Aims: We screened 378 late-onset Alzheimer's disease (LOAD) patients and 308 matched controls for the presence of the common MTHFR 677C>T, MTRR 66A>G, MTR 2756 A>G, and TYMS 28bp repeat polymorphisms, searching for association with disease risk and age at onset. Moreover, we searched for correlation between each of the studied polymorphisms and available data on plasma homocysteine (Hcy), serum folate, and vitamin B12 values. Results: We observed a significant increased frequency of the MTHFR 677T allele (0.48 vs. 0.42; p=0.019) and of MTHFR 677CT (OR=1.46; 95%CI=1.03–2.06) and TT genotypes (OR=1.62; 95%CI=1.05–2.49) in LOAD subjects with respect to controls. We also observed a significant increased frequency of the MTRR 66G allele (0.49 vs. 0.43; p=0.044) and of the MTRR 66GG genotype (OR=1.57; 95%CI=1.01–2.46) in the LOAD group. Significantly increased mean plasma Hcy levels (22.7±1.7 vs 14.5±1.7 μmol/L; p=0.037) and decreased serum folate values (5.7±0.5 vs. 7.8±0.8 ng/mL; p=0.005) were observed in LOAD subjects with respect to controls, whilst the difference in serum vitamin B12 values did not reach statistical significance. Several interactions between the studied polymorphisms and biochemical biomarkers were observed. None of the studied polymorphisms was associated with disease age at onset. Innovation: The present study suggests that the MTRR 66G allele might contribute to LOAD risk and confirms an increased frequency of the MTHFR 677T allele in LOAD. Conclusion: Overall, present results support a contribution for one-carbon metabolism to LOAD pathogenesis. Antioxid. Redox Signal. 17, 195–204.

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.

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal enzyme in the one-carbon metabolism, a metabolic pathway required for DNA synthesis and methylation reactions. MTHFR hypermethylation, resulting in reduced gene expression, can contribute to several human disorders, but little is still known about the factors that regulate MTHFR methylation levels. We performed the present study to investigate if common polymorphisms in one-carbon metabolism genes contribute to MTHFR methylation levels. MTHFR methylation was assessed in peripheral blood DNA samples from 206 healthy subjects with methylation-sensitive high-resolution melting (MS-HRM); genotyping was performed for MTHFR 677C>T (rs1801133) and 1298A>C (rs1801131), MTRR 66A>G (rs1801394), MTR 2756A>G (rs1805087), SLC19A1 (RFC1) 80G>A (rs1051266), TYMS 28-bp tandem repeats (rs34743033) and 1494 6-bp ins/del (rs34489327), DNMT3A -448A>G (rs1550117), and DNMT3B -149C>T (rs2424913) polymorphisms. We observed a statistically significant effect of the DNMT3B -149C>T polymorphism on mean MTHFR methylation levels, and particularly CT and TT carriers showed increased methylation levels than CC carriers. The present study revealed an association between a functional polymorphism of DNMT3B and MTHFR methylation levels that could be of relevance in those disorders, such as inborn defects, metabolic disorders and cancer, that have been linked to impaired DNA methylation.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease due to motor neuron loss variably associated with frontotemporal dementia (FTD). Next generation sequencing technology revealed an increasing number of rare and novel genetic variants and interpretation of their pathogenicity represents a major challange in the diagnosis of ALS. We selected 213 consecutive patients with sporadic or familial (16%) ALS, tested negative for SOD1, FUS, TARDBP, and C9orf72 mutations. To reveal rare forms of genetic ALS, we performed a comprehensive multi-gene panel screening including 46 genes associated with ALS, hereditary motor neuronopathies, spastic paraplegia, and FTD. Our study allowed the identification of pathogenic or likely pathogenic variants in 4.2% of patients. The genes with the highest percentage of pathogenic variants were OPTN (1%), VCP (1%) SQSTM1(1%), SETX (0.4%), FIG4 (0.4%), and GARS1 (0.4%) genes. We also found 49 novel or rare gene variants of unknown significance in 30 patients (14%), 44 unlikely pathogenic variants (39%), and 48 variants in ALS susceptibility genes. The results of our study suggest the screening of OPTN, VCP, and SQSTM1 genes in routine diagnostic investigations for both sporadic and familial cases, and confirm the importance of diagnosis and couselling for patients and their relative family members.

Are DNMT3B promoter polymorphisms among maternal risk factors for the birth of a child with Down syndrome (DS)? Present results suggest that combinations of functional DNMT3B promoter polymorphisms might modulate maternal risk of birth of a child with DS. The DNMT3B gene codes for DNA methyltransferase 3b (DNMT3b), a protein required for genome-wide de novo methylation, for the establishment of DNA methylation patterns during development and for regulating the histone code and DNA methylation at centromeric regions. Two common functional DNMT3B promoter polymorphisms, namely –149 C > T (rs2424913) and −579 G > T (rs1569686), have been extensively investigated in cancer genetic association studies but less is known about their role in non-cancer diseases. Early in 1999, it was supposed that impaired DNA methylation of pericentromeric regions might represent a maternal risk factor for having a baby with DS. We aimed to investigate DNMT3B –149 C > T and −579 G > T polymorphisms as maternal risk factors for the birth of a child with DS. The study was performed on DNA samples from 172 mothers of DS individuals (135 aged <35 years when they conceived) and 157 age-matched mothers of unaffected individuals. Genotyping was performed by means of the PCR-RFLP technique. The DNMT3B −579T allele [odds ratio (OR) = 0.68; 95% confidence interval (CI) = 0.48–0.94, P = 0.02], the DNMT3B −579 GT genotype (OR = 0.55; 95% CI = 0.35–0.87 , P = 0.01) and the combined DNMT3B −579 GT + TT genotype (OR = 0.55; 95% CI = 0.36–0.86 , P = 0.008) were associated with reduced risk of birth of a child with DS. A joint effect of the two polymorphisms was observed and the combined −579 GT/–149 CC genotype resulted in decreased DS risk (OR = 0.22; 95% CI = 0.08–0.64, P = 0.003). The effect remained statistically significant after Bonferroni's correction for multiple comparisons. Similar results were obtained when the analysis was restricted to women who conceived a DS child before 35 years of age. To the best of our knowledge, this is the first genetic association study aimed at evaluating DNMT3B polymorphisms as maternal risk factors for DS. Replication of the findings in other populations is required. If confirmed in subsequent studies, DNMT3B promoter polymorphisms might be additional markers to be taken into account when evaluating the contribution of one-carbon (folate) metabolism to the maternal risk of birth of a child with DS. None of the authors has any competing interest. This work was partially supported by the Italian Ministry of Health and ‘5 per mille’ funding.