Folate Metabolism Genes Research Articles

Genetic variants in folate metabolism-related genes, serum folate and hepatocellular carcinoma survival: the Guangdong Liver Cancer Cohort study.

Folate metabolism is involved in the development and progression of various cancers. We investigated the association of single nucleotide polymorphisms (SNP) in folate-metabolising genes and their interactions with serum folate concentrations with overall survival (OS) and liver cancer-specific survival (LCSS) of newly diagnosed hepatocellular carcinoma (HCC) patients. We detected the genotypes of six SNPin three genes related to folate metabolism: methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) and 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR). Cox proportional hazard models were used to calculate multivariable-adjusted hazard ratios (HR) and 95 % CI. This analysis included 970 HCC patients with genotypes of six SNP, and 864 of them had serum folate measurements. During a median follow-up of 722 d, 393 deaths occurred, with 360 attributed to HCC. In the fully-adjusted models, the MTRR rs1801394 polymorphism was significantly associated with OS in additive (per G allele: HR = 0·84, 95 % CI: 0·71, 0·99), co-dominant (AG v. AA: HR = 0·77; 95 % CI: 0·62, 0·96) and dominant (AG + GG v. AA: HR = 0·78; 95 % CI: 0·63, 0·96) models. Carrying increasing numbers of protective alleles was linked to better LCSS (HR10–12 v. 2–6 = 0·70; 95 % CI: 0·49, 1·00) and OS (HR10–12 v. 2–6 = 0·67; 95 % CI: 0·47, 0·95). Furthermore, we observed significant interactions on both multiplicative and additive scales between serum folate levels and MTRR rs1801394 polymorphism. Carrying the variant G allele of the MTRR rs1801394 is associated with better HCC prognosis and may enhance the favourable association between higher serum folate levels and improved survival among HCC patients.

Higher Incidence of Common Polymorphisms in the Genes of Folate and Methionine Cycles in Children With Orofacial Clefs and Congenital Heart Defects Compared to their Unaffected Siblings.

Uninterrupted folate metabolism plays a vital role in embryonic development, ensuring a supply of one-carbon-activated folate cofactors for essential processes. Folate deficiency has been implicated in the development of orofacial clefts (OFC) and congenital heart disease (CHD). Although both malformations have been extensively studied in lieu of folate deficiency, the results of corresponding studies are ambiguous due to the interplay of maternal and fetal genomes controlling folate metabolism in the developing fetus. We used the innovative study design to compare affected and unaffected siblings from the same mother, thus minimizing the effect of the maternal genome. Thus, it might be possible to identify genetic markers of congenital malformations that pertain exclusively to the child. This study compared demographic and environmental factors between OFC or CHD-affected and unaffected pregnancies as well as the presence of polymorphisms in genes of folate metabolism between OFC or CHD-affected and unaffected siblings. Only the maternal fever in the first trimester was a risk factor for OFC, whereas the maternal advanced age, medication administration, and common polymorphism in the FPGS gene increased the risk of CHD formation. Both OFC and CHD formation were associated with a higher number of variant loci in genes of folate-methionine cycles. Both OFC and CHD formation were associated with a higher number of mutated loci in genes of folate-methionine cycles, indicating polygenic and possibly multifactorial inheritance.

Standardization and application of ARMS TaqMan real-time PCR for screening of folate metabolism genes in Han Chinese.

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG+GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

Polymorphism of Folate Metabolism Genes among Ethnic Kazakh Women with Preeclampsia in Kazakhstan: A Descriptive Study.

