Improved real-time multiplex polymerase chain reaction detection of methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C polymorphisms using nearest neighbor model-based probe design.

The disorders of folate metabolism caused by methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms may lead to several disease states including coronary heart disease, venous thrombosis, and several types of cancer. We have developed a real-time multiplex single-tube polymerase chain reaction procedure on the LightCycler for the detection of the two most commonly occurring variants, 677C>T and 1298A>C, in the MTHFR gene. An improved probe design, based on the nearest neighbor model for nucleic acid-probe duplex stability, resulted in a better separation (DeltaTm approximately 10 degrees C) of melting peaks of the wild-type and mutant alleles than that by the existing method (DeltaTm approximately 3 degrees C) for specimens heterozygous for the 1298A>C polymorphism. Of the 333 blood specimens analyzed by this procedure, we did not find any samples that gave ambiguous results. The specimens with homozygous mutation for one polymorphism were of the wild type for the other variant. The assay was validated by the comparison of the genotyping results of 50 blood specimens from the LightCycler polymerase chain reaction with the conventional restriction fragment length polymorphism procedures. There was 100% concordance of the test results obtained by the two techniques. This assay is reliable, economical, and can be performed by less trained technologists compared with the procedure performed by the conventional restriction fragment length polymorphism technique.

Fetal genotype for specific inherited thrombophilias is not associated with severe preeclampsia.

OBJECTIVE: Little is known about the association between fetal thrombophilias and severe preeclampsia. The objective of this study was to examine the association between fetal genotype for factor V Leiden, prothrombin, and methylene tetrahydrofolate reductase (MTHFR) mutations and severe preeclampsia. METHODS: Patients with severe preeclampsia or HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome admitted to Georgetown University Hospital were retrospectively identified. Controls were patients with uncomplicated, term deliveries. Fetal DNA was extracted from placental specimens and amplified by polymerase chain reaction (PCR) with locus-specific primers. The presence of polymorphisms was determined by enzymatic digestion with specific enzymes, and analyzed by polyacrylamide gels. Statistical analysis used Student t test for continuous variables and Fisher exact test for categorical data. RESULTS: Patients with preeclampsia (n = 27) and controls (n = 17) were similar for maternal age, but, as expected, they were significantly different for gestational age at delivery, birth weight, Apgar scores at 5 minutes, rate of preterm delivery less than 37 weeks, and fetal growth restriction (all P <.05). DNA extraction was successful in 25 of 27 cases from the severe preeclampsia group and 14 of 17 controls. None of the placentas analyzed in the preeclamptic or control group revealed mutations in the factor V Leiden or prothrombin genes. There was no significant difference in the rate of fetuses heterozygous for MTHFR in the preeclampsia versus control group (48% vs 43%, P >.05). CONCLUSION: In our study, fetal genotype for specific inherited thrombophilias does not appear to be associated with severe preeclampsia.