C3435T Single Nucleotide Polymorphisms Research Articles

Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR. Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay. We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits. We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.

Prevalence of C677T single nucleotide polymorphism of methylene tetrahydrofolate reductase gene and its relationship with serum levels of homocysteine, vitamin B12, folate and cholesterol in Alzheimer’s patients

Prevalence of C677T single nucleotide polymorphism of methylene tetrahydrofolate reductase gene and its relationship with serum levels of homocysteine, vitamin B12, folate and cholesterol in Alzheimer’s patients

Multidrug resistance Gene-1 polymorphisms (C3435T and G2677T) and the risk of inflammatory bowel disease in Egyptian patients

Background and study aimsThe multidrug resistance 1 (MDR1) gene is a gene involved in the pathogenesis of inflammatory bowel disease (IBD).The aim of the study is to investigate the association of MDR-1 gene polymorphisms (C2345T and G2677T) and IBD incidence in Egyptian patients, and its relation with disease severity. Patients and methodsThis is a case-control study where genotyping of MDR-1 gene C3435T and G2677T single nucleotide polymorphisms (SNPs) were assayed. ResultsForty naïve IBD patients, who were composed of 25 UC and 15CD, were compared to 60 healthy controls. They were young aged with significant female predominance, particularly in CD (P = 0.004). UC was mainly (48 %) presented in moderate severity while CD was mainly (53.3 %) presented with mild severity. MDR-1 gene C3435T SNP was not statistically related to IBD, whether in terms of genotypes or alleles, yet its T allele was significantly related to moderate cases of UC (P = 0.014). However, GG genotype of G2677T SNP was significantly low in IBD (P = 0.013), while TT genotype and T allele were significantly related to CD (P = 0.011, and 0.012 respectively). Moreover, G allele proved to be associated significantly with moderate cases of UC (P = 0.001) and mild cases of CD (P = 0.002). ConclusionsMDR-I gene G2677T SNP GG genotype proved to be protective against IBD, thus may be considered in diagnostic workup of IBD including its severity.

Methylenetetrahydrofolate reductase polymorphic variants C677T and A1298C in rectal cancer in Slavic population: significance for cancer risk and response to chemoradiotherapy.

Background: Methylenetetrahydrofolate reductase (MTHFR) single nucleotide polymorphisms (SNPs) have been suggested as risk, prognostic, and predictive factors for colorectal cancer in various populations, but have not been validated so far. The aim of this study was to examine the association of MTHFR C677T (rs1801133) and A1298C (rs1801131) single nucleotide polymorphisms with the risk of rectal cancer as well as the response to neoadjuvant chemoradiotherapy (nCRT) based on 5-Fluorouracil (5-FU)/leucovorin (LV) in the locally advanced setting. Patients and methods: This case-control study included 119 healthy controls and 97 patients with locally advanced rectal cancer (LARC). For MTHFR genotyping, restriction fragment length polymorphism analysis (PCR-RFLP) was employed. Results: In silico analysis highlighted that SNPs C677T and A1298T correlate with MTHFR gene expression, and that gene expression profile correlates with cancer risk and stage. Using dominant and recessive models, it was found that the MTHFR 677CC vs. 677CT+677TT have increased risk of cancer development (odds ratio (OR): 2.27; 95% confidence interval (CI): 1.30-3.95, p = 0.002) as well as 677CC+677CT compared to 677TT (OR: 4.18, 95% CI: 1.16-14.99, p = 0.014). MTHFR 1298AA also shown increased risk for cancer development compared to 1298AC+1298CC (OR:2.0, 95% CI: 1.20-3.59, p = 0.035) Statistical analysis of combined genotypes highlighted the protective role of CT/AC combined genotype (OR: 3.15 95% CI: 1.576-6.279, p = 0.002) while the CC/AA genotype showed an increased risk for rectal cancer development (OR: 2.499, 95% CI: 1.246-5.081, p = 0.016) The carriers of the 677C/1298A haplotype had the highest risk for developing rectal cancer (OR: 1.74; 95% CI: 1.198-2.530, p = 0.002) while the 677T/1298C haplotype seems to provide a protective effect. (OR: 0.44; 95%CI 0.248-0.795, p = 0.003). No significant association with response to chemoradiotherapy was found. Conclusion: Our data point to MTHFR 667C allele and 1298A alleles as low-penetrance risk factors for rectal cancer in our population. To the best of our knowledge, this is the first study of this type performed on the Slavic population in the Western Balkan, as various population-based factors might also be significant our findings can be used for future meta-analyses and the construction of genetic cancer risk prediction panels.

