Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome

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Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome Maria E. Bernabéu-Herrero a,d , Miguel Jiménez-Alcázar a,1 , Jaouad Anter c,d , Sheila Pinto c,d , Daniel Sánchez Chinchilla c,d , Sofía Garrido b,d , Margarita López-Trascasa b,d , Santiago Rodríguez de Córdoba c,d , Pilar Sánchez-Corral a,d,∗ a

Unidad de Investigación, Hospital Universitario La Paz-IdiPAZ, Paseo de la Castellana 261, 28046 Madrid, Spain Unidad de Inmunología, Hospital Universitario La Paz-IdiPAZ, Paseo de la Castellana 261, 28046 Madrid, Spain c Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain d Ciber de Enfermedades Raras (CIBERER), Madrid, Spain b

a r t i c l e

i n f o

Article history: Received 20 April 2015 Received in revised form 5 June 2015 Accepted 10 June 2015 Available online xxx Keywords: Complement Factor H Factor H-related protein 1 Factor H-related protein 3 Atypical hemolytic uremic syndrome Haplotypes

a b s t r a c t Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy affecting the renal microvasculature and is associated with complement dysregulation caused by mutations or autoantibodies. Disease penetrance and severity is modulated by inheritance of “risk” polymorphisms in the complement genes MCP, CFH and CFHR1. We describe the prevalence of mutations, the frequency of risk polymorphisms and the occurrence of anti-FH autoantibodies in a Spanish aHUS cohort (n = 367). We also report the identification of a polymorphism in CFHR3 (c.721C>T; rs379370) that is associated with increased risk of aHUS (OR = 1.78; CI 1.22–2.59; p = 0.002), and is most frequently included in an extended risk haplotype spanning the CFH-CFHR3-CFHR1 genes. This extended haplotype integrates polymorphisms in the promoter region of CFH and CFHR3, and is associated with poorer evolution of renal function and decreased FH levels. The CFH-CFHR3-CFHR1 aHUS-risk haplotype seems to be the same as was previously associated with protection against meningococcal infections, suggesting that the genetic variability in this region is limited to a few extended haplotypes, each with opposite effects in various human diseases. These results suggest that the combination of quantitative and qualitative variations in the complement proteins encoded by CFH, CFHR3 and CFHR1 genes is key for the association of these haplotypes with disease. © 2015 Elsevier Ltd. All rights reserved.

Abbreviations: aHUS, atypical hemolytic uremic syndrome; AMD, age-related macular degeneration; ANOVA, analysis of variance; C3, complement component 3; C3, C3 gene; C3G, C3-glomerulopathy; C4, complement component 4; C4, C4 gene; CGH, comparative genomic hybridization; CFH, complement factor H gene; CFI, complement factor I gene; CFHR1, complement factor H-related 1 gene; CFHR3, complement factor H-related 3 gene; CI, confidence interval; DGKE, diacylglycerolkinase epsilon gene; ESRD, end-stage renal disease; FH, factor H; fHbp, factor H-binding protein; FHR, factor H-related; FHR-1, factor H-related protein 1; FHR-2, factor H-related protein 2; FHR-3, factor H-related protein 3; FHR-4, factor Hrelated protein 4; FHR-5, factor H-related protein 5; FI, factor I; HRP, horseradish peroxidase; HW, Hardy–Weinberg; MCP, membrane cofactor protein; MLPA, multiplex ligation-dependent probe amplification; OR, odds ratio; PBLs, peripheral-blood leukocytes; PCR, polymerase chain reaction; RCA, regulators of complement activation; SCR, short consensus repeat; SNP, single nucleotide polymorphism; STEC, Shiga toxin-producing E. coli; Stx, Shiga toxin; THBD, thrombomodulin gene; tHUS, typical hemolytic uremic syndrome; UTR, untranslated region. ∗ Corresponding author at: Unidad de Investigación, Hospital Universitario La PazIdiPAZ, Madrid, Spain. Tel.: +34 912071026. E-mail address: [email protected] (P. Sánchez-Corral). 1 Present address: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany.

1. Introduction Hemolytic uremic syndrome (HUS) is a rare disease clinically defined by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. Most cases result from infections with Shiga toxin-producing bacteria, particularly Escherichia coli O157:H7, and are accordingly referred to as STEC-HUS, or Stx-HUS. This presentation primarily affects children and generally has a good prognosis, with full hematological and renal recovery within a few weeks. In approximately 5–10% of cases, however, HUS is not directly caused by a bacterial toxin. This atypical form (aHUS) presents in children and adults and has a poor prognosis, with a mortality/end stage renal disease (ESRD) rate of 10–25% in acute episodes, and approximately 50% of the surviving patients developing ESRD in the long term (Noris and Remuzzi, 2009; Loirat and Frémeaux-Bacchi, 2011).

http://dx.doi.org/10.1016/j.molimm.2015.06.021 0161-5890/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Bernabéu-Herrero, M.E., et al., Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.06.021

