Mutations in Complement Factor I Predispose to Development of Atypical Hemolytic Uremic Syndrome

David Kavanagh,* Elizabeth J. Kemp,* Elizabeth Mayland,* Robin J. Winney,† ʈ Jeremy S. Duffield,‡ Graham Warwick,§ Anna Richards,* Roy Ward, Judith A Goodship,* and Timothy H.J. Goodship* *Institute of Human Genetics, University of Newcastle upon Tyne, United Kingdom; †Department of Renal Medicine, Royal Infirmary, Edinburgh, United Kingdom; ‡Renal Division, Brigham and Women’s Hospital, Boston, ʈ Massachusetts; §John Walls Renal Unit, University Hospitals of Leicester, Leicester, United Kingdom; and Department of Immunology, Newcastle upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, United Kingdom

Mutations in the plasma complement regulator factor H (CFH) and the transmembrane complement regulator membrane co-factor protein (MCP) have been shown to predispose to atypical hemolytic uremic syndrome (HUS). Both of these proteins act as co-factors for complement factor I (IF). IF is a highly specific serine protease that cleaves the ␣-chains of C3b and C4b and thus downregulates activation of both the classical and the alternative complement pathways. This study looked for IF mutations in a panel of 76 patients with HUS. Mutations were detected in two patients, both of whom had reduced serum IF levels. A heterozygous bp change, c.463 G>A, which results in a premature stop codon (W127X), was found in one, and in the other, a heterozygous single base pair deletion in exon 7 (del 922C) was detected. Both patients had a history of recurrent HUS after transplantation. This is in accordance with the high rate of recurrence in patients with CFH mutations. Patients who are reported to have mutations in MCP, by contrast, do not have recurrence after transplantation. As with CFH- and MCP- associated HUS, there was incomplete penetrance in the family of one of the affected individuals. This study provides further evidence that atypical HUS is a disease of complement dysregulation. J Am Soc Nephrol 16: 2150–2155, 2005. doi: 10.1681/ASN.2005010103

emolytic uremic syndrome (HUS) is characterized by bound complement regulator membrane co-factor protein the triad of thrombocytopenia, Coomb’s test–nega- (MCP; CD46) also predispose to atypical HUS (12,13). MCP acts H tive microangiopathic hemolytic anemia, and acute as a co-factor for IF-mediated cleavage of C3b and C4b. renal failure (1). HUS is classified as either typical when it is IF consists of a light and a heavy chain linked by a disulphide bond associated with a preceding diarrheal illness, which is most (Figure 1). There are four domains in the heavy chain: a factor I commonly caused by infection with Escherichia coli O157, or, domain, a scavenger receptor domain, and two LDL receptor less commonly, atypical, which is rarely associated with diar- (LDLr-1 and LDLr-2) domains. There is one domain in the light rhea. Atypical HUS may be sporadic or familial. chain, a serine protease (SP) domain (14). The IF gene is located on Recent findings suggest that atypical HUS is a disease of com- chromosome 4q25 and spans 63 kb (15). It comprises 13 exons, and plement dysregulation. Mutations in the complement regulatory there is a strong correlation between the exonic organization of the protein factor H (CFH) have been described in both sporadic and gene and the modular structure of the protein. IF is a highly specific familial HUS (2–7). CFH is a serum protein that binds to C3b, serine protease and cleaves the ␣ chains of C3b and C4b. This func- accelerates the decay of the alternative pathway convertase tion is dependent on co-factors: CFH for cleavage of C3b in the fluid (C3bBb), and acts as a co-factor for factor I (IF)-mediated proteo- phase (8) and MCP (16) on host cells. Inactivation of C3b and C4b lytic inactivation of C3b (8). The majority of mutations in CFH in prevents the formation of the C3/C5 convertases. IF cleaves C3b at atypical HUS are missense heterozygous changes that tend to two positions in the ␣ chain to yield iC3b and C3f, whereas it cleaves cluster in the C-terminal end of the molecule, an area that is C4 on either side of the thioester to yield C4c and C4d. known to be important for binding to anionic surfaces and C3b (9). Fremeaux-Bacchi et al. (17) recently reported the finding of IF Such mutations result in impaired protection of host surfaces mutations in atypical HUS. We screened 76 patients with atypical against complement activation (10,11). HUS for IF mutations and identified two cases of heterozygous IF Recent studies have shown that mutations in the membrane- deficiency, which, like CFH-associated HUS, were associated with incomplete penetrance and disease recurrence after transplantation.

