Lack of association between polymorphisms in C4b-binding protein and atypical haemolytic uraemic syndrome in the Spanish population

Abstract

Dysregulation of the alternative pathway of complement activation, caused by mutations or polymorphisms in the genes encoding factor H, membrane co-factor protein, factor I or factor B, is associated strongly with predisposition to atypical haemolytic uraemic syndrome (aHUS). C4b-binding protein (C4BP), a major regulator of the classical pathway of complement activation, also has capacity to regulate the alternative pathway. Interestingly, the C4BP polymorphism p.Arg240His has been associated recently with predisposition to aHUS and the risk allele His240 showed decreased capacity to regulate the alternative pathway. Identification of novel aHUS predisposition factors has important implications for diagnosis and treatment in a significant number of aHUS patients; thus, we sought to replicate these association studies in an independent cohort of aHUS patients. In this study we show that the C4BP His240 allele corresponds to the C4BP*2 allele identified previously by isoelectric focusing in heterozygosis in 1.9-3.7% of unrelated Caucasians. Crucially, we found no differences between 102 unrelated Spanish aHUS patients and 128 healthy age-matched Spanish controls for the frequency of carriers of the His240 C4BP allele. This did not support an association between the p.Arg240His C4BP polymorphism and predisposition to aHUS in the Spanish population. In a similar study, we also failed to sustain an association between C4BP polymorphisms and predisposition to age-related macular degeneration, another disorder which is associated strongly with polymorphisms in factor H, and is thought to involve alternative pathway dysregulation.

Fig. 1

Sequence analysis of the C4BPA isoelectric focusing (IEF) alleles. IEF on polyacrylamide gels under completely denaturing conditions of neuraminidase-treated affinity-purified C4b-binding protein (C4BP) proteins from C4BP*1/C4BP*3, C4BP*1/C4BP*1 and C4BP*1/C4BP*2 individuals is shown on the left side of the figure. Each C4BPα polypeptide chain discloses a banding pattern composed of three bands, the cathodal band being the more heavily stained. The cathodal bands corresponding to the three C4BPA alleles are indicated by arrows. On the right side fragments of the electropherograms corresponding to the regions of exon 7 (SCR4) and exon 11 (SCR8) of C4BPA gene from the same C4BP*1/C4BP*3, C4BP*1/C4BP*1 and C4BP*1/C4BP*2 individuals are shown to illustrate the nucleotide substitutions that identify the C4BP*2 (c.719G>A; Arg240His) and C4BP*3 (c.1615G>C; Glu539Gln) alleles.

Fig. 2

Segregation analysis of the C4BPA alleles. Five pedigrees carrying the C4BP*2 or the C4BP*3 isoelectric focusing (IEF) protein variants are shown to illustrate the perfect segregation of these alleles with the c.719G>A (His240; C4BP*2) and c.1615G>C (Gln539; C4BP*3) nucleotide substitutions. The numbers on the right of the pedigrees are the codes for the samples.