Preeclampsia is a severe multifactorial complication of pregnancy. Studies found associations between folate metabolism genes' polymorphisms and preeclampsia. However, investigations in this field are limited among Asian populations. Thus, the study's aim was to evaluate the prevalence of methionine synthase (MTR), methionine synthase reductase (MTRR), and methylenetetrahydrofolate reductase (MTHFR) genes' polymorphisms among ethnic Kazakh women with preeclampsia. This was a retrospective study involving 4246 patients' data for the period of 2018-2022. Identification of MTR, MTRR, and MTHFR genes' polymorphism was performed via PR-PCR. Peripheral blood samples were obtained for the analyses. In total, 4246 patients' data of Kazakh ethnicity with preeclampsia at >20 weeks gestational age who had undergone an investigation to identify polymorphisms of the folate metabolism pathway genes for the period of 5 years were included in this study. The most common and prevalent mutation was the MTRR A66G polymorphism: 24.5% of all tested patients with preeclampsia had the MTRR A66G polymorphism. It was highest among the 35-39 age group participants. The second most prevalent was the MTHFR C677T polymorphism: 9% of women with preeclampsia had the MTHFR C677T mutation. It was highest among women aged 30-34. There was a rare association of the MTR A2756G mutation with preeclampsia among the study participants. The identified levels of MTRR A66G and MTHFR C677T polymorphisms among the study participants suggest the importance of evaluating MTRR and MTHFR polymorphisms in women with preeclampsia. The role of the MTR A2756G polymorphism in the development of preeclampsia needs to be further investigated.

Genetic polymorphisms and their association with methotrexate polyglutamates during maintenance treatment in Korean children and young adults with acute lymphoblastic leukemia

The aim of this study was to investigate the impact of genetic polymorphisms on methotrexate (MTX) metabolism in Korean children and young adults with acute lymphoblastic leukemia, specifically focusing on MTX polyglutamates (MTX-PGs) in erythrocytes, which have been rarely studied. Korean children and young adult patients undergoing maintenance therapy for acute lymphoblastic leukemia, who were receiving weekly oral MTX doses of 20 mg/m²/week, were prospectively included. We investigated erythrocyte MTX-PG (PG1 to PG5) levels, MTX-PG/MTX dose ratios, and 222 genetic polymorphisms spanning 78 genes and three intergenic areas related to MTX transport, folate cycle metabolism, purine/pyrimidine pathways, and non-pathway genes (including TPMT and NUDT15 genotypes) to explore their association with MTX metabolism. MTX-PG levels were associated with MTX dose (p < 0.05), and MTX-PG3 comprised the majority of the total MTX-PGs, with a median of 39.3 %. Various polymorphisms within the same gene demonstrated differing associations with each type of MTX-PG, underscoring the complexity of MTX pharmacogenetics. Among the polymorphisms examined, 14 across 13 genes showed significant associations with MTX-PG2–5 levels, even after adjusting for the false discovery rate (ABCC5, ATG16L1, CEP72, FSTL5, GMPS, HTR3A, IMPDH1, NT5C2, SLC28A3, SLCO1B3, SUCLA2, TPMT, and TYMS). This study enhances our understanding of genetic polymorphisms in MTX metabolism and therapeutic monitoring for MTX maintenance, promoting personalized medicine in acute lymphoblastic leukemia patients.

Analysis of the contribution of polymorphisms of hemostasis system genes and folate cycle genes to the genetic presposition to the development of thrombosis in patients with Ph-negative myeloproliferative neoplasia in the Republic of Uzbekistan

Thrombotic complications often complicate the course of the underlying disease in patients with chronic myeloproliferative neoplasia. However, a thromboembolic condition is not observed in all patients.The aim of the study was to assess the carriage frequency of polymorphic genes of the blood coagulation system and folate metabolism genes and the contribution of genetic variants to the development of thrombosis in patients with chronic Ph-negative myeloproliferative neoplasia (CMPN).Materials and methods. Molecular genetic testing for the presence of genetic variants with an assessment of their frequency of occurrence and allelic load was carried out in 142 patients with CMPN. We studied polymorphisms of the folate cycle genes – A2756G (Asp919Gly) of the MTR gene, C677T (Ala22Val) of the MTHFR gene, A66G (ILe22Met) of the MTRR gene (rs1801394), as well as mutations of the genes of blood coagulation factors – G(455)A of the FGB gene, G20210A of the F2 gene, G10976A (Arg353Gln) of the F7 gene, G1691A (Arg506Gln) of the F5 gene. The study was carried out using real-time polymerase chain reaction (PCR); The biological material for the test was whole blood.Results and discussion. The study showed that only 6 out of 50 (12%) patients with thrombotic complications had no changes in the genes studied, while 88% of patients had one or another genotypic variant, which may indicate a high probability of involvement of a hereditary genetic factor in the development of hypercoagulability and thrombotic complications in patients with chronic MPN.Conclusions. A comprehensive study of the role, interaction and operating conditions of genes that control blood coagulation processes in patients with Ph-negative chronic MPN will make it possible to understand the causes of hemostatic system disorders and develop effective measures to prevent thrombotic complications in this category of patients.