Received January 25, 2005. Accepted April 14, 2005. Published online ahead of print. Publication date available at www.jasn.org. Materials and Methods Address correspondence to: Dr. Timothy H.J. Goodship, Institute of Human Genet- Patients ics, University of Newcastle upon Tyne, Tyne and Wear NE1 3BZ, UK. Phone: We studied 76 patients with atypical HUS; of these, six were known 44-191-241-8632; Fax: 44-191-241-8666; E-mail: [email protected] to have a CFH mutation (3,9) and two an MCP mutation (12). In 17 of

she returned to hemodialysis. For the subsequent 18 yr, she has been treated with both peritoneal dialysis and hemodialysis. During this time, she has had only two episodes of severe infection. She had an episode of severe peritonitis while on peritoneal dialysis, necessitating catheter removal. This was complicated by persistent intra-abdominal sepsis. There was also an episode of methicillin-resistant Staphylococcus aureus septicemia related to a hemodialysis catheter. There has been no infection with an encapsulated organism. Patient B (Figure 2B, II:4) presented at the age of 31 with hematuria and proteinuria. Plasma creatinine at that time was normal, and a renal biopsy was not undertaken. Two years later, he presented again with general malaise and oliguria. Plasma creatinine was 1023 ␮mol/L, and lactate dehydrogenase (LDH) was 1320 IU/L. Hemoglobin was 7.6 g/dl, platelet count was 61 ϫ 109/L, and there was evidence of hemolysis on a blood film. C3 on presentation was 0.3 g/L (normal range, Figure 1. Domain structure of factor I (IF). Adapted from refer- 0.73 to 1.4g/L), and C4 was 0.2 g/L (normal range, 0.12 to 0.3g/L). ence 37 with permission from Elsevier. Histologic findings on renal biopsy were consistent with HUS. He was initially treated with cyclophosphamide 150 mg daily, prednisolone 60 mg daily, and plasma exchange (eight daily exchanges with 40 ml/kg the 76, at least one other family member was affected. Ethical approval fresh-frozen plasma replacement fluid). With this, there was an im- for the study was given by the Northern and Yorkshire Multi-Centre provement in both the LDH level and the platelet count. However, after Research Ethics Committee. plasma exchange was stopped, the LDH level increased and the platelet count fell; despite reinstating plasma exchange, he did not recover renal Case Reports function. Patient A (Figure 2A, II:1) presented with acute renal failure, throm- Two years later, he received a live related transplant from his bocytopenia, and microangiopathic hemolytic anemia at the age of 32, brother. Immunosuppression consisted of prednisolone, cyclosporin, during the third trimester of her first pregnancy. A renal biopsy con- and azathioprine. The transplant functioned immediately, and creati- ␮ firmed HUS. Complement assays were not performed at her initial nine fell to 105 mol/L. One year after transplantation, his plasma ␮ presentation. Plasma exchange was not undertaken, renal function did creatinine rose to 157 mol/L. Biopsy showed acute rejection, which not recover, and hemodialysis was commenced. Seventeen months responded to three daily intravenous pulses of methylprednisolone. later, she received a cadaveric renal transplant with excellent initial Twenty months after transplantation, there was another episode of function. Immunosuppression was with cyclosporin A and pred- acute graft dysfunction, but biopsy on this occasion showed recurrent nisolone. After 2 mo, there was deterioration in transplant function, HUS. He was treated with plasma exchange (23 exchanges with 40 and biopsy showed recurrent HUS. The transplant was removed, and ml/kg fresh-frozen plasma replacement fluid), but despite this renal function continued to deteriorate with evidence of ongoing hemolysis. Graft nephrectomy was undertaken, and the hemolysis resolved. For the past 6 yr, he has continued to be treated with hemodialysis. Since his initial presentation with HUS, he has had only one severe infection, a S. viridans peritoneal dialysis–related peritonitis, which settled with antibiotics. His sister (Figure 2B, II:3) presented with end-stage renal failure and malignant hypertension at the age of 9. A biopsy was not undertaken. Subsequently, she received two cadaveric renal transplants, both of which failed rapidly. She died in 1973 at the age of 12. No tissue samples were available for analysis. Both parents are deceased.

Complement Assays Convalescent EDTA plasma samples from patients with factor I mutations were obtained and stored at Ϫ80°C. C3 and C4 levels were measured by rate nephelometry (Beckman Array 360). CFH and IF levels were measured by radioimmunodiffusion (Binding Site, Bir- mingham, UK).