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aHUS is a multifactorial disease in which both environmental and genetic factors concur. Infections, immunosuppressants, antitumoral drugs, oral contraceptives and pregnancy are thought to initiate the pathogenic cascade that can lead to aHUS in genetically susceptible individuals. Approximately 50% of patients with aHUS have mutations or autoantibodies in the alternative pathway of the complement system that amplify the initial endothelial damage and favor HUS development (Sánchez-Corral and Melgosa, 2010; Kavanagh et al., 2013). Thus, single or combined mutations in the complement genes complement factor H (CFH), membrane cofactor protein (MCP), complement factor I (CFI), complement component 3 (C3) and complement factor B (CFB) have been found in 44% of 795 aHUS patients from 4 European cohorts (Bresin et al., 2013). Other genes that can be mutated in some patients are thrombomodulin (THBD) (Delvaeye et al., 2009), which is a cofactor for thrombin, and diacylglycerolkinase epsilon (DGKE) (Lemaire et al., 2013), which is an enzyme that catalyzes the conversion of diacylglycerol to phosphatidic acid. On the other hand, anti-factor H (anti-FH) autoantibodies have been observed in 6–11% of pediatric cases and in a few adult cases (Dragon-Durey et al., 2010). Approximately 90% of these patients are homozygous for the genomic deletion of Complement CFHR1 and CFHR3 genes, coding for the FHRelated (FHR) proteins FHR-1 and FHR-3, respectively (Zipfel et al., 2007; Józsi et al., 2008; Dragon-Durey et al., 2009; AbarrateguiGarrido et al., 2009; Moore et al., 2010; Noris et al., 2010). Complement mutations in patients with aHUS generally present as heterozygous, with approximately 50% disease penetrance in mutation carriers. Genetic variants in CFH (Caprioli et al., 2003), MCP (Esparza-Gordillo et al., 2005) and CFHR1 (Abarrategui-Garrido et al., 2009) are associated with a higher risk of aHUS, suggesting they are genetic co-predisposing factors. In fact, the CFHtgtggt haplotype (known as CFH(H3), Hageman et al., 2005) and the MCPggaac haplotype have been shown to increase aHUS penetrance in complement mutation carriers (Bresin et al., 2013; Esparza-Gordillo et al., 2005; Sansbury et al., 2014). The effect of the CFHR1*B allele on aHUS penetrance has not been properly addressed. The CFH, CFHR1 and MCP genes are located within the regulator of complement activation (RCA) gene cluster on human chromosome 1q32 (ReyCampos et al., 1988). CFH and CFHR1 are separated by only 72.25 kb, so there is the possibility that the aHUS risk variants CFH(H3) and CFHR1*B are in linkage disequilibrium. Along these lines, a recent report on a large family with many aHUS cases revealed that all chromosomes with the CFH(H3) haplotype also carried the CFHR1*B allele (Sansbury et al., 2014). We have analyzed complement mutations, anti-FH autoantibodies, and CFH, MCP and CFHR1 haplotypes in a Spanish cohort of aHUS patients (n = 367) to determine their frequency and to establish potential genotype-phenotype relationships with demographic and clinical data. We have also identified another aHUS risk variant in the CFHR3 gene, which confirms the presence of an extended CFH-CFHR3-CFHR1 haplotype conferring increased genetic susceptibility to aHUS. A comprehensive genetic and immunological analysis of aHUS patients, and an appropriate understanding of the clinical consequences of the diseaseassociated genetic variations is becoming essential to anticipate patient evolution and individualize therapeutic strategies.

2. Patients, materials and methods 2.1. Patients and controls Blood samples from atypical HUS (aHUS, n = 367), typical HUS (tHUS, n = 43) and C3 glomerulopathy (C3G, n = 32) patients were centrifuged to obtain serum and EDTA-plasma and were stored at −80 ◦ C until used. Peripheral-blood leukocytes (PBLs) were used to

prepare genomic DNA by standard procedures. Blood samples from patients with aHUS were collected at HUS onset and/or at relapses. All the patients or their relatives gave written informed consent, as approved by the ethical committees from University Hospital “La Paz” or the Biological Research Center. Serum, EDTA-plasma and DNA were also obtained from a total of 92 healthy Spanish adult volunteers as controls. 2.2. Protein studies Serum or plasma samples from the 367 patients with aHUS were analyzed by Western blot with various sets of polyclonal antibodies recognizing FH and the FHR proteins (Abarrategui-Garrido et al., 2009) to identify homozygous FH/FHR deficiencies or abnormal bands. These samples were also checked for the presence of circulating anti-factor H autoantibodies by using the original ELISA test (Dragon-Durey et al., 2005). Levels of FH in plasma samples were measured by a sandwich ELISA, using “in house” polyclonal and monoclonal (moAb214) antibodies. The epitope recognized by moAb214 was localized to SCRs 10–11 of FH by Western-blot, using recombinant fragments of FH expressed in Pichia pastoris; this monoclonal antibody does not cross react with any of the FHRs. Plates with 96 wells were coated with 100 ␮L of the “in house” polyclonal rabbit anti-human FH antibody diluted in 0.1 M NaHCO3 , pH 9.5, and incubated at 4 ◦ C overnight. The plates were washed and blocked with 50 mM Tris, pH 7.4, 150 mM NaCl, 0.2% Tween 20 and 1% BSA for 1 h at room temperature. Two serial dilutions (1:3000, 1:6000) of each plasma sample were added, and incubated for 1 h. FH binding was then detected with moAb214 and HRP-conjugated goat anti-mouse IgG antibody (DAKO). Upon addition of the peroxidase substrate ophenylene-diamine (Kem-En-Tec Diagnostics), a colored reaction was developed; the reaction was stopped with 0.1 M H2 SO4 , and the optical density read at 492 nm. The concentration of plasma FH was estimated by comparison with reference plasma containing a known FH amount. 2.3. Genetic studies Studies to identify mutations and risk variants were performed on the patients with aHUS. Each exon of the CFH (Pérez-Caballero et al., 2001), MCP (Richards et al., 2003), CFI (Fremeaux-Bacchi et al., 2004), CFB (Goicoechea de Jorge et al., 2007), C3 (MartínezBarricarte et al., 2012), THBD (Delvaeye et al., 2009) and DGKE (Sánchez Chinchilla et al., 2014) genes was amplified from genomic DNA using specific primers derived from the 5 and 3 intronic sequences as described. Polymerase chain reaction (PCR) fragments were sequenced to identify mutations and to determine the frequency of the CFH(H3) and MCPggaac aHUS-risk haplotypes. The CFHR1*B allele, conferring increasing susceptibility to aHUS, was identified by PCR generation and direct sequencing of CFHR1 exon 6 (Abarrategui-Garrido et al., 2009). The presence of the common CFHR3-CFHR1 allele, as well as copy number variations in the CFH-CFHR1 to CFHR5 genomic region were analyzed by Multiplex ligation-dependent probe amplification (MLPA), using the P236 A1 ARMD mix 1 from MRC-Holland (Amsterdam, Netherlands). Copy number variations of the CFH and CFHRs genes were also analyzed in some cases, using a recently developed, in-house CGH microarray (Tortajada et al., 2013). CFHR3 exon 5 was generated from genomic DNA by using forward (5 -TTGAAAATGCAGATGTCTTCC-3 ) and reverse (5 -GAACTCCTGACCTCATGG-3 ) PCR primers and FideliTaq DNA polymerase (USB). PCR conditions were as follows: 94 ◦ C for 5 min; 35 cycles (94 ◦ C/30 s, 56 ◦ C/15 s, 68 ◦ C/10 s); 68 ◦ C for 7 min. Excess primers and deoxyribonucleotides (dNTPs) were digested with Exonuclease I and shrimp alkaline phosphatase (USB). Direct

