1 Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, S-413 45 Gothenburg, 2 Institute of Clinical Neuroscience, Psychiatry Section, Sahlgrenska University Hospital, Göteborg University, S-431 80 Mölndal, Sweden, 3 Department of Virology, Medical School, University of Crete, Heraklion, Crete, P.O. Box 1393, Greece and 4 Institute of Clinical Neuroscience, Department of Experimental Neuroscience, Sahlgrenska University Hospital, Göteborg University, S-431 80 Mölndal, Sweden
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Abstract |
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Key words: B12/homocysteine/polymorphism/spontaneous abortion/transcobalamin
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Introduction |
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Materials and methods |
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DNA extraction and genotyping
Genomic DNA was extracted as previously described (Field et al., 1995). The study and control materials were analysed for the TC Pro259Arg polymorphism by the solid-phase minisequencing method (Syvänen et al., 1993
) as previously described (McCaddon et al., 2001
).
Statistical analysis
TC Pro259Arg allele and genotype frequencies were determined for the study and control groups and compared by a 2-test. Statistical significance was defined as P < 0.05. Odds ratios (OR) and 95% confidence intervals (CI) were calculated (Altman, 1991
). All analyses were performed using Systat (SPSS Inc., Chicago, IL, USA).
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Results |
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Discussion |
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We observed a low frequency of the 259-Pro allele in spontaneously aborted embryos compared with adult controls. The difference between cases and controls with regard to the frequency of 259-Pro homozygote subjects was even more pronounced, suggesting that the 259-Pro allele may have protective effects during embryogenesis, conceivably through its positive effects on TC plasma levels (Afman et al., 2001; Namour et al., 2001
), which may be important for efficient vitamin B12 delivery to the cells of the growing embryo. The frequency of Pro259Arg heterozygotes was significantly increased suggesting that this genotype may have negative effects during embryogenesis, while no significant difference in the frequency of 259-Arg homozygotes was observed. It has previously been shown that 259-Arg homozygous subjects have lower TC concentrations than Pro259Arg heterozygotes, who have lower TC concentrations than 259-Pro homozygotes (Afman et al., 2001
; Namour et al., 2001
). However, Namour et al. also found that the lower TC concentration in 259-Arg homozygotes was not associated with an increase in plasma homocysteine, whereas the heterozygous genotype was (Namour et al., 2001
). One explanation, suggested by Namour et al., would be that the binding of TC to the dimeric TC receptor previously described (Bose et al., 1996
) is associated with dimerization of TC and that the heterodimer of Pro/Arg isoforms may have a lower affinity than Pro/Pro or Arg/Arg homodimers. The results presented here are compatible with this model, which may explain why heterozygosity for the Pro259Arg polymorphism was associated with a significantly decreased fetal viability, whereas 259-Arg homozygosity was not.
One common limitation in studies of risk factors for congenital defects and/or compromised fetal viability is bias toward late-pregnancy outcomes. Since exposure to folic acid antagonists during the first or second months of pregnancy, which most likely has a similar pathophysiological mechanism as functional vitamin B12 deficiency, more than doubled the risk of NTD (Hernández-Díaz et al., 2001) and since the relative risk of having an infant with a congenital cardiovascular defect peaked by the maternal use of folic acid antagonists during the second and third months of pregnancy (Hernández-Díaz et al., 2000
), it is clear that fetal needs for the essential components of one-carbon metabolism (folate and vitamin B12) are especially high early in embryogenesis. In addition, Martinelli et al. demonstrated that the methylenetetrahydrofolate reductase C677T polymorphism does not add to the risk of unexplained late fetal loss (fetal death after
20 weeks gestation) (Martinelli et al., 2000
). Thus we focused on fetuses spontaneously aborted earlier than the 20th week, 87% of them in the first trimester of pregnancy, so bias is unlikely to be present in this study. Moreover, exclusion of the 10 samples of spontaneous abortions later than the 12th week of pregnancy did not change the results significantly.
One limitation of our study is that neither embryos nor parents were karyotyped. Aneuploidy is a common cause of spontaneous abortion and this was not excluded in our study group. However, the molecular mechanisms behind chromosomal abnormalities, produced in the germ line of either parent through an error in meiosis or in the early embryo through an error in mitosis, are largely unknown. It is tempting to hypothesize that disrupted one-carbon metabolism might play a role in the generation of chromosomal abnormalities, because of its involvement on many levels in DNA and protein synthesis, but clearly this hypothesis would need to be tested in further studies.
There are numerous investigations on the association between low concentrations of vitamin B12 and/or TC and neural tube defects (Magnus et al., 1991; Kirke et al., 1993
; van der Put et al., 1997
; Steen et al., 1998
). This is the first study assessing the role of fetal TC codon 259 genotypes in the pathogenesis of spontaneous abortion. In conclusion, our data indicate that the 259-Pro allele may have protective influences during embryonic development, emphasizing the biological significance of appropriate vitamin B12 delivery to peripheral tissues in states of rapid cell proliferation. Our results warrant additional investigations exploring the potential beneficial effects of periconceptional supplementation with vitamin B12, in addition to folic acid, in the prevention of spontaneous abortion. Moreover, there may be an interaction between maternal and fetal TC genotypes, which would be interesting to explore in future studies.
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Acknowledgements |
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Notes |
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References |
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Submitted on March 14, 2002; resubmitted on June 18, 2002; accepted on August 8, 2002.