Effects of methionine supplementation in a reduced protein diet on growth performance, oxidative status, intestinal health, oocyst shedding, and methionine and folate metabolism in broilers under Eimeria challenge

BackgroundThis study investigated effects of different methionine (Met) supplementation levels in a reduced protein diet on growth performance, intestinal health, and different physiological parameters in broilers under Eimeria challenge. A total of 600 fourteen-day-old Cobb500 male broilers were challenged with E. maxima, E. tenella, and E. acervulina, and randomly allocated in a 2 × 5 factorial arrangement. Birds received normal protein diets (20% crude protein, NCP) or reduced protein diets (17% crude protein, LCP), containing 2.8, 4.4, 6.0, 7.6, and 9.2 g/kg of Met.ResultsOn 6 and 9 days post inoculation (DPI), increasing Met level linearly improved the growth performance (P < 0.05). Total oocyst shedding linearly increased as Met level increased (P < 0.05). Duodenal villus height (VH):crypt depth (CD) in the LCP groups were higher on 6 DPI (P < 0.01) while lower on 9 DPI (P < 0.05) compared to the NCP groups. Jejunal CD and duodenal VH:CD changed quadratically as Met level increased (P < 0.05). On 6 DPI, liver glutathione (GSH) and glutathione disulfide (GSSG) linearly increased as Met level increased (P < 0.05). On 9 DPI, GSSG quadratically increased, whereas GSH:GSSG quadratically decreased as Met levels increased (P < 0.05). The expression of amino acid transporters linearly decreased as Met level increased (P < 0.05). The expression of zonula occludens 2 and claudin-1 linearly increased on 6 DPI whereas decreased on 9 DPI as Met level increased (P < 0.05). The expressions of cytokines were lower in the LCP groups than the NCP groups (P < 0.05). Interaction effects were found for the expression of IL-10 and TNFα on 6 DPI (P < 0.05), where it only changed quadratically in the NCP group as Met level increased. The expression of Met and folate metabolism genes were lower in the LCP groups than the NCP groups on 9 DPI (P < 0.05). The expression of these genes linearly or quadratically decreased as Met level increased (P < 0.05).ConclusionThese results revealed the regulatory roles of Met in different physiological parameters including oxidative status, intestinal health, and nutrient metabolism in birds fed reduced protein diet and challenged with Eimeria.

Effect of Methylfolate, Pyridoxal-5'-Phosphate, and Methylcobalamin (SolowaysTM) Supplementation on Homocysteine and Low-Density Lipoprotein Cholesterol Levels in Patients with Methylenetetrahydrofolate Reductase, Methionine Synthase, and Methionine Synthase Reductase Polymorphisms: A Randomized Controlled Trial.

Exploring the link between genetic polymorphisms in folate metabolism genes (MTHFR, MTR, and MTRR) and cardiovascular disease (CVD), this study evaluates the effect of B vitamin supplements (methylfolate, pyridoxal-5'-phosphate, and methylcobalamin) on homocysteine and lipid levels, potentially guiding personalized CVD risk management. In a randomized, double-blind, placebo-controlled trial, 54 patients aged 40-75 with elevated homocysteine and moderate LDL-C levels were divided based on MTHFR, MTR, and MTRR genetic polymorphisms. Over six months, they received either a combination of methylfolate, P5P, and methylcobalamin, or a placebo. At the 6 months follow-up, the treatment group demonstrated a significant reduction in homocysteine levels by 30.0% (95% CI: -39.7% to -20.3%) and LDL-C by 7.5% (95% CI: -10.3% to -4.7%), compared to the placebo (p < 0.01 for all). In the subgroup analysis, Homozygous Minor Allele Carriers showed a more significant reduction in homocysteine levels (48.3%, 95% CI: -62.3% to -34.3%, p < 0.01) compared to mixed allele carriers (18.6%, 95% CI: -25.6% to -11.6%, p < 0.01), with a notable intergroup difference (29.7%, 95% CI: -50.7% to -8.7%, p < 0.01). LDL-C levels decreased by 11.8% in homozygous carriers (95% CI: -15.8% to -7.8%, p < 0.01) and 4.8% in mixed allele carriers (95% CI: -6.8% to -2.8%, p < 0.01), with a significant between-group difference (7.0%, 95% CI: -13.0% to -1.0%, p < 0.01). Methylfolate, P5P, and methylcobalamin supplementation tailored to genetic profiles effectively reduced homocysteine and LDL-C levels in patients with specific MTHFR, MTR, and MTRR polymorphisms, particularly with homozygous minor allele polymorphisms.

