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Comprehensive Genetic Analysis of Complement and Coagulation in Atypical Hemolytic Uremic Syndrome

† † † ‡ † Fengxiao Bu,* Tara Maga,* Nicole C. Meyer, Kai Wang, Christie P. Thomas, § † † Carla M. Nester, § and Richard J. H. Smith* §

*Interdisciplinary PhD Program in Genetics, †Molecular Otolaryngology and Renal Research Laboratories, ‡Department of Biostatistics, College of Public Health, and §Rare Renal Disease Clinic, Departments of Pediatrics and Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa

ABSTRACT Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy caused by uncontrolled activation of the alternative pathway of complement at the cell surface level that leads to microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. In approximately one half of affected patients, pathogenic loss-of-function variants in regulators of complement or gain-of-function variants in effectors of complement are identified, clearly implicating complement in aHUS. However, there are strong lines of evidence supporting the presence of additional genetic contributions to this disease. To identify novel aHUS-associated genes, we completed a comprehensive screen of the complement and coagulation pathways in 36 patients with sporadic aHUS using targeted genomic enrichment and mas- sively parallel sequencing. After variant calling, quality control, and hard filtering, we identified 84 reported or novel nonsynonymous variants, 22 of which have been previously associated with disease. Using computational prediction methods, 20 of the remaining 62 variants were predicted to be dele- terious. Consistent with published data, nearly one half of these 42 variants (19; 45%) were found in genes implicated in the pathogenesis of aHUS. Several genes in the coagulation pathway were also identified as important in the pathogenesis of aHUS. PLG, in particular, carried more pathogenic var- iants than any other coagulation , including three known plasminogen deficiency mutations and a predicted pathogenic variant. These data suggest that mutation screening in patients with aHUS should be broadened to include genes in the coagulation pathway.