Association Between Multiple Single Nucleotide Polymorphisms in Folate Metabolism Pathway and Breast Cancer Risk in Georgian Women: A Case-Control Study.

The folate metabolism pathway plays an integral part in DNA synthesis, methylation, and repair. Methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase (MTHFD1) are both enzymes that are involved in this pathway, and the single nucleotide polymorphisms (SNPs) in genes coding for them have modulatory effects on DNA expression. This study aimed to investigate the relationship between MTHFR C677T (rs1801133) and MTHFD1 G1958A (rs2236225) polymorphisms and the risk of developing breast cancer in Georgian women. A case-control study was performed examining the MTHFR C677T and MTHFD1 G1958A SNP in breast cancer-confirmed cases and healthy matched controls. Real time-polymerase chain reaction (PCR) was used to genotype SNPs. The case individuals' pathology reports were obtained following surgeries for cancer characteristic data. Statistical analysis was performed to investigate the significance of the acquired data. Statistical analysis of MTHFR C677T SNP revealed that the CT genotype increased the risk of breast cancer by 2.17 folds in the over-dominant model. Statistical analysis of MTHFD1 G1958A SNP showed that the GA genotype increased the risk of breast cancer by 4.12 folds in the codominant model and 2.41 folds in the over-dominant model. No statistically significant link was found between genotypes and lymph node status, however, patients with the CT genotype had higher percentages of proliferative activity. Breast cancer seems to have a statistically significant association with the CT genotype in MTHFR C677T and the GA genotype in MTHFD1 G1958A in Georgian women.

Impact of MTHFR Polymorphisms C677T and A1298C in Acute Lymphoblastic Leukemia and Burkitt Lymphoma Patients Exposed to Methotrexate and Asparaginase

Background: Asparaginase (Asp) and high-dose methotrexate (HDMTX) are pivotal drugs in the treatment of aggressive hematological malignancies as acute lymphoblastic leukemia (ALL) and Burkitt lymphoma (BL). Asp and MTX present specific drug-related toxicities; for Asp, prothrombotic alteration of the coagulation profile, hepatobiliary and pancreatic toxicities, for MTX, renal, hepatobiliary, hematologic, gastro-intestinal and neuro- toxicity. The 5,10-methylenetetrahydrofolate reductase (MTHFR) is the enzyme involved in conversion of homocysteine into methionine. MTHFR most frequent single nucleotide polymorphisms C677T and A1298C are associated with reduced enzymatic activity up to 60%, that implies hyperhomocysteinemia and increased toxicity of MTX. Numerous studies have explored the relationship between C677T and A1298C polymorphisms of MTHFR and MTX toxicity and/or outcome in children ALL, but results are controversial. Nevertheless, it is common practice to reduce MTX dosage in adult patients in homozygosis. Aims: To explore the relationship between the MTHFR C677T and A1298C polymorphisms in homozygosis and development of MTX toxicity in a cohort of adult patients; furthermore, to evaluate the relationship with thrombotic risk in patients exposed to Asp. Materials and Methods: Data from consecutive adult onco-hematologic patients for whom MTHFR C677T and A1298C polymorphism analysis was available, treated with chemotherapeutic regimens containing HDMTX and/or Asp was retrospectively collected at a single center Italian institution. Adverse events (AEs) were graded according to CTCAE 5.0. Statistical analysis was performed with R language. The impact of MTHFR polymorphisms was evaluated by mean of a multivariate analysis adjusting Odds Ratio (OR) estimates for drug dosage and disease status. Results: Data from 78 patients, 57 affected by ALL, 3 by mixed phenotype leukemia (MPAL) and 18 by BL, was retrospectively collected. Overall, 13 (16.7%) of patients were 677TT and 8 (10.3%) were 1298CC. Patients with ALL/MPAL received mostly pediatric-inspired chemotherapy regimens (containing MTX 2.5 - 5 g/m 2 and Asp 1000-2000 UI/m 2), patients with BL received modified Rituxumab (R)-BFM protocol (MTX 1.5 g/m2); age/comorbidities-adjusted dose reduction was performed according to institutional guidelines. Median age for ALL and BL patients was 37.5 (IQR 27.0-54.2) and 33.5 (26.0-49.7) years, respectively; 54/78 (69.2%) of patients were male. Patients received a median of 3 (IQR 2-6) cycles containing HDMTX and/or Asp, for a total number of 299 cycles (94 containing Asp, 184 HDMTX and 24 both); 142 (48.0%) cycles were administered for active disease (AD), at diagnosis/relapse, and 154 (52.0%) in complete remission (CR). The impact of different dosages of Asp < 1000 UI/m 2 and ≥ 1000 UI/m² and of HDMTX <1.5 g/m 2 and ≥ 1.5 g/m 2 as well as the impact of disease status (AD vs CR) on AEs was explored. Concerning Asp therapy and thrombotic risk, 13 deep vein thrombosis (DVT) were observed, but no association between 677TT nor 1298CC was appreciated. AD status was strongly related to DVT (21.4% vs 1.8%, p 0.003). Coherently with the MTHFR-independent drug's mechanism of action, the polymorphisms had no impact on the development of Asp-related toxicities. Diversely, concerning HDMTX, in multivariate analysis, patients with 677TT were more likely to experience AST increase grade ≥3 (OR 10.55, 95%CI 1.34-83.09) while a protective role versus any grade GGT increase was observed (OR 0.09, 95%CI 0.01-0.71) (Figure 1A). Patients with 1298CC exhibited a superior incidence any grade AST/ALT increase, not confirmed for grade ≥3 (Figure 1B). No significant superior toxicity was observed in terms of renal, gastro-intestinal, hematologic AEs. The impact of a MTX dose reduction >25% of expected dose was then explored in a homogeneous population of 45 front-line pediatric-regimen treated ALL patients (35 received MTX dose ≥75% and 10 < 75%) and no impact in terms of OS and RFS was appreciated. Conclusions: The analysis ofMTHFR polymorphisms in adult patients is useful in identifying patients at higher risk for transaminitis, providing a biological parameter to integrate with hepatosteatosis, liver stiffness evaluation and BMI, for a comprehensive hepatic baseline evaluation. Patients at higher risk for hepatic complications may benefit from MTX dose reduction with no impact on disease outcome.

