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Articles |
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Department of Pharmacology and Center for Pharmacogenetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.
a Address correspondence to this author at: Department of Pharmacology and Center for Pharmacogenetics, University of Pennsylvania School of Medicine, 153 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Fax 215-573-9135; e-mail aswhitehead{at}pharm.med.upenn.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Sequence-specific heteroduplex generators for each of the MTHFR and MS single nucleotide polymorphisms were generated by site-directed mutagenesis. These were subcloned into a single construct, pHcyHG-1, which could be multiplexed with a simple PCR amplification across the CBS 844ins68 polymorphic site to generate composite genotype-specific banding patterns from individual genomic DNA samples that could be electrophoretically resolved.
Results: The "multiplex heteroduplexing" method yielded unambiguous MTHFR, MS, and CBS genotypes in a single-tube reaction that could be analyzed in a single gel run.
Conclusions: This method permits unambiguous genotyping of the four most common functional variants of enzymes involved in folate/homocysteine metabolism. It is rapid, reproducible, and inexpensive, and requires no special preparative or analytic facilities; consequently, it will facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and the many pathologies that have been associated with this phenotype.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The etiology of hyperhomocysteinemia involves both nutritional (e.g., folate, vitamin B6, and vitamin B12 concentrations) and genetic factors (6)(7). The methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), and cystathionine ß-synthase (CBS) genes encode enzymes that control Hcy metabolism and have been surveyed for polymorphisms that modulate plasma Hcy concentrations.
The MTHFR C677T transition (A222V), the first such polymorphism identified, specifies a functionally altered "thermolabile" enzyme (8). Homozygotes for the 677T allele are predisposed to hyperhomocysteinemia (8), particularly in the context of suboptimal folate status (9), and the TT genotype is associated with increased risk of cardiovascular diseases in some (10)(11)(12), but not all (13), populations.
Three other polymorphisms [A1298C (E429A) in MTHFR, A2756G (D919G) in MS, and a 68-bp insertion (844ins68) in CBS] with allele frequencies of ~30%, 20%, and 8%, respectively, in Caucasian populations (14)(15)(16), influence enzyme activity and/or plasma Hcy concentrations (14)(16)(17) and therefore are candidate genetic risk factors for pathologies in which hyperhomocysteinemia may be an etiologic factor.
Large-scale studies of patients and controls that are genotypically and biochemically well characterized will be needed to determine the contribution of each polymorphism to the above pathologies. Technical considerations are, however, a limiting factor in the implementation of such studies. The most widely used method for genotyping the above polymorphisms has been PCR amplification followed by restriction enzyme analysis (PCR-restriction fragment length polymorphism analysis), an approach that often is compromised by partial fragment digestion and, consequently, genotyping errors (18). For both MTHFR C677T and MS A2756G, partial digestion or digestion failure leads to underestimation of the mutant 677T and 2756G allele frequencies, respectively, whereas for A1298C, it leads to underestimation of the wild-type 1298A allele frequency.
Genotyping methods based on heteroduplex generator (HG) technology do not require restriction enzymes (19). A HG is a synthetic DNA molecule, identical in sequence to a short genomic fragment spanning the polymorphism of interest, with a microdeletion adjacent to the polymorphic site. Coamplification of the HG with test genomic DNA and subsequent mixed hybridization of complementary strands produces allele-specific heteroduplexes with different mismatched protruding loops and different electrophoretic properties.
To facilitate MTHFR, MS, and CBS genotyping in large cohorts, we have developed a "multiplex heteroduplexing" method that generates products in a single-tube reaction that can be resolved in a single electrophoretic run.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HGs
HGs for analyzing the MTHFR A1298C and MS
A2756G polymorphisms were each synthesized by PCR-mediated
site-directed mutagenesis, according to the general strategy outlined
in Fig. 1
. The 160-bp MTHFR A1298C HG contains a 3-bp
microdeletion located 2 bp upstream of the polymorphic site. The 236-bp
MS A2756G HG contains a 4-bp microdeletion located 2 bp
downstream of the polymorphic nucleotide. The MTHFR C677T HG
was a gift of Dr. Ian McDowell (University of Wales, Cardiff, United
Kingdom), and contains a 3-bp microdeletion located 1 bp upstream of
the polymorphic site (20). All primers used are listed in
Table 1
A.
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cloning
The MTHFR C677T, MTHFR A1298C, and
MS A2756G HGs were individually amplified by PCR, purified
by gel electrophoresis (Qiagen), and inserted into the pGEM-T Easy II
vector (Promega) by direct TA cloning to yield plasmids p677,
p1298, and p2756, respectively. The integrity of these constructs was
verified by automated DNA sequencing. Subsequently, the
MTHFR C677T and MS A2756G HGs were excised from
their respective plasmids and tandemly ligated into p1298 to yield
pHcyHG-1. The organization and sequence of
pHcyHG-1 were verified as above (Fig. 2
).
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pcr amplification and gel resolution
Genomic DNA was extracted from leukocytes by an established method
(21). PCR amplifications were performed using 1.5 U of Taq
polymerase (Boehringer Mannheim) in the manufacturer’s buffer
containing 1.5 mmol/L MgCl2 and 240 µmol/L
dNTPs (Amersham Pharmacia Biotech) in a final volume of 50 µL.
Genomic DNA (100 ng) and 1.5 pg of pHcyHG-1 were coamplified
in the presence of 2 µmol/L primers MTHFR-1 and MTHFR-0, 2 µmol/L
primers MTHFR-D and MTHFR-E, 3 µmol/L primers CBS-1 and CBS-2, and 3
µmol/L primers MS-A and MS-B. All primer sequences and positions
relative to target genes are summarized in Table 1
B. Cycling conditions
were as follows: 5 min of initial denaturation at 94 °C, and 1 min
at 94 °C, 1 min at 55 °C, 1 min at 72 °C for 35 cycles,
followed by 30 min at 35 °C, in a PTC-0200 thermocycler (MJ
Research).
PCR products (25 µL) were mixed with 5 µL of loading buffer (containing, per liter, 300 mL of glycerol, 2.5 g of bromphenol blue, and 2.5 g of xylene cyanol) and separated on 12% polyacrylamide gels (20 x 20 cm; Bio-Rad) with 50 mL/L glycerol for 16 h at 150 V in 1x Tris-borate-EDTA buffer and stained with ethidium bromide (1 mg/L).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). Under these
amplification conditions, both HGs generated specific heteroduplex
patterns. The MTHFR A1298C HG yielded two bands for both the
A and C alleles. Although one band out of each
pair comigrated, the other unique bands allowed the A and
C alleles to be unambiguously identified. The MS
A2756G HG yielded distinct two-band patterns for both the A
and G alleles, thereby allowing their unambiguous
identification (data not shown).
A silent MTHFR polymorphism, T1317C, recently has been
identified in Canadian and African-American populations
(22). To avoid potential interference from this
polymorphism, the MTHFR-D primer (Table 1
B) was designed to overlap
position 1317. A rare loss-of-function mutation at nucleotide 2758 of
the MS gene (15) has not been observed in any of
our subjects (S. Barbaux, unpublished results) and is very unlikely to
confound MS A2756G genotyping.
analysis of multiplex genotyping
To facilitate the generation of definitive composite patterns for
the MTHFR C677T, MTHFR A1298C, MS
A2756G, and CBS 844ins68 polymorphisms, primers were
redesigned to generate nonoverlapping homoduplexes and heteroduplexes
(Table 1
B and Fig. 2
). An example of such a composite genotyping
analysis, and its schematic interpretation as definitive genotypes, are
shown in Fig. 3
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A range of annealing temperatures was tested for each PCR to identify the one that optimally supports the simultaneous amplification of all four products. In addition, the concentration of each primer pair was adjusted empirically to compensate for the different product intensities (because of different sizes and amplification characteristics), observed after gel electrophoresis. The ratio of pHcyHG-1 plasmid to genomic DNA was optimized to obtain almost equal amplification for the two homoduplexes, thereby maximizing the subsequent generation of heteroduplexes. Parallel genotyping of >100 individuals by this multiplex heteroduplexing method and by rigorously controlled conventional PCR-restriction fragment length polymorphism methods gave identical results (data not shown).
Occasional faint bands, from chimeric products amplified across adjacent HGs within the pHcyHG-1 plasmid, appeared in the 300–400 bp range but did not impinge on the pHcyHG-1-generated genotyping patterns that migrate in the 65–225 bp size range.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The assay design permits amplification of all PCR fragments in a single tube; the subsequent spatial separation of all heteroduplexes and homoduplexes during electrophoresis makes it amenable to multiplexing. Furthermore, the products may be visualized immediately after gel electrophoresis and ethidium bromide staining, and no other post-PCR manipulations are needed. In our hands, similar heteroduplex patterns are always observed for each polymorphism, and each run allows the four genotypes to be unambiguously derived. The assay is reproducible, accurate, rapid, and amenable to automation, and is therefore suitable for high-throughput genotyping.
A key determinant of the optimal formation of heteroduplexes is the genomic DNA:HG ratio. Both should be present at approximately equimolar concentrations to allow similar amounts of both the genome-derived and HG-derived homoduplexes to be amplified. The consolidation of all three HGs into a single construct ensures that they are present at equimolar concentrations in genotyping reactions, thereby eliminating quantitative and qualitative variations between the HGs themselves. Therefore, the amount of pHcyHG-1 can be readily adjusted to allow optimal genotyping of all DNA samples in a given series after pilot studies using only a few DNA samples. Even low-concentration DNA samples, such as those extracted from buccal swabs, can be accurately genotyped using this method after appropriate adjustment of the HG concentration.
By empirical testing of reaction conditions, we have established standard conditions under which similar heteroduplex patterns are always observed, i.e., the readout is easy to interpret visually and is accurate and reproducible. Optimization included adjustments of oligonucleotide lengths to allow primers to anneal at the same temperature. In addition, the concentrations of the oligonucleotides for amplifying across the CBS 844ins68 and MS A2756G variants were increased 50% over those used to amplify both of the smaller MTHFR fragments; using this 2:3 ratio, almost equal proportions of the four PCR products were consistently obtained.
The novel genotyping method reported here may be useful for acquiring genetic data from large study cohorts to assess the roles of the MTHFR C677T, MTHFR A1298C, MS A2756G, and CBS 844ins68 polymorphisms in hyperhomocysteinemia-associated pathologies. The imminent completion of the Human Genome Project is likely to lead to the identification of new genetic factors that contribute to hyperhomocysteinemia. Incorporation of the underlying polymorphisms into similar multiplex heteroduplexing systems may extend our capacity to investigate the multifactorial basis of hyperhomocysteinemia and the various pathologies in which increased plasma Hcy concentrations are considered to be etiologically involved.
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Footnotes |
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2 Nonstandard abbreviations: Hcy, homocysteine; MTHFR, methylenetetrahydrofolate reductase; MS, methionine synthase; CBS, cystathionine ß-synthase; and HG, heteroduplex generator.
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References |
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The following articles in journals at HighWire Press have cited this article:
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  K. Robien and C. M. Ulrich 5,10-Methylenetetrahydrofolate Reductase Polymorphisms and Leukemia Risk: A HuGE Minireview Am. J. Epidemiol., April 1, 2003; 157(7): 571 - 582. [Abstract] [Full Text] [PDF]
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) adjacent to the single nucleotide polymorphism site
(•) and generate a modified product. (c) after gel
purification to remove the residual primers, the PCR A and PCR B
products are combined as templates for PCR amplification using only
external Ext F and Ext R primers. The
resulting product is the HG, the sequence of which is
identical to the genomic target sequence except for the microdeletion.
Ext, external; Int, internal;
F, forward; R, reverse