Folate gene polymorphisms CBS 844ins68 and RFC1 A80G and risk of Down syndrome offspring in young Iranian women: A cross-sectional study

Background: Cytogenetics and association studies showed that folate gene polymorphisms can increase the risk of chromosomal nondisjunction and aneuploidies. The folate-metabolizing gene polymorphisms in Down syndrome mothers (DSM) have been assessed in a variety of populations. Reduced folate carrier 1 (RFC1) and cystathionine beta-synthase (CBS) are key enzymes in folate metabolism.&#x0D; Objective: 2 common polymorphisms, CBS 844ins68 and RFC1 A80G, were analyzed to determine their probable risk for having Down syndrome (DS) babies in young mothers of Khuzestan province, Iran.&#x0D; Materials and Methods: This study was conducted on 100 mothers who had trisomy 21 DS children. 100 age- and ethnic-matched mothers with at least 2 healthy children and no history of abnormal pregnancies were considered as control. The samples were collected from all the mothers from June 2019 to April 2021. Genomic DNA was extracted from peripheral blood. The CBS-844ins68 and RFC1-A80G were genotyped using polymerase chain reaction-electrophoresis and restriction fragment length polymorphism, respectively.&#x0D; Results: The frequency of RFC1 AG and GG genotypes in DSM was significantly higher than the control mothers (odds ratio [OR] of 2.38 and 3.07, respectively). The heterozygote genotype of CBS 844ins68 was significantly more prevalent among DSM than the control (OR: 2.419). The OR was significantly increased to 6.667 when the homozygote of both variants was found together.&#x0D; Conclusion: Studying polymorphisms possibly increases the susceptibility of having a DS child. However, ethnicity, nutrition, and epistatic interactions are considerable factors to be evaluated in future studies.&#x0D; Key words: Down syndrome, Folic acid, Polymorphism, CBS, RFC1.

Association Study of Polymorphisms in Folate Metabolism and Mothers of Down Syndrome Offsprings in the Southwest of Iran

Background: Down syndrome (DS) is a complex genetic disease that is caused by having three copies of chromosome 21. A possible association between polymorphisms in maternal folate metabolism genes and DS has been evaluated. Objectives: It was aimed to first investigate the influence of C677T and A1298C polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR) and plasma homocysteine (Hcy) on the maternal risk for DS in the southwest of Iran. Methods: The MTHFR C677T and A1298C polymorphisms were genotyped using restriction fragment length polymorphism and Sanger sequencing, respectively. Allele and genotype frequencies and the dominant model of the MTHFR C677T and A1298C polymorphisms were evaluated in 80 mothers of children with DS and 80 control mothers. Eventually, the ELISA test was used to compare the concentration of plasma Hcy in both groups. Results: A significant association was observed in the 677T and 1298C alleles between the mothers of DS and control groups (P = 0.00077 and P = 0.01248, respectively). Further, the median concentrations of Hcy were significantly higher in mothers with DS babies compared to the control group (P &lt; 0.05). Conclusions: There was an association between MTHFR C677T, A1298C, and plasma Hcy concentrations as the maternal risk of mothers with DS children.

Constructing a folate metabolism gene signature for predicting prognosis in pulmonary neuroendocrine carcinomas.