Prevalence of genetic polymorphisms ABCB1/ C3435T in patients with epilepsy in Iranian population

Introduction: Epilepsy is a common neurological disorder affecting approximately 1% of the global population. A significant proportion of patients develop drug-resistant epilepsy (DRE), which leads to poor outcomes for treatments and low quality of life. The multidrug transporter P-glycoprotein, encoded by the ABCB1 gene, plays a key role in limiting brain penetration of antiepileptic drugs. The C3435T single nucleotide polymorphism (SNP) in ABCB1 has been linked to variable P-glycoprotein expression and AED response. In this study, the prevalence of C3435T genotypes among epilepsy patients and healthy controls in Fars Province, Iran was investigated. Materials and methods: This study involved 50 patients diagnosed with epilepsy and 100 healthy individuals without a history of seizures or epilepsy. Genotyping was conducted using PCR-RFLP analysis. Results: The variant T allele occurred at a frequency of 52% in cases and 54% in controls. However, no significant association between the C3435T polymorphism and epilepsy risk was observed. Interestingly, it was found that the CT genotype was overrepresented in drug-responsive patients compared to those with drug-resistant cases. This suggests that individuals with the CT genotype may have a more favorable response to antiepileptic drug therapy. Conclusion: These findings suggest that the ABCB1 C3435T variant may play a role in influencing seizure control specifically among Iranian patients, but its effects on susceptibility are minimal. Moreover, individuals with the CT genotype may have a more favorable response to antiepileptic drug therapy. Further research involving larger cohorts is needed to fully understand the impact of ABCB1 genetics on determining individual responses to antiepileptic drugs. Such research could also pave the way for personalized management strategies based on pharmacogenomic testing.

