Autoantibodies to folate receptor during pregnancy and neural tube defect risk

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Abstract

Periconceptional folic acid can reduce the occurrence of neural tube defects (NTDs) by up to 70%, and autoantibodies for folate receptors (FRs) have been observed in serum from women with a pregnancy complicated by an NTD. This population-based cohort study has examined serum from pregnant mothers for autoantibodies to FRs, antibodies to bovine folate binding protein (FBP), and inhibition of folic acid binding to FR and FBP in association with NTD risk. The mid-gestational maternal serum specimens used for this study were collected during the 15–18th week of pregnancy. Samples were obtained from the California Birth Defects Monitoring Program; 29 mothers had a pregnancy complicated by spina bifida and 76 mothers had unaffected children. The presence of IgG and IgM antibodies to human FR, bovine FBP, and inhibition of folic acid binding to FR and FBP was determined. Higher activity of IgM to FBP in cases verses controls was observed (P = 0.04). Higher activity of IgM and IgG autoantibodies to FR was observed (P < 0.001 and P = 0.04, respectively). Risk estimates at two standard deviations above average control antibody concentrations were OR = 2.07 (CI = 1.02, 4.06) for anti-FBP IgM, OR = 2.15 (CI = 1.02, 4.69) for anti-FR IgG and OR = 3.19 (CI = 1.47, 6.92) for anti-FR IgM. These data support the hypothesis that high titers of antibodies and blocking of folic acid binding to FRs by maternal serum should be regarded as risk factors for NTDs.

Introduction

Over the last two decades, periconceptional folic acid supplementation has been shown to significantly reduce the risk of neural tube defects (NTDs) by as much as 70% (Berry et al., 1999, Czeizel and Dudas, 1992, Milunsky et al., 1989, MRC, 1991, Steegers-Theunissen et al., 1994, Werler et al., 1993). However, most pregnant women carrying an NTD-affected fetus do not have serum folate deficiency (Kirke et al., 1993, Yates et al., 1987). No polymorphisms have been observed in folate receptor-α to account for effect of folic acid on NTD risk (Barber et al., 1998). However, multiple polymorphisms for a variety of folate pathway enzymes have been identified, some of which have been proposed as risk factors for NTDs (Botto and Yang, 2000, Shaw et al., 1998, van der Put et al., 1995, Zhu et al., 2003), but they account for only a fraction of the reduction in NTD risk following folate supplementation. The other underlying mechanisms by which folic acid supplementation decreases NTD risk are poorly understood (Cabrera et al., 2004). However, evidence has emerged that maternal immunological responses can have a substantive impact on embryonic development. When antibodies to rat placenta, kidney, heart and other tissues are generated and administered to pregnant rats, they bind to the yolk sac and contribute to congenital abnormalities and embryonic death (da Costa and Rothenberg, 1996, da Costa et al., 2003). It was hypothesized that the presence of antibodies binding to the yolk sac impaired the delivery of critical nutrients to the embryo (da Costa et al., 2003, Rothenberg et al., 2004). Similarly, studies have also indicated that competitive inhibition of proteins such as the FR can result in cellular growth inhibition by blocking the uptake of folate (Ebel et al., 2007, Henderson and Strauss, 1990).

Rothenberg et al. (2004) reported autoantibodies to the FR in 75% of 12 mothers who had given birth to NTD-affected infants, but in only 8.3% of mothers of 24 non-malformed infants. Unfortunately, this provocative but small study examined only three women during pregnancy, all of whom had a pregnancy complicated by an NTD. Maternal autoantibodies to FR that produce immune responses against, or inhibit folate uptake by, the developing embryo may explain the beneficial effect of periconceptional folic acid supplementation on NTD risk. That is, supplemental folic acid may reduce the level of serum autoantibodies or compensate for blocking of the receptors caused by FR autoantibodies. Consequently nullizygous mutations for, or immunologically targeting of, folate binding protein has been demonstrated to increase risk for folate-responsive congenital anomalies in mouse and rat models (da Costa et al., 2003, Piedrahita et al., 1999, Tang and Finnell, 2003).

Several etiologies have been suggested for the presence of FR autoantibodies in human serum. For example, bovine folate binding protein (bFBP) (GI: 110282963) may be antigenic, and some antibodies cross-react with the endogenous receptor (GI: 544337) due to >80% protein homology as determined by the Protein Basic Local Alignment Search Tool (blastp) (Tatusova and Madden, 1999). It is possible also that degradation or cleavage of FR, or the binding of an antigenic ligand, makes the receptor antigenic. The de novo production of anti-idiotypes by the variable region of an antibody could also explain the presence of maternal autoantibodies to the FR (Schwartz, 2005).

We hypothesized that, during pregnancy, blocking of folic acid binding to FR and serum autoantibodies to FR are risk factors for NTDs. Here, we report the results of anti-FR antibodies and folic acid blocking in serum from expectant mothers. Specifically, two preparations of human placental FR and exogenous bovine milk FBP proteins were assessed for interactions with folic acid and antibodies in maternal serum, and their measure of NTD risk was determined.

Section snippets

Study design

Between January 2003 and December 2004, more than 140,000 serum specimens were collected and banked from women during the 15–18th week of pregnancy. These sera were collected from women who live in selected regions in California (Orange and San Diego counties, and Central Valley counties). The specimens were collected from women as part of the Expanded Alpha-Fetoprotein (XAFP) Screening Program. Once diagnostic screening was complete, a proportion of the residual serum sample was stored frozen

Antibodies, autoantibodies, folic acid blocking, and NTD risk

Functional testing of the antibody assay and folate binding assay indicated that both are functional and detect their respective interactions (Fig. 1). Data analysis indicated a low coefficient of variance (average CV < 10%) for antibody detection, and a minimum detectable fold change of two could accurately be determined for both assays (t-test, P < 0.01). The specificity of the assay was confirmed also by using other antigens, in place of folate receptors, with high expected rates of exposure (

Discussion

This study of mid-gestational serum samples from NTD-affected pregnancies compared to pregnancies involving non-malformed fetuses indicates that high concentrations of IgG or IgM antibodies to FRs or FBPs are risk factors for NTDs. During the fourth week of gestation, the neural tube closes and the human embryo is interstitially implanted into the uterine wall, and migration of syncitiotrophoblasts and cytotrophoblasts has formed chorionic villi that cover the entire surface of the blastocyst (

Acknowledgments

The authors are grateful to Drs. Jacob Selhub and Bart Kamen for providing folate receptors for these experiments. This work was partially supported by the NIH/NHLBI P30 HL66398, and by funds from the Centers of Disease Control and Prevention, Centers of Excellence Award no. U50/CCU913241.

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