Folate metabolism is a crucial biological process in cell proliferation and exhibits its pro-tumorigenic functions in multiple tumor types. However, its role in pulmonary neuroendocrine carcinomas remains uncertain. Folate metabolism related genes were obtained from previous studies, and the gene expression data and clinical data were collected from GEO database. The expression patterns of folate metabolism related genes were measured across normal and tumor tissues. We subsequently assessed their prognostic role using Kaplan-Meier and univariate Cox regression analysis. The core genes were isolated from 16 prognostic genes through four algorithms. Based on the expression of core genes, patients were divided into two clusters employing consensus clustering algorithm. Furthermore, we evaluated immune infltration level, biological mechanisms, and drug sensitivity. ALDH1L2 was finally identified through qRT-PCR and its pro-tumorigenic function was confirmed via in vitro experiments. The expression patterns of 26 folate metabolism related genes were evaluated between normal lung tissues and PNEC tumor tissues, and 20 of them exhibited differential expression. All of folate metabolism related genes were related to the prognosis of PNECS and 16 genes were identified as prognostic genes. Using SVM-RFE, RF, Xgboost and LASSO algorithm, three core genes were isolated from 16 prognostic genes. Based on the expression patterns of core genes, PNECs patients were divided into two clusters through consensus clustering algorithm. Cluster 1 was characterized by the worse survival, higher immune infiltration level, and sensitivity to chemotherapy. Compared with the HBEC cells, ALDH1L2 was notably overexpressed in NCI-H446 cells (SCLC cell line). ALDH1L2 knockdown significantly repressed the proliferation and migration capacity of tumor cells and increased the cell proportion in S phase. Our results indicated that folate metabolism gene signature is a reliable biomarker for PNECs. Classification based on this signature could be utilized to guide the treatment of PNECs patients and improve its prognosis.

Novel Role of Mitochondrial Folate Metabolism in Cell Fate Decisions and Leukaemogenesis

Folate-mediated one carbon (1C) metabolism sustains proliferation of rapidly dividing cells through its direct contribution to nucleotide biosynthesis. In brief, new 1C units are donated from the amino acid serine (the primary source of 1C units), while folate molecules function as 1C unit carriers. Accordingly, antifolates have been used in the treatment of several malignancies, particularly in the field of haemato-oncology. Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder that arises in a single haematopoietic stem cell with the introduction of the BCR::ABL1 oncoprotein. Although it is generally accepted that CML is driven by leukaemic stem cells (LSCs) that are insensitive to standard therapy due to their quiescent/slow-cycling status, the role of folate metabolism in LSCs remains undescribed. Transcriptomic analysis of patient-derived CML LSCs (CD34 +38 -) revealed a significant upregulation of folate metabolism genes, including the mitochondrial hydroxymethyltransferase (SHMT2; p≤0.05), when compared with normal counterparts. Furthermore, CML stem/progenitor (CD34 +) cells displayed a significant increase in the exchange rate of formate (folate intermediate necessary for nucleotide synthesis) compared to the secretome of normal CD34 + cells (p&amp;lt;0.05), suggesting an upregulation of 1C metabolism in therapy resistant CML cells. Following SHMT2 knockout (KO), we observed a strong antiproliferative effect and significantly impaired tumour formation in CML cell line xenograft models (p&amp;lt;0.01), verifying that mitochondrial 1C metabolism is not dispensable for CML cells. Metabolic characterisation using liquid chromatography-mass spectrometry revealed that both genetic and pharmacological inhibition (using the SHMT1/2 inhibitor, SHIN1) of 1C metabolism results in a significant decrease in de novo purine synthesis. Furthermore, we uncovered activation of AMPK signalling and suppression of mTORC1 activity following mitochondrial 1C metabolism inhibition. Notably, formate supplementation or reconstitution of purine levels was sufficient to reverse the effect on AMPK and mTORC1. Phenotypically, SHIN1 treatment induced the expression of erythropoiesis markers CD71 and Glycophorin A in CML cells, including patient derived CD34 + cells. AMPKα1/α2 KO revealed that the increased expression of these markers was independent of AMPK activity. Mechanistically, we discovered that reconstitution of purine levels (adenine) and mTORC1 activity prevented erythroid differentiation following SHMT1/2 inhibition, highlighting that depletion of 1C units drives differentiation of leukaemic cells through mTORC1-mediated purine sensing. Of clinical relevance, combination of SHIN1 with imatinib, a frontline treatment for CML patients, decreased the number of therapy-resistant CML LSCs in a patient-derived xenograft model (p&amp;lt;0.05). Lastly, stable isotope-assisted metabolomics using 13C 315N 1-serine revealed that imatinib reduced labelling into purine nucleotides, but it maintained labelling into glutathione (GSH), the most abundant antioxidant in the cell, and its oxidised derivate (GSSG). Through generation of glycine, folate metabolism directly contributes to the GSH pool. These data suggest that standard therapy does not affect the incorporation of serine into GSH, implying that the synergistic effect with folate metabolism inhibition might be related to impeded antioxidant defence. Overall, our findings highlight a novel role for mitochondrial folate metabolism in stem cell fate decisions and leukaemogenesis. This work is available as a preprint: https://www.biorxiv.org/content/10.1101/2022.12.21.521404v1

