Pyrosequencing assay for genotyping of the Transcobalamin II 776C>G polymorphism

Abstract

The 776C>G polymorphism of the Transcobalamin II gene is located in a GC-rich region and TaqMan real-time polymerase chain reaction (PCR) does not yield satisfactory genotyping results. We therefore hypothesized that a method based on DNA sequencing would be needed for this single nucleotide polymorphism (SNP) analysis; Pyrosequencing technology was tested for this purpose. A Pyrosequencing protocol was developed, optimized and applied to a sample of 389 Swedish senior citizens. The three genotypes CC, CG, and GG consistently yielded typical programs that were readily distinguishable from each other. The prevalence of the mutated allele in the studied Swedish population was q=0.445. It is concluded that the TC II 776C>G polymorphism can be reliably genotyped by Pyrosequencing technology.

Transcobalamin C776G genotype modifies the association between vitamin B12 and homocysteine in older Hispanics

“…In addition, it is not known whether these Hispanic cohorts are genetically similar. Reported prevalence of the 776GG variant in other ethnic groups ranges from ~3% in West Africans to ~11% in Italian Caucasians; ~13% in Afro-Americans; ~13% in Brazilians; ~20% in French, Scandinavian, and American Caucasians; and ~42% in ethnic Hans from Central China (Bowen et al , 2004; Gueant et al , 2007; Miller et al , 2002; Bottiger and Nilsson et al , 2007; Pereira et al , 2007; Alessio et al , 2007). Gueant et al (2007) have proposed that environmental factors, in particular malaria, may have imposed selective pressures that contribute to the wide variation in prevalence of the TC 776 polymorphism in different populations.…”

Variations in folate pathway genes are associated with unexplained female infertility

“…The control group consisted of 1,079 individuals from a cross-sectional population studied in central Sweden, the same area in which our group of infertile women was recruited. The data concerning the control individuals have already been published (19)(20)(21)(22).…”

“…Previously described Pyrosequencing assays (19)(20)(21)(22) (Biotage AB, Uppsala, Sweden) were used to genotype the polymor-…”

“…Data from women with unexplained infertility were compared with data from cross-sectional population studies conducted in the same region (19)(20)(21)(22). All genotype distributions in the study subjects were in Hardy-Weinberg equilibrium.…”

Folate-metabolizing gene variants and pregnancy outcome of IVF

“…PCR reactions were performed using the HotStarTaq DNA Polymerase Kit (Qiagen, Venlo, The Netherlands). Genotyping of the single-nucleotide polymorphisms (SNP) MTHFR 677 C/T (rs1801133), MTHFR 1298 A/C (rs1801131), MTHFR 1793 G/A (rs2274976), FOLR1 1314 G/A (rs2071010), FOLR1 1816 C/delC (rs3833748), FOLR1 1841 G/A (rs1540087), FOLR1 1928 C/T (rs9282688), TCN2 776 C/G (rs1801198), CTH 1208 G/T (rs1021737) and SLC19A1 80 G/A (rs1051266) was conducted by pyrosequencing using Pyro Gold Reagents and the PSQ 96 MA system (Biotage, Uppsala, Sweden) as described previously (Böttiger et al, 2007a,b;Böttiger and Nilsson, 2007;Nilsson et al, 2008). Pyrosequencing results were automatically analysed using the PSQ 96 MA 2.0.2 software (Biotage).…”