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sequencing of the PCR products was performed with BigDye Terminator v1.1 (Applied Biosystems) on an ABI Prism 3100-Avant Genetic Analyzer.

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treatment, transplantation or pregnancy/postpartum condition preceding HUS onset was described, and these patients were considered as secondary HUS cases (Fremeaux-Bacchi et al., 2013). Most patients from our cohort are European (305 from Spain and 21 from other countries), 21 are North African (Morocco, Tunisia, Algeria, Mauritania, Libya, Nigeria and Senegal), 19 are American (13 from Central or South America, and 6 from the USA) and 1 patient is from Pakistan. Our aHUS cohort includes 124 pediatric cases (i.e. below 18 years at disease onset) and 221 adult cases; age at HUS onset was not available for 22 additional patients. A familial history of aHUS was documented in 47 patients. C3, C4, FH and FI plasma levels, as well as MCP expression in PBLs, were determined in blood samples from each patient. These samples were also screened for the presence of anti-FH autoantibodies by ELISA, and the integrity of the FH and FHR proteins was analyzed by Western blot. Anti-FH autoantibodies were detected in 14 patients, all but one children (1–8 years) at disease onset (Table 1). This detection represents 10.5% (13/124) of our pediatric cases. Most (11/14) of our patients with detected anti-FH autoantibodies carry a complete deficiency of FHR-1, which confirms the strong association between this deficiency and the generation of anti-FH antibodies (Józsi et al., 2008; Dragon-Durey et al., 2009). Three patients also showed mutations in CFH, MCP or THBD, as already observed in other anti-FH-positive patients (Moore et al., 2010). Deficiencies in FHR proteins were observed in 9.8% of the patients with aHUS (36/367). The most frequent situation was the combined deficiency of FHR-1 and FHR-3 (29 patients), followed by single FHR-3 deficiency (4 patients), single FHR-1 deficiency (1 patient), single FHR-4 deficiency (1 patient), and combined FHR-1 and FHR-4 deficiency (1 patient). To date, we have not observed isolated deficiencies of FHR-3 or FHR-4 in the control subjects. The aHUS patients were screened for mutations and polymorphisms in the complement genes CFH, MCP, CFI, CFB and C3; the frequency of single or combined mutations in these 5 genes is 33.04% (13.35% CFH, 6.85% MCP, 5.34% CFI, 3.27% combined mutations, 2.68% C3, and 1.48% CFB). Mutations in THBD and/or DGKE

2.4. Statistical analyses A chi-squared test was used to ascertain Hardy–Weinberg (HW) equilibrium in genotypes corresponding to the following polymorphisms: CFH (rs3753394, rs800292, rs1061170, rs3753396, rs1410996, rs1065489), CFHR3 (rs138675433) and CFHR1 (rs4230, rs414628). The allele frequencies of these SNPs in the controls and the patients were compared using a chi-squared test of association; odds ratio (OR) and 95% confidence interval (CI) were also calculated, and statistical significance was set at p < 0.05. To assess the degree of SNP linkage, Pearson’s r was calculated in the control and patient populations. CFH-CFHR3-CFHR1 haplotype frequencies in both groups were estimated using the expectation maximization algorithm implemented in the R software (haplo.stats package), and compared by using a Pearson chi-squared test of association. The frequencies of the CFH(H3)-CFHR3* B-CFHR1* B haplotype according to sex, age at onset, and evolution of renal function were also compared using a Pearson chi-squared test. The levels of plasma FH were compared taking into account the number of CFH(H3)-CFHR3* B-CFHR1* B haplotype copies, by using a one-way analysis of variance (ANOVA) test, followed by Tukey’s post hoc test. 3. Results 3.1. Prevalence of mutations, risk variants and anti-FH antibodies in the Spanish aHUS cohort Between 1999 and 2014, we collected blood samples and clinical data from 367 patients (195 females and 172 males) who had been diagnosed with aHUS according to the clinical criteria of acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia. In about 25% of patients a pharmacological

Table 1 Frequency of genetic susceptibility factors and anti-FH antibodies in the Spanish aHUS cohort. Females (n = 195)

AutoAbs With FHR-1 deficiency Without FHR-1 deficiency Mutations Complement genes CFH MCP CFI CFB C3 Combined Mutations THBDa Mutations DGKEb MCPggaac HET HOM CFH(H3) HET HOM CFHR1*B HET HOM

Males (n = 172)

Whole cohort (n = 367)

Children (n = 56)

