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Although the methylenetetrahydrofolate reductase (MTHFR) gene was cloned in 1994,1 the role of MTHFR mutations in the etiology of hyperhomocysteinemia, coronary heart disease, or inherited thrombophilias remains controversial.2–11 The variants 677CT (alanine to valine) in the catalytic domain and 1298AC (glutamate to alanine) in the regulatory domain of the MTHFR gene have been shown to decrease MTHFR activity.12–15 The 677TT homozygote and 677CT/1298AC compound heterozygote genotypes have been shown to be contributing factors in the development of hyperhomocysteinemia.4,5,9,12,15–18 Hyperhomocysteinemia has previously been implicated in neural tube defects, stillbirths, recurrent miscarriages, ocular and skeletal abnormalities, stroke, cerebrovascular disease, arteriosclerotic vascular disease, coronary artery disease, and thrombophilic disease.

The role of hyperhomocysteinemia in thrombophilic disease is still not clearly defined. It has been suggested that homocysteine interferes with anticoagulant and fibrinolytic pathways.7 It also has been suggested that hyperhomocysteinemia in combination with the Factor V Leiden mutation (R506Q) results in resistance to activation by protein C, thereby facilitating a predisposition to thrombosis.19–21 Double and triple variant MTHFR mutations of the 677CT and 1298AC loci in the MTHFR gene have been implicated in thrombophilic disease.5,11

Genotyping 37,026 individuals in a commercial laboratory for a thrombophilia evaluation identified 30 individuals with triple variant MTHFR mutations. Our genotype frequencies and the frequency of each combination of triple MTHFR mutations were compared with a previously published meta-analysis.5 Based on this comparison, our observed genotypic frequencies and the frequencies of each combination of triple variant MTHFR mutations may be more representative of the general population than a high-risk population. Double or triple variant MTHFR mutations in certain chromosome phase (cis or trans) may be inappropriately labeled as high risk for thrombotic episodes. Further clinical investigations are needed to correlate whether certain allelic combinations containing MTHFR mutations may have any clinical significance when associated with thrombophilic disease.

MATERIALS AND METHODS

Patient population

We performed MTHFR genotype analysis on 37,026 consecutive samples submitted to Quest Diagnostics. We assume that the overwhelming majority of samples were taken from individuals after a thrombotic episode or those with a family history of thrombosis. Analysis of our patient population for the 677CT and 1298AC mutations in the MTHFR gene were performed by either a laboratory-developed test involving PCR amplification and restriction digestion utilizing the ABI 3100 capillary electrophoresis apparatus (Applied Biosystems Inc) or by using an Analyte Specific Reagent (ASR) supplied by Third Wave Technologies.

PCR/restriction digest/3100 ABI capillary electrophoresis platform

DNA was extracted from EDTA or ACD-anticoagulated whole-blood specimens using the MagAttract DNA Blood M96 Kit (Qiagen) according to the recommendations of the manufacturer. A polymerase chain multiplex reaction was performed with fluorescently labeled primers for exons 4 (677CT) and 7 (1298AC) of the MTHFR gene. The sequences were as follows: Exon 4: forward 5′-(6-FAM)-CCAGCCTCTCCTGACTGTCATC-3′; reverse 5′-GCGGTCCCAAAAGAGTCAGTAGCTGCGTGATGATGAAATCG-3′; Exon 7: forward 5′-CAAGGAGGAGCTGCTGAAGATG-3′; reverse 5′-(HEX)-CTTTGTGACC-ATTCCGGTTTGG-3′. The 25-μL reaction volume consisted of 12.5 μL PCR master mix (Promega Corp), 3 μmol/L exon 4 primers, 4 μmol/L exon 7 primers, 9.5 μL molecular-biology grade water (Sigma), and 2 μL DNA. PCR amplifications were performed in an MJR PTC-200 thermocycler (MJ Research) under the following conditions: 2 minutes at 94°C, 31 cycles of 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 40 seconds, and 72°C for 10 minutes. Expected amplicon sizes are 151 bp for exon 4 and 110 for exon 7. Detection of the 677CT and 1298AC mutations was accomplished by digestion of PCR products in a 10-μL reaction mix consisting of 2.5 units of MboII (New England Biolabs), 5 units of HinfI (NEB), 1 μL 10X NEB Buffer 2, 3 μL molecular-biology grade water (Sigma), and 5 μL PCR product. Digestion products (2 μL) were separated on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Fragment analysis was performed using Genescan and Genotyper software (Applied Biosystems). Expected labeled digestion products are as follows: 677CT (FAM): wild type, 138 bp; wt/677CT, 110 and 138 bp; 677CT/677CT, 110 bp. 1298AC (HEX): wild type, 54 bp; wt/1298AC, 54 and 82 bp; 1298AC/1298AC, 82 bp.

