NATIONAL REFERENCE LABORATORY Methotrexate Sensitivity (MTHFR) 2 Mutations Identifies patients who may experience intolerance to antifolate medications such as methotrexate Indications for Ordering Clinical Background • Disease Overview ◦◦ Methotrexate is an antimetabolite drug used in treatment of cancer and autoimmune diseases. As a structural analogue of folate, methotrexate interferes with folate metabolism by inhibiting dihydrofolate reductase, which leads to depletion of cellular folate. Supplementation with folate or folinic acid (leucovorin) reduces the efficacy and toxicity of methotrexate. ◦◦ Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in maintaining cellular folate pools, and MTHFR gene variants associated with reduced enzyme function and hyperhomocysteinemia may affect methotrexate sensitivity and contribute to toxicity. ◦◦ Adverse effects of methotrexate therapy may include cardiovascular, neurological, dermatologic, hematologic, hepatic, and gastrointestinal symptoms. ◦◦ An association between MTHFR variants and methotrexate toxicity supports dose adjustment and limitation/discontinuation of therapy in affected individuals. ◦◦ MTHFR mutations are associated with an increased risk for arteriosclerotic coronary disease and venous thrombosis. They may also contribute to the pathophysiology of neural tube defects, cardiovascular disease, and a variety of malignancies. • Epidemiology ◦◦ US allele frequency of C677T = 0.39 and A1298C = 0.17. ◦◦ The frequency of homozygotes for C677T or A1298C is 8–12 percent in most populations. • Genetics ◦◦ The common MTHFR mutations, C677T and A1298C, produce reduced function enzymes with altered catalytic activity and thermolability. ◦◦ Homozygotes for C677T have about 30 percent of normal MTHFR enzyme activity; heterozygotes for C677T have approximately 60 percent normal activity. ◦◦ The C677T mutation is associated with elevated plasma homocysteine levels, whereas the A1298C mutation is not. ◦◦ Clinical relevance is associated with homozygosity for C677T or A1298C, and the compound heterozygous state (C677T/ A1298C). • • Identification of individuals at risk for methotrexate sensitivity due to prolonged administration of methotrexate as management for the following conditions: ◦◦ Acute lymphoblastic leukemia (ALL) ◦◦ Chronic myelogenic leukemia (CML) ◦◦ Juvenile idiopathic arthritis ◦◦ Rheumatoid arthritis ◦◦ Immune disease Optimization of therapy for individuals requiring methotrexate or other folate antimetabolites, particularly when a family history of intolerance to methotrexate exists. Contraindication for Ordering For assessment of homocysteine levels and risk for arteriosclerotic coronary disease and venous thrombosis, refer to Methylenetetrahydrofolate Reductase (MTHFR) 2 Mutations (ARUP test # 0055655). Interpretation • • • Negative: No mutations detected; genotype is consistent with normal MTHFR enzyme activity. Positive: ◦◦ Heterozygous C677T or A1298C: decreased enzyme activity but no correlation with methotrexate intolerance ◦◦ Homozygous C677T: associated with increased plasma homocysteine levels and methotrexate intolerance; dosing adjustments/discontinuation may be required ◦◦ Homozygous A1298C: associated with decreased enzyme activity and lower dose requirements for methotrexate ◦◦ Compound heterozygote (C677T/ A1298C): associated with increased plasma homocysteine levels and methotrexate intolerance Genotype should be interpreted with clinical information; consultation with a clinical pharmacist is recommended. Methodology • • Analysis of the common MTHFR mutations, C677T and A1298C, by polymerase chain reaction and fluorescence monitoring using hybridization probes. Analytical sensitivity and specificity are >99 percent. ARUP LABORATORIES • JANUARY 2009 500 Chipeta Way, Salt Lake City, UT 84108 • (800) 522-2787 • (801) 583-2787 • www.aruplab.com Limitations • • • • • References Specific genotype or haplotype-based dosing guidelines are not currently available. MTHFR mutations, other than C677T and A1298C, will not be detected. Testing will not determine haplotypes. Mutations in other genes and non-genetic factors that may affect drug sensitivity are not detected. Rare diagnostic errors may occur due to primer-site mutations. 1. 2. 3. 4. Chiusolo P, et al. Preponderance of methylenetetrahydrofolate reductase C677T homozygosity among leukemia patients intolerant to methotrexate. Ann Oncol 2002;13:1915–8. Donnelly JG. Pharmacogenetics in cancer chemotherapy: balancing toxicity and response. Ther Drug Monit 2004; 26:231–5. Krajinovic M, et al. Role of polymorphisms in MTHFR and MTHFD1 genes in the outcome of childhood acute lymphoblastic leukemia. Pharmacogenomics 2004;4:66–72. Urano W, et al. Polymorphisms in the methylenetetrahydrofolate reductase gene were associated with both the efficacy and the toxicity of methotrexate used for the treatment of rheumatoid arthritis, as evidenced by single locus and haplotype analyses. Pharmacogenetics 2002;12:183–90. Test Information 0051286 Methotrexate Sensitivity by MTHFR Genotyping For specific collection, transport, and testing information, refer to the ARUP Web site at www.aruplab.com. For information on test selection, ordering, and interpretation, refer to ARUP Consult® at www.arupconsult.com. ARUP LABORATORIES • JANUARY 2009