Adults (n = 127)

Age unknown (n = 12)

Children (n = 68)

Adults (n = 94)

Age unknown (n = 10)

Total/ analyzed

Frequency (%)

4 4 0 17 5 5 1 1 4 2 4 2 25 14 11 25 17 8 33 21 12

1 0 1 37 18 4 7 1 3 4 1 0 84 52 32 69 46 23 78 43 35

0 0 0 4 3 1 0 0 0 0 0 0 7 6 1 5 4 1 6 4 2

9 7 2 23 8 11 1 2 0 1 2 2 43 34 9 27 24 3 41 30 11

0 0 0 28 10 2 8 1 2 4 1 0 54 35 19 47 40 7 58 36 22

0 0 0 2 1 0 1 0 0 0 0 0 3 3 0 3 3 0 3 3 0

14/367 11 3 111/336 45 23 18 5 9 11 8/227 4/98 216/346 144 72 176/349 134 42 219/319 137 82

3.81 2.99 0.82 33.04 13.35 6.85 5.34 1.48 2.68 3.27 3.52 4.08 62.43 41.62 20.81 50.42 38.4 12.03 68.66 42.95 25.71

Number of patients with anti-FH autoantibodies, mutations and risk haplotypes (MCP, CFH, CFHR1) in the whole cohort. The distribution according to sex (female/male) and age at onset (children, adults, unknown age) is also shown. a 3 of these patients also have mutations in CFH or CFB. b 3 of these patients also have mutations in C3 or THBD.

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Table 2 Mutation frequency in primary and secondary aHUS cases. Number of patients (percentage) Whole cohort Primary aHUS Secondary aHUS Post-transplantation Drugs Pregnancy/postpartum

367 (100%) 273 (74.39%) 94 (25.61%) 47 (12.81%) 30 (8.17%) 17 (4.63%)

Mutation frequency within each group 33.04% 37.35%a 20.69%a 4.76%b 41.38%b 25%b

Number of primary and secondary aHUS cases in the Spanish aHUS cohort; secondary cases are further subgrouped according to the triggering/underlying condition. The percentage of mutated patients within each group or subgroup is also shown. a Differences in mutation frequency were statistically significant (Fisher’s exact test, p = 0.005). b Differences in mutation frequency were statistically significant (Pearson’s chisquared test, p = 0.001).

were observed in 12 patients; 4 of these patients also have mutations in one complement gene. As it could be expected, we observed that the proportion of patients with mutations was significantly higher in primary aHUS than in secondary aHUS cases (37.35% vs. 20.69%, p = 0.004) (Table 2). Within secondary aHUS cases the highest mutation frequency corresponds to the subgroup of patients having oral contraceptives or other drugs at HUS onset. The MCPggaac haplotype, conferring increased susceptibility to aHUS, was present in 62.4% of patients (Table 1), a frequency very similar to the 68.3% from our original observation (Esparza-Gordillo et al., 2005), with 41.6% of MCPggaac heterozygotes and 20.8% homozygotes. The CFH(H3) risk haplotype, defined by the combination of rs3753394 T, rs800292 G, rs1061170 T, rs3753396 G, rs1410996 G and rs1065489 T SNPs, is carried by 50.4% of patients (38.4% heterozygotes; 12% homozygotes). We previously described two CFHR1 alleles and showed that the CFHR1*B allele was associated with increased risk of aHUS (Abarrategui-Garrido et al., 2009), whereas the CFHR1*A allele was associated with increased risk of AMD (Martínez-Barricarte et al., 2012). The frequency of the CFHR1*B allele in the 319 patients with aHUS currently studied is 68.7% (43% heterozygotes and 25.7% homozygotes), which is nearly identical to the 69% frequency (41% heterozygotes and 28% homozygotes) from our original observation in 151 patients. 3.2. The c.721 T variant in CFHR3 is specifically associated with aHUS We observed that 91% of patients who were homozygous for the CFH(H3) haplotype were also homozygous for the CFHR1*B variant. These data strongly suggested the existence of an extended CFH(H3)-CFHR1*B haplotype conferring increased risk of aHUS. To test this hypothesis, we selected polymorphism c.721C>T in CFHR3 exon 5 (rs138675433), which generates a proline to serine change at amino acid 241 in the SCR4 domain of FHR-3. The analysis of the CFHR3 c.721C>T polymorphism was performed on a total of 326 aHUS patients and 92 control individuals who had previously been analyzed by MLPA to determine the gene dosage of CFHR3. We also included 43 typical HUS patients and 32 patients with C3 glomerulopathy. We observed (Fig. 1) that the CFHR3 c.721C>T polymorphism was associated with polymorphisms c.614-3insT (intron 4; rs201265523), c.786A>T (exon 5; rs149352569) and c.796+22T>A (intron 5; rs200264114). As detailed in Table 3, the frequency of the minor allele (c.721T) in the control individuals was 0.24; a similar allele frequency was observed in the patients with typical HUS (0.23), and in the patients with C3 glomerulopathy (0.19), but none of these differences were statistically significant. Among the patients with aHUS, however,

