorni, Mussa, Riccio, Ferrero, Russo, Larizza and Finelli.)

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder characterized by growth retardation, skeletal anomalies and intellectual disability, caused by heterozygous mutations in either CREBBP (RSTS1) or EP300 (RSTS2) genes. We characterized 3 iPSC lines generated by Sendai from blood of RSTS1 patients with unique non sense c.4435G > T, p.(Gly1479*), c.3474G > A, p.(Trp1158*) and missense c.4627G > T, p.(Asp1543Tyr) CREBBP mutations. All lines displayed iPSC morphology, pluripotency markers, trilineage differentiation potential, stable karyotype and specific mutations. Western-blot using a CREB-Binding Protein N-terminus antibody demonstrated the same amount of full length protein as control in the missense mutation line and reduced amount in lines with stop mutations., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are two imprinting disorders associated with opposite molecular alterations in the 11p15.5 imprinting centres. Their clinical diagnosis is confirmed by molecular testing in 50-70% of patients. The authors from different reference centres for BWS and SRS have identified single patients with unexpected and even contradictory molecular findings in respect to the clinical diagnosis. These patients clinically do not fit the characteristic phenotypes of SRS or BWS, but illustrate their clinical heterogeneity. Thus, comprehensive molecular testing is essential for accurate diagnosis and appropriate management, to avoid premature clinical diagnosis and anxiety for the families.

Rubinstein-Taybi syndrome (RSTS) is a rare neurodevelopmental disorder characterized by distinctive facial features, growth retardation, broad thumbs and toes and mild to severe intellectual disability, caused by heterozygous mutations in either CREBBP or EP300 genes, encoding the homologous CBP and p300 lysine-acetyltransferases and transcriptional coactivators. No RSTS in vitro induced Pluripotent Stem Cell (iPSC)-neuronal model is available yet to achieve mechanistic insights on cognitive impairment of RSTS patients. We established iPSC-derived neurons (i-neurons) from peripheral blood cells of three CREBBP- and two EP300-mutated patients displaying different levels of intellectual disability, and four unaffected controls. Pan neuronal and cortical-specific markers were expressed by all patients' i-neurons. Altered morphology of patients' differentiating neurons, showing reduced branch length and increased branch number, and hypoexcitability of differentiated neurons emerged as potential disease biomarkers. Anomalous neuronal morphology and reduced excitability varied across different RSTS patients' i-neurons. Further studies are needed to validate these markers and assess whether they reflect cognitive and behavioural impairment of the donor patients., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Angelman syndrome (AS, MIM 105830) is a rare neurodevelopmental disorder affecting 1:10-20,000 children. Patients show moderate to severe intellectual disability, ataxia and absence of speech. Studies on both post-mortem AS human brains and mouse models revealed dysfunctions in the extra synaptic gamma-aminobutyric acid (GABA) receptors implicated in the pathogenesis. Taurine is a free intracellular sulfur-containing amino acid, abundant in brain, considered an inhibiting neurotransmitter with neuroprotective properties. As taurine acts as an agonist of GABA-A receptors, we aimed at investigating whether it might ameliorate AS symptoms. Since mice weaning, we orally administered 1 g/kg/day taurine in water to Ube3a -deficient mice. To test the improvement of motor and cognitive skills, Rotarod, Novel Object Recognition and Open Field tests were assayed at 7, 14, 21 and 30 weeks, while biochemical tests and amino acid dosages were carried out, respectively, by Western-blot and high-performance liquid chromatography (HPLC) on frozen whole brains. Treatment of Ube3a m -/p+ mice with taurine significantly improved motor and learning skills and restored the levels of the post-synaptic PSD-95 and pERK1/2-ERK1/2 ratio to wild type values. No side effects of taurine were observed. Our study indicates taurine administration as a potential therapy to ameliorate motor deficits and learning difficulties in AS., Competing Interests: The authors declare no conflict of interest.

Objectives: Oxidative stress seems to be involved in Rett syndrome (RTT). The aim of this study was to assess the antioxidant status in RTT children with MECP2 gene mutations with respect to healthy controls, and to explore novel blood antioxidant markers for RTT severity., Methods: In erythrocytes from RTT females aged 2-14 years (n = 27) and age-matched controls (n = 27), we measured the levels of malonaldehyde and the activity of two antioxidant enzymes, Cu/Zn-superoxide dismutase and catalase, by spectrophotometric assays. In leukocytes, the expression of metallothioneins, the main non-enzymatic antioxidants, was assessed by real-time RT-PCR. In nine selected RTT children, methylome analysis was also performed., Results: Blood of RTT patients showed increased lipid peroxidation and a dysregulated pattern of MT expression, while enzymatic activities did not change significantly with respect to controls. Moreover, we observed no epigenetic dysregulation in CpG-enriched promoter regions of the analysed genes but significant hypomethylation in the random loci., Conclusions: As the haematic level of MT-1A directly correlates with the phenotype severity, this metallothionein can represent a marker for RTT severity. Moreover, the attempt to link the level of blood oxidative stress with MECP2 mutation and specific clinical features led us to draw some interesting conclusions.

