A novel mutation in the complement regulator clusterin in recurrent hemolytic uremic syndrome

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Abstract

A novel heterozygous mutation in the clusterin gene, nucleotide position A1298C (glutamine > proline Q433P), was detected in exon 7 of a child with recurrent hemolytic uremic syndrome (HUS). The same mutation was found in the child's two siblings and mother but not in 120 controls. In addition, a previously described heterozygous mutation was detected in the gene encoding membrane cofactor protein (MCP) causing a 6 base-pair deletion 811-816delGACAGT in exon 6. It was found in the patient, both siblings and the father. One sibling had recovered from post-streptoccocal glomerulonephritis. Clusterin levels in the patient, siblings and parents were normal as was the migration pattern in a gel. Patient serum induced C3 and C9 deposition on normal washed platelets, and platelet activation, as detected by flow cytometry. The same phenomenon was found in serum taken from the siblings and the mother but not in the sample from the father and controls. Addition of clusterin to patient serum did not inhibit complement activation on platelets. The Q433P mutant, in isolated form, was further studied by binding to the components of the terminal complement complex. The mutant did not bind to C5b-7 that was immobilized onto a BIAcore chip, whereas wild-type clusterin did, indicating that the mutation could lead to defective inhibition of formation of the membrane attack complex under these conditions. Hemolysis of rabbit erythrocytes was inhibited by wild-type clusterin but not by the mutant. Mutated clusterin could thus not prevent assembly of the membrane attack complex on platelets and erythrocytes.

Introduction

Mutations in complement regulators factor H (Warwicker et al., 1998, Ying et al., 1999), factor I (Kavanagh et al., 2005, Nilsson et al., 2007) and membrane cofactor protein (MCP/CD46) (Richards et al., 2003) have been associated with recurrent atypical hemolytic uremic syndrome (aHUS). In addition, mutations in C3 have been associated with HUS (Fremeaux-Bacchi et al., 2008) as well as gain-of-function mutations in factor B (Goicoechea de Jorge et al., 2007). Mutations in factor H are usually localized at the C terminal in aHUS (Caprioli et al., 2001). These mutations have been shown to affect host recognition and allow uninhibited complement activation via the alternative pathway on endothelial cells (Manuelian et al., 2003) and platelets (Ståhl et al., 2008), which could explain the endothelial injury and platelet consumption seen during this condition. This phenomenon has been characterized in mice with a homozygous deletion of the C terminal of factor H causing a condition resembling human HUS (Pickering et al., 2007).

The defective activity of mutated MCP (Richards et al., 2003), factor I (Kavanagh et al., 2005, Nilsson et al., 2007), C3 (Fremeaux-Bacchi et al., 2008) and factor B (Goicoechea de Jorge et al., 2007) leading to alternative pathway activation have also been documented. Most mutations are heterozygous and certain patients may have more than one mutation and/or risk-associated polymorphisms in these factors (Caprioli et al., 2003). A pattern of incomplete penetrance has been demonstrated in families (Esparza-Gordillo et al., 2006) suggesting that other factors, which may be environmental, infectious and/or genetic, may trigger disease induction. The known mutations account for disease in approximately 50% of aHUS patients (Loirat et al., 2008). All mutations found to date affect the initial steps of the alternative pathway. So far, no mutations in regulators of membrane attack complex (MAC) assembly have been reported.

Formation of the MAC can result from activation of the classical, lectin or the alternative pathways of the complement system. MAC assembly is initiated by the generation of C5b, followed by sequential addition of one C6, C7, C8 and numerous C9 molecules. Host cells are protected from cell lysis by regulatory proteins that inhibit the formation or integration of the MAC into the cell membrane. S protein (vitronectin) (Podack and Muller-Eberhard, 1979) and clusterin (Murphy et al., 1989) are the two major inhibitors in the fluid phase while CD59 regulates MAC formation on cell membranes (Meri et al., 1990).

Clusterin is transcribed from a single copy gene of nine exons located on chromosome 8 (8p21) (Fink et al., 1993). Clusterin is a widely expressed and multifunctional protein (Jenne and Tschopp, 1992). There are two isoforms, nuclear and secreted. The nuclear form is pro-apoptotic, while the secretory form is considered pro-survival with properties that can regulate DNA repair (Shannan et al., 2006). Clusterin is mainly produced by the liver (Högåsen et al., 1996) but also by megakaryocytes and thus released from platelets upon activation (Tschopp et al., 1993b). Clusterin circulates in association with lipoproteins (de Silva et al., 1990) at a concentration of approximately 250–420 μg/mL (Högåsen et al., 1993). Mutations in clusterin have, as yet, not been associated with human disease although disease-associated polymorphisms have been described (Miwa et al., 2005, Tycko et al., 1996).

In this study a novel mutation in clusterin was demonstrated in one family in which one child suffered from recurrent aHUS and another had recovered from one episode of post-streptococcal glomerulonephritis. The study investigated the function of this mutated protein with regard to regulation of MAC assembly leading to complement deposition and activation of platelets.

Section snippets

Subjects

The proband is a currently 11-year-old boy admitted at 4 years of age to the University Hospital of Leuven due to fever, edema, pallor, petechiae, reduced consciousness and macroscopic hematuria. He was normotensive and laboratory assays revealed hemoglobin 7.7 g/dL, platelet count 40,000/μL, haptoglobin 0.2 g/L (reference value 0.3–2.0), negative direct antiglobulin (Coombs’) test, urea 257 mg/dL, creatinine 4.95 mg/dL, C3 0.57 g/L (0.79–1.52) and normal C4. Urinalysis showed hematuria and

Detection of a clusterin mutation

Mutations in the genes for factor H, factor I, C3 or factor B were not found in the patient. Likewise, risk-associated polymorphisms were not found in the patient's factor H gene. In the MCP gene a heterozygous mutation previously described in a Belgian family (Richards et al., 2003) was detected causing a 6 base-pair deletion 811-816delGACAGT in exon 6. It was found in the patient, both siblings and the father. In addition, a novel heterozygous mutation in the clusterin gene, nucleotide

Discussion

A novel mutation in clusterin was found in one family in which one child suffered from recurrent episodes of aHUS and his brother recovered after one episode of post-streptococcal glomerulonephritis. Activation of the alternative pathway of complement is known to occur in both conditions. In addition to the clusterin mutation, a known mutation in the MCP gene was identified which could, in itself, explain the occurrence of HUS in this child, especially as the course of disease was rather mild

Acknowledgements

This study was supported by grants from The Swedish Research Council (K2007-64X-14008-07-3), Sven Jerring Foundation, the Crafoord Foundation, Crown Princess Lovisa's Society for Child Care, The Magnus Bergvall Foundation, Region Skåne research grants, Fanny Ekdahl Foundation and King Gustav Vs 80th Birthday Fund. Diana Karpman is the recipient of a clinical-experimental research fellowship from the Royal Swedish Academy of Sciences.

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