the c.721T allele frequency was significantly higher than in the controls (0.36 vs. 0.24; p = 0.002). The differences between the patients with aHUS and the control volunteers were particularly important in c.721T homozygous individuals (0.17 in aHUS vs. 0.01 in controls; p = 0.000), further supporting the strong association of the CFHR3 c.721C>T polymorphism with aHUS. Analysis of two other polymorphisms in CFHR3 exon 1 (rs385390 and rs446868) in a total of 92 patients with aHUS demonstrated a complete linkage disequilibrium between these variants and the CFHR3 c.721C>T polymorphism in exon 5 (rs138675433), revealing two primary CFHR3 haplotypes, defined by a combination of exon 1 to exon 5 SNPs. We have named these haplotypes as CFHR3*A (the more frequent allele in control individuals, tagged by rs385390 A, rs446868 C and rs138675433 C) and CFHR3*B (the more frequent allele in aHUS patients, tagged by rs385390 C, rs446868 A and rs138675433 T). 3.3. The CFH(H3), CFHR3*B and CFHR1*B variants are linked in an extended aHUS-risk haplotype To determine whether the CFH(H3), CFHR3*B, and CFHR1*B aHUS risk variants represent a unique haplotype, we first confirmed that individual SNPs in CFH (rs3753394, rs800292, rs1061170, rs3753396, rs1410996, rs1065489), CFHR3 (rs138675433), and CFHR1 (rs4230, rs414628) were in Hardy–Weinberg equilibrium in our control and aHUS populations. We then compared the allelic frequencies of these CFH, CFHR3 and CFHR1 SNPs in the controls and the patients with aHUS, and observed that they were in strong linkage disequilibrium (Supplementary Table 1). Finally, we calculated the frequency of the various CFH-CFHR3-CFHR1 haplotypes in the patients with aHUS (n = 240) and in the control individuals (n = 65). A total of 14 different SNP combinations with a frequency higher than 1% was obtained, revealing significant differences between patients and controls for two extended haplotypes (Fig. 2). The most frequent combination of CFH-CFHR3-CFHR1 variants in the controls was haplotype H1, which does not contain any of the aHUS-risk variants. Conversely, in the patients with aHUS the combination CFH(H3)-CFHR3* B-CFHR1*B (haplotype H3) was the most frequent situation (30% vs. 16% of controls) showing significant association with aHUS (p = 0.01; OR 2.40; 95% CI = 1.16–4.98). Therefore, we conclude that the CFH(H3)-CFHR3* B-CFHR1* B haplotype is strongly associated with aHUS. Interestingly, 62% of the aHUS patients with mutations carry one or two copies of each of the three aHUS risk variants (CFH(H3), CFHR3*B and CFHR1*B), while this frequency is only 30% in patients without mutations (p < 0.001). These results strongly suggest a higher frequency of the risk CFH(H3)-CFHR3*B-CFHR1*B haplotype in patients with mutations, in line with was already observed for the MCPggaac aHUS-risk variant (Esparza-Gordillo et al., 2005). 3.4. Haplotype CFH(H3)-CFHR3*B-CFHR1*B is associated with a poorer evolution of renal function and increases disease penetrance of mutations To explore the clinical relevance of the CFH(H3)-CFHR3*BCFHR1*B haplotype, we compared patients with 0, 1 or 2 copies of this haplotype. We observed a higher frequency of the CFH(H3)-CFHR3*B-CFHR1*B haplotype in females than in males, in homozygosis (Fig. 3a), but female vs. male differences in haplotype frequency were not observed in the control group (not shown). The CFH(H3)-CFHR3*B-CFHR1*B haplotype was also more frequent in patients developing the disease between 18 and 40 years of age than in patients with pediatric (0–17 years) or late adult (>40 year) onset (Fig. 3B). Because the highest proportion of secondary HUS cases is found in the 18–40 years patient subgroup, these results suggest that the haplotype has a higher contribution to

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Fig. 1. Analysis of c.271C>T polymorphism in CFHR3 Exon 5. Electropherograms of the CFHR3 Exon 5 region, including SNP c.721T>C (rs138675433) and 3 surrounding SNPs that are inherited in the same haplotype. The homozygous and heterozygous sequences obtained with the PCR forward primer are shown. Because SNP rs201265523 introduces an extra nucleotide that generates a double sequence in the heterozygote, the sequence obtained with the PCR reverse primer is also shown.

Table 3 CFHR3 c.271 T variant confers increased risk of aHUS.

Allele C T Del Genotype C/C C/Del C/T T/Del T/T Del/Del

Controls (n = 92)

aHUS (n = 326)

tHUS (n = 43)

C3G (n = 32)

p-Value

0.53 0.24 0.23

0.42 0.36 0.22

0.57 0.23 0.20

0.64 0.19 0.17

0.008 0.002 0.762

0.24 0.26 0.33 0.13 0.01 0.03

0.22 0.14 0.27 0.11 0.17 0.09

0.26 0.33 0.30 0.07 0.05 0.00

0.41 0.25 0.22 0.03 0.06 0.03

0.660 0.001 0.260 0.650 0.000 0.078

OR (95%CI)

0.64 (0.46–0.89) 1.78 (1.22–2.59) 0.94 (0.64–1.39) 0.89 (0.51–1.53) 0.48 (0.27–0.83) 0.75 (0.46–1.24) 0.85 (0.43–1.71) 18.47 (2.52–135.37) 2.9 (0.86–9.73)

Frequency of the c.271C and c.271T variants and the CFHR3-CFHR1 deletion (Del) in controls, aHUS patients, tHUS patients and C3G patients. Genotype frequencies in the four groups are also included. Statistical analyses were performed using a chi-squared test of association; the results from the comparison between the controls and the patients with aHUS are included in the table. The differences between the tHUS and C3G patients and the controls were not significant.

secondary than to primary aHUS. Interestingly, the patients with the CFH(H3)-CFHR3*B-CFHR1*B haplotype had a poorer recovery of renal function at HUS onset (Fig. 3C); thus, while 36% of patients without the risk haplotype developed terminal renal insufficiency during the first HUS episode, this figure increased to 54% in the CFH(H3)-CFHR3*B-CFHR1*B homozygous patients. Analysis of the CFH-CFHR3-CFHR1 haplotypes in the relatives of patients with aHUS provided important insights into disease penetrance in complement mutation carriers. Two examples from our cohort are illustrated in Fig. 4. In family H142, (Román-Ortiz et al., 2014) a mutated factor H resulting from a hybrid CFH:CFHR1 gene was present in several members of the maternal lineage, but the only member thus far developing HUS also carried a paternal