J Am Soc Nephrol 25: 55–64, 2014. doi: 10.1681/ASN.2013050453

Hemolytic uremic syndrome (HUS) is a thrombotic Uncontrolled activation of the alternative path- microangiopathy characterized by microangio- way of complement at the level of the cell surface has pathic hemolytic anemia, thrombocytopenia, and been closely linked to disease.5,6 In about one half of acute kidney failure that is most frequently caused aHUS patients, pathogenic mutations are identified by Shiga-toxigenic Escherichia coli (STEC) serogroup in complement genes, including loss-of-function O157. About 10% of HUS cases, however, are atypical mutations in regulators of complement activity and not caused by an antecedent STEC infection.1 The prevalence of atypical HUS (aHUS) is estimated at 1 per 2 and 1 per 3.3 million children in the United Received May 3, 2013. Accepted June 23, 2013. 2,3 States and Europe, respectively. Until the recent Published online ahead of print. Publication date available at availability of Eculizumab, aHUS carried significant www.jasn.org. morbidity and mortality, with 25% of patients dying Correspondence: Dr. Richard J. H. Smith, Iowa Institute of Hu- of disease, 50% of patients progressing to end stage man Genetics, 5270 CBRB, Iowa City, IA 52242. Email: richard- renal failure, and 40%–80% of patients experiencing [email protected] disease recurrence after transplantation.1,4 Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: 55–64, 2014 ISSN : 1046-6673/2501-55 55 BASIC RESEARCH www.jasn.org like complement (CFH),7 RESULTS (CFI),8 and membrane cofactor (CD46)9,10 and gain-of-function mutations in effectors of complement like Patients factor B (CFB)11 and (C3).12,13 In total, 36 European-American patients with sporadic aHUS In a recent large genetic screen of 794 aHUS patients, single (24 men and 12 women) were recruited into this study from mutations in CFH, C3, CFI, CFB,orCD46 were identified in 2007 to 2011 (Figure 1). The diagnosis of aHUS was based on 41% of patients, and combined mutations were found in 3% of the presence of acute renal failure, thrombocytopenia, patients.14 Additional genetic contributors include single nucle- and microangiopathic hemolytic anemia after excluding an- otide polymorphisms and haplotypes in CFH and CD46,14–17 tecedent STEC infection. All procedures were approved by the copy number variation of the complement factor H-related Institutional Review Board of the University of Iowa Carver three and one genetic intervals (homozygosity for deletion of College of Medicine. CFHR3-CFHR1), and novel fusion genes of the factor H-related region caused by nonallelic homologous recombination.18–20 Sequencing Quality Metrics and Variant Calling Although these studies clearly implicate mutations in We performed TGE+MPS on 36 European-American aHUS complement genes in the pathogenesis of aHUS, additional patients using an RNA-bait panel that targets coding sequence genetic contributions to this disease are likely to exist. There are of genes in the complement and coagulation pathways three lines of evidence that strongly support this hypothesis. (hereafter referred to as capture and sequencing of comple- First, in a large percentage of cases, no mutations are found in ment-associated disease exons [CasCADE]). The mean total the commonly implicated complement genes. Second, there is reads generated per sample approximated 9,000,000, with an extremely high rate of incomplete penetrance in familial more than 97% of reads mapping to the reference genome and aHUS, which is consistent with the existence of additional more than 50% of reads mapping to targeted regions. The modifying/contributory genetic factors. Third, current screens average coverage per targeted base was 1670 reads; 99% of have narrowly focused on fewer than a dozen genes, although targeted regions were covered by more than 30 reads (Table 1). the is substantially larger and integrated Of 7486 variants identified in 36 aHUS patients, 7071 with numerous other signaling pathways. (95.46%) variants passed quality control (Figure 2). Each sub- In this study, we sought to address these limitations by using ject carried a mean of 196 variants. After filtering by minor targeted genomic enrichment and massively parallel sequenc- allele frequency and functional effect, 18 novel and 66 re- ing (TGE+MPS) to screen the coding sequence and splice sites ported nonsynonymous rare variants (nsRVs) remained. All of all genes in both the complement and coagulation pathways. nsRVs were present in heterozygosity, with each patient We selected the coagulation pathway because of its role in carrying a mean of 2.33 nsRVs (range=0–5). thrombotic thrombocytopenic purpura (TTP), a thrombotic microangiopathy in which extensive microscopic thrombi form in the microvasculature. aHUS and TTP share the clinical features of microangiopathic hemolysis, thrombocytopenia, and renal involvement, but in TTP,patients also have fever and neurologic symptoms.21,22 The fact that the coagulation path- way warrants detailed scrutiny is also supported by the finding of pathogenic aHUS mutations in two anticoagulant-related genes, THBD23 and DGKE.24 THBD encodes thrombomodu- lin, which functions as a cofactor for thrombin to reduce blood coagulation and also regulates CFI-induced inac- tivation.23,25 DGKE encodes diacylglycerol kinase-« and is im- plicated in regulation of thrombus formation by modulating protein kinase C activity in endothelial cells and platelets.24 In total, we sequenced 85 genes in 36 European-American sporadic aHUS patients, and we filtered and prioritized var- iants based on frequency and functional effects. As expected, we found novel deleterious variants in multiple complement genes, but we also identified deleterious nonsynonymous rare variants in several genes that play important roles in coag- ulation, definitely implicating this pathway in aHUS. The most frequently mutated gene in the coagulation pathway was PLG, Figure 1. Diagnostic age distributes similarly in male and female which encodes plasminogen, a zymogen that is secreted by the aHUS patients. Although a trend toward higher diagnostic age liver and converted into plasmin by a variety of enzymes on was observed in women, this difference was not significant binding to clots. (Wilcoxon rank sum test P50.067).

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Table 1. Quality of sequencing results Exome Variant Server (EVS29; 3 DRV counts in 36 aHUS Sequencing Metrics Mean 6 SD Median cases versus 76 DRV counts in 4300 EVS European-Ameri- Total reads 9,195,90761,294,156 9,176,081 can subjects; Fisher exact test P=0.03). Other interesting Mapped reads 9,112,15161,215,320 9,008,634 ultrarare DRVs included a G-to-A transition that alters a Mapped reads, % 97.84 98.39 canonical splice site in F12 (c.1681–1G.A) and leads to Reads overlapping targets 5,025,5946875,441 5,053,139 factor XII deficiency.30 Reads overlapping targets, % 54.84 55.92 6 Average coverage 1670.65 290.82 1677.48 fi Bases covered 13,% 99.60 99.67 Novel nsRV Identi cation fi Bases covered 103,% 99.17 99.35 Among 18 novel variants that we identi ed, there were 15 Bases covered 303,% 98.97 99.29 missense, 2 frame-shift, and 1 splice site change variants (Supple- mental Table 1). These variants included a G-to-C transversion in VWF,c.4165G.C (p.Glu1389Gln), that altersthesamenucleotideasaDRV (c.4165G.A, p.Glu1389Lys), which is re- ported to cause type 2 von Willebrand dis- ease by reducing amounts of high-molecular weight VWF and VWF-platelet binding.31 One novel variant, c.3221A.G, p.Asn1074Ser in F5, was shared by two aHUS patients and alters a potential N-link glycosylation site.

