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Case Reports
. 2003 Oct 28;100(22):12966-71.
doi: 10.1073/pnas.2135497100. Epub 2003 Oct 17.

Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome

Anna Richards  1 , Elizabeth J Kemp, M Kathryn Liszewski, Judith A Goodship, Anne K Lampe, Ronny Decorte, M Hamza Müslümanoğlu, Salih Kavukcu, Guido Filler, Yves Pirson, Leana S Wen, John P Atkinson, Timothy H J Goodship
Affiliations
Case Reports

Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome

Anna Richards et al. Proc Natl Acad Sci U S A. .

Abstract

Membrane cofactor protein (MCP; CD46) is a widely expressed transmembrane complement regulator. Like factor H it inhibits complement activation by regulating C3b deposition on targets. Factor H mutations occur in 10-20% of patients with hemolytic uremic syndrome (HUS). We hypothesized that MCP mutations could predispose to HUS, and we sequenced MCP coding exons in affected individuals from 30 families. MCP mutations were detected in affected individuals of three families: a deletion of two amino acids (D237/S238) in family 1 (heterozygous) and a substitution, S206P, in families 2 (heterozygous) and 3 (homozygous). We evaluated protein expression and function in peripheral blood mononuclear cells from these individuals. An individual with the D237/S238 deletion had reduced MCP levels and approximately 50% C3b binding compared with normal controls. Individuals with the S206P change expressed normal quantities of protein, but demonstrated approximately 50% reduction in C3b binding in heterozygotes and complete lack of C3b binding in homozygotes. MCP expression and function was evaluated in transfectants reproducing these mutations. The deletion mutant was retained intracellularly. S206P protein was expressed on the cell surface but had a reduced ability to prevent complement activation, consistent with its reduced C3b binding and cofactor activity. This study presents further evidence that complement dysregulation predisposes to development of thrombotic microangiopathy and that screening patients for such defects could provide informed treatment strategies.

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Figures

Fig. 1.
Fig. 1.
(A) The pedigree of family 1. WT DNA sequence (B) and sequence (cloned PCR product) (C) from an affected individual from family 1. The nucleotide sequence is shown above and the corresponding amino acids below the sequence. There is a 6-bp deletion in the affected individual (GACAGT) resulting in deletion of an aspartic acid and serine (ΔD237/S238), denoted by the bar under B.(D) Shown are the pedigrees of families 2 and 3 (filled areas indicate an affected individual, • within □ or ○ indicates an unaffected heterozygote, and the double connecting line indicates consanguinity). (E) WT sequence. (F) A heterozygote T822C. (G) Homozygous T822C. This transition leads to an amino acid change, S206P. PCR products were directly sequenced.
Fig. 2.
Fig. 2.
(A) Western blot of CHO cell lysates probed with a rabbit polyclonal Ab to MCP. Lane 1 contains the size markers. Lane 2 is a CHO cell not expressing MCP. Lane 3 shows the phenotype of WT MCP as expressed by transfected CHO cells. Lanes 4 and 5 are the mutations in WT MCP identified in the HUS families. Lane 4 represents the cell line expressing the mutant observed in family 1. Its lower migration pattern is similar to a precursor form of MCP (see below). S206P is the single proline substitution for the native serine identified in families 2 and 3 (lane 5) that migrates similarly to WT. (B) Fluorescence-activated cell sorting analysis of expression of the MCP mutants ΔD237/S238 versus S206P shows low expression levels of the deletion mutant. (C) Biotinylation of cell surface proteins followed by immunoprecipitation with a mAb to MCP shows only a trace of deleted protein expressed on the cell surface. (D) Pulse–chase analysis of the deletion mutant (ΔD237/S238) versus WT MCP. The precursor of the deletion mutant does not chase into the mature protein. (E) Glycosidase digestion of deletion mutant (ΔD237/S238) versus WT MCP (see text for explanations).
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