DNA Analysis Direct sequencing of all IF exons and the promoter was undertaken in all 76 patients. Genomic DNA was prepared from peripheral blood according to standard procedures. Genomic DNA was amplified by PCR using intronic oligonucleotides. The minimal promoter region Figure 2. (a) Pedigree of patient A (II.1). (b) Pedigree of patient identified by Paramaswara et al. (18) was also amplified using oligo- B (II.4). nucleotides flanking the region. The sequences of the oligonucleotides, 2152 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2150–2155, 2005 annealing temperature, and magnesium concentration for each PCR were heated to 95°C in nonreducing sample buffer (Pierce, Rockford, reaction are shown in Table 1. IL) for 5 min. Protein electrophoresis was performed on 12% SDS- PCR products were purified by treatment with shrimp alkaline phos- PAGE before transfer to nitrocellulose (Hybond ECL; Amersham). The phatase (Amersham, Little Chalfont, UK) and Exonuclease (New En- membrane then was stained with a mouse anti-human factor I mAb gland Biolabs, Beverly, MA). Direct DNA sequencing was performed (MCA507; Serotec, Oxford, UK), which recognizes the 56-kD heavy using Megabase Dyenamic ET (Amersham) on a Megabase1000 (Am- chain (20). ersham). Sequence analyses were performed using Sequencher soft- ware (Gene Codes Corp., Ann Arbor, MI). To clarify the mutation in patient B, we cloned the PCR product spanning exon 7 using the Results pGEMTeasy Cloning Kit (Promega, Madison, WI) and sequenced it as Patient A had a heterozygous substitution c.463 GϾA, which above. Nucleotide and amino acid numbering was according to the results in a premature stop codon W127X in exon 3 (Figure 3). published cDNA sequence (accession number M25615) (19). Nucleotide Her unaffected father (I:1) has the same change. This change 1 is located 29 nucleotides before the start of the peptide signal sequence. The start codon of the sequence after the peptide signal was was not identified in 106 control subjects. IF levels in both used as the first amino acid of the protein. This numbering corresponds patient A (29.2 mg/L; normal range 38–58 mg/L) and her to that used by Fremeaux-Bacchi et al. (17). father (33.7 mg/L) were low. C3 levels in patient A (0.87 g/L; normal range 0.73 to 1.4g/L) and her father (1.4 g/L) were Western Blots normal. The CFH level in patient A was normal (0.50 g/L; Serum samples were stored at Ϫ80°C. Samples first were treated with normal range 0.35 to 0.59 g/L). Mutation screening of CFH (3) an Albumin and IgG removal kit (Amersham Biosciences). They then and MCP (12) did not reveal a mutation in either of these genes

Table 1. Primer sequences and PCR conditions

Annealing Primer Sequence MgCl (mmol) Temperature (°C) 2

Promoter F-tcttccctggaatacagaattga 56.4 1.5 R-ttgctgactatagagtggcattg Exon 1 F-tctctctggatttcagccaaa 56.4 1.5 R-ttgctgactatagagtggcattg Exon 2 F-cttgaagccaccagacaaca 56.4 1.5 R-ggcaacccctgatttgttta Exon 3 F-tcgtcatgatgttcaaagctc 56.4 1.5 R-tgatgcacatagttaattttcttag Exon 4 F-cttgcccaagctgtaactcc 56.4 1.5 R-gcaacgaggcatcaatcat Exon 5 F-cctccataagccaggtttga 58.9 1.5 R-ggcatgctgtgcaaacataa Exon 6 F-tttctgactaaaagaggaaaagcaa 56.4 1.5 R-agttctgcaaatgcccactc Exon 7 F-aataaggtgcaatggttttca 56.4 1.5 R-gctgggttattactaaaatgtggtc Exon 8 F-catgccttggggattttgta 58.9 1.5 R-tccagttttaaaagcaattctcaa Exon 9 F-tccagcctgtcttgtactgg 61.0 1.5 R-tttgtgtggtcactgccatt Exon 10 F-tgcccagaaacagcctaaat 61.0 1.5 R-tttggtctgtggaggacaca Exon 11 F-ttctggggaaatgaaaaagg 56.4 2.0 R-tgcttctctctgagtgctagg Exon 12 F-ccctttcataatcccaatggt 58.9 1.5 R-ctgatgtggtgggaggagat Exon 13a F-agagcccatgctattgctgt 52.5 1.5 R-aaatggaactcttgagagaaaaaga Exon 13b F-tgcctgtaaaggggactctg 61.0 2.0 R-ggcataaactctgtggagacc Exon 13c F-tggccaattattttgactgga 56.4 1.5 R-ctgggatttgaacccaacct J Am Soc Nephrol 16: 2150–2155, 2005 Factor I Deficiency and Atypical HUS 2153

Figure 3. (a) Sequence of patient A. The nucleotide sequence is shown above, and the corresponding amino acid sequence is shown below. There is a single base pair change C.463GϾA, which results in a premature stop codon (W127stop). (b) Wild- Figure 5. (a) Sequence of patient B (cloned PCR product). A type DNA sequence. single base pair deletion (922delC) in exon 7 results in nine amino acid substitutions followed by a premature stop codon. (b) Wild-type DNA sequence (cloned PCR product). in patient A. Western blot of serum from patient A did not show any abnormal bands (Figure 4). Our report confirms the recent findings of Fremeaux-Bacchi et Patient B had a single base pair deletion (922delC) in exon 7, al. (17), who described two patients with heterozygous IF pre- which results in nine amino acid substitutions followed by a mature stop codons associated with half-normal IF levels. They premature stop codon (Figure 5). This change was not identified in also described a patient with a heterozygous missense muta- 100 control subjects. IF (29 mg/L; normal range 38 to 58 mg/L) tion, which results in an amino acid change (D506V) in the and C3 (0.3 g/L; normal range 0.73 to 1.4 g/L) levels were low. C4 serine protease domain, the functional significance of which (0.2 g/L; normal range 0.12 to 0.45/L), and CFH (0.50 g/L; normal remains to be established. range 0.35 to 0.59 g/L) levels were normal. Mutation screening of The first case of IF deficiency was described by Alper et al. CFH (3) and MCP (12) did not reveal a mutation in either of these (21) in 1970. Since then, a total of 31 cases have been described. genes in patient B. Western blot of serum from patient B did not These patients all have had virtual complete deficiency of IF, show any abnormal bands (Figure 4). presumably as a result of mutations in both IF alleles. The clinical manifestations are present from early childhood with Discussion increased susceptibility to recurrent infection with encapsu- We report two HUS patients with IF mutations, both of lated microorganisms (Neisseria meningitidis, S. pneumoniae, and which led to heterozygous premature termination codons. Pos- Haemophilus influenzae). This is secondary to functional C3 defi- sible effects of these changes include nonsense mediated decay ciency as a result of uncontrolled activation of the alternative of the RNA transcript or impaired secretion of the protein, both pathway. C3 opsonizes encapsulated microorganisms, enhancing of which are consistent with the low levels of serum IF ob- phagocytosis (22). The two patients in our study showed no served and the absence of an abnormal band on Western blot. evidence of increased susceptibility to infection. In these previous reports, no clinical phenotype was reported in the majority of family members who were found to have partial IF deficiency. Two patients with complete IF deficiency have been reported to have renal disease. One patient presented with a multisys- tem inflammatory disorder characterized by hepatitis, pneumo- nitis, myositis, and histologic evidence of a microangiopathic vasculitis; he subsequently developed focal segmental glomer- ulosclerosis (23). The other patient had serologic evidence of systemic lupus erythematosus and diffuse proliferative glomerulonephritis on renal biopsy (24). The molecular basis of complete IF deficiency in association Figure 4. Western blot of unreduced IF in human serum with albumin and IgG removed. Five lanes from left to right are with recurrent infection was described in two pedigrees by molecular weight marker, control serum from a single unaf- Vyse et al. (20). In one, a missense mutation resulted in a fected subject, serum from patient B, serum from patient A, and homozygous change (H400L); in the other, the proband was a purified IF (Sigma C5938). The open arrow represents the po- compound heterozygote with H400L on one allele and a donor sition of unreduced IF. splice site change on the other. 2154 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2150–2155, 2005

