Abstract
Miscarriage is the most common complication of pregnancy. Approximately 1% of couples trying to conceive will experience recurrent miscarriages, defined as three or more consecutive pregnancy losses and many of these cases remain idiopathic. Complement is implicated both in the physiology and pathology of pregnancy. Therefore, we hypothesized that alterations in the C3 gene could potentially predispose to this disorder. We performed full Sanger sequencing of all exons of C3, in 192 childless women, with at least two miscarriages and without any known risk factors. All exons carrying non-synonymous alterations found in the patients were then sequenced in a control group of 192 women. None of the identified alterations were significantly associated with the disorder. Thirteen identified non-synonymous alterations (R102G, K155Q, L302P, P314L, Y325H, V326A, S327P, V330I, K633R, R735W, R1591G, G1606D, and S1619R) were expressed recombinantly, upon which C3 expression and secretion were determined. The L302P and S327P were not secreted from the cells, likely due to misfolding and intracellular degradation. Y325H, V326A, V3301I, R1591G, and G1606D yielded approximately half C3 concentration in the cell media compared with wild type (WT). We analyzed the hemolytic activity of the secreted C3 variants by reconstituting C3-depleted serum. In this assay, R1591G had impaired hemolytic activity while majority of remaining mutants instead had increased activity. R1591G also yielded more factor B activation in solution compared with WT. R1591G and G1606D showed impaired degradation by factor I, irrespectively if factor H, CD46, or C4b-binding protein were used as cofactors. These two C3 mutants showed impaired binding of the cofactors and/or factor I. Taken together, several alterations in C3 were identified and some of these affected the secretion and/or the function of the protein, which might contribute to the disorder but the degree of association must be evaluated in larger cohorts.
Highlights
Spontaneous pregnancy loss, or miscarriage, is the most common complication of pregnancy and includes all pregnancy losses from conception until 24 weeks of gestation
The exons carrying identified alterations were analyzed in a control group of 192 women and a total of 13 heterozygous non-synonymous alterations were found in both groups
Four of these (L302P, Y325H, R1591G, and G1606D) were novel mutations only found in the patient group and three (V326A, S327P, and V330I) were novel ones only found in the control group
Summary
Spontaneous pregnancy loss, or miscarriage, is the most common complication of pregnancy and includes all pregnancy losses from conception until 24 weeks of gestation. It is believed that as many as 50% of all conceptions and 15% of clinically recognized pregnancies end up in miscarriage [1]. The definition of recurrent spontaneous pregnancy loss (RSPL) differs among international societies. The American Society for Reproductive Medicine defines RSPL as two or more pregnancy losses [2], affecting around 5% of couples trying to conceive, while The European Society for Human Reproduction and Embryology defines it as three or more consecutive losses [3], affecting around 1% of couples trying to conceive. Various etiologies have been identified, such as genetic, structural, autoimmune, endocrine, thrombophilic abnormalities, together with infections. Approximately 40–50% of the cases still remain idiopathic [1]
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