Increased MTHFR promoter methylation in mothers of Down syndrome individuals

Despite that advanced maternal age at conception represents the major risk factor for the birth of a child with Down syndrome (DS), most of DS babies are born from women aging less than 35 years. Studies performed in peripheral lymphocytes of those women revealed several markers of global genome instability, including an increased frequency of micronuclei, shorter telomeres and impaired global DNA methylation. Furthermore, young mothers of DS individuals (MDS) are at increased risk to develop dementia later in life, suggesting that they might be "biologically older" than mothers of euploid babies of similar age.Mutations in folate pathway genes, and particularly in the methylenetetrahydrofolate reductase (MTHFR) one, have been often associated with maternal risk for a DS birth as well as with risk of dementia in the elderly. Recent studies pointed out that also changes in MTHFR methylation levels can contribute to human disease, but nothing is known about MTHFR methylation in MDS tissues.We investigated MTHFR promoter methylation in DNA extracted from perypheral lymphocytes of 40 MDS and 44 matched control women that coinceived their children before 35 years of age, observing a significantly increased MTHFR promoter methylation in the first group (33.3 ± 8.1% vs. 28.3 ± 5.8%; p = 0.001). In addition, the frequency of micronucleated lymphocytes was available from the women included in the study, was higher in MDS than control mothers (16.1 ± 8.6‰ vs. 10.5 ± 4.3‰ p = 0.0004), and correlated with MTHFR promoter methylation levels (r = 0.33; p = 0.006).Present data suggest that MTHFR epimutations are likely to contribute to the increased genomic instability observed in cells from MDS, and could play a role in the risk of birth of a child with DS as well as in the onset of age related diseases in those women

Artificial Neural Networks Link One-Carbon Metabolism to Gene-Promoter Methylation in Alzheimer's Disease

Background: There is increasing interest in DNA methylation studies in Alzheimer's disease (AD), but little is still known concerning the relationship between gene-promoter methylation and circulating biomarkers of one-carbon metabolism in patients. Objective: To detect the connections among circulating folate, homocysteine (hcy) and vitamin B12 levels and promoter methylation levels of PSEN1, BACE1, DNMT1, DNMT3A, DNMT3B, and MTHFR genes in blood DNA. Methods: We applied a data mining system called Auto Contractive Map to an existing database of 100 AD and 100 control individuals. Results: Low vitamin B12 was linked to the AD condition, to low folates, and to high hcy. Low PSEN1 methylation was linked to low folate levels as well as to low promoter methylation of BACE1 and DNMTs genes. Low hcy was linked to controls, to high folates and vitamin B12, as well as to high methylation levels of most of the studied genes. Conclusions: The present pilot study suggests that promoter methylation levels of the studied genes are linked to circulating levels of folates, hcy, and vitamin B12

Association study between the DNMT3A -448A>G polymorphism and risk of Alzheimer's disease in Caucasians of Italian origin

Increasing evidence points to an epigenetic contribution in Alzheimer's disease (AD) pathogenesis. In this regard, variants and polymorphisms of DNA methyltransferase genes (DNMTs) are being investigated for their contribution to cognitive decline and dementia, but results are still scarce or controversial. In the present study we genotyped 710 Caucasian subjects of Italian descent, including 320 late-onset AD (LOAD) patients, 70 individuals with amnestic Mild Cognitive Impairment (MCI), and 320 matched healthy controls, for the presence of a functional DNMT3A -448A>G (rs1550117) polymorphism, searching for association with disease risk. In addition, we searched for correlation between the studied polymorphism and circulating levels of folate, homocysteine (hcy) and vitamin B12, all involved in DNA methylation reactions and available from 189 LOAD patients and 186 matched controls. Both allele and genotype frequencies of rs1550117 were closely similar between MCI, LOAD and control subjects, and no association with dementia or pre-dementia conditions was observed. Plasma hcy levels were significantly higher (p = 0.04) and serum folate levels significantly lower (p = 0.01) in LOAD than in controls, but no difference in circulating folate, hcy or vitamin B12 levels was seen between carriers and non-carriers of the minor DNMT3A -448A allele. Collectively, present results confirmed previous associations of increased hcy and decreased folate with LOAD risk, but do not support an association between the DNMT3A -448A>G polymorphism and AD in our population

