Methylenetetrahydrofolate Reductase (MTHFR) 2 Mutations Indications for Ordering • Determine genetic cause for early-onset hyperhomocysteinemia • Predict methotrexate sensitivity which may be associated with o Toxicity in individuals with a family history of intolerance o Individuals taking prolonged administration of methotrexate therapy (eg, ALL, CML, rheumatoid arthritis) • Limited utility o For identification of early onset arteriosclerotic vascular disease o In determining risk for deep vein thrombosis (DVT) • Not recommended testing for women who have o Pregnancy complications o Recurrent miscarriages o A previous child with a neural tube defect (NTD) Test Description • PCR followed by fluorescence monitoring • Hybridization probes detect c.665C>T; p.Ala222Val and c.1286A>C; p.Glu429Ala (previously known as c.677C>T and c.1298A>C, respectively) Tests to Consider Primary test Methylenetetrahydrofolate Reductase (MTHFR) 2 Mutations 0055655 • To determine potential sensitivity to antifolate medications and genetic cause for early-onset hyperhomocysteinemia Related tests Homocysteine, Total 0099869 • Acceptable screening test for disorders of methionine metabolism (congenital hyperhomocysteinemia) • Not recommended for risk assessment of cardiovascular disease or venous thromboembolism Methotrexate, Sensitive 2005405 • Monitor methotrexate concentration in rheumatoid arthritis Thrombotic Risk, DNA Panel 0056200 • Acceptable panel to detect the 2 most common inherited thrombophilias Disease Overview Prevalence – most common inherited risk factor for hyperhomocysteinemia • Allelic frequency (based on data from dbSNP, February 2013) o c.665C>T − 0.35 in European Caucasians, 0.12 in African Americans o c.1286A>C − 0.31 in European Caucasians, 0.15 in African Americans • Homozygosity for c.665C>T is 1-15% in the United States Related conditions Hyperhomocysteinemia • Moderately elevated plasma homocysteine o Independent risk factor for atherosclerotic vascular disease and venous thrombosis ▪ 10% of total risk may be attributable to elevated plasma homocysteine ▪ For each 5 μmol/L homocysteine elevation, risk of coronary artery disease increased by 60% for men and 80% for women o Affected by genetic and environmental factors (eg, dietary) o Folic acid supplementation reduces homocysteine levels, but effect on atherosclerosis and thrombosis risk is not clearly defined o Genetic testing is generally not useful for preventing or predicting atherosclerosis NTDs • Defects in folate metabolism may play a role in NTDs but risk is largely dependent on nutritional status and homocysteine level Thrombophilia • MTHFR mutations − may interact with other inherited risk factors for thrombosis (eg, factor V Leiden) o Co-inheritance does not further increase the thrombotic risk associated with factor V Leiden FEBRUARY 2014 | © 2013 ARUP LABORATORIES | ARUP is a nonprofit enterprise of the University of Utah and its Department of Pathology. 500 Chipeta Way, Salt Lake City, UT 84108 | (800) 522-2787 | (801) 583-2787 | www.aruplab.com | www.arupconsult.com Methotrexate sensitivity • MTHFR mutations − associated with methotrexate toxicity o Dose adjustment or discontinuation of therapy may be advised for individuals with high-risk MTHFR genotypes Pregnancy loss • MTHFR mutations − not associated with recurrent pregnancy loss o Conflicting evidence regarding role in pregnancy complications (eg, pre-eclampsia, placental abruption, intrauterine growth restriction) Genetics Gene – MTHFR Inheritance – autosomal recessive Structure/function − MTHFR mutations (c.665C>T and c.1286A>C) correlate with reduced MTHFR enzyme activity • MTHFR enzyme is involved in folate metabolism o Catalyzes 5,10-methylenetetrahydrofolate to 5methyltetrahydrofolate o Necessary cofactor for the re-methylation of homocysteine o Reduced enzyme function increases plasma homocysteine Test Interpretation Sensitivity/specificity • Clinical sensitivity – undefined; dependent upon multiple contributing factors • Analytical sensitivity/specificity – 99% Results • Negative – no mutations detected o Normal MTHFR enzyme activity expected • Positive o Homozygosity for c.665C>T ▪ Associated with increased plasma homocysteine levels and increased risk for arteriosclerotic coronary disease and venous thrombosis ▪ At risk for methotrexate intolerance and may require dosing adjustments/discontinuation o Homozygosity for c.1286A>C ▪ Associated with intermediate enzyme activity, but not with increased plasma homocysteine levels ▪ No increased risk for premature cardiovascular disease/venous thrombosis ▪ May require lower dose requirements for methotrexate o Compound heterozygosity (c.665C>T/c.1286A>C) ▪ Associated with increased plasma homocysteine levels and increased risk for arteriosclerotic coronary disease and venous thrombosis ▪ At risk for methotrexate intolerance and may require dosing adjustments/discontinuation o Heterozygosity for either c.665C>T or c.1286A>C ▪ Associated with intermediate levels of enzyme activity, but NOT with increased plasma homocysteine levels ▪ No increased risk for premature cardiovascular disease/venous thrombosis or methotrexate intolerance • Genotypes should be interpreted with clinical information o Specific genotype- or haplotype-based methotrexate dosing guidelines are not currently available ▪ Consultation with a clinical pharmacist is recommended Limitations • MTHFR mutations other than c.665C>T and c.1286A>C are not evaluated by this test • Rare diagnostic errors may occur due to primer-site mutations FEBRUARY 2014 | © 2013 ARUP LABORATORIES