Allelic variants of folate metabolism genes among students in Arctic Russia

AIM: To assess the prevalence of allelic variants of folate metabolism genes in Russian and Indian students living in the Arctic region of Russia.&#x0D; METHODS: A cross-sectional study was performed on a sample of ethnic Indians, students at the Faculty of General Practitioner of the Northern State Medical University, living in Arkhangelsk and ethnic Russians who were born in Arkhangelsk region. The study included 117 ethnic Indians and 195 Russian participants. A survey of participants and a molecular genetic study of the nucleotide sequence in the genes involved in the exchange of folate by real-time PCR were carried out. A group of ethnic Indians was compared with Russians according to 4 variants of the nucleotide sequence in the genes involved in folate metabolism.&#x0D; RESULTS: Statistically significant differences were observed in the frequency distribution of the T allele of the MTHFR gene polymorphism (rs1801133) (p=0.001). Among Russians, the occurrence of the T allele was found to be 26.3%, whereas among Indians, it was only 7.7%. At the same time, no statistically significant differences were found in the distribution of other polymorphic variants of the folate cycle genes between the groups. The mutant allele G of the MTRR gene polymorphism (rs1801394) was the most frequently occurring polymorphism among the participants.&#x0D; CONCLUSION: Greater prevalence of the unfavorable allele T of the MTHFR gene (rs1801133) compared to the group of ethnic Indians may suggest an elevated susceptibility to folate metabolism disorders in the Russian population

Phase 1 Trial of Orelabrutinib in Combination with Rituximab, Methotrexate, and Dexamethasone in Patients with Newly Diagnosed Primary CNS Lymphoma Implementing Bayesian Design for Dose-Seeking