Both of the patients described here had recurrence of HUS to partial IF deficiency are associated with a predisposition to after transplantation, as did the deceased sibling of patient B. the development of atypical HUS. The description of mutations One of the patients reported by Fremeaux-Bacchi et al. (17) had in IF, CFH, and MCP has established that atypical HUS is a recurrence in two transplants. Thus, all four transplants in disease of complement dysregulation. Understanding the mo- patients with IF mutations reported to date have been lost to lecular mechanisms that are responsible for this disease allows recurrent HUS. This is not unexpected as IF, like CFH, is us now to examine the potential of complement inhibition as a predominantly synthesized by the liver and thus a renal allo- means of therapy. graft will not correct the underlying defect. From individuals reported in the literature and unpublished from our own co- hort, the recurrence rate in those with CFH mutations and/or Acknowledgments CFH deficiency is approximately 80% (2,4,6,7,25–30). This study was supported by grants from the National Kidney Re- search Fund, the Northern Counties Kidney Research Fund, the New- In contrast, the three patients who have MCP mutations and castle Healthcare Charity, the Peel Medical Research Trust, and the have received a transplant have had no recurrence of HUS (12). Robin Davies Trust. E.M. was supported by a Wellcome Trust Vacation MCP is a transmembrane protein, and a renal allograft there- Scholarship. fore should correct the underlying defect. In patients with CFH and MCP mutations, C3 levels may be normal. In patients with asymptomatic partial IF deficiency, C3 References levels have also been reported in some to be normal (31–35). In 1. Richards A, Goodship J, Goodship T: The genetics and our study, one patient had normal C3 levels and the other had pathogenesis of haemolytic uraemic syndrome and throm- low levels. Therefore, a normal C3 does not rule out heterozy- bocytopenic purpura. Curr Opin Nephrol Hypertens 11: 431– gous IF deficiency. This was also seen in the patients who were 436, 2002 reported by Fremeaux-Bacchi et al. (17). 2. Warwicker P, Goodship TH, Donne RL, Pirson Y, Nicholls In both our study and that of Fremeaux-Bacchi et al. (17), A, Ward RM, Turnpenny P, Goodship JA: Genetic studies there is incomplete penetrance. This suggests that IF mutations, into inherited and sporadic hemolytic uremic syndrome. like CFH and MCP, do not directly cause a thrombotic microan- Kidney Int 53: 836–844, 1998 giopathy but rather predispose to it. In this situation, endothe- 3. Richards A, Buddles M, Donne R, Kaplan B, Kirk E, Ven- lial activation secondary to injury is maintained by excessive ning M, Tielemans C, Goodship J, Goodship T: Factor H complement activation. Moreover, genetic variation in comple- mutations in hemolytic uremic syndrome cluster in exons 18–20, a domain important for host cell recognition. Am J ment regulatory genes may modify disease penetrance in pa- Hum Genet 68: 485–490, 2001 tients with IF, CFH, and MCP mutations. Caprioli et al. (7) 4. Caprioli J, Bettinaglio P, Zipfel P, Amadei B, Daina E, reported an association between CFH alleles and HUS, and Gamba S, Skerka C, Marziliano N, Remuzzi G, Noris M: Esparza-Gordillo et al. (36) also recently reported that a specific The molecular basis of familial hemolytic uremic syn- single nucleotide polymorphism haplotype block spanning drome: Mutation analysis of factor H gene reveals a hot MCP is overrepresented in patients with atypical HUS. This spot in short consensus repeat 20. J Am Soc Nephrol 12: may also explain why HUS has not previously been described 297–307, 2001 in other families with IF deficiency. Another possibility is that 5. Perez-Caballero D, Gonzalez-Rubio C, Gallardo M, Vera what we and Fremeaux-Bacchi et al. have reported is an ascer- M, Lopez-Trascasa M, Rodriguez de Cordoba S, Sanchez- tainment artifact. We think that this is unlikely, as neither we Corral P: Clustering of missense mutations in the C-termi- nor Fremeaux-Bacchi et al. have found the same changes in nal region of factor H in atypical hemolytic uremic syndrome. Am J Hum Genet 68: 478–484, 2001 Ͼ400 chromosomal analyses from regionally matched normal 6. Dragon-Durey M-A, Fremeaux-Bacchi V, Loirat C, Blouin control subjects. J, Niaudet P, Deschenes G, Coppo P, Fridman W, Weiss E: This study has important clinical implications. We suggest Heterozygous and homozygous factor H deficiencies asso- that an IF level be measured routinely in all patients who ciated with haemolytic uraemic syndrome or membrano- present with atypical HUS. It is important in patients who are proliferative glomerulonephritis: Report and genetic anal- being considered for transplantation that it be known whether ysis of 16 cases. J Am Soc Nephrol 15: 787–795, 2004 they have a CFH, IF,orMCP mutation so that they can be 7. Caprioli J, Castelletti F, Bucchioni S, Bettinaglio P, Bresin E, informed appropriately of the risks for recurrence. 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Even if the living related donor is known not complement C3b inactivator: Isolation, characterisation, to carry the same mutation, the risk for recurrence in the and demonstration of an absolute requirement for the se- recipient is likely to be as high as observed with cadaveric rum protein beta1H for cleavage of C3b and C4b in solu- donors, a risk that some would consider to be unacceptably tion. J Exp Med 146: 257–270, 1977 high for any transplant. 9. Perkins SJ, Goodship THJ: Molecular modelling of the In conclusion, this study confirms that IF mutations leading C-terminal domains of factor H of human complement. A J Am Soc Nephrol 16: 2150–2155, 2005 Factor I Deficiency and Atypical HUS 2155

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