Background: Multiple (epi)genetic defects affecting the expression of the imprinted genes within the 11p15.5 chromosomal region underlie Silver-Russell (SRS) and Beckwith-Wiedemann (BWS) syndromes. The molecular diagnosis of these opposite growth disorders requires a multi-approach flowchart to disclose known primary and secondary (epi)genetic alterations; however, up to 20 and 30 % of clinically diagnosed BWS and SRS cases remain without molecular diagnosis. The complex structure of the 11p15 region with variable CpG methylation and low-rate mosaicism may account for missed diagnoses. Here, we demonstrate the relevance of complementary techniques for the assessment of different CpGs and the importance of testing multiple tissues to increase the SRS and BWS detection rate., Results: Molecular testing of 147 and 450 clinically diagnosed SRS and BWS cases provided diagnosis in 34 SRS and 185 BWS patients, with 9 SRS and 21 BWS cases remaining undiagnosed and herein referred to as "borderline." A flowchart including complementary techniques and, when applicable, the analysis of buccal swabs, allowed confirmation of the molecular diagnosis in all borderline cases. Comparison of methylation levels by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in borderline and control cases defined an interval of H19/IGF2:IG-DMR loss of methylation that was distinct between "easy to diagnose" and "borderline" cases, which were characterized by values ≤mean -3 standard deviations (SDs) compared to controls. Values ≥mean +1 SD at H19/IGF2: IG-DMR were assigned to borderline hypermethylated BWS cases and those ≤mean -2 SD at KCNQ1OT1: TSS-DMR to hypomethylated BWS cases; these were supported by quantitative pyrosequencing or Southern blot analysis. Six BWS cases suspected to carry mosaic paternal uniparental disomy of chromosome 11 were confirmed by SNP array, which detected mosaicism till 10 %. Regarding the clinical presentation, borderline SRS were representative of the syndromic phenotype, with exception of one patient, whereas BWS cases showed low frequency of the most common features except hemihyperplasia., Conclusions: A conclusive molecular diagnosis was reached in borderline methylation cases, increasing the detection rate by 6 % for SRS and 5 % for BWS cases. The introduction of complementary techniques and additional tissue analyses into routine diagnostic work-up should facilitate the identification of cases undiagnosed because of mosaicism, a distinctive feature of epigenetic disorders.

Beckwith-Wiedemann syndrome (BWS) is characterized by cancer predisposition, overgrowth and highly variable association of macroglossia, abdominal wall defects, nephrourological anomalies, nevus flammeus, ear malformations, hypoglycemia, hemihyperplasia, and organomegaly. BWS molecular defects, causing alteration of expression or activity of the genes regulated by two imprinting centres (IC) in the 11p15 chromosomal region, are also heterogeneous. In this paper we define (epi)genotype-phenotype correlations in molecularly confirmed BWS patients. The characteristics of 318 BWS patients with proven molecular defect were compared among the main four molecular subclasses: IC2 loss of methylation (IC2-LoM, n=190), IC1 gain of methylation (IC1-GoM, n=31), chromosome 11p15 paternal uniparental disomy (UPD, n=87), and cyclin-dependent kinase inhibitor 1C gene (CDKN1C) variants (n=10). A characteristic growth pattern was found in each group; neonatal macrosomia was almost constant in IC1-GoM, postnatal overgrowth in IC2-LoM, and hemihyperplasia more common in UPD (P<0.001). Exomphalos was more common in IC2/CDKN1C patients (P<0.001). Renal defects were typical of UPD/IC1 patients, uretheral malformations of IC1-GoM cases (P<0.001). Ear anomalies and nevus flammeus were associated with IC2/CDKN1C genotype (P<0.001). Macroglossia was less common among UPD patients (P<0.001). Wilms' tumor was associated with IC1-GoM or UPD and never observed in IC2-LoM patients (P<0.001). Hepatoblastoma occurred only in UPD cases. Cancer risk was lower in IC2/CDKN1C, intermediate in UPD, and very high in IC1 cases (P=0.009). In conclusion, (epi)genotype-phenotype correlations define four different phenotypic BWS profiles with some degree of clinical overlap. These observations impact clinical care allowing to move toward (epi) genotype-based follow-up and cancer screening.