Prevalence of C677T Single Nucleotide Polymorphism of Methylenetetrahydrofolate Reductase Gene and its Relationship With Serum Levels of Homocysteine, Vitamin B12, Folate, and Cholesterol in Alzheimer’s Patients

Prevalence of C677T Single Nucleotide Polymorphism of Methylenetetrahydrofolate Reductase Gene and its Relationship With Serum Levels of Homocysteine, Vitamin B12, Folate, and Cholesterol in Alzheimer’s Patients

#3145 ASSOCIATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE POLYMORPHISM WITH ENDOTHELIAL DYSFUNCTION IN DIABETIC NEPHROPATHY AMONG AZERBAIJANIS

Abstract Background and Aims Vascular endothelial dysfunction resulting from impaired nitric oxide synthase (NOS) activity in the endothelial cells of blood vessels has been suggested as playing an important role in the pathogenesis of Diabetic nephropathy (DN). It regulates endothelial function and maintains endothelial-dependent vasodilation in multiple organs, including the kidney. The study was carried out to investigate the association of T-786C single nucleotide polymorphisms with the flow mediated dilation (FMD) in DN among type 2 diabetic subjects. Method 90 patients (45 male and 45 female) with diabetic nephropathy were recruited from Nephrology and Endocrinology departments of the Azerbaijan Medical University hospital. As a control group the results were compared with the findings in 100 healthy Azerbaijani individuals (50 males and 50 females). The study was approved by ethics committee and informed consent was obtained from all patients in written form. Brachial endothelial function was determined by brachial artery vasodilator response or brachial FMD in the dominant arm according to previously validated techniques. T786C polymorphism was detected using polymerase chain reaction-restriction fragment length polymorphism analysis. Results The results showed significant differences between DN group and control group regarding the genotype and allele distributions of the T786C single nucleotide polymorphisms. The CC genotype was significantly more frequent in diabetics nephropathy patients than in control group (42% vs. 8%, p = 0.01). FMD in allele homozygote T786C(TT), T786C(CC) and heterozygote T786C(CT) were respectively 13.5 ±0.16%, 12.3±0.19%,13.9 ±0,28%. In the study groups, Azerbaijani patients showed dominance of the C allele and a mutant homozygous genotype CC of the T786C gene of eNOS. Subjects with the 786C(CC) (n = 48) showed significantly reduced FMD with those with T786C (TT) (n = 7) (p = 0.021) and T786C (CT) (n = 35) (p = 0.023), whereas there was no significant difference in endothelial-independent vasodilation between these groups. Although subjects with homozygote T786C(TT), did not show significantly reduced levels of FMD. Who were carriers of the T786C allele demonstrated markedly reduced levels of FMD compared with noncarriers (p = 0.019). Conclusion The data suggest that in diabetic nephropathy, the eNOS T786C allele may be involved in endothelial dysfunction. Azerbaijani patients showed dominance of the C allele and a mutant homozygous genotype CC of the T786C gene of eNOS.

Association of Methylenetetrahydrofolate Reductase Gene Polymorphism in Mothers With Adverse Clinical Outcomes in Neonates.

The presence of polymorphic methylenetetrahydrofolate reductase(MTHFR) in mothers poses a risk for numerous detrimental outcomes in neonates. The present study investigated the association of maternal MTHFR A1298C and C677T single nucleotide polymorphisms (SNPs) with the clinical outcomes in their neonates. The cross-sectional study included 60 mothers and their neonates. Blood samples from mothers were analyzed for MTHFR A1298C and C677T SNP genotyping by real-time polymerase chain reaction. Clinical details of mothers and neonates were documented. Study groups were stratified based on wild, heterozygous, and mutant genotypes for the respective polymorphisms observed in mothers. Multinomial regression was applied for the association, followed by gene model formulation to estimate the impact of the genetic variants on the outcomes. The frequency percentages of mutant CC1298 and TT677 genotypes were 25% and 8.06%, respectively, and the mutant allele frequencies (MAF) were 42.5% and 22.5%. Percentages of adverse outcomes such as intrauterine growth restriction, sepsis, anomalies, and mortality were higher in neonates born to mothers with homozygous mutant genotypes. Maternal C677T MTHFR SNPs revealed a significant association with neonatal anomalies (p = 0.001). The multiplicative risk model depicted OR (95% CI) for CT vs. CC+TT as 3.0 (95% CI: 0.66-13.7), and for TT vs. CT+CC was 15 (95% CI: 2.01-112.12). The C677T SNP in mothers predicted a dominant model for neonatal death (OR (95% CI): 5.84 (0.57-60.03), p = 0.15), whereas the A1298C reported recessive model for 1298CC mothers (OR (95% CI): 11 (1.05-115.5), p = 0.02). Both the genotypes assumed a recessive model for adverse neonatal outcomes: OR (95%CI) for CC vs. AA+ACwas 3.2 (0.79-12.9, p = 0.1), and for TT vs. CC+CT was 5.48 (0.57-175.7, p = 0.2). The risk for sepsis in neonates was nearly six times higher in those born from mothers with homozygous CC1298 and TT677 than in the wild and heterozygous variants. Mothers with C677T and A1298C SNPs are highly susceptible to adverse outcomes in their neonates. Hence, screening the SNPs during the antenatal period can purposefully serve as a better predictive marker, following which proper clinical management could be planned.