Case report: a typical Silver-Russell syndrome patient with hand dystonia: the valuable support of the consensus statement to the wide syndromic spectrum

The amount of Insulin Growth Factor 2 (IGF2) controls the rate of embryonal and postnatal growth. The IGF2 and adjacent H19 are the imprinted genes of the telomeric cluster in the 11p15 chromosomal region regulated by differentially methylated regions (DMRs) or imprinting centers (ICs): H19/IGF2:IG-DMR (IC1). Dysregulation due to IC1 Loss-of-Methylation (LoM) or Gain-of-Methyaltion (GoM) causes Silver–Russell syndrome (SRS) or Beckwith–Wiedemann syndrome (BWS) disorders associated with growth retardation or overgrowth, respectively. Specific features define each of the two syndromes, but isolated asymmetry is a common cardinal feature, which is considered sufficient for a diagnosis in the BWS spectrum. Here, we report the case of a girl with right body asymmetry, which suggested BWS spectrum. Later, BWS/SRS molecular analysis identified IC1_LoM revealing the discrepant diagnosis of SRS. A clinical re-evaluation identified a relative macrocephaly and previously unidentified growth rate at lower limits of normal at birth, feeding difficulties, and asymmetry. Interestingly, and never previously described in IC1_LoM SRS patients, since the age of 16, she has developed hand-writer’s cramps, depression, and bipolar disorder. Trio-WES identified a VPS16 heterozygous variant [NM_022575.4:c.2185C>G:p.Leu729Val] inherited from her healthy mother. VPS16 is involved in the endolysosomal system, and its dysregulation is linked to autosomal dominant dystonia with incomplete penetrance and variable expressivity. IGF2 involvement in the lysosomal pathway led us to speculate that the neurological phenotype of the proband might be triggered by the concurrent IGF2 deficit and VPS16 alteration

Clinical and molecular characterization of patients affected by Beckwith-Wiedemann spectrum conceived through assisted reproduction techniques

The prevalence of Beckwith-Wiedemann spectrum (BWSp) is tenfold increased in children conceived through assisted reproductive techniques (ART). More than 90% of ART-BWSp patients reported so far display imprinting center 2 loss-of-methylations (IC2-LoM), versus 50% of naturally conceived BWSp patients. We describe a cohort of 74 ART-BWSp patients comparing their features with a cohort of naturally conceived BWSp patients, with the ART-BWSp patients previously described in literature, and with the general population of children born from ART. We found that the distribution of UPD(11)pat was not significantly different in ART and naturally conceived patients. We observed 68.9% of IC2-LoM and 16.2% of mosaic UPD(11)pat in our ART cohort, that strongly differ from the figure reported in other cohorts so far. Since UPD(11)pat likely results from post-fertilization recombination events, our findings allows to hypothesize that more complex molecular mechanisms, besides methylation disturbances, may underlie BWSp increased risk in ART pregnancies. Moreover, comparing the clinical features of ART and non-ART BWSp patients, we found that ART-BWSp patients might have a milder phenotype. Finally, our data show a progressive increase in the prevalence of BWSp over time, paralleling that of ART usage in the last decades

Trans-acting genetic variants causing multilocus imprinting disturbance (MLID): common mechanisms and consequences.

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

Multi-locus imprinting disturbance (MLID): interim joint statement for clinical and molecular diagnosis

Background: Imprinting disorders are rare diseases resulting from altered expression of imprinted genes, which exhibit parent-of-origin-specific expression patterns regulated through differential DNA methylation. A subgroup of patients with imprinting disorders have DNA methylation changes at multiple imprinted loci, a condition referred to as multi-locus imprinting disturbance (MLID). MLID is recognised in most but not all imprinting disorders and is also found in individuals with atypical clinical features; the presence of MLID often alters the management or prognosis of the affected person. Some cases of MLID are caused by trans-acting genetic variants, frequently not in the patients but their mothers, which have counselling implications. There is currently no consensus on the definition of MLID, clinical indications prompting testing, molecular procedures and methods for epigenetic and genetic diagnosis, recommendations for laboratory reporting, considerations for counselling, and implications for prognosis and management. The purpose of this study is thus to cover this unmet need. Methods: A comprehensive literature search was conducted resulting in identification of more than 100 articles which formed the basis of discussions by two working groups focusing on clinical diagnosis (n = 12 members) and molecular testing (n = 19 members). Following eight months of preparations and regular online discussions, the experts from 11 countries compiled the preliminary documentation and determined the questions to be addressed during a face-to-face meeting which was held with the attendance of the experts together with four representatives of patient advocacy organisations. Results: In light of available evidence and expert consensus, we formulated 16 propositions and 8 recommendations as interim guidance for the clinical and molecular diagnosis of MLID. Conclusions: MLID is a molecular designation, and for patients with MLID and atypical phenotypes, we propose the alternative term multi-locus imprinting syndrome. Due to the intrinsic variability of MLID, the guidelines underscore the importance of involving experts from various fields to ensure a confident approach to diagnosis, counselling, and care. The authors advocate for global, collaborative efforts in both basic and translational research to tackle numerous crucial questions that currently lack answers, and suggest reconvening within the next 3–5 years to evaluate the research advancements and update this guidance as needed

Folate metabolism, Alzheimer's Disease and Epigenetics

The possible contribution of folate metabolism in modulating the methylation profile and the expression of disease-related genes has attracted substantial research in the field of complex diseases, such as Alzheimer’s disease (AD), likely resulting from still not well understood gene-environment interactions. Several hypotheses have been formulated linking impaired folate metabolism to AD risk, among them one of the most arguing suggests that it could result in altered DNA methylation and expression of genes involved in disease pathogenesis. Aim of this project is to understand the correlation between folate metabolism and epigenetic modifications of genes related to AD. For this purpose, we screened 205 late onset Alzheimer’s disease (LOAD) patients, 74 subjects with Mild Cognitive Impairment (MCI) and 175 healthy controls for the presence of the common polymorphisms in folate metabolism gene, searching for association with disease risk. Moreover, we searched for correlation between each of the studied polymorphisms and available data on plasma homocysteine (Hcy), serum folate and vitamin B12 values. Finally, we analyzed analyzed the methylation levels of genes involved in amyloid-beta peptide production (PSEN1 and BACE1), in DNA methylation (DNMT1, DNMT3A and DNMT3B), and in one-carbon metabolism (MTHFR), searching for correlation with age and gender, with biomarkers of one-carbon metabolism. We observed a significant increase frequency of MTRR 66G allele and MTRR 66GG genotype in AD patients with respect to controls and a significant increase frequency of MTR 2756G allele in MCI subjects. Several interactions between the studied polymorphisms and biochemical biomarkers were observed. Methylation analysis revealed no difference in mean methylation levels between AD and control groups, but MCI patients showed higher methylation levels with respect the other groups in almost all the studied genes. MTHFR methylation showed an inverse correlation with age. Inverse correlation between plasma homocysteine and MTHFR, DNMT1 and PSEN1 methylation was observed. Positive correlation was observed between serum folate levels and MTHFR methylation

Polimorfismi dei geni della via metabolica dei folati e livelli ematici di folati, omocisteina e vitamina B12 in pazienti affetti da Malattia di Alzheimer

La Malattia di Alzheimer (MA) è un disordine neurodegenerativo ad esordio tardivo e rappresenta la più comune forma di demenza (circa l’80% dei casi). La MA è caratterizzata clinicamente da una progressiva riduzione di volume a livello dei lobi frontale e temporale, dell’ippocampo e dell’amigdala, come conseguenza della degenerazione delle sinapsi e della morte dei neuroni, la quale porta alla perdita di memoria associata a cambiamenti nel comportamento e nella personalità. Tali regioni sono inoltre caratterizzate dalla presenza di placche amiloidi extracellulari (placche senili) e di grovigli neurofibrillari composti da aggregati intracellulari della proteina tau iperfosforilata. Sebbene le manifestazioni cliniche e le caratteristiche neuropatologiche sono state ben illustrate, l’eziologia della malattia è ancora sconosciuta. Negli anni passati è stato dimostrato che sia fattori genetici che fattori ambientali possono portare allo sviluppo di questa malattia; i dati epidemiologici identificano infatti due tipi di MA: una forma autosomica dominante, che comprende circa il 5-15% dei casi e della quale si conoscono 3 geni deterministici (APP, PSEN1 e PSEN2), e una forma sporadica, la quale mostra una patogenesi complessa e risulta da un’interazione tra fattori genetici, epigenetici, ambientali e stocastici. Recentemente, vari studi clinici ed epidemiologici hanno posto l’attenzione su una possibile associazione tra la MA e la via metabolica dei folati. È stato infatti dimostrato che individui affetti dalla MA mostrano un decremento dei livelli plasmatici dei folati e un aumento dei livelli di omocisteina sia nel plasma che nel liquor. I folati derivano interamente dalla dieta e sono nutrienti essenziali per l’organismo in quanto vengono utilizzati come cofattori e come donatori di unità carboniose attivate in vari processi biosintetici, come la sintesi dei nucleotidi e degli amminoacidi. I folati entrano nel nostro organismo sotto forma di una miscela complessa di composti di poliglutammato. Tali composti vengono convertiti nell’intestino ad acido folico, che può essere assorbito dalle cellule con meccanismo di trasporto attivo che coinvolge il carrier dei folati ridotto (RFC); tuttavia tale composto non è biologicamente attivo, per cui esso viene trasformato da una serie di enzimi citoplasmatici in tetraidrofolato (THF), il quale entra a far parte di vari pathway biosintetici. Lo scopo di questa tesi è quello di valutare una possibile associazione tra polimorfismi dei geni della via metabolica dei folati, quali MTHFR C677T, TYMS ripetizione di 28 bp, MTR A2756G e MTRR A66G, e il rischio di sviluppare la malattia di Alzheimer. In questo studio è stata valutata la frequenza di tali polimorfismi in una popolazione di 378 pazienti e 308 controlli sani mediante la tecnica della PCR-RFLP. Inoltre, in un sottogruppo della popolazione, è stata valutata la correlazione tra ognuno dei polimorfismi oggetto dello studio ed i valori ematici di acido folico, omocisteina e vitamina B12. Lo studio ha evidenziato un incremento significativo nella frequenza dell’allele MTHFR 677T (0.48 vs. 0.42; p=0.019) e dei genotipi MTHFR 677CT (OR=1,46; 95%CI=1,03-2,06) e MTHFR 677TT (OR=1,62; 95%CI=1,05-2,49), nei soggetti AD rispetto ai controlli. È stato inoltre osservato un aumento significativo della frequenza dell’allele MTRR 66G (0.49 vs. 0.43; p=0.044) e del genotipo MTRR 66GG (OR=1,57; 95%CI=1,01-2,46) nel gruppo dei pazienti. I soggetti AD presentano, rispetto ai controlli, un aumento dei livelli plamastici di omocisteina (22.7+15.3 vs 14.5+5.7 μmol/L; p=0.037) e una diminuzione dei livelli di folati (5.7+4.9 vs. 7.8+7.2 ng/mL; p=0.005). È inoltre emerso che esistono varie interazioni tra questi marker biochimici e i polimorfismi presi in esame