Human Reproduction, Vol. 17, No. 12, 3033-3036,
December 2002
© 2002 European Society of Human Reproduction and Embryology
The transcobalamin codon 259 polymorphism influences the risk of human spontaneous abortion
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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BACKGROUND: The remethylation cycle of methionine is folate and vitamin B12 (cobalamin) dependent and appears to be crucial for embryonic development, probably through effects on synthesis of DNA, proteins and polyamines. Transcobalamin (TC) transports vitamin B12 to the tissues. The objective of the present investigation was to explore the putative association between the major TC genetic polymorphism (Pro259Arg) and human spontaneous abortion. METHODS: The prevalence of the TC Pro259Arg polymorphism was determined in DNA samples from embryos that had been spontaneously aborted between the 6th and 20th week after conception, and adult controls using solid-phase minisequencing technique. RESULTS: The 259-Pro allele was significantly less frequent in the spontaneous abortion group than in the control group (42.2 and 57.0% respectively; P = 0.005), while the frequency of 259-Arg was significantly increased. There was a lower prevalence of 259-Pro homozygotes in the spontaneous abortion group compared with the control group (9.1 and 32.2% respectively; P < 0.001). CONCLUSIONS: The 259-Pro allele seems to have beneficial influences during embryogenesis, conceivably through its positive effect on vitamin B12 intracellular bioavailability. Our results warrant additional investigations addressing the question if vitamin B12 supplementation in addition to folic acid supplementation may prevent spontaneous abortion in women planning a pregnancy.
Key words: B12/homocysteine/polymorphism/spontaneous abortion/transcobalamin
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Vitamin B12 (cobalamin) is an essential co-factor in folate-dependent homocysteine metabolism and is frequently deficient in pregnant women (Baker et al., 2002
). Methionine synthase (EC 2.1.1.13) requires vitamin B12 to transfer the methyl group of 5-methyltetrahydrofolate to homocysteine. The products formed are the essential amino acid methionine and tetrahydrofolate, which is required for DNA synthesis. Vitamin B12 deficiency is commonly associated with hyperhomocysteinaemia (Allen et al., 1993), which is a risk factor for neural tube defects (NTD) (Steegers-Theunissen et al., 1991; Mills et al., 1995) and recurrent embryo loss (Steegers-Theunissen et al., 1992; Wouters et al., 1993; Nelen et al., 2000). It is unclear how abnormalities of folate- and vitamin B12-dependent homocysteine and methionine metabolism would cause compromised fetal viability, although both insufficient methylation of crucial metabolites and direct toxicity of homocysteine have been suggested as possible mediators of teratogenesis (Rosenquist et al., 1996). Two proteins bind vitamin B12 in serum: haptocorrin and transcobalamin (TC). The latter is the critical transporter that delivers vitamin B12 to peripheral tissues. Polyacrylamide gel electrophoresis reveals four common phenotypic isotypes of TC: M, X, S and F (Daiger et al., 1978; Frater-Schroder et al., 1979), which influence vitamin B12 levels in healthy Caucasians. The different isotypes most likely also influence the cellular availability of vitamin B12 and consequently homocysteine metabolism, especially in individuals with a limited vitamin B12 status (Namour et al., 1998, 2001). The phenotypic variability of TC is a multifactorial phenomenon that probably includes cell type-specific processing of translated TC (Namour et al., 2001). However, the substitution of proline for arginine at codon 259 of the TC gene is the major determinant of the TC variability, at least in Caucasians (McCaddon et al., 2001; Namour et al., 2001) and affects TC levels in plasma (Afman et al., 2001; Namour et al., 2001). In the present investigation, we hypothesized that there may be a relation between fetal TC Pro259Arg genotypes and spontaneous abortion, because of the differential effects of the genotypes on TC levels, vitamin B12 bioavailability and homocysteine metabolism. We analysed DNA samples from 77 spontaneously aborted human embryos and 115 adult controls for the TC Pro259Arg polymorphism by using the solid-phase minisequencing technique (Syvänen et al., 1993). This technique directly detects the TC Pro259Arg polymorphism and is not sensitive to silent polymorphisms. |
Materials and methods |
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Clinical materials
The study was approved by the Ethics Committee at the University Hospital of Heraklion and written informed consent was obtained from the relatives of all participants. The final study group consisted of 77 fetal tissue samples from spontaneous abortions, collected at the Department of Obstetrics and Gynecology, Medical School, University of Crete, Heraklion, either at the time of rejection or after suction of the uterus in cases of incomplete abortions. None of the abortions included were fetal deaths diagnosed in utero on ultrasound. The samples consisted in most cases of indusium (the membranous cover surrounding the embryonic tissue), chorion and chorionic villuses, while in some there were parts of embryonic organs. Spontaneous abortion occurred between the 6th and 20th weeks of pregnancy (10.4 ± 2.9), with the majority (87%) occurring earlier than the 12th week. Maternal tissue contamination was excluded as previously described (Spandidos et al., 1998
). Briefly, DNA paternity test was performed using a class of genetic markers at the four hyper-polymorphic short tandem repeat (STR) loci, HUMVWA31/1, HUMTHO1, HUMF13A1 and HUMFES/FPS (Kimpton et al., 1993; Lygo et al., 1994). The four STR in fetal tissue were compared with those in maternal blood. Three samples with identical banding patterns, which indicated maternal contamination, were excluded from the study. Moreover, two cases were excluded because of a complete maternal STR pattern alongside a paternally derived STR pattern, since it was impossible to determine whether the pattern was due to chromosomal abnormality or maternal contamination. Unfortunately, neither embryos nor parents were karyotyped. Laboratory tests for rubella virus, cytomegalovirus (CMV), herpes simplex virus (HSV), Toxoplasma species and syphilis (group STORCH) were performed for all women at the beginning of their pregnancy. Moreover, all tissue specimens were examined by PCR for CMV, human papilloma virus, Parvo B19 (Sifakis, et al., 1998a) and HSV (Sifakis, et al., 1998b). All included cases had negative tests. Altogether, 23 cases were excluded from the study because of a positive test for any of these infections. None of the women had a medical record of serious systemic disease, and no anatomic defects of the reproductive tract were found on ultrasound examination. Seventy-three women reported one or more previous miscarriages. Thus, the large majority of the investigated cases were unexplained recurrent abortions. Four women did not have a history of previous miscarriages. Of these four women, three were primigravidas and one had undergone an elective termination of pregnancy. Seventeen women had one or more children. The control group consisted of 115 DNA samples from randomly chosen healthy blood donors from Crete.
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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The TC Pro259Arg allele frequencies are presented in Table I
. The 259-Pro allele frequency was significantly decreased (42.2 versus 57.0%, P = 0.005) while the 259-Arg allele frequency was significantly increased in the spontaneous abortion group. The OR, a measure of the relative risk of spontaneous abortion between embryos with or without a given allele, were 0.552 (95% CI 0.362–0.840) and 1.81 (95% CI 1.19–2.76) for the 259-Pro and 259-Arg alleles respectively. The TC Pro259Arg genotype distributions for the control and spontaneous abortion groups are presented in Table II. The TC Pro259Arg genotype frequencies found in our control group agreed well with those reported in other Caucasian populations (McCaddon et al., 2001; Namour et al., 2001) and did not deviate significantly from Hardy–Weinberg equilibrium in any of the groups. Only 9.1% of spontaneously aborted embryos were homozygous for the 259-Pro allele as compared with 32.2% of the control group (P < 0.001), resulting in an OR of 0.211 (95% CI 0.087–0.512). There was a higher prevalence of Pro259Arg heterozygotes in the spontaneous abortion groups compared with the control group (P = 0.022), resulting in an OR of 2.00 (95% CI 1.08–3.67). The frequency of 259-Arg homozygotes was not significantly different between control and spontaneous abortion groups. Since the pathophysiological mechanisms between spontaneous abortions occurring before and after the 12th week of pregnancy may be different, we also excluded the 10 samples of spontaneous abortions that had occurred after the 12th week of pregnancy from the statistical analyses. The genotype frequencies in the reduced spontaneous abortion group remained largely unchanged (10.4% for 259-Pro homozygotes, 67.2% for Pro259Arg heterozygotes and 22.4% for 259-Arg homozygotes) and the differences between the study and control groups remained significant (P = 0.004). Likewise, when excluding the four spontaneous abortions that had occurred in women without a history of previous miscarriages, the differences remained significant.
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Discussion |
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The pathogenesis of spontaneous abortion is complex, presumably involving the interaction of several genetic and environmental factors. The association between recurrent spontaneous abortion and raised plasma homocysteine concentrations (Steegers-Theunissen et al., 1992
; Wouters et al., 1993; Nelen et al., 2000) and the report of increased plasma homocysteine in TC Pro259Arg heterozygote subjects (Namour et al., 2001), were cues to the present investigation in which we determined the TC codon 259 genetic polymorphism in DNA samples from spontaneously aborted embryos and adult controls.
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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This work was supported by grants from the Swedish Medical Research Council (project #12103) and the Sahlgrenska University Hospital.
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5 To whom correspondence should be addressed. E-mail: henrik.zetterberg{at}clinchem.gu.se
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Submitted on March 14, 2002; resubmitted on June 18, 2002; accepted on August 8, 2002.
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