MTHFR Gene Polymorphisms in Iraqi Kurdish Rheumatoid Arthritis Patients: Relation to Methotrexate Response and Toxicity

Methotrexate (MTX) is still one of the gold standard treatments for rheumatoid arthritis (RA). It shows diverse outcomes in blood level and clinical response, this was demonstrated by its relation to the genetic polymorphism in the pharmacogenetic study. This study aimed to investigate the role of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms in relation to MTX efficacy and toxicity in Iraqi Kurdish RA patients. Sixty-four RA patients were involved in this study with an average age of 47.78 ±14.08 and female to male ratio of (8.1). Diagnosis and disease activity were confirmed. Blood analyses, including those of laboratory markers of disease activity, were done. The 28 joint disease activity score (DAS28-CRP) was calculated. MTHFR gene polymorphisms were analyzed by real-time polymerase chain reaction. The most frequent genotypes which were identified in RA patients were the CT genotype of the C677T single nucleotide polymorphism (SNP) (51.6%) and the AC genotype of the A1298C SNP (48.4%). Patients with non-response to treatment had high frequencies of genotypes CT and TT (58.0% and 12.0%) of the C677T SNP respectively, as compared to those in the responder group; 28.6% and 0.0%); T-allele was associated with drug non-responding OR=4.17, P value=.0.009, meanwhile; genotypes AC and CC of the A1298C SNP were seen in (54.0% and 16.0%) in non-responder group. Patients with active RA had increased frequencies of CT and TT genotypes of the C677T SNP (60.0% and16.0%) respectively as compared to those who were in remission (26.6% and 0.0%); T-allele was associated with high disease activity; OR = 5.11. No association was found between C677T SNP and A1298C SNP, and MTX level status (P> 0.05). However, the variant alleles (T and C) were associated with the MTX toxic level (OR: 2.05, 95% CI [0.97 – 4.32]) and (OR: 1.99, 95% CI [0.96 – 4.18]) respectively. This study suggests that genetic polymorphisms of MTHFR SNP (C677T and A1298C) are associated with MTX efficacy but not toxicity in RA patients. This may assist the physicians in personalizing RA treatment in Iraqi patients.

Pharmacogenetic Study of the Impact of ABCB1 Single Nucleotide Polymorphisms on the Response to Cyclosporine in Psoriasis Patients.

Psoriasis is a chronic, T cell-mediated skin disease affecting 2-3% of the Caucasian population. Cyclosporine A is a calcineurin inhibitor that acts selectively on T cells. The cyclosporine A treatment response has been suggested to be modulated by single-nucleotide polymorphisms (SNPs) in the ABCB1 gene. The aim of this research was to evaluate the effect of ABCB1 genetic variants that could affect the response to a cyclosporine treatment in Russian psoriasis patients with the ABCB1 genotype status. The ABCB1 T-129C, G1199A, C1236T, G2677T/A and C3435T SNPs in the 168 patients with psoriasis were genotyped by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and TaqMan SNP genotyping assays. The ABCB1 C1236T, G2677T/A and C3435T SNPs were significantly associated with a negative response to cyclosporine therapy. A very strong association was evident for the C3435T SNP in the ABCB1 gene in the allele, dominant and recessive models (OR = 2.58, OR = 4.01, OR = 2.50, respectively). ABCB1 C1236T and G2677T/A polymorphisms were significantly associated with a negative response to the cyclosporine therapy in the codominant, dominant and recessive models (p ˂ 0.05). Additionally, the haplotype analysis identified that the TGC haplotype is significantly associated with a negative response to cyclosporine therapy in psoriasis patients (p ˂ 0.05). The current study to the best of our knowledge is the first of its kind to be performed in the Russian population. In conclusion, the present results suggest an association between the ABCB1 genetic variants and unresponsiveness to cyclosporine in the Russian population. Further, larger studies are necessary to confirm our findings and replicate them in various ethnic populations before its implementation in the clinical practice.

Can we predict unresponsiveness to methotrexate in rheumatoid arthritis? A pharmacogenetic study.

Methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis (RA) and the therapeutic response to MTX has been observed to vary widely among these patients. The aim of this study was to investigate ABCB1 gene (the multidrug resistant 1 gene; MDR1 gene) polymorphism in patients with RA and to evaluate the relation between MTX unresponsiveness and this polymorphism. Forty-five patients with RA administered MTX were included in this pharmacogenetic cross-sectional study. The gender, age, body mass index (BMI), rheumatoid factor (RF) positivity, anti-cyclic citrullinated peptide (anti-CCP) positivity, doses of MTX and glucocorticoids were recorded. In addition, initial and third month disease activity (DAS28, Simplified and Clinical Disease Activity Index; SDAI and CDAI) scores were evaluated. We also examined frequencies of two single-nucleotide polymorphisms (SNPs), G2677T and C3435T, within the gene encoding ABCB1. 22 patient's responsive and 20 patients unresponsive to MTX were enrolled. Initial demographic and disease related factors were similar between patients responsive or nonresponsive to MTX. In the third month evaluation, disease activity scores were significantly higher in patients unresponsive to MTX (p < 0.05). In addition, almost all patients unresponsive to MTX (19 of the 20 patients) presented heterozygosity in C3435T (p < 0.000). We determined heterozygosity in C3435T SNP of ABCB1 gene (multidrug resistant 1 gene) in almost all patients with RA who were non-responders to MTX. This result may contribute to predict unresponsiveness to MTX in RA. Individualized treatment strategies based on the pharmacogenetic characteristics of MTX may lead to optimization of the treatment.

P-43 C677T and A1298C MTHFR gene polymorphisms and response to fluoropyrimidine-based chemotherapy in mestizo patients with metastatic colorectal cancer

The objective of this study was to assess the association between C677T and A1298 methylenetetrahydrofolate reductase (MTHFR) single nucleotide polymorphisms (SNPs) and response to first-line fluoropyrimidine-based chemotherapy for metastatic colorectal adenocarcinoma.

Drug Transporter ABCB1 SNP Predicts Outcome in Patients with Acute Myeloid Leukemia Treated with Gemtuzumab Ozagamicin: A Report from Children's Oncology Group AAML0531 Trial

Background: Gemtuzumab Ozogamicin (GO), is first ever approved immunotherapy for relapsed AML patients. GO is a humanized anti-CD33 antibody linked with the toxin calicheamicin gamma. Calicheamicin induces DNA damage and cell death once the linked CD33 antibody facilitates its uptake. Previous studies have shown that the drug efflux transporter PgP1, which is encoded by gene ABCB1, influences the cellular accumulation of calicheamicin. PgP1 activity in leukemic cells is also inversely correlated with GO response in patients with AML. Although GO was withdrawn from market due to lack of benefit and increase in early mortality observed in SWOG-S0106 study subsequent multiple randomized studies have shown benefit of GO with significant improvement observed in patients with favorable cytogenetic risk features. In light of these results and recent favorable voting by FDA oncology drug advisory committee based on results of ALFA-0701 trail demonstrating a favorable risk:benefit profile for GO there is increased enthusiasm among AML investigators. Given the potential comeback of GO, it is very timely to focus efforts on identifying genetic biomarkers that can improve our ability to develop personalized treatment with GO. In this study evaluate whether single nucleotide polymorphisms (SNPs) in ABCB1 gene are predictive of GO response in patients with AML.Method: Genomic DNA samples from 942 patients enrolled in the COG trial AAML0531 who gave consent for the study were genotyped for 12 SNPs in the ABCB1 gene . Since AAML0531 was designed to test the benefit of GO in a randomized manner, we analyzed the effect of ABCB1 genotypes on outcomes and risk of relapse (RR) between patients treated with or without addition of GO (GO vs. No-GO respectively).Results: The ABCB1 SNP C3435T (rs1045642) was significantly associated with outcome when analyzed by GO vs. No-GO arm. Presence of C3435T change has been previously associated with lower PgP1 expression. For AML patients in the GO arm, presence of variant allele (CT and TT genotype) was significantly associated with lower risk of relapse (RR) from the end of course 1 (GO vs. No-GO arm: RR: 30 ± 6%, n = 259 vs. 46 ± 7%, n= 235; P < 0.0001) and better 5-year event free survival (EFS) from study than for those in the No-GO arm (GO vs. No-GO arm: 55 ± 6%, n = 340 vs. 45 ± 5%, n= 337; P= 0.006). However, patients with the CC genotype showed no differences in RR (GO and No-GO arms: 45 ± 10%, n = 97 vs. 40 ± 11%, n= 91; P= 0.514) and EFS (GO and No-GO arms: 44 ± 9%, n = 128 vs. 50 ± 9%, n= 132; P= 0.425) between the GO and No-GO arms (Fig 1). Further the rs1045642 genotype was significantly associated with EFS (CC vs. CT/TT: 44 ± 9% vs. 55 ± 6%; P=0.022) and RR (CC vs. CT/TT 45 ± 10% vs. 30 ± 6%, P=0.007) for patients in the GO arm but for those in the No-GO arm no difference in outcome between the genotypes was observed (RR: P=0.278; EFS P=0.466).Conclusion: Our results show that for the nonsynonymous SNP rs1045642 (C>T) in an drug efflux transporter ABCB1, the presence of T allele is associated with better response in patients treated with GO based as compared to standard arm. Since ABCB1 is involved in efflux of calicheamicin the presence of a low expression T allele might result in higher intracellular accumulation of calicheamicin and thus better response in patients treated with GO. [Display omitted] DisclosuresLoken:Hematologics Inc: Employment, Equity Ownership. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding.