Background: Primary central nervous system lymphoma (PCNSL) is an aggressive extranodal subtype of non-Hodgkin's lymphoma (NHL). High-dose methotrexate (HD-MTX)-based regimens are the first-line treatment for PCNSL, but remain suboptimal due to response instability and short effective remission duration. Orelabrutinib is a novel, potent, highly selective BTK inhibitor with a high CSF concentration. This study aims to investigate optimal combination dose of Orelabrutinib in combination with Rituximab, Methotrexate, and Dexamethasone (ORMD) as well as safety and efficacy of the regimen. Methods: This was a phase 1, investigator-initiated, dose-escalation study of ORMD, being conducted at Huashan Hospital, Fudan University (NCT05036577). Eligible patients must have histopathologically confirmed PCNSL-DLBCL by biopsy of brain lesions and be aged 18-75 years with adequate organ functions. Patients were treated for 6-8 cycles of induction therapy with 21 days per cycle, receiving rituximab (375mg/m 2 on day 1), dexamethasone (10-15mg on d1-d4), MTX (d2, 3.5g/m 2 or 5g/m 2), and orelabrutinib (once daily, after MTX clearance, 150mg/d, or 200mg/d), followed by orelabrutinib maintenance up to one year among CR/CRu patients. The primary objective was to determine the maximum tolerated dose (MTD) of the combination of orelabrutinib and MTX with R and D and investigate the safety and tolerability of this regimen using Bayesian Optimal Interval (BOIN) waterfall design to determine rule of dose escalation and movement among dose combination matrix to identify MTD contour. DLT was defined by the occurrence of severe toxicities during the first cycle: any grade 4 hematologic toxicity, grade 3 febrile neutropenia and grade 3 thrombocytopenia with hemorrhage, or any grade 3 non-hematologic toxicity that failed to respond to supportive therapy and possibly related to orelabrutinib and/or MTX (assessed according to NCI CTCAE V5.0). The secondary objectives included ORR/CR（u） and PFS/OS. Results: From October 2021 to July 2023, the study completed enrollment of all 13 patients. The median age was 60 years, and other basic characteristics are shown in Figure 1. All the patients had completed at least one cycle of ORMD treatment and were evaluable for DLT. The median cycle of ORMD was seven (range: 2.5-8). No DLT occurred among first 3 patients in cohort 1 (Orelabrutinib: 150mg/MTX: 3.5g/m 2) and dose escalation was allowed to cohort 2 (Orelabrutinib: 200mg/MTX: 3.5g/m 2). In cohort 2, 5 patients were dosed in total, and Gr 3 lung infection was observed in patient #4 during cycle 1, was deemed as a DLT. Another four patients were enrolled in the same cohort according to BOIN design algorithm and no DLT was identified, allowing to escalate to cohort 3 (Orelabrutinib:200mg/MTX:5g/m 2). In cohort 3, 5 patients were dosed in total, and Gr 3 elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was observed in patient #9 during cycle 1, was deemed as a DLT. The causality of this AE was certain to methotrexate due to folate metabolic gene polymorphism in the assessment of the investigator. The toxicity probability from 3 dose combo cohort is 0.13, 0.13 and 0.21, all of which is below predefined threshold 0.3. So MTD is not reached and 200mg+5g/m 2 dose combo remains to be RP2D candidate. No Gr 4 AE had been observed. The most common adverse events were lymphopenia (Gr 3 in 8 case), neutropenia(Gr 3 in 2 case), Covid-19(Gr 3 in 2 case) and hyperglycemia (Gr 3 in 2 case). No atrial fibrillation or bleeding was observed by the cut-off date. ORR and CR/CRu in 12 patients possessed at least one efficacy evaluation were both 83.3%, the PFS and OS rate were 56.6% (95% CI, 29.6% ,100%) and 100%(95% CI, 100% ,100%) at 1 year. Among nine patients completed ≥6 cycles of induction therapy, ORR and CR/CRu were both 100%. PFS and OS rate were 75.0% (95% CI, 42.6% ,100%) and 100%(95% CI, 100% ,100%) at 1 year. All patients in remission achieved CR/CRu, the high rate of deep remission may be related to the addition of orelabrutinib. Conclusions: As a novel therapeutic combination of induction therapy, ORMD therapy has shown deep remission potential as well as a predictable and manageable safety profile in patients with newly diagnosed PCNSL. MTD contour not reached. The combination of this induction therapy with consolidation therapies such as ASCT deserves further investigation and may provide greater survival benefit for patients.

71P Single nucleotide polymorphisms in the folate metabolic pathway genes and global DNA methylation in ovarian cancer

71P Single nucleotide polymorphisms in the folate metabolic pathway genes and global DNA methylation in ovarian cancer

Marine bacteria harbor the sulfonamide resistance gene sul4 without mobile genetic elements.

Marine bacteria are possible reservoirs of antibiotic-resistance genes (ARGs) originating not only from clinical and terrestrial hot spots but also from the marine environment. We report here for the first time a higher rate of the sulfonamide-resistance gene sul4 in marine bacterial isolates compared with other sul genes. Among four sulfonamide-resistance genes (sul1, sul2, sul3, and sul4), sul4 was most abundant (45%) in 74 sulfonamide-resistant marine isolates by PCR screening. The order of abundance was sul4 (33 isolates) >sul2 (6 isolates) >sul3 (5 isolates) >sul1 (1 isolate). Whole-genome sequencing of 23 isolates of sul4-expressing α- and γ-proteobacteria and bacilli revealed that sul4 was not accompanied by known mobile genetic elements. This suggests that sul4 in these marine isolates is clonally transferred and not horizontally transferable. Folate metabolism genes formed a cluster with sul4, suggesting that the cluster area plays a role in folate metabolism, at which sul4 functions as a dihydropteroate synthase. Thus, sul4 might be expressed in marine species and function in folate synthesis, but it is not a transferable ARG.

Correlation between single nucleotide polymorphisms of folate metabolism genes and ethnic distribution in pregnant women.