CFH(H3)-CFHR3*B-CFHR1*B haplotype. A similar situation is observed in patient H364, who presented combined MCP and CFI mutations inherited from his mother and the extended risk haplotype inherited from his father. 3.5. Haplotype CFH(H3)-CFHR3*B-CFHR1*B is associated with lower FH levels, and includes CFH and CFHR3 SNPs previously associated with protection against meningococcal disease Because the CFH(H3)-CFHR3*B-CFHR1*B haplotype includes SNPs located in the promoter region of the CFH gene (rs3753394; Caprioli et al., 2003) and in the 5 UTR of CFHR3 exon 1 (rs385390 and rs446868; this study), there is the possibility that these SNPs

Please cite this article in press as: Bernabéu-Herrero, M.E., et al., Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.06.021

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CFH

CFHR3

Ex1(UTR)

(1)

(1)

C

H1

C/T

G

C

A

G

G

A

H2

C

A

T

A

A

G

A

H3

T

G

T

G

G

T

C

H4

C/T

G/A

T

A

A

G

DEL

C

G

A

C

C

T

T

A

T

T

T

DEL

DEL

DEL DEL

CONTROLS (n=65)

Ex 6

c.906G>T R302R (rs4230) c.942A>T R314R (rs414628)

c.1-53 C/A 5’UTR (rs446868)

Ex 5

c.1-90 A/C 5’UTR (rs385390)

c.2808 G/T E963D (rs1065489)

c.2237-543G/A IVS15 (rs1410996)

Ex 14 Ex 15 Ex 19

c.2016A/G Q672Q (rs3753396)

Ex 9

c.1204T/C Y402H (rs1061170)

c.184G/A V62I (rs800292)

c.1-331C/T Promoter (rs3753394)

Haplotype

Ex 2

c.721C/T P241S (rs138675433)

Promoter

CFHR1

aHUS Patients (n=240) P

FREQ

FREQ

OR (95% CI)

0.31

0.19

0.53 (0.29-0.99)

0.10

0.61 (0.28-1.35)

NS

0.30

2.40 (1.16-4.98)

0.01

0.22

1.01 (0.52-1.96)

NS

0.15

0.16

0.22

0.04

(1) These two SNPs were not considered for the statistical analysis of extended haplotypes

Fig. 2. CFH-CFHR3-CFHR1 extended haplotypes. Frequencies of extended haplotypes in the CFH-CFHR3-CFHR1 genomic region in control individuals and patients with aHUS, estimated with the R software (haplo.stats package). Each extended haplotype carries specific variants in CFH (6 SNPs), CFHR3 (3 SNPs) and CFHR1 (2 SNPs). Only haplotypes with a frequency higher than 3% are shown. Haplotype frequencies in the controls and patients were compared using a Pearson chi-squared test of association; significant differences are boxed in gray.

modulate the transcriptional activity of the CFH and CFHR3 genes, resulting in variations in the plasma levels of FH and FHR-3. We have determined the levels of FH in plasma samples from individuals carrying 0, 1 or 2 copies of the CFH(H3)-CFHR3*B-CFHR1*B haplotype; we have found (Fig. 5) that carriers of 2 copies presented significantly lower FH levels than individuals with 0 copies (152 ␮g/ml vs. 185 ␮g/ml; p = 0.039), suggesting that the risk to aHUS conferred by the CFH(H3)-CFHR3*B-CFHR1*B haplotype could be related to lower complement regulatory activity by FH in carriers of this haplotype. Unfortunately, there are no reagents to measure FHR-3 levels to perform similar experiments on individuals with different CFHR3 genotypes. Interestingly, the CFH(H3)-CFHR3*B-CFHR1*B haplotype also carries the CFH c.2808 T variant (rs1065489) that was associated with a reduced susceptibility to meningococcal disease (Davila et al., 2010). This CFH variant, which was in linkage disequilibrium with three additional variants in the CFHR3 gene (c.−90C, rs385390; c796+1060G, rs426736; c.14193C, rs371075), is characteristic of the CFH(H3)-CFHR3*B-CFHR1*B haplotype, strongly suggesting that the same haplotype is associated with protection against meningococcal disease and an increased risk of aHUS. To check this possibility, we selected 37 individuals homozygotes for the CFH(H3)-CFHR3*B-CFHR1*B haplotype and 28 individuals without it, and genotyped them for the three CFHR3 SNPs associated with meningococcal disease. Our results (Table 4) demonstrate that in these patients the three CFHR3 variants associated with protection against meningococcal disease are included in the CFH(H3)-CFHR3*B-CFHR1*B haplotype, and therefore, it is likely that this haplotype confers both an increased risk of aHUS and protection from meningococcal disease. 4. Discussion Complement dysregulation is a significant pathogenic mechanism in atypical hemolytic uremic syndrome (aHUS) (Rodríguez de

