Mutations in Proteins of the Alternative Pathway of Complement and the Pathogenesis of Atypical Hemolytic Uremic Syndrome

IN TRANSLATION Mutations in Proteins of the Alternative Pathway of Complement and the Pathogenesis of Atypical Hemolytic Uremic Syndrome Cynthia Abarrategui-Garrido,1 Marta Melgosa, MD,2 Antonia Peña-Carrión, MD,2 Elena Goicoechea de Jorge, PhD,3 Santiago Rodríguez de Córdoba, PhD,3 Margarita López-Trascasa, PhD,4 and Pilar Sánchez-Corral, PhD1 Atypical hemolytic uremic syndrome is associated with mutations in the complement proteins factor H, factor I, factor B, C3, or membrane cofactor protein in about 50% of patients. The evolution and prognosis of the disease in patients carrying mutations in factor H is particularly poor, and renal transplantation most often fails because of recurrence of the disease in the graft. The risk of rapid loss of renal function in patients with functional mutations in factor H requires that effective treatment be initiated as soon as possible, but identification of these patients relies on genetic studies that are time consuming. We describe a case in which an in vitro hemolytic assay proved useful for rapidly assessing factor H dysfunction and for testing whether this dysfunction could be corrected with fresh frozen plasma. In the context of this case, we summarize recent advances in understanding the molecular mechanisms contributing to atypical hemolytic uremic syndrome, including descriptions of DNA- and protein-based analysis. We conclude that functional analysis of factor H should help rationalize the plasma treatment of patients with atypical hemolytic uremic syndrome. Am J Kidney Dis 52:171-180. © 2008 by the National Kidney Foundation, Inc. INDEX WORDS: Hemolytic uremic syndrome; complement factor H; hemolytic assays; plasma therapy.

typical hemolytic uremic syndrome (aHUS) has a poor prognosis and most often leads to end-stage renal disease (ESRD). Plasma exchange or infusion usually is used for management of acute episodes and to prevent relapses, with variable results. Mutations in proteins of the alternative pathway of the immune complement system have been found in 50% of patients with aHUS, and the presentation, response to treatment, and outcome of the disease is influenced by the patient’s genotype. Patients with mutations in the plasma complement protein factor H have a very poor prognosis and can benefit from early establishment of plasma treatment, which may prevent the complete loss of renal function.

of 45 mL/min/1.73 m2 (0.75 mL/s/1.73 m2). Microhematuria and non-nephrotic proteinuria were present. HUS was diagnosed, and conservative treatment was initiated. In the following days, the clinical course worsened and the patient was referred to our hospital, where hemodialysis therapy was initiated 25 days after HUS onset (Fig 1). The patient had normal levels of C3, C4, and the complement regulators factor H, factor I, and membrane cofactor protein (MCP). An in vitro factor H–dependent hemolytic assay detected anomalous regulation of the alternative pathway, suggesting dysfunction of factor H. Subsequent genetic analysis showed that the pa-

CASE VIGNETTE An 18-month-old previously healthy girl presented at her local hospital with anorexia, weakness, and abdominal pain. She had received diptheria-tetanus-pertussis vaccine 48 hours before, and erythema infectiosum had been diagnosed the previous week. On admission, she was pale and blood pressure was 110/70 mm Hg. Blood chemistry showed Coombs-negative hemolytic anemia (hemoglobin, 5.2 g/dL [52 g/L]) with thrombocytopenia (platelets, 93 ⫻ 109/L). Serum creatinine level was 1.09 mg/dL (96 ␮mol/ L), corresponding to a glomerular filtration rate

From the 1Research Unit and 2Pediatric Nephrology Unit, Hospital Universitario La Paz; 3Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas; and 4Immunology Unit. Hospital Universitario La Paz, Madrid, Spain. Received June 4, 2007. Accepted in revised form January 2, 2008. Originally published online as doi: 10.1053/j.ajkd.2008.01.026 on April 16, 2008. Address correspondence to Pilar Sánchez-Corral, PhD, Unidad de Investigación, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046-Madrid, Spain. E-mail: [email protected] © 2008 by the National Kidney Foundation, Inc. 0272-6386/08/5201-0023$34.00/0 doi:10.1053/j.ajkd.2008.01.026