Invader analyte specific reagent

Invader ASRs were purchased from Third Wave Technologies. Automated DNA extraction from EDTA or ACD anticoagulated whole-blood specimens was performed utilizing a Biomek FX liquid handling system (Beckman) and the Generation DNA Purification Capture plate Kit (Gentra Systems). All procedures utilized for Invader detection were followed by published procedures.22,23

DNA sequencing

Genomic DNA purification from whole blood, methods and materials used for DNA amplification, and direct sequencing of the three individual's samples possessing the triple MTHFR mutations was performed as previously described.24 Briefly, PCR products were amplified by primers listed in the PCR/restriction digest/3100 ABI capillary electrophoresis platform section. PCR products were then sequenced with primers 677CT Exon 4: forward 5′-CCAGCCTCTCCTGACTGTCATC-3′; reverse 5′-AAGTGATGCCCATGTCGGTG-3′; A1298C Exon 7: forward 5′-CTCTTCTACCTGAAGAGCAAGTC-3′; reverse 5′-CTTTGCC-ATGTCCACAGCATG-3′ and electrophoresed on the ABI 3100.

RESULTS

Screening 37,026 individuals as part of a thrombophilia evaluation, 30 patients were identified to contain the triple variant MTHFR mutations (Table 1). Nineteen individuals were homozygous for 677TT and heterozygous for 1298AC, and 11 were heterozygous for 677CT and homozygous for 1298CC. The triple variant MTHFR frequencies of 677TT/1298AC and 677CT/1298CC genotypes were 0.0005 and 0.0003, respectively. These genotypic frequencies from the triple variant MTHFR mutations were significantly lower than findings of Ogino and Wilson,5 meta-analysis. Each type of triple variant MTHFR mutations 677TT/1298AC or 677CT/1298CC gave a frequency of 0.633 and 0.367, respectively. The additional MTHFR frequencies of the 677CT/1298AA (Heterozygous/WT), 677CC/1298AC (WT/Heterozygous), 677CT/1298AC (Heterozygous/Heterozygous), 677CC/1298AA (WT/WT), 677TT/1298AA (Homozygous/WT), and 677CC/1298CC (WT/Homozygous) genotypes were 0.228, 0.208, 0.198, 0.153, 0.122, and 0.088, respectively (Table 1). Direct sequencing was used to confirm 3 separate samples containing triple variant MTHFR mutations. Sequencing electropherograms of each type of triple variant MTHFR mutations (samples 18 and 19) are shown in Figures 1 and 2, respectively.

Table 1 MTHFR genotypes and genotypic frequencies
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Fig 1
figure 1

Direct sequencing electropherogram of sample 18 (677TT/1298AC): 1A: 677TT homozygous, 1B: 1298AC heterozygous.

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Fig 2
figure 2

Direct sequencing electropherogram of sample 19 (677CT/1298CC): 1A: 677CT heterozygous, 1B: 1298CC homozygous.

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DISCUSSION

Previous investigations have implicated the homoyzgote (677TT) and compound heterozygote (677CT/1298AC) MTHFR genotype in the causation of hyperhomocysteinemia and subsequent development of cardiovascular disease.4,5,9,12,15–17,21,25–32 The homozygote 677TT and compound heterozygote 677CT/1298AC MTHFR genotypes decrease MTHFR enzyme activity, leading to hyperhomocysteinemia.8,15,16,18,33,34 Controversy remains regarding the role of hyperhomocysteinemia as a risk factor for deep-vein thrombosis.6,11,18,35,36 The high prevalence of the homozygote 677TT and compound heterozygote 677CT/1298AC MTHFR mutations in the general population is represented by our genotypic frequencies of 0.122 and 0.198 (Table 1), supporting other previously published findings.5,8 The two variant MTHFR mutations, associated with the compound heterozygotes (677CT/1298AC), have previously been reported to be predominantly on different alleles (in trans) rather than on the same allele (in cis).4,5,16,35,37 The overall chromosomal effect of the triple variant mutations predisposes both variant MTHFR mutations to be on the same chromosomal allele (in “cis”) (677TT/1298AC or 677CT/1298CC). A meta-analysis of the general population showed the genotypic frequencies of 677TT/1298AC and 677CT/677CT/1298CC triple variant MTHFR mutations to be 0.0046 and 0.0025, respectively.4 Screening 37,026 individuals as part of a thrombophilia evaluation, we identified 30 individuals with triple variant MTHFR mutations. Our analysis of the triple variant MTHFR mutations identified 19 patients with 677TT/1298AC genotype and 11 patients with 677CT/1298CC genotype. Our findings indicated that 677TT/1298AC and 677CT/1298CC triple variant MTHFR mutation genotypic frequencies were 0.0005 and 0.0003, respectively. Our larger study may be more representative of the general US population than previously studied populations, thereby causing the lower genotypic frequencies identified in our series. Previous studies have reported an unusually high genotypic frequency of the triple MTHFR mutations in their control population, indicating that a cis T/C allele may have a founder effect in the Canadian and United Kingdom population.11,12,36,38,39 Isotalo and Donnelly11 have suggested a trend between triple variant MTHFR mutations and their association with the occurrence of a venous thrombosis. A larger venous thrombosis population needs to be ascertained in order to further support this hypothesis. Our lower genotypic frequencies of the triple variant MTHFR mutations could also be perceived as supporting an association with venous thrombosis. In theory, as the high-risk, nonthrombotic population increases, the frequencies of the triple variant MTHFR mutations should decrease, due to the nonthrombotic population analyzed. The frequencies of each combination of triple variant MTHFR mutation (677TT/1298AC or 677CT/1298CC) were 0.633 and 0.367, respectively. These findings correlated with Ogino and Wilson,5 showing that a higher frequency of the 677TT/1298AC than the 677CT/1298CC triple variant MTHFR mutations does exist in the general population. Theoretically, the double (677CT/1298AC) and triple variant (677TT/1298AC) MTHFR mutations predisposes an etiology of hyperhomocysteinemia, which has been previously implicated in the literature with thrombolytic disease.2,4–9,16,19–21,25,36,40 To date, we believe this is the largest population evaluation of the triple variant MTHFR mutations in the literature. The overall clinical significance of the double and triple variant MTHFR mutations related to thrombophilic disease is still unknown. Further clinical investigations are needed to clarify the role these double and triple variant MTHFR mutations have on thrombophilic disease. The most important implication from our observations of individuals who possess a single allele with two variant MTHFR mutations is that a proportion of patients who appear to be compound heterozygotes for 677CT and 1298AC will actually be heterozygotes for this double mutant allele and may not be at increased risk of thrombosis. When such an individual is discovered, linkage analysis may be indicated to determine the phase of two variant alleles before an accurate genetic risk assessment can be given.