English

BACKGROUND Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C variants are considered as potential genetic risk factors for vaso-occlusive complications in sickle cell disorder (SCD). The purpose of the study was to determine the interaction of the combined haplotypes on the clinical presentations in children with sickle cell disorder. METHODS A cross-sectional study was conducted on 249 children, confirmed for sickle cell disorder. Clinical details and frequencies of clinical episodes in the past one year were noted and a severity index number was allotted to each child and evaluated for their relationship with the combined haplotypes of C677T and A1298C single nucleotide polymorphisms genotyped by real-time PCR. RESULTS The frequency for 677T / 1298A haplotype was 46.4 % and that of 677C / 1298C was 12.4 %. The three variant combined haplotypes had higher plasma homocysteine values than the wild 677C / 1298A haplotypes (P &lt; 0.001). Clinical events like vasoocclusive crisis (VOC), homocysteinemia, hospitalization frequency and SI were found significantly related among the children in sickle cell trait (SCT) group (P &lt; 0.001) but not so in SCD group. Chances for anemia was 1.93 times more in presence of dual variant alleles (95 %CI: 0.95 to 3.92, P = 0.07) in SCT. The 677T / 1298C haplotype accounted for higher SI was 7.85 times more than the wild haplotypic children even in SCT children and 1.67 times in SCD children. CONCLUSIONS Presence of the variant haplotypes had significant implication on crisis events in children with sickle cell trait and make them more prone for the clinical severity. A preliminary allelic screening might be helpful in them. KEY WORDS Dual Variant Alleles, Heterozygous, Homozygous, MTHFR, Variant Haplotypes

C677T and A1298C MTHFR gene polymorphisms and response to fluoropyrimidine-based chemotherapy in Mestizo patients with metastatic colorectal cancer.

To assess the association between C677T and A1298C methylenetetrahydrofolate reductase (MTHFR) single-nucleotide polymorphisms (SNPs) and response to first-line fluoropyrimidine-based chemotherapy for metastatic colorectal adenocarcinoma. A total of 68 patients were prospectively followed up in San Juan de Dios Hospital (San José, Costa Rica) from January 2019 to November 2020. Patients received first-line therapy with capecitabine or 5-fluorouracil in combination with oxaliplatin or irinotecan. Germline and somatic DNA was extracted from blood samples and paraffin-embedded tissue, respectively. Overall response rate (partial response + complete response) was assessed according to RECIST 1.1 criteria. Cox regression models were performed to identify the effect of MTHFR C677T and A1298C SNPs on progression-free survival (PFS) and overall survival (OS) (NCT registration number: 03852290). Patients harboring one or both T alleles of the MTHFR C677T SNP had better overall response than homozygous wild-type individuals [odds ratio (OR): 3.21; 95% confidence interval (CI), 1.05-9.81; P = 0.03]. No association was found between the MTHFR A1298C genotypes and overall response (OR: 0.75; 95% CI, 0.26-2.20; P = 0.60). Patients with the MTHFR 677 TT and CT genotypes had longer PFS than CC individuals (hazard ratio: 0.53; 95% CI, 0.28-0.98; P = 0.045), even after adjustment for confounders (hazard ratio: 0.50; 95% CI, 0.25-0.98; P = 0.04). We found no association between the MTHFR A1298C SNP and PFS (hazard ratio: 1.35; 95% CI, 0.72-2.55; P = 0.34). None of the SNPs was associated with OS. Patients carrying at least one mutant allele of the MTHFR C677T SNP had a better overall response and longer PFS than wild-type homozygous patients.