Córdoba et al., 2014), and patients with single or combined mutations in complement genes, or with circulating anti-FH antibodies, have been described (Maga et al., 2010; Strobel et al., 2011; Blanc et al., 2012; Bresin et al., 2013). In this study, we describe the frequency of genetic and acquired complement defects in the Spanish aHUS cohort, and report a novel genetic predisposing factor that will likely influence disease penetrance and evolution in mutation carriers. Currently, the cohort includes 367 aHUS patients selected according to clinical criteria (273 primary cases, and 94 secondary cases). The overall frequency of mutations in complement genes in our cohort is 33.04%. This frequency is much lower than the 50–60% reported in the French cohort (Fremeaux-Bacchi et al., 2013), but is not very different from the 40% observed in a series of 795 patients with aHUS from several European cohorts (Bresin et al., 2013). The differences between cohorts could be partially due to the selection criteria: the French cohort does not include patients with aHUS triggered by immunosuppressants, oral contraceptives or other drugs (referred to as “secondary MAT cases”), whereas the Spanish cohort includes all the patients with a clinical diagnosis of aHUS. If we only consider the primary aHUS cases, the mutation frequency in our cohort increases up to 37.35%, significantly higher than the 20.69% observed in secondary cases. These data reveal that complement genetic studies should also be performed in secondary aHUS cases, particularly in women developing the disease while having oral contraceptives, who present the highest mutation frequency (9 out of 20 patients). Complement mutations in patients with aHUS from all cohorts are mostly heterozygous, and genetic risk variants in the complement genes CFH (Caprioli et al., 2003) and MCP (Esparza-Gordillo et al., 2005) have been described, which increase disease penetrance in mutation carriers (Bresin et al., 2013). In our aHUS cohort, the frequency of patients carrying the risk MCPggaac haplotype is 62%, whereas 50% carry the CFH(H3) haplotype. The CFHR1*B allele, which we identified as a third aHUS-risk

Please cite this article in press as: Bernabéu-Herrero, M.E., et al., Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.06.021

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Fig. 3. Clinical relevance of the CFH(H3)-CFHR3*B-CFHR1*B haplotype. aHUS patients were grouped according to the number of the CFH(H3)-CFHR3*B-CFHR1*B haplotype copies (0, 1 or 2). The female/male frequency was determined in each group. Similar comparisons were established for Age at HUS onset, and Evolution of renal function.

Please cite this article in press as: Bernabéu-Herrero, M.E., et al., Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.06.021

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Family HUS142

H142P

H142M (44y)

(46y)

HUS142 (11y)

H142P1 (15y)

H142P5 (34y)

H142P3 (45y)

H142 P2 H142P2

H142P4

(16y)

(12y)

Carrier of Hybrid gene on CFH 402His allele Carrier of 890Ile and 1007Leu CFH variants Carrier of CFH(H3)-CFHR3*B-CFHR1*B haplotype

Family HUS364

H364P H364P (54y)

HUS364 (26y)

H364M (52y)

H36 4P1 H364P1 (24y)

Carrier of CFI mutation Carrier of MCP mutation Carrier of CFH(H3)-CFHR3*B-CFHR1*B haplotype Fig. 4. CFH(H3)-CFHR3*B-CFHR1*B haplotype and HUS penetrance. Segregation of mutations and the CFH(H3)-CFHR3*B-CFHR1*B haplotype in pedigrees from two Spanish patients with aHUS. Patient H142 has one copy of the CFH:CFHR1 hybrid gene (Román et al., Pediatr Nephrol 2014), and patient H364 has combined mutations in MCP and CFI (unpublished data). The two patients also carry one copy of the CFH(H3)-CFHR3*B-CFHR1*B haplotype. The current age of each individual (in years) is shown in brackets.

variant (Abarrategui-Garrido et al., 2009), is present in 69% of patients. We have found that the CFHR3 c.721T variant, which generates a serine to proline change at amino acid 241 in FHR-3, is associated with increased risk of aHUS, but not with typical HUS or with C3 glomerulopathy, a renal pathology also associated with dysregulation of the complement alternative pathway (Pickering et al., 2013). Our analyses also reveal that the c.721T variant is linked to other CFHR3 SNPs, defining a haplotype that extends at least from exon 1 to intron 5 in CFHR3. Therefore, there are two main CFHR3 alleles with a different distribution in the control volunteers and the patients with aHUS. Allele CFHR3*A (coding for proline 241) is more frequent in the control volunteers than in the patients with aHUS (0.50 vs. 0.42), and allele CFHR3*B (coding for serine 241) is more frequent in the patients with aHUS than in the controls (0.36 vs. 0.24). Additional analyses allowed us to confirm that the CFH(H3), CFHR3*B and CFHR1*B aHUS-risk variants segregate together in an extended haplotype that is present in 30% of patients with aHUS but only in 16% of controls, thus conferring increased susceptibility to aHUS (OR = 2.40; 95% CI 1.16–4.98; p = 0.01). Our results also show that the CFH(H3)-CFHR3*B-CFHR1*B risk haplotype appears to favor

a poorer prognosis for renal function at HUS onset and explains disease penetrance in some families. Why the CFH(H3)-CFHR3*BCFHR1*B haplotype is aHUS-specific and does not associate with other complement dysregulation diseases such as AMD or C3G is currently unknown. The CFH gene codes for FH, the main regulator of complement activation in plasma, and aHUS patients with a mutated FH have a particularly bad prognosis (Bresin et al., 2013). The CFHR3 and CFHR1 genes code for FHR-3 and FHR-1, which are evolutionarily and structurally related to FH, but whose precise function on complement activation and regulation is incompletely understood. We have observed that the CFH(H3)-CFHR3*B-CFHR1*B haplotype is associated with decreased levels of FH in plasma, suggesting a reduced complement regulation in haplotype carriers. It is possible that the CFHR3*B and/or CFHR1*B variants also influence complement activation on the renal endothelial surface, and that it is the combination of the decreased FH levels and the functional alterations in FHR-3 and FHR-1 that increase the risk of aHUS and determine disease evolution. Importantly, the CFH(H3)-CFHR3*B-CFHR1*B haplotype also carries the rs426736 CFHR3 variant that was shown to confer protection against meningococcal disease (Davila et al., 2010). A major strategy of Neisseria meningitidis to avoid killing by the

Please cite this article in press as: Bernabéu-Herrero, M.E., et al., Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.06.021

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Table 4 The CFH(H3)-CFHR3*B-CFHR1*B haplotype confers protection against meningococcal disease. CFH(H3)-CFHR3*B-CFHR1*B carriers Number of patients

rs385390 c.-90 (A>C)

rs426736 c.796+1060 (A>G)

rs371075 c.14193 (T>C)

35

C C C A C C

G G G G G G

C C C C C T

Number of patients

rs385390 c.-90 (A>C)

rs426736 c.796+1060 (A>G)

rs371075 c.14193 (T>C)

18

A A A CFHR3-CFHR1 A C A A A C

A A A CFHR3-CFHR1 A G A A A A

T T T CFHR3-CFHR1 T C T C T C

1 1

CFH(H3)-CFHR3*B-CFHR1*B non-carriers

5 2 2 1

Analysis of the three CFHR3 variants associated with meningococcal disease (Davila et al., 2010) in 75 patients with aHUS from a Spanish cohort; 37 carriers and 28 non-carriers of the CFH(H3)-CFHR3*B-CFHR1*B haplotype were studied.

effect against meningococcal disease; other interesting possibilities, which should be tested in future experiments, are that the SNPs in the promoter/5 UT region of the CFHR3 gene result in increased expression of FHR-3, whereas those in the coding region increase its affinity for fHbp and the competition with FH. In conclusion, we have determined the prevalence of mutations, risk polymorphisms and anti-FH antibodies in the Spanish cohort of patients with aHUS (n = 367), and have identified a CFHR3 allele that is another genetic susceptibility factor to this disease. We describe a CFH(H3)-CFHR3*B-CFHR1*B extended haplotype specifically associated with aHUS that gives rise to reduced FH levels, but whether it also determines functional and/or quantitative defects in FHR-3 and FHR-1 remains to be determined. These data significantly advances our understanding of the clinical and functional consequences of the aHUS risk polymorphisms, which will facilitate a comprehensive genetic analysis of the patients and the implementation of a personalized medicine, particularly Complement-inhibiting treatments such as Eculizumab (Legendre et al., 2013). Conflict of interest Fig. 5. CFH(H3)-CFHR3*B-CFHR1*B haplotype and FH levels. Levels of FH in plasma samples from individuals with 0, 1 or 2 copies of the CFH(H3) haplotype. The mean value and the standard deviation within each group are illustrated with horizontal bars. FH levels in individuals with 2 copies of CFH(H3)-CFHR3*B-CFHR1*B were significantly lower (p = 0.039) than in individuals without any CFH(H3) copy.

human complement system is the incorporation of human FH to its surface protein fHbp (Schneider et al., 2006; Madico et al., 2006). This is a high affinity interaction that will be favored in individuals with higher levels of FH in plasma. It has been recently shown that FHR-3 competes with FH for binding to fHbp on the bacterial surface, influencing its survival in plasma (Caesar et al., 2014). These data strongly suggest that the ability of the meningococcus to evade the host complement system is determined by the relative levels of FH and FHR-3 on the bacterial surface, and therefore, both qualitative and quantitative variants of FH and FHR-3 are crucial for developing meningococcal disease. In this context, the observation that the CFH(H3)-CFHR3*B-CFHR1*B haplotype determines decreased FH levels might help explain the protective

SRdeC has received honoraria from Alexion Pharmaceuticals for giving lectures and participating in advisory boards. None of these activities has had any influence on the results or interpretation in this article. The other authors declare no conflicts of interest. Acknowledgments We are grateful to all patients and their relatives for their participation in this study. We thank the technical assistance of Elena ˜ (Unidad de Investigación, IdiPAZ) in manAlgarra and Carolina Pena agement of biological samples, as well as the statistical help of Rosario Madero and Carlos Iniesta (Unidad de Bioestadística, IdiPAZ). We greatly appreciate the helpful comments from Dr. Marta Melgosa (Nephrology Unit, Hospital La Paz-IdiPAZ) about primary and secondary aHUS cases. Work in this report has been funded by the Spanish “Ministerio de Economía y Competitividad” to PS-C (PI1200597), SRdeC (SAF2011-26583), and MLT (SAF2012-38636), and by the Autonomous Region of Madrid (S2010/BMD-2316) and

Please cite this article in press as: Bernabéu-Herrero, M.E., et al., Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.06.021

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the Spanish Society of Nephrology (SENEFRO) to SRdeC, MLT and PS-C. SRdeC received additional support from the Fundación Renal ˜ Inigo Alvarez de Toledo and the 7FP European Union project EURenOmics. Some grants were also co-funded by the European program FEDER (Fondo Europeo de Desarrollo Regional). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molimm.2015. 06.021 References Abarrategui-Garrido, C., Martínez-Barricarte, R., López-Trascasa, M., Rodríguez de Córdoba, S., Sánchez-Corral, P., 2009. Characterization of complement factor Hrelated (CFHR) proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome. Blood 114, 4261–4271. 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Please cite this article in press as: Bernabéu-Herrero, M.E., et al., Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.06.021

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Pérez, R., López Trascasa, M., Pickering, M.C., Harris, C.L., Sánchez-Corral, P., Llorca, O., Rodríguez de Córdoba, S., 2013. C3 glomerulopathy-associated CFHR1 mutation alters FHR oligomerization and complement regulation. J. Clin. Invest. 123, 2434–2446. Zipfel, P.F., Edey, M., Heinen, S., Jòzsi, M., Richter, H., Misselwitz, J., Hoppe, B., Routledge, D., Strain, L., Hughes, A.E., Goodship, J.A., Licht, C., Goodship, T.H., Skerka, C., 2007. Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome. PLoS Genet. 3, e41.

Please cite this article in press as: Bernabéu-Herrero, M.E., et al., Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.06.021