A type III complement factor D deficiency: Structural insights for inhibition of the alternative pathway

[First paragraph] We investigated an alternative complement pathway (AP) deficiency in a patient with absent alternative pathway hemolytic activity but normal classical pathway hemolytic activity recovering from invasive meningococcal infection (for patient and sibling details, see Patient details in this article's Online Repository at www.jacionline.org). Serum reconstitution with proximal AP components suggested a factor D (FD) deficiency (Fig 1, A). Sanger sequencing of CFD identified a rare homozygous missense mutation (c.602G>C) in exon 4 in the patient (II-1) and sibling (II-2), resulting in an arginine to proline substitution (p. R176P) (see Fig E1, A, and reference E9 in this article's Online Repository at www.jacionline.org). This genotype cosegregated with an alternative pathway hemolytic activity–null phenotype, as the parents, both heterozygotes, had normal alternative pathway hemolytic activity (Fig 1, B). In contrast to previously confirmed FD deficiencies,1, 2, 3 all members of the pedigree had normal levels of circulating FD, as corroborated by Western blot (see Fig E1, B). Meanwhile, identical circular dichroism spectra and melting curves of recombinant wild-type (WT) and R176P FD precluded gross changes in FD structure or stability, suggesting a functional deficiency (Fig 1, C, and see Fig E1, C). We assessed the cleavage of C3b-bound factor B (FB) by recombinant WT and mutant FD (R176P, R176A, R176Q). WT FD could cleave C3b-bound FB to produce fragments Bb and Ba. Conversely, R176P FD demonstrated diminished in vitro catalytic activity at all concentrations and had negligible activity at physiological concentration (0.04 μmol/L) (Fig 1, D, and see Fig E1, D). Reconstitution of FD-depleted serum with R176P FD also demonstrated impaired AP-mediated hemolysis (see Fig E1, E).