This retrospective study aims to identify the single nucleotide polymorphisms (SNPs) of 5,10-methylenetetrahydrofolate reductase (MTHFR) (C677T, A1298C), methionine synthase reductase (MTRR) (A66G) and ethnic distribution characteristics in pregnant women, and to explore the risk correlation with folate metabolism. The demographic data of 8735 pregnant women aged 15 to 47 years were retrospectively analyzed, and peripheral blood samples were collected and tested. Reverse transcription-quantitative polymerase chain reaction was applied to determine the genotype and allele frequency of MTHFR C677T, A1298C and MTRR A66G in blood samples. Sperman correlation analysis, univariate and multivariate logistic regression analysis were used to verify the correlation between SNPs of MTHFR (C677T, A1298C), MTRR (A66G), different ethnic groups and the susceptibility and risk levels of folate metabolism. The relative risk of the SNPs was further determined by calculating the odds ratio (OR) at a 95% confidence interval (CI). The average age of 8735 pregnant women was 28.87 ± 4.20 years old. The evaluation of risk levels for folate metabolism was relative high, including 2296 cases with low risk, 3971 cases with medium risk, and 752 cases with high risk. Among the MTHFR C677T locus, the CC genotype had the highest frequency, MTHFR A1298C locus had the highest frequency of the AA genotype, and MTRR A66G locus had the highest frequency of the AA genotype. The frequency distribution of SNPs in different ethnic groups revealed that the frequency of CT genotype among the MTHFR C677T locus, AA genotype among the MTHFR A1298C locus and the MTRR A66G locus was the highest in Han, Buyi, Miao and Dong ethnic groups. The results of logistic regression analysis showed that the Han, Buyi, Miao and other ethnic groups (including Yi, Bai, Zhuang, Chuanqing) had the possibility of increasing the risk levels of folate metabolism. The CC genotype of MTHFR C677T (adjusted OR = 2.46, 95% CI: 2.14-2.84, P < .001) and the AG genotype of MTRR A66G (adjusted OR = 1.89, 95% CI: 1.61-2.22, P < .001) were significantly related to the risk levels of folate metabolism, which is an independent risk factor for the susceptibility of folate metabolism.

Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages.

Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.

АNALYSIS OF THE ROLE OF FOLATE CYCLE GENES IN THE PATHOGENESIS OF ROSACEA

The polymorphism of the genes of the folate cycle was studied. The study identified genes that are presumably involved in the occurrence and development of rosacea, as well as affecting the severity of the disease. Unfavorable variants of the folate metabolism genes: MTHFR: 1298 A&gt;C rs1801131, MTHFR: 677C&gt;T rs1801133. An increase in the level of homocysteine in the blood of patients with rosacea turned out to be significant. The identified loci provide specificity of inflammatory mechanisms in rosacea, and identify potential pathways for therapeutic intervention.

Polymorphism of folate metabolism genes in breast cancer patients

Breast cancer is a widespread oncological disorder, which is considered one of the leading causes of mortality among women. DNA repair processes a well as its methylation involving genes of folate cycle play a crucial role in cancerogenesis. The aim of the study is to generalize the current data about association between polymorphic variants of folate cycle genes MTHFR С677Т (rs1801133), MTR A2756G (rs1805087), MTRR А66G (rs1801394) with risk of breast cancer development. The search of scientific papers has been conducted using PubMed and elibrary.ru sources. Original and randomized surveys published from 2008 till 2022 have been included in this review.&#x0D; The study has accumulated sufficient data on MTHFR С677Т polymorphic variant in breast cancer patients, having significant influence on disease development. However, the data about MTR A2756G and MTRR A66G variants is quite limited. According to the findigs of many scientific papers, there is no link between polymorphic variants of these genes and breast cancerogenesis manifestation. Such factors like ethniсity and sufficient consumption of folates with nutrition can have significant impact on results and conclusions of studies about role of folate cycle genes in breast cancer patients.&#x0D; Further investigation of MTHFR С677Т (rs1801133), MTR A2756G (rs1805087) and MTRR А66G (rs1801394) genetic polymorphic variants with consideration of gene-environment and gene-gene interactions could explain the presence of individual differences of breast cancer risk.