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Figure 1. Time course of therapeutic interventions and hematologic parameters in the patient. Asterisks denote red blood cell transfusions, and arrows point to the 4 hemolytic uremic syndrome (HUS) episodes. Periods of plasma therapy (plasma exchange [PE] or infusion [PI]) and supportive treatment to restore renal function (hemodialysis [HD] or peritoneal dialysis [PD]) also are indicated. To convert serum hemoglobin in g/dL to g/L, multiply by 10; creatinine in mg/dL to ␮mol/L, multiply by 88.4.

tient was heterozygous for a factor H missense mutation. Plasma therapy was immediately established, although it was later interrupted because of hemodynamic problems. The patient experienced improvement in renal and hematologic parameters, leading to discontinuation of hemodialysis and plasmapheresis therapy. However, the patient experienced 3 subsequent HUS episodes. Although the patient’s condition stabilized between each episode, renal function never recovered, and a switch from hemodialysis to peritoneal dialysis therapy became necessary because of serious hemodynamic instability. About 6.5 months after the initial HUS episode, the patient experienced acute respiratory insufficiency and died a few hours later.

PATHOGENESIS HUS is a clinical entity defined by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure resulting from endothelial damage in the renal microvasculature.1 In most cases, known as typical HUS, the disease develops after infection caused by enterohemorrhagic

Escherichia coli strains producing toxins that damage the renal endothelium. Patients with typical HUS generally are children, who normally recover after a few weeks of supportive treatment and do not experience relapses of the disease. However, in 5% to 10% of patients with HUS, the initial endothelial damage in the microvasculature is not related to an E coli infection and the prognosis is very poor, with a high percentage of patients developing ESRD, which is sometimes fatal. This atypical form of HUS also appears in adults, often after immunosuppressive or antitumoral treatments, consumption of oral contraceptives, or during the postpartum period. Research work performed in the last 7 years has revealed that approximately 50% of patients with aHUS have mutations in the complement regulatory proteins factor H,2-6 MCP (CD46),7,8 factor I,9,10 or 2 of these proteins simultaneously.11 Some mutations decrease the levels of these proteins, whereas others cause functional defects that affect normal regulation of the alternative pathway. Factor H and MCP polymor-

Factor H in Atypical HUS

phisms associated with the disease have also been described12-14 and are considered to be additional genetic susceptibility factors. Anti– factor H autoantibodies have also been found in a few patients.15 A possible involvement of the factor H–related proteins 1 and 3 (CFHR1 and CFHR3) has been suggested because deficiency of these proteins is more frequent in patients with HUS than control individuals.16 Recently, gainof-function mutations in the complement activating protein factor B have been described and shown to increase alternative pathway activation.17 Mutations in C3 identified in some patients with HUS are expected to have a similar effect.18 These findings show that an improper balance between activation and regulation in the alternative pathway of complement is involved in the pathogenesis of aHUS. Moreover, full expression of the disease in some patients may require the presence of several genetic and/or acquired susceptibility factors. Mutations in the complement regulator factor H are the most frequent and have a very poor prognosis, with most patients developing ESRD after the initial HUS episode or as a consequence of additional relapses, and 80% recurrence in the transplanted kidney.19 These adverse effects of factor H mutations are particularly conspicuous in pediatric cases. In a recent study of 46 children with HUS, 60% of patients with mutations in factor H reached ESRD or died within the first year after disease onset.20 This rapid evolution toward loss of renal function requires that effective treatment be initiated as soon as possible in patients with mutations in factor H, but identification of these patients relies on genetic studies that are time consuming. Factor H is the most important regulator of the alternative pathway of complement in plasma, and its deficiency provokes consumption of the central complement component C3.21 It also is able to control complement activation on cellular surfaces, and this is an important mechanism to protect host cells from autologous complement attack. Factor H is a 150-kDa polypeptide organized into 20 short consensus repeat (SCR) domains, also known as complement control protein modules. SCR1 to SCR4 are the most relevant for factor H function in plasma, whereas SCR16 to SCR20 are specifically involved in the control of complement activation on cellular

