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Development of specific DNA methylation patterns is required for normal spermatogenesis. DNA methyltransferases (DNMTs) use S-adenosylmethionine (SAM) produced in a pathway requiring 5,10-methylenetetrahydrofolate reductase (MTHFR). This thesis describes: testicular phenotype differences derived from Mthfr-deficiency in different mouse strains; the cellular Mthfr expression pattern during male germ cell development; and finally, changes to the DNA methylation of Mthfr-deficient sperm. Mthfr-deficient BALB/c, but not C57BL/6, mice have reduced neonatal germ cell proliferation but both have abnormal germ cells as adults. Germ cell MTHFR expression differed developmentally in parallel with DNMTs associated with de novo methylation. Sperm from mice with reduced Mthfr levels or dietary folate deficiency had differential DNA methylation at multiple loci, compared to wildtype mice, indicating that maintenance as well as acquisition of methylation can be altered by SAM-reduction. These results highlight the important role of folate in sperm development throughout life.

Orientadores: Ricardo Barini, Egle Cristina Couto de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T19:49:25Z (GMT). No. of bitstreams: 1 Soligo_AdrianadeGoeseSilva_M.pdf: 464907 bytes, checksum: c9378bf0a2d1fe337908abb53f773dbe (MD5) Previous issue date: 2007 / Resumo: Objetivo: determinar a prevalência dos fatores trombofílicos em mulheres inférteis. Método: estudo de corte transversal, no qual foram admitidas mulheres inférteis (atendidas em clínica privada) e submetidas à investigação de trombofilia, conforme protocolo da referida clínica, no período de março de 2003 a março de 2005. Foram incluídas mulheres em idade fértil com história de infertilidade, definida como um ano de coito sem método contraceptivo e sem concepção. Foram excluídas mulheres com hepatopatia e dados incompletos em prontuário, obtendo-se a amostra de 144 mulheres. Os fatores trombofílicos avaliados foram: o anticorpo anticardiolipina (ACL) e o anticoagulante lúpico (ACGL); a deficiência de proteína C (DPC), a deficiência de proteína S (DPS), a deficiência de antitrombina III (DAT), a presença do fator V de Leiden, uma mutação no gene da protrombina e a mutação da metileno tetrahidrofolato redutase (MTHFR). Resultados: os valores de prevalência obtidos para ACL e ACGL foram de 2%. A prevalência dos fatores trombofílicos hereditários foram: DPC 4%, DPS 6%, DAT 5%, fator V de Leiden 3%, mutação da protrombina 3%, mutação MTHFR 57%. Conclusões: das 144 pacientes selecionadas, 105 mulheres, ou seja, 72,9% apresentavam pelo menos um fator trombofílico presente. Isto reforça a importância e justifica a necessidade da investigação neste grupo / Abstract: Purpose: to establish the prevalence of thrombophilic factors in infertile women. Methods: a cross-sectional study was performed, in which infertile women were included, seen in a private clinic with investigation for thrombophilia, according to the protocol of the clinic, between March 2003 and March 2005, after the approval of the Research Ethics Committee of UNICAMP. One hundred and forty four infertile women without any liver disease were evaluated. Infertility is defined as one year of unprotected sexual intercourse without contraception and with no conception. The acquired and/or inherited thrombophilic factors are: anticardiolipin antibody (aCL) and lupus anticoagulant (LA); protein C deficiency (PCD), protein S deficiency (PSD), antithrombin III deficiency (ATD), presence of the factor V Leiden, mutation in the prothrombin gene, and mutation of Methylene tetrahydrofolate reductase (MTHFR). Results: the prevalence values obtained for aCL and LA were 2%. The prevalence of hereditary thrombophilic factors were: PCD 4%, PSD 6%, ATD 5%, factor V Leiden 3%, prothrombin mutation 3%, MTHFR mutation 57%. Out of the selected 144 patients, 105 women (72, 9%) presented at least one thrombophilic factor. This reinforces the importance and justifies the need of investigation in this grou / Mestrado / Tocoginecologia / Mestre em Tocoginecologia

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Statement of problem: We have shown that the C677T 5,10 methylenetetrahydrofolate reductase (MTHFR) variant is associated with elevated maternal serum α-fetoprotein (MSAFP), the most common screening test for neural tube defects (NTD). Therefore, past contradictory studies of NTDs and C677T MTHFR may have been biased because of changes in case populations after prenatal diagnosis and termination of pregnancy. Further, an unexplained elevation of MSAFP is known to increase the risk for later pregnancy complications. Is the C677T MTHFR variant a predisposing genetic variant for both NTDs and later complications of pregnancy? Methods: A retrospective study of women with pregnancies resulting in NTD outcome and women with unexplained elevations of MSAFP was undertaken. Women and their partners were genotyped for the C677T MTHFR allele. Couples with a pregnancy resulting in a NTD outcome were compared to couples whose pregnancy outcome did not involve. Couples with unexplained elevations of MSAFP who did and did not have later complications of pregnancy were also compared. Allele frequencies for all groups were then compared against the previously established Manitoba population allele frequency (based on 977 consecutive newborn metabolic screening bloodspots). A review of all studies of NTDs and association with the C677T MTHFR variant was undertaken to determine if the association between the variant and MSAFP is a source of bias. NTD incidence was examined before and after folic acid food fortification introduced in Canada in 1999. Results: There is an increase in the allele frequency of the C677T MTHFR variant in parents with an unexplained elevated MSAFP followed by later complications of pregnancy. The C677T MTHFR variant is also a contributing genetic factor to NTDs worldwide. The incidence of NTDs in Manitoba has decreased by 37% since food fortification with folic acid was introduced. Conclusions: The C677T MTHFR variant is a contributing genetic factor to both later complications of pregnancy after an unexplained elevation of MSAFP and to NTDs. This variant is folate sensitive and folic acid fortification has reduced the incidence of NTDs. / February 2005

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 85-105).

Methylenetetrahydrofolate reductase (MTHFR) synthesizes 5-methyltetrahydrofolate, a methyl donor for conversion of homocysteine to methionine. A common thermolabile variant causes mild MTHFR deficiency, induces mild hyperhomocysteinemia when plasma folate levels are low and increases risk for neural tube defects (NTD) and pregnancy loss. To increase our understanding of Mthfr regulation, the 5' and 3' regions of the mouse cDNA and gene were characterized. These studies revealed two major promoters, an internal coding exon in the 5'UTR, alternative transcriptional and translational start sites and alternative splicing and polyadenylation. These data suggest that Mthfr regulation is likely to be complex. To investigate the role of Mthfr in NTD, several approaches were taken. First, folate and MTHFR co-factor, flavin adenine dinucleotide, were shown to stabilize normal and thermolabile MTHFR during heat inactivation, suggesting that folate might prevent hyperhomocysteinemia in individuals with thermolabile enzyme through protein stabilization. Next, in situ hybridization of neurulating mouse embryos showed that Mthfr is expressed in the forebrain, hindbrain, branchial arches, blood vessels, gut, and importantly, in the ventral part of the neural tube. Mthfr+/- mice were then used as a model of mild deficiency to address the effects of maternal and embryonic Mthfr deficiency on development. When combined with inadequate dietary folate, Mthfr +/- pregnant females showed a two-fold higher rate of pregnancy loss than Mthfr+/+ pregnant females. As well, a percentage of day 10.5 embryos from only the Mthfr+/- pregnant females were underdeveloped by 2 days. These effects were not apparent when dietary folate was sufficient, consistent with a genetic-nutritional interactive effect. Finally, folate metabolism was investigated in an NTD model, the curly-tail (ct) mouse, since the ct defect and Mthfr were mapped in close proximity. However, Mthfr sequence in ct mice was simila

Hyperhomocysteinemia is a risk factor for cardiovascular disease and stroke. Nutritional and/or genetic disruptions in homocysteine metabolism can cause hyperhomocysteinemia. Mild methylenetetrahydrofolate reductase (MTHFR) deficiency due to the 677C → T mutation in the MTHFR gene is the most common genetic cause of hyperhomocysteinemia. The 677C → T variant is associated with an increased risk for neural tube defects, pregnancy complications, schizophrenia and Down syndrome, and with a decreased risk for colon cancer and leukemia. This variant is also a potential risk factor for vascular disease. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. We have generated mice with a knockout of the Mthfr gene. The Mthfr-deficient mice exhibit hyperhomocysteinemia and decreased methylation capacity. The Mthfr+/- mice appear normal, whereas the Mthfr-/- mice are smaller and have reduced survival. Abnormal external granule neuron development associated with increased cell death in the cerebellum was observed in the Mthfr-/- mice. / Evidence for cardiovascular pathology was obtained in several ways. Impaired aortic relaxation response to acetylcholine was seen in the Mthfr +/- mice fed a high methionine diet. Both Mthfr+/- and Mthfr-/- mice fed a low folate high methionine diet developed myocardial fibrosis in the left ventricle. Abnormal lipid deposition in the proximal portion of the aorta was observed in older Mthfr+/- and Mthfr-/- mice. After crossing Mthfr -deficient mice with apoE-null mice, we demonstrated that MTHFR deficiency promoted atherogenesis and its progression in the apoE-null mice. / Gene expression in brain of Mthfr-deficient mice was investigated via microarray analysis. Five genes with altered expression in the brain of Mthfr-/- mouse were validated by RT-PCR. In biochemical studies of human MTHFR, both FAD and folate were shown to stabilize the purified recombinant wild type and mutant MTHFRs from the baculovirus expression system against heat inactivation. The effect of folate appeared to be secondary to that of FAD, and S-adenosylmethionine (SAM) inhibited purified wild type and mutant MTHFRs with similar efficiency. / This dissertation will significantly contribute to our understanding of the role of MTHFR in human disease.