For personal use. Only reproduce with permission from The Lancet. MECHANISMS OF DISEASE 1542 THE LANCET • Vol 362 • November 8, 2003 • www.thelancet.com Summary Background Mutations in factor H (HF1) have been reported in a consistent number of diarrhoea-negative, non-Shiga toxin-associated cases of haemolytic uraemic syndrome (D - HUS). However, most patients with D - HUS have no HF1 mutations, despite decreased serum concentrations of C3. Our aim, therefore, was to assess whether genetic abnormalities in other complement regulatory proteins are involved. Methods We screened genes that encode the complement regulatory proteins—ie, factor H related 5, complement receptor 1, and membrane cofactor protein (MCP)—by PCR- single-strand conformation polymorphism (PCR-SSCP) and by direct sequencing, in 25 consecutive patients with D - HUS, an abnormal complement profile, and no HF1 mutation, from our International Registry of Recurrent and Familial HUS/TTP (HUS/thrombotic thrombocytopenic purpura). Findings We identified a heterozygous mutation in MCP, a surface-bound complement regulator, in two patients with a familial history of HUS. The mutation causes a change in three aminoacids at position 233–35 and insertion of a premature stop-codon, which results in loss of the transmembrane domain of the protein and severely reduced cell-surface expression of MCP. Interpretation Results of previous studies on HF1 indicate an association between HF1 deficiency and D - HUS. Our findings of an MCP mutation in two related patients suggest that impaired regulation of complement activation might be a factor in the pathogenesis of genetic forms of HUS. MCP could be a second putative candidate gene for D - HUS. The protein is highly expressed in the kidney and plays a major part in regulation of glomerular C3 activation. We propose, therefore, that reduced expression of MCP in response to complement-activating stimuli could prevent restriction of complement deposition on glomerular endothelial cells, leading to microvascular cell damage and tissue injury. Lancet 2003; 362: 1542–47 See Commentary *Members listed at end of paper Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Villa Camozzi-Ranica, Bergamo, Italy (M Noris Chem Pharm D, S Brioschi Biol Sci D, J Caprioli Biol Sci D, M Todeschini, E Bresin MD, F Porrati Biol Sci D, S Gamba RN, Prof G Remuzzi MD); Department of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Italy (Prof G Remuzzi) Correspondence to: Dr Marina Noris, Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases “Aldo e Cele Daccò” 24020, Ranica (BG), Italy (e-mail: noris@marionegri.it) Introduction Haemolytic uraemic syndrome (HUS) is a rare disease of microangiopathic haemolysis, thrombocytopenia, and renal failure. 1,2 The most common form of HUS in children, with predominant renal failure, is associated with infection by Escherichia coli, which produce a powerful Shiga-like toxin. 1 This form of the disease (D + HUS) usually presents with a diarrhoea prodrome and has an excellent prognosis in most cases. 3 By contrast, non-Shiga toxin-associated and diarrhoea-negative forms of HUS (D - HUS) have a much poorer outcome (often end-stage renal failure or death 4–6 ), with patients prone to relapse. There is sometimes a clustering of affected individuals within families, suggesting a genetic predisposition to the disease. Both autosomal dominant and autosomal recessive forms of inheritance have been noted, with precipitating events such as pregnancy, virus-like disease, or sepsis reported in some instances. 7–11 Low serum concentrations of the third component of complement (C3) have been identified in patients with D - HUS. 12,13 Among such patients, a subgroup—between 13% and 30%—carry mutations in the gene encoding for factor H (HF1), a plasma protein that inhibits the activation of the alternative pathway of complement. 14–19 However, two thirds of patients with D - HUS have no HF1 mutations, despite decreased C3 concentrations, 1,18,19 indicating a role for genetic abnormalities in other complement regulatory proteins. Methods Participants Between, 1996, and May, 2003, we enrolled consecutive patients with familial, recurrent, or sporadic D - HUS with no HF1 mutations but an abnormal serum complement profile (defined as C3 serum concentrations <0·83 g/L 12 or a plasma C3d/serum C3 ratio >0·015) through the International Registry of Recurrent and Familial HUS/TTP (HUS/thrombotic thrombocytopenic purpura), a network of 60 Haematology and Nephrology Units established under the coordination of the Clinical Research Centre for Rare Diseases “Aldo e Cele Daccò’’. We also recruited healthy blood donors as controls. For protein expression studies in peripheral blood mononuclear cells (PBMC), healthy female controls and uraemic female controls on chronic haemodialysis for causes other than HUS, were recruited. All participants received detailed information on the purposes and design of the study and provided informed written consent, according to the guidelines of the Declaration of Helsinki. The protocol was approved by the institutional review board of the “Mario Negri” Institute for Pharmacological Research. Procedures With respect to complement profile assessment, we quantified serum C3 and C4 concentrations by kinetic nephelometry, and ascertained serum concentrations of Familial haemolytic uraemic syndrome and an MCP mutation Marina Noris, Simona Brioschi, Jessica Caprioli, Marta Todeschini, Elena Bresin, Francesca Porrati, Sara Gamba, Giuseppe Remuzzi for the International Registry of Recurrent and Familial HUS/TTP* Mechanisms of disease