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surfaces. The majority of factor H mutations identified in patients with aHUS are located in the SCR16 to SCR20 region and do not decrease the level of the protein.22 In these patients, C3 and C4 levels also are normal or subnormal. Functional studies have revealed that mutations in this region of the factor H molecule decrease its binding to surface-bound C3b and/or autologous endothelial cells,23,24 suggesting anomalous complement activation on the surface of these cells that will contribute to the pathogenic mechanism of HUS. These conclusions have been further supported by an in vitro hemolytic assay using sheep erythrocytes and serum samples from patients with HUS.25 Patients with mutations in the SCR20 domain of factor H showed an anomalous capacity to lyse the sheep erythrocytes, showing that the functional alterations associated with these factor H mutations are relevant for appropiate control of the alternative pathway of complement on cellular surfaces. The patient described in this report had normal levels of factor H and the other complement components associated with HUS, but her serum was capable of lysing sheep erythrocytes by means of the alternative pathway, as previously reported for patients with HUS with mutations in the SCR20 domain of factor H.25,26 Moreover, addition of normal factor H prevented this anomalous lysis, providing evidence that the underlying defect was either directly related to a factor H dysfunction or, alternatively, overlapped with normal factor H regulatory activity. This finding suggested the existence of a mutation in her factor H gene and provided a rationale for the prompt initiation of plasma therapy.

RECENT ADVANCES Complement Studies Genetic or acquired disorders of complement regulation define a subgroup of patients with HUS with known cause, classified as type I.3 patients.27 A comprehensive diagnostic approach to identifying those patients has recently been suggested.28 This protocol includes quantification of the plasma proteins factor H, factor I, factor B, C3, CFHR1, and CFHR3; analysis of MCP expression on the surface of peripheralblood leukocytes; quantification of antifactor H autoantibodies; and analysis of factor H, factor I,

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and MCP genes. To determine whether the patient with aHUS described in this report had a complement disorder, we essentially followed this protocol, but before performing the genetic analyses, we carried out an in vitro hemolytic assay that we previously showed to be useful for detecting factor H dysfunctions.25 As we show next, introduction of this assay in the diagnostic approach allowed prompt functional screening of a factor H mutation in the patient. Quantification of Complement Components Serum and EDTA-plasma samples obtained from the patient and her parents were either immediately analyzed or aliquoted and frozen at ⫺80°C until used. C3 and C4 levels were determined in freshly drawn serum samples by using standard nephelometry. Levels of the plasma regulators factor H, factor I, and factor B were established by means of sandwich enzymelinked immunosorbent assay, and expression of the membrane-bound regulators MCP (CD46) and decay accelerating factor (CD55) in peripheral-blood leukocytes was determined by means of flow cytometry.11 The presence of antifactor H autoantibodies was tested by means of an enzymelinked immunosorbent assay identical to that previously described.15 As listed in Table 1, the patient showed normal levels of complement components C3, C4, factor H, factor I, and factor B in plasma, and expression of the membrane regulator MCP also was normal. No factor H autoantibodies were found, and the banding pattern of factor H, CFHR1, and CFHR3 by means of Western blot was similar to that of control individuals (not shown).

Hemolytic Assay for Factor H The sheep erythrocyte lysis assay has previously been described.25 It is performed under conditions specific to activation of the alternative pathway of the complement system. When sheep erythrocytes are added to human serum, a small number of C3b molecules spontaneously generated through the alternative pathway are deposited on the surface of sheep erythrocytes. In normal human serum, factor H will then bind these C3b molecules through its N-terminal domains (SCR1 to SCR4), and it will also bind to polyanionic molecules on the surface of the sheep erythrocytes through its C-terminal domains (SCR19 to SCR20). These 2 kinds of interactions result in efficient protection of the sheep red blood cells against complement attack, and no lysis will be observed (Fig 2A). We showed in our previous report that serum samples from 6 patients with aHUS with mutations in the SCR20 domain of factor H were able to lyse the sheep erythrocytes, and we interpreted this finding as the consequence of decreased binding of the mutated factor H to the cell surface. The assay has also been used for functional studies in patients with other mutations in the SCR20 of factor H26,29 and in a patient with autoantibodies against the C-terminal region of factor H.30 When we performed the hemolytic assay in a serum sample from the patient with aHUS described here that was drawn upon admittance to the hospital, we observed that this serum was able to lyse sheep erythrocytes in a dose-dependent manner (Fig 2B), and this anomalous lysis could be prevented by the addition of exogenous purified factor H. We therefore suspected the existence of

Table 1. Complement Profile in the Patient and Her Parents

Patient Father Mother

C3 (77-210 mg/dL)

C4 (14-47 mg/dL)

Factor H (12-56 mg/dL)

Factor I (75%-115%)

Factor B (7.5-28 mg/dL)

MCP (91%-109%)

Lysis (1%-5%)

85.5 172 104

34.3 25 27.7

33.4 41.7 29.7

109 128 139

14.6 14.2 13.9

90 ND ND

22 2 2

Note: For each variable, the normal range of variation in controls is shown in parentheses. Factor I levels were referred to a reference serum, and MCP values, to a series of 17 control samples. For the hemolytic assay, 20 ␮L of serum samples from the patient or her parents were mixed with sheep erythrocytes in a final volume of 200 ␮L, and the degree of lysis was determined after 30 minutes of incubation at 37°C. Results expressed as percentage of sheep erythrocytes lysed; the normal range was obtained with 16 control samples. To convert C3, C4, factor H, and factor B in mg/dL to g/L, multiply by 0.01. Abbreviations: MCP, membrane cofactor protein; ND, not determined.

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Figure 2. Anomalous hemolytic activity in the patient=s serum. (A) Hemolytic activity observed after incubation of sheep erythrocytes with a control human serum, or (B) serum sample from the patient drawn before the beginning of plasma therapy. The percentage of sheep erythrocytes lysed is shown in the Y axis. Inset: hemolysis observed with 15 ␮L of the patient=s serum in the presence or absence of increasing amounts of factor H purified from control sera. A schematic representation of sheep erythrocytes, C3b, and factor H is included in the right part of the figure to illustrate the molecular basis for the results observed in A and B. White circles indicate polyanionic molecules on the erythrocyte surface.

a mutation in the patient’s factor H and decided to begin plasma exchange. We previously performed the hemolytic assay using serum samples from all patients with aHUS in our registry, which currently includes 14 patients with mutations in factor H; 22 patients with mutations in MCP, factor I, or factor B; and 4 patients with factor H autoantibodies. Anomalous lysis of sheep erythrocytes was observed in the 10 patients with mutations in factor H that cause functional defects in factor H and the 4 patients with factor H autoantibodies, but not in the 4 patients with mutations causing decreased levels of factor H or the 22 patients with mutations in other complement components. In all cases, the anomalous lysis was reverted by the addition of factor H purified from human serum. According to these results, we believe the hemolytic assay detects factor H mutations that do not provoke factor H deficiency, but that have strong functional consequences on its ability to control complement activation on cellular surfaces, and these mutations seem to be localized in the C-terminal domains of factor H. The assay might not be sensitive enough to detect mutations outside this region of factor H. Factor H dysfunctions provoked by the presence of factor H auto-

antibodies also can be detected by using the hemolytic assay. As stated, the assay cannot be used with serum samples with very low C3 and/or C4 levels because they will not be able to generate lysis and could be interpreted as falsenegative results. It also is possible that defective C3 activation caused by low levels of factor B could give a false-negative result, but factor B deficiency has not been described, so it must be extremely infrequent. Genetic Studies of Complement Genes Genomic DNA from the patient and her parents was obtained from peripheral-blood leukocytes and used to amplify all exons of the factor H (CFH), MCP (MCP), factor I (CFI), and factor B (CFB) genes by means of polymerase chain reaction, as previously described.11,17 Direct sequencing of polymerase chain reaction products was performed using the BigDye Terminator V1.1 sequencing kit (Applied Biosystems, Foster City, CA) on an ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems). Sequence analysis showed the presence of a heterozygous mutation in exon 22 of the CFH gene, consisting of an A to G substitution at nucleotide 3425 of the complementary DNA

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Figure 3. Genetic analysis of factor H in the patient and her parents. The patient has a missense mutation inherited from her mother in exon 22 of the CFH gene (c.3425G¡A) that substitutes a cysteine residue in short consensus repeat (SCR) 19 for a tyrosine. The figure also illustrates inheritance of the CFH haplotypes H1 and H3 from the mother and father, respectively.

sequence (c.3425A¡G; nucleotide numbering is based on the translation start site: A in ATG is ⫹1). This change would be expected to lead to a tyrosine to cysteine substitution at amino acid 1142 (p.Tyr1142Cys), which is found in the SCR19 domain of factor H (Fig 3). A mutation affecting this same amino acid was found in another patient with HUS, although in that case, the change generated an aspartic residue.5 No mutations were found in the MCP, CFI, and CFB genes. Sequencing and genotyping of CFH showed that the patient was also a heterozygote for the CFH haplotypes H1 and H3. CFH haplotype H1 (c.⫺332C, c.184G, c.1204C, c.2016A, and c.2808G) and CFH haplotype H3 (c.⫺332T, c.184G, c.1204T, c.2016G, and c.2808T) are defined by specific combinations of singlenucleotide polymorphisms, as described.31 CFH haplotype H3 has been shown to be an important aHUS-associated at-risk allele in several different independent aHUS patient cohorts.12-14 As also shown in Fig 3, the patient inherited the mutated CFH allele from her mother and the CFH at-risk allele from her father. We hypothesize that the concurrence of these 2 genetic susceptibility factors was critical for the development of the disease in the patient. Detection of the Mutant Factor H Polypeptide in Plasma The normal factor H levels found in the patient=s serum suggested that the p.Tyr1142Cys

mutation in the SCR19 domain did not affect the synthesis or secretion of the mutated factor H polypeptide. To assess that hypothesis, we purified factor H from a blood sample drawn before the beginning of plasma therapy by using affinity chromatography and polyacrylamide gel electrophoresis. An immunosorbent was prepared by covalently coupling rabbit polyclonal anti–factor H antibodies generated in the laboratory to cyanogen bromide-activated Sepharose beads (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), following the manufacturer’s instructions. A 25-␮L plasma sample from the patient, drawn before initiating plasma treatment, was incubated with 50-␮L beads for 1 hour at room temperature. The beads were centrifuged and the supernatant was discarded. After several washes in phosphate-buffered saline buffer, proteins bound to the immunosorbent were eluted in sodium dodecyl sulfate sample buffer and loaded onto 8% polyacrylamide gels. Proteins were visualized after electrophoresis with Coomassie Colloidal staining (GE Healthcare), and several spots from the band corresponding to factor H were picked and trypsin digested for mass spectrometry analysis. As shown in Fig 4, 2 different peptides corresponding to amino acids 389 to 405 of the factor H molecule were identified. Peptide 1 (m/z 2031.8890) contains histidine at position 402 and therefore is derived from the maternal allele that also carries the mutated cys-

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Figure 4. Detection of the mutant factor H polypeptide (containing cysteine at amino acid 1142) in the patient=s plasma. (A) Electrophoretic separation of the proteins eluted from the Sepharose anti– human factor H immunosorbent incubated with plasma from the patient. Proteins were visualized by using Coomassie staining, and several spots from the band corresponding to factor H were picked, digested with trypsin, and analyzed further by using mass spectrometry. Positions of molecular-weight markers are shown at the left. (B) Matrix-assisted laser desorption-ionization (MALDI) time-of-flight (TOF) spectrum of the tryptic digest of factor H isolated from the patient. Two peptides potentially containing histidine (m/z 2031.8890) or tyrosine (m/z 2057.8933) at position 402 were fragmented further and analyzed to determine their amino acid sequence. Peptides from both the paternal and maternal alleles were detected in factor H isolated from the patient=s plasma. (C) Amino acid differences between factor H polypeptides of paternal and maternal origin, deduced from genetic analyses. The paternal polypeptide has tyrosine (Y) at position 402, whereas the maternal polypeptide has histidine (H) and also contains the mutated cysteine (C) residue at position 1142. Identification of factor H tryptic peptides was performed in the Proteomics Unit of the UCM-PCM (Universidad Complutense, Madrid, Spain) by using MALDI-TOF/TOF spectrometry (4700 Proteomics Analyzer; PerSeptives Biosystems, Framingham, MA) and ESI-IT (Electrospray Ionization-Ionic Trap spectrometer; Finnigan-LTQ, Thermo Electron Corp, Waltham, MA). Abbreviation: SCR, short consensus repeat.

teine residue; peptide 2 (m/z 2057.8933) contains tyrosine at position 402 and is derived from the paternal allele. Therefore, the 2 factor H alleles in the patient were expressed, and their products were secreted into plasma. It seems reasonable to attribute the anomalous hemolytic capacity of the patient’s serum to the p.Tyr1142Cys mutation in the SCR19 domain of factor H; the mutated factor H would have decreased ability to bind to sheep erythrocytes and protect them from complement attack. Nonetheless, the p.Tyr1142Cys mutation was also present in the patient’s maternal allele, but no anomalous lysis was observed with the mother’s serum, suggesting that additional factors in the patient contribute to this phenotype. In this context, it is relevant to indicate that the patient also inherited an important aHUS-associated at-risk CFH allele from her father, CFH haplotype H3. Although the functional consequences of this allele have not yet been determined, it is possible that

all factor H molecules in the plasma of the patient had decreased complement regulatory function on cellular surfaces. It also is possible that in the mother’s serum, the reduced function of the mutated factor H is compensated for by higher activity of other complement regulators. Alternatively, the effect of mutation p.Tyr1142Cys might not become evident until there is a situation (eg, endothelial damage) requiring effective regulation of complement activation by factor H. Evaluation of Response to Plasma Therapy Patients with factor H–associated HUS have been treated with fresh frozen plasma (FFP) infusion or exchange. Clinical results are variable, but normally there is a better hematologic than renal response. In patients with complete factor H deficiency, FFP alone has proven useful in the acute phase and to prevent relapses,32,33 although its efficacy in long-term treatment is uncertain.34 FFP infusions also were successful

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in 2 patients with half-normal levels of factor H,35,36 whereas a protocol of plasma exchange with FFP infusion worked properly in 2 other patients.37,38 The requirement for plasma exchange seems to be stronger in patients with mutations in factor H that do not change the protein level, but presumably alter its complement regulatory function.39,40 In our patient with HUS, early treatment with plasma exchange proved to be effective at improving renal and hematologic function, but when it was reinitiated after recurrence of the disease, hemodynamic problems in the patient forced us to interrupt it when renal insufficiency remained unchanged. When the patient experienced the third HUS episode, with severe hypertension provoking cardiac insufficiency, a protocol of FFP infusion (up to 20 mL/kg) combined with hemodialysis could be established. This treatment restored hemoglobin and platelet levels to normal, but was unable to recover renal function, and again, treatment had to be interrupted because of hemodynamic instability in the patient. To evaluate the effect of plasma treatment on the patient’s complement, we determined C3, C4, and factor H concentrations in serum samples obtained during the 6-month period that the patient remained in our hospital. As shown in Fig 5, levels of the central complement components C3 and C4 remained stable, with some small fluctuations that cannot be ascribed to any of the therapies applied (plasma exchange or plasma infusion). Also, antigenic levels of factor H were not greatly affected by plasma infusion, as reported in another patient with HUS who was administered several series of plasma infusions.32 When we performed the hemolytic assay in these serum samples, we observed that the anomalous lysis of sheep erythrocytes attributed to the factor H dysfunction was not observed during the plasma infusion period, suggesting that the factor H administered with the plasma was capable of preventing this lysis. These results provide the first experimental evidence that factor H dysfunction can be corrected by plasma therapy. It is tempting to speculate that administration of small volumes of purified factor H could have prevented HUS relapses and further deterioration of the kidneys in the patient.

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Figure 5. Time course of complement components and hemolytic activity. Levels of C3, C4, and factor H and degree of hemolytic activity observed in serum samples from the patient drawn at different dates. Lysis denotes anomalous complement regulation. Plasma exchange (PE) and plasma infusion (PI) periods are indicated by boxes. To convert C3, C4, and factor H in mg/dL to g/L, multiply by 0.01. Abbreviation: HUS, hemolytic uremic syndrome.

SUMMARY Patients with aHUS carrying genetic disorders in the alternative pathway of the complement system have a poor prognosis. Prompt initiation of plasma therapy can be beneficial for the management of acute HUS episodes in most patients, particularly in patients with mutations in plasma protein factor H. There are no clear guidelines to determine whether the plasma protocol is effective in every patient, and side effects of plasma therapy require stopping the treatment in some patients. In this report, we describe a pediatric patient with HUS carrying a factor H mutation of maternal origin and an HUS-associated at-risk factor H allele inherited from her father. Factor H polypeptides corresponding to the 2 alleles were present in the patient’s plasma, confirmed by using mass spectrometry. Anomalous complement activity attributed to factor H was detected early in the patient=s serum with the use of an in vitro hemolytic assay, allowing prompt initiation of plasma therapy. The factor H alteration was

Factor H in Atypical HUS

corrected by infusion of FFP, but plasma therapy was not well tolerated by the patient, thus limiting its potential benefits. We believe that in patients with HUS with normal factor H levels, this hemolytic assay can be used for the screening of factor H dysfunction, the most frequent situation in factor H–associated HUS. The assay allows prompt identification of patients who will benefit from early establishment of plasma therapy. It provides another tool for monitoring the efficacy of treatment during the acute phase of the disease and also can help establish a long-term therapy regimen to prevent further relapses or avoid recurrence of the disease in the transplanted kidney. The patient we describe here also illustrates the necessity of getting preparations of factor H that can be administered to patients with HUS with factor H deficiency or dysfunction and that will avoid the undesirable effects of plasma therapy. We propose that it is likely that small amounts of factor H will be enough to correct the alteration, and the in vitro hemolytic assay could help determine the dosage in individual patients.

ACKNOWLEDGEMENTS We appreciate the technical assistance and advice of Lola Gutiérrez, from the Proteomics facility of the Parque Científico de Madrid-UCM. The study has the approval of the Ethics Committee from the Hospital Universitario La Paz. Written informed consent was obtained from the patient=s parents. Support: This work was funded by the Spanish Ministerio de Sanidad y Consumo (grants FIS 03/0621 and 06/0625 to P.S.C.) and the Spanish Ministerio de Educación y Cultura (grant SAF2005-00913 to S.R.C.). Financial Disclosure: None.

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