MTHFR (methylenetetrahydrofolate reductase: EC 1.5.1.20) SNPs (single-nucleotide polymorphisms) and homocysteine in patients referred for investigation of fertility

PurposeMTHFR, one of the major enzymes in the folate cycle, is known to acquire single-nucleotide polymorphisms that significantly reduce its activity, resulting in an increase in circulating homocysteine. Methylation processes are of crucial importance in gametogenesis, involved in the regulation of imprinting and epigenetic tags on DNA and histones. We have retrospectively assessed the prevalence of MTHFR SNPs in a population consulting for infertility according to gender and studied the impact of the mutations on circulating homocysteine levels.MethodsMore than 2900 patients having suffered at least two miscarriages (2 to 9) or two failed IVF/ICSI (2 to 10) attempts were included for analysis of MTHFR SNPs C677T and A1298C. Serum homocysteine levels were measured simultaneously.ResultsWe observed no difference in the prevalence of different genetic backgrounds between men and women; only 15% of the patients were found to be wild type. More than 40% of the patients are either homozygous for one SNP or compound heterozygous carriers. As expected, the C677T SNP shows the greatest adverse effect on homocysteine accumulation. The impact of MTHFR SNPs on circulating homocysteine is different in men than in women.ConclusionsDetermination of MTHFR SNPs in both men and women must be seriously advocated in the presence of long-standing infertility; male gametes, from MTHFR SNPs carriers, are not exempted from exerting a hazardous impact on fertility. Patients should be informed of the pleiotropic medical implications of these SNPs for their own health, as well as for the health of future children.

The association between MDR1 C3435T genetic polymorphism and the risk of multidrug-resistant epilepsy in Egyptian children

BackgroundEpilepsy is a chronic disease affecting about 2% of the population and is considered a serious neurological disease. Despite its good prognosis, 20–30% of epileptic patients were not cured of their seizures even with the many trials of antiepileptic drug (AED) therapy. The resistance mechanism is still unclear, maybe due to the effect of the genetic factors on the bioavailability of the drugs. Consequently, the association between therapy resistance and the presence of a gene called “multidrug resistance 1 (MDR1)” had been proposed. Thus, the present study aimed to understand the relationship between the genetic polymorphism of MDR1C3435T and the resistance to AEDs.ResultA non-significant association was found between MDR1 C3435T single-nucleotide polymorphism (SNP) and drug-resistant epilepsy. However, there was statistical significance in the association between the drug type and the genotype distribution, in cases that were maintained on sodium valproate and MDR1C3435T genotype.ConclusionPossible involvement of the MDR1 gene C 3435T polymorphism with sodium valproate resistance clarifies the importance of genetic variability in response to the drug and may help to find novel genetic therapy for epilepsy, by targeting the biological mechanisms responsible for epilepsy in each specific individual. Future studies with bigger sample sizes and in other racial populations will be necessary.

Single probe PCR melting curve analysis MTHFR C677T SNP sites

BackgroundThe detection and analysis of methylene tetrahydrofolate reductase (MTHFR) C677T single nucleotide polymorphism (SNP) from blood samples is time-consuming and costly. We aimed to establish a method to detect these SNPs by direct whole blood PCR and without DNA extraction. MethodsProbes modified by different fluorescent groups on the same sequence were designed. Various MTHFR genotypes from direct blood PCR experiments were used to verify the similarity of the obtained and sequencing results. The SNP sites adjacent to the MTHFR C677T SNP were used to verify whether the method can accurately distinguish these sites. ResultsThe ROX probe was found to be the most suitable for this study. We tested 291 samples with 1 μL whole blood as a template, and obtained 126, 43, and 122 cases of C677C, C677T, and C677 C/T genotypes, respectively. The melting curve was consistent with the sequencing results. The detection limit was approximately 1000 white blood cells/μL. Through PCR and the melting curve method, the adjacent sites were accurately distinguished. ConclusionWe established a reliable, simple, rapid, and low-cost direct blood PCR method for the detection of MTHFR C677T SNPs. This could also be used as a potential diagnostic tool for a variety of diseases.