Prediction of Deleterious Variants To assess possible pathogenicity of 62 variants not previously implicated in dis- ease (58 missense, 1 splice site, and 3 frame-shift indels variants), we first used combined computational prediction meth- ods (PhyloP, SIFT, PolyPhen2, LRT, Muta- tionTaster, and GERP++) to calculate the mean pathogenicity score (PS) of the re- ported missense DRVs. Based on this PS, Figure 2. Application of hard filtering uncovers pathogenic variants. Variants were which was 3.81, we accepted as likely dele- fi ltered by focusing on nonsynonymous, frame shift, and indel changes. Rare variants terious all novel and nsRVs with a PS of at fi , are de ned as variants with a minor allele frequency 1% in European-American and least 4 (Table 2). The average PS of 16 mis- African-American populations based on data from the EVS database. Predicted del- sense novel and nsRVs meeting this minimal eterious mutations or rare variants are stop-gain mutations, indels, or missense var- iants with a high pathogenicity score (PS) (.4). PS for missense variants was calculated threshold was 4.57 (Table 3). Three frame- using PhyloP, SIFT, PolyPhen2, LRT, MutationTaster, and GERP++, and it ranged from shift indels (c.356_357insG in C3AR1, zero to six. The mean PS of reported DRVs was 3.41; the mean PS of predicted del- c.443_453del in , eterious missense variants was 4.57. *DRVs have been previously reported to be and c.1770delG in mannan-binding lectin disease-causing or -associated. DRVs may or may not be recorded in public single serine protease 1 [MASP1]) and a splice site nucleotide polymorphism databases. QC, quality control; UTR, untranslated region. change (c.1160–2A.GinCFH) were also considered possibly damaging. In aggregate, 42 deleterious variants (22 Reported Disease-Related Variants DRVs and 20 predicted deleterious variants) were identified in Twenty-two of eighty-four nsRVs were reported disease-re- 25 patients (69.4%) (Figure 3). Not surprisingly, of these var- lated variants (DRVs), most frequently being causally related to iants, 20 variants were found in genes commonly implicated in aHUS (15 DRVs in CD46, CFB, CFH,andCFI)(Table2).Re- aHUS (CFH, CD46, C3, CFI, CFB,andTHBD). However, 15 markably, however, there were three variants in PLG (c.112 variants were found in other complement-related genes, and 7 A.G, p.Lys38Glu; c.2134 G.A, p.Gly712Arg; c.758 G.A, variants were found in coagulation pathway genes. The greatest p.Arg253His) associated with plasminogen deficiency,26–28 number of deleterious variants was found in CFH (11 variants in which is a significant enrichment of DRVs in this gene com- 10 patients), but the second greatest number was in PLG (4 pared with European-American data from the National variants in 4 patients), suggesting that this gene makes an im- Heart, Lung, and Blood Institute Exome Sequencing Project portant contribution to the aHUS phenotype.

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Table 2. Reported disease-related variants Gene Nucleotide AA Change PSa Related Disease MAFb (EVS_EA) dbSNP 137 C9 c.460 C.T p.R154X — C9 deficiency50 0.0002 rs144138616 CD46 c.692 C.G p.P231R 6 aHUS51 ND ND CD46 c.648 G.C p.W216C 6 aHUS51 ND ND CD46 c.565 T.G p.Y189D 5 aHUS52 ND ND CFB c.967 A.C p.K323Q 2 aHUS51 ND ND CFH c.1825 G.A p.V609I 1 aHUS51 0.0006 rs148165372 CFH c.1873 G.T p.E625X — aHUS51 0.0002 rs150694809 CFH c.2745 C.A p.C915X — aHUS51 ND ND CFH c.3514 G.T p.E1172X — aHUS53 ND rs121913060 CFH c.3583 G.T p.E1195X — aHUS51 ND ND CFH c.3592 G.A p.E1198K 4 aHUS54 ND ND CFH c.3643 C.G p.R1215G 4 aHUS7 ND rs121913051 CFH c.3644 G.Ac p.R1215Q 2 aHUS55 ND ND CFI c.355 G.A p.G119R 3 aHUS51 0.0013 rs141853578 CFI c.859 G.A p.G287R 5 aHUS51 0.0001 rs182078921 F12 c.1681–1G.ANA — Factor XII deficiency30 0.0006 ND PLG c.112 A.G p.K38E 5 Plasminogen deficiency26 0.0062 rs73015965 PLG c.2134 G.A p.G712R 3 Plasminogen deficiency27 0.0007 ND PLG c.758 G.A p.R253H 5 Plasminogen deficiency28 0.0006 rs143034754 THBD c.1502 C.T p.P501L 3 aHUS23 0.0029 rs1800579 VWF c.2561 G.A p.R854Q 4 Von Willebrand disease56 0.0056 rs41276738 All variants are heterozygous. AA, African-American; EA, European-American; C9, ; F12, coagulation factor XII; VWF,VonWillebrand factor; NA, not available; ND, no data. aPathogenicity score (PS) was calculated using PhyloP, SIFT, PolyPhen2, LRT, MutationTaster, and GERP++. The average PS of reported disease-related variants was 3.81. bMinor allele frequency (MAF) values in the European-American population are from the Exome Variant Server (EVS). Novel variants are those variants not reported in the EVS, 1000 Genomes, or dbSNP databases. cMutation is carried by two patients.

Copy Number Variants in CFHRs patients with sporadic aHUS. After variant calling, quality We identified copy number variants (CNVs) in the CFHR geno- control, and hard filtering, we identified 84 reported or novel mic region in 15 patients (41.7%). Homozygosity for delCFHR3- nsRVs, 22 of which have been previously associated with dis- CFHR1 was found in three patients who carried no deleterious ease. Using computational prediction methods (PhyloP, SIFT, nsRVs. Ten patients were heterozygous for this deletion, and in PolyPhen2, LRT, MutationTaster, and GERP++), 20 additional two other patients, we identified heterozygous duplications in variants were predicted to be deleterious. As expected and the CFHR1 and CFHR4 genes. The frequency of these variations consistent with published data, nearly one half of these 42 was not statistically different than the frequency seen in 314 variants (19; 45%) were found in genes implicated in the healthy controls (unpublished data; Fisher exact test P=0.16; val- pathogenesis of aHUS (CFH, CD46, CFB, CFI, and C3). How- idated Hardy–Weinberg Equilibrium in patients and controls). ever, PLG, a gene in the coagulation pathway, also emerged as an important gene in the pathogenesis of aHUS. Multiple Variants Thefourvariantsthatweidentified in PLG included three Twelve patients (33.3%) carried two or more deleterious nsRVs reported plasminogen deficiency-related variants: c.112 A.G (Supplemental Table 2). In six of these patients, the deleterious (p.Lys38Glu), c.758 G.A (p.Arg253His), and c.2134 G.A(p. nsRVs were present only in complement genes, but in the Gly712Arg). Of these known variants, c.112 A.G, p.Lys38Glu remaining six patients, deleterious nsRVs were found in both (rs73015965; also referred to as Lys19Glu27)isnoteworthy complement and coagulation genes. Although not statistically for being the first variant identified in sporadic plasminogen- significant, there was a trend to negative correlation between deficient patients (two men and one woman) and being asso- CNVs of the CFHR region and deleterious nsRVs (Figure 4). ciated with low plasminogen levels in the unaffected parental In five patients (13.9%), we were unable to identify genetic al- carrier.26 Subsequent studies have shown that this missense terations in any complement or coagulation gene. variant is found in one third of patients with plasminogen de- ficiency type I and associated with less severe reduction in plasma plasminogen levels and milder disease.27,28 The fourth DISCUSSION variant that we identified, c.505 C.T, p.Pro169Ser, is novel and predicted to be disease-causing based on genetic conser- In this study, we used TGE+MPS to screen 85 genes in the com- vation, bioinformatics (PS=4), and frequency data (Figure 5 plement and coagulation pathways in 36 European-American and Table 2).

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Table 3. Predicted deleterious nsRVs reduction of fibrinolysis is important in 34 Gene Nucleotide AA Change PSa MAFb (EVS_EA) dbSNP 137 aHUS. Plasminogen-related studies on Reported typical HUS, however, have yielded equiv- 35–39 C1S c c.943 G.A p.D315N 6 0.0053 rs117907409 ocal results. Some studies have reported C3 c.4855 A.C p.S1619R 4 0.0028 rs2230210 that inhibition of fibrinolysis precedes and C3AR1 c.356_357insG p.D119fs — 0.0004 ND may be the cause of renal injury,35 with C8A c.1331 G.A p.R444H 6 0.0050 rs143908758 plasma levels of plasminogen activator in- CFHR5c c.832 G.A p.G278S 4 0.0088 rs139017763 hibitor-1 inversely correlating with improve- CR2 c.524 C.T p.P175L 4 0.0074 rs75282758 ment in renal function.39 Other studies, FCN1 c.866 A.G p.N289S 4 0.0016 rs138055828 however, report that evidence of impaired . MASP2 c.467 G A p.C156Y 6 0.0097 rs41307788 fibrinolysis in children with typical HUS is . PHB c.128 G T p.R43L 4 0.0088 rs2233665 lacking.37 PLG c.505 C.T p.P169S 4 0.0004 rs143256245 Novel deleterious variants were found in Novel C2 c.443_453del p.148_151del — known complement genes as well as com- C3 c.3085 G.A p.D1029N 6 plement genes not previously associated CFH c.595 A.G p.S199G 4 with aHUS, like complement component 2, CFH c.1160–2A.G —— 1, ficolin 1, and MASP1 CR1 c.4750 C.T p.R1584W 4 and -2 (Supplemental Table 1). Although MASP1 c.1770delG p.G590fs — these findings need to be replicated, they MASP2 c.1633 A.G p.N545D 4 do suggest that aHUS can be caused by a d VWF c.4165 G.C p.E1389Q 4 broad range of complement abnormalities. All variants are heterozygous. AA, African-American; EA, European-American; C1S, complement A single novel variant was also identified in component 1 s subcomponent; C3AR1, complement component 3a receptor 1; C8A, complement . component 8 alpha polypeptide; CR2, complement component receptor 2; FCN1, ficolin 1; PHB, ADAMTS13 (c.3056 T C or p.M1019T), a prohibitin; C2, complement component 2; CR1, ; ND, no data. finding that is noteworthy for the afore- a Pathogenicity score (PS) was calculated using PhyloP, SIFT, PolyPhen2, LRT, MutationTaster, and mentioned clinical similarity between GERP++. The mean PS of predicted deleterious missense nsSNVs was 4.57. bMinor allele frequency (MAF) values in the European-American population are from the Exome Variant aHUS and TTP. This variant is predicted Server (EVS). Novel variants are those variants not reported in the EVS, 1000 Genomes, and dbSNP to have limited impact on protein structure databases. and function (PS=0), which we expected cMutation is carried by two patients. dMutation (c.4165 G.A) in VWF causing Von Willebrand disease was found at the same position given the aHUS phenotype of the patient. (21094983) but leads to a different alteration in the amino acid sequence (p.E1389K versus p.E1389Q). Functional studies would be required to de- termine whether the variant has a minor effect on ADAMTS13 activity. Interestingly, The enrichment of PLG for plasminogen deficiency DRVs is this patient also carries a deleterious variant in MASP2 (c.1633 statistically significant (P,0.05) and suggests that subnormal A.G or p.N545D, PS=4). activity of plasminogen or plasmin may compromise the deg- Multiple variants in common aHUS genes were identified in radation of thrombi in aHUS. We also identified other DVRs two patients, which is in agreement with expected frequencies linked to thrombosis and fibrinolysis, including a variant in (3%–12%) (Supplemental Table 2).14 However, these num- THBD (c.1502 C.T [p.Pro501Leu]) that has been associated bers are almost certainly an underestimate, because one third with aHUS23 and thromboembolic disease.32 In vitro,this of patients carried multiple deleterious nsRVs. We also found missense change reduces cell surface expression of that most identified coagulation variants (seven of eight) were thrombomodulin, which may contribute in vivo to suppres- accompanied by one or two complement mutations (Supple- sion of the alternative pathway through CFI-mediated C3b mental Table 2). In all cases, these variants were found in inactivation23 and venous thrombosis.33 Another variant, heterozygosity, suggesting a role as disease modifiers. Our c.1681–1G.AinF12, alters a canonical splice acceptor, lead- data also suggest that CNVs of the CFHR genetic region may ing to a truncated transcript, reduced protein expression, in- contribute to the aHUS phenotype. Although it is well known creased partial thromboplastin time, and recurrent superficial that homozygosity for deletion of CFHR3-CFHR1 is more venous thrombosis.30 commonintheaHUSpatientpopulationandcorrelates The role of coagulation activity in aHUS patients has been with the presence of CFH autoantibodies,40 our results show evaluated at the mRNA level in one study, in which gene that del(CFHR3-CFHR1) is disease-contributory, whereas pa- expression was quantitated in glomeruli from archival paraffin- tients with aHUS who carry more mutations are less likely to embedded renal biopsies.34 Expression of antifibrinolytic, have CFHRs deletions and vice versa. prothrombotic plasminogen activator inhibitor-1 and antith- Figure 6 shows a modified model of aHUS pathogenesis rombotic thrombomodulin were increased, and expression of that integrates our results and recent findings in heme as a profibrinolytic, antithrombotic tissue plasminogen activator secondary hit.41 The key concept in this model is the dysregu- was decreased compared with controls, suggesting that lation loop, which can be activated spontaneously or by trigger

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thus closing the loop. Dysregulation of com- plement and/or coagulation typically caused by genetic abnormalities maintains the cycle. We noted two important limitations in this study. First, the relatively small sample size reduces statistical power to uncover minor genetic contributions. Second, we focused on exonic variants and indels at the exclusion of intronic and untranslated re- gionvariants, which could be used to identify haplotypes that may contribute to aHUS. Al- though a more comprehensive genetic screen in large cohort of aHUS patients will reme- diate these shortcomings, this study is note- worthy for implicating the coagulation pathway and PLG in particular as important contributors to aHUS.

Figure 3. Distribution of reported and predicted deleterious variants per gene implicates CONCISE METHODS PLG in aHUS pathogenesis. The second greatest number of deleterious variants was identified in PLG. This enrichment of deleterious variants is statistically significant DNA Extraction, Targeted Genomic (P,0.05). A detailed description of the variants is given in Supplemental Table 3. Enrichment, and Massively Parallel Sequencing Genomic DNA was extracted from peripheral blood using the Gentra Puregene Kit (Qiagen Inc., Valencia, CA), and quality was verified by 1% agarose gel electrophoresis and NanoDrop 1000 spectrophotometry (Thermo Scientific Inc., Wilmington, DE). Before sequence cap- ture, DNA concentration was determined using the Qubit dsDNA BR Assay Kit (Invitrogen Inc., Carlsbad, CA). Targeted genomic enrichment was per- formed using CasCADE, an RNA-bait panel based on the Agilent SureSelect Target Enrichment System (Agilent Technologies Inc., Santa Clara, CA) that targets coding sequence of genes in the complement and coagulation pathways. CasCADE was developed and optimized to capture 1071 exons of 85 complement and coagulation genes representing250kBgenomicDNA(Supplemental Figure 4. Copy number of complement factor H related (CFHR) genes negatively Figure 1). In CasCADE versions 1 and 2, baits correlates with number of deleterious variants in aHUS patients. Less frequent copy were balanced based on sequencing results of number variation of CFHR genes was observed in patients with more deleterious test samples to optimize efficiency and accuracy variants. nsRV, deleterious nonsynonymous rare variant; hom, homozygous; het, het- erozygous; del, deletion; dup, duplication. of targeted enrichment, and in this study, we used CasCADE version 3 to perform capture and en- richment of 2 mg genomic DNA using a solution- events at either point: overactivation of complement or for- based targeted genomic enrichment protocol as previously described.42 mation of thrombi in microvessels. Overactivation of comple- Multiplexing indexes were used to barcode prepared libraries. Quality ment causes endothelial cells lysis, which leads to thrombi and concentration were evaluated using a Bioanalyzer 2100 (Agilent formation. Thrombi in microvessels cause mechanical damage Technologies Inc., Santa Clara, CA). After pooling, libraries were se- to erythrocytes with release of heme. Heme directly activates the quenced with a 23 100-bp paired-end module in one lane on a HiSeq alternative pathway and represses membrane cofactor protein 2000 (Illumina Inc., San Diego, CA) at the DNA Core Facility of the and decay-accelerating factor expression in endothelial cells,41 University of Iowa’s Institute of Human Genetics.

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Figure 5. Deleterious variants of PLG are conserved. The four identified deleterious variants in PLG localize to different domains of plasmin- ogen. The amino acids are highly conserved across multiple species, suggesting that these variants may impair normal function of the protein.

Variant Calling and Filtering Sequence data were analyzed using a local in- stallation of the open-source Galaxy software running on a high-performance computing clus- ter at the University of Iowa. A dedicated compute queue for Galaxy as well as shared compute resources were leveraged for minimized job wait time and continuous availability. We use a com- bination of custom and publicly available tools on the pipeline running in Galaxy: read mapping was performed with Burrows–Wheeler Alignment, duplicate removal was performed with Picard, lo- cal realignment and variant calling were per- formed with GATK, enrichment statistics were performed with NGSRich, and variant annotation was performed with ANNOVAR.43,44 During hard filtering, we used quality control metrics that included a phred-scaled quality score$30, depth$10, and Q/D$5, and then fo- cused on the following variants: nonsynonymous, Figure 6. Modified model implicates dysregulation of both the complement and coagulation frame shift, splicing site changes, and indels. 29 pathways in the pathogenesis of aHUS. This model suggests three stages in the development Common variants from either the EVS or the of aHUS. (1) The disease is initiated by trigger events, such as nonenteric bacterial infections, 1000 Genomes Project (April of 2012)45 with a viruses, drugs, malignancies, transplantation, pregnancy, and medical conditions like sclero- minor allele frequency value.1% in any popula- derma, antiphospholipid syndrome, and systemic lupus erythematosus. The complement tion were excluded. We defined as novel those cascade is activated, and/or the coagulation pathway leads to thrombus formation. (2)A variants not reported in EVS, 1000 Genomes, Na- dysregulation loop develops, which is driven by overactivation of complement that causes tional Center for Biotechnology Information endothelial cells stimulation and lysis. Lysis leads to thrombi formation, with altered function dbSNP (build 137), aHUS mutation database of the coagulation pathway caused by genetic variants in coagulation genes, such as PLG, (www.fh-hus.org), or our in-house database. To PLAT (encoding tissue plasminogen activator, the main plasminogen activator), PLAU (en- assign pathogenicity, we combined multiple func- coding urokinase plasminogen activator, primarily responsible for pericellular plasmin acti- tional prediction methods, including PhyloP, vation), F12, ADAMTS13,andVWF. The consequence is enhanced formation or decreased degradation of thrombi, promoting thrombosis. Thrombi in small vessels cause mechanical SIFT, PolyPhen2, LRT, MutationTaster, and damage to erythrocytes releasing peptides (such as heme41) or overexpression of other GERP++. Prediction results for each variant unidentified factors that activate the complement system. Genetic variants in complement were integrated to generate a pathogenicity score, genes and/or acquired factors like CFH autoantibodies lead to inadequate/ineffective com- which was the sum of tools predicting the variant plement regulation. (3) This dysregulation loop induces the clinical onset of aHUS. to be deleterious. Missense nonsynonymous

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single nucleotide variants were considered likely deleterious when the hemolytic uremic syndrome. Proc Natl Acad Sci U S A 100: 12966– PS$4. Splicing site variants, nonsense mutations, and indels were not 12971, 2003 assigned a PS. 10. Noris M, Brioschi S, Caprioli J, Todeschini M, Bresin E, Porrati F, Gamba S, Remuzzi G; International Registry of Recurrent and Familial HUS/TTP: Familial haemolytic uraemic syndrome and an MCP mutation. Lancet Multiplex Ligation-Dependent Probe Amplification 362: 1542–1547, 2003 CNVs were evaluated over the contiguous CFH-CFHR genomic 11. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, Carreras L, region using multiplex ligation-dependent probe amplifica- Arranz EA, Garrido CA, López-Trascasa M, Sánchez-Corral P, Morgan tion. Probes were designed according to MRC-Holland’sguide- BP, Rodríguez de Córdoba S: Gain-of-function mutations in comple- ment factor B are associated with atypical hemolytic uremic syndrome. lines (MRC-Holland, Amsterdam, The Netherlands) or obtained Proc Natl Acad Sci U S A 104: 240–245, 2007 18,19,46,47 from the literature. CNVs were assigned by data com- 12. Frémeaux-Bacchi V, Miller EC, Liszewski MK, Strain L, Blouin J, Brown parison of normalized peak areas generated in five control AL, Moghal N, Kaplan BS, Weiss RA, Lhotta K, Kapur G, Mattoo T, Nivet samples.48,49 H, Wong W, Gie S, Hurault de Ligny B, Fischbach M, