The single nucleotide polymorphism (80GA) of reduced folate carrier gene in trisomy

ISAB - Unity QUASH
Laboratory of Nutritional Genomics
BP.30313
Rue Pierre Waguet
60026-Beauvais
France
E-mail: abalo.chango{at}isab.fr

Nathalie Fillon-Emery and Jean Pierre Nicolas

Faculty of Medicine
Laboratory of Medical Biochemistry
Vandoeuvre-Les-Nancy
France

Henri Bléhaut

Jerome Lejeune Institute
Paris
France

Dear Sir:

The reduced folate carrier gene (RFC1 or SLC19A1) located on chromosome 21 (21q22.3) codes for the reduced folate carrier, which is responsible for 5-methyltetyrahydrofolate internalization within cells. We published earlier the 80GA single-nucleotide polymorphism (SNP) in human reduced folate carrier cDNA and hypothesized that it influences folate metabolism or the plasma total homocysteine concentration (1). The SNP 80GA polymorphism (rs1051266; http://www.ncbi.nlm.nih.gov at the SNP database) is a guanine-to-adenine exchange at nucleotide 80 in RFC1 cDNA. Because of the extra copy of chromosome 21 in persons with trisomy 21, or Down syndrome, a dosage effect of RFC1 and a functional effect of the SNP 80GA polymorphism may contribute to a imbalance of one-carbon-derived metabolites and DNA methylation. Allelic ratios are 3:0, 2:1, 1:2, and 0:3 for the 80GGG, 80GGA, 80GAA, and 80AAA genotypes, respectively, in trisomy 21 compared with 2:0, 1:1, and 0:2 for the 80GG, 80GA, 80AA genotypes, respectively, in control subjects.

In a study published in this issue of the Journal, we analyzed the 80G ± SD; range: 15–46 y) with full trisomy 21 confirmed by karyotype and in 160 healthy, unrelated control subjects (2). With the standard restriction fragment length polymorphism method using the HhaI (or CfoI) restriction enzyme (1, 3), we were not able to distinguish heteroallelic individuals containing 1 or 2 copies of each allele among the persons with Down syndrome. We have now applied the pyrosequencing technology ( The principle of pyrosequencing is based on the polymerization of single-stranded amplified DNA fragments and the detection of de novo incorporation of nucleotides, which leads to the generation of visible light in proportion to the number of incorporated nucleotides (Figure 1). Briefly, DNA samples were amplified by polymerase chain reaction with the sense primer HsRFC 4.3 5'-TGC AGA CCA TCT TCC AAG G-3' and the antisense primer HsRFC 4.3 5'-CCA TGA AGC CGT AGA AGC-3'. The amplified DNA samples were purified by using streptavidin-Sepharose HP beads (Amersham Biosciences, Orsay, France) and a pyrosequencing sample preparation kit (Pyrosequencing AB, Uppsala, Sweden) according to the manufacturer's instructions. Purified samples were run on a PSQ 96MA instrument containing a pyrosequencing cartridge filled with dATPS, dTTP, dCTP, dGTP, substrate, and enzyme as supplied in a PSQ reagent kit (Pyrosequenring). The 4 nucleotides are added stepwise to the primed DNA template. Analysis of sequences was performed automatically by the ALLELE QUANTIFICATION software (Pyrosequencing). The intensity of the light signal is directly proportional to the number of nucleotides incorporated. Genotypes were determined by comparison of the peak heights of allele positions with the theoretical results predicted by the software.

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FIGURE 1.. Quantification of the reduced folate carrier 80GA single-nucleotide polymorphism (SNP) by pyrosequencing. The sequence to be analyzed was GGCA/GCCT. The sequencing primer was 5'-CTC CGG TCC TGG C. The SNP position was denoted A/G when entered in the PSQ 96MA software (Pyrosequencing AB, Uppsala, Sweden). The T and A at the first and sixth positions, respectively (TGCA/GACCTC), were used as controls that should not bind to the DNA template and therefore should not result in a peak; the appearance of small peak heights at these positions is an indication of background in the assay. High peak heights at the G and C positions indicate the presence of the same adjacent nucleotide (GG or CC).

 
The pyrosequencing results of our initial samples (2) show that among the persons with trisomy 21, 37 persons (23.7%) were homozygous 80GGG, 52 persons (33.5%) were heterozygous 80GGA, 44 persons (28.4%) were heterozygous 80GAA, and 22 persons (14.2%) were homozygous 80AAA. Among the control subjects, the numbers of subjects were as follows: 53 persons (33.4.%) were homozygous 80GG, 74 persons (46.5%) were heterozygous 80GA, and 32 persons (20.0%) were homozygous 80AA. In conclusion, genotyping SNP and determination of allele frequency in trisomy need specific techniques based on allele quantification. Pyrosequencing is an appropriate technique that allows one to distinguish heteroallelic individuals containing 1 or 2 copies of each allele.

ACKNOWLEDGMENTS

Funded by the Jerome Lejeune Foundation, Paris. We are grateful to Severine Barry-Charret and Severine Ferre for expert laboratory analysis.

AC was the principal investigator and was responsible for developing the pyrosequencing technique and writing the manuscript. NFE contributed to collecting samples and analyzing the laboratory data. HB was the initial protocol designer responsible for collecting and managing the patients and samples during the study. JPN was the co-principal investigator and is the head of the medical biochemistry research group. FW developed the Agrohealth research program in the ISAB. None of the authors had any conflicts of interest.

REFERENCES

1. Chango A, Emery-Fillon N, Potier de Courcy G, et al. A polymorphism (80G->A) in the reduced folate carrier gene and its associations with folate status and homocysteinemia. Mol Genet Metab 2000;70:310–5.
2. Fillon-Emery N, Chango A, Mircher C, et al. Homocysteine concentrations in adults with trisomy 21: effect of B vitamins and genetic polymorphisms. Am J Clin Nutr 2004;80:1551–7.
3. Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 1980;32:314–31.
4. Rickert AM, Premstaller A, Gebhardt C, Oefner PJ. Gentyping of SNPs in a polyploidy genome by pyrosequencing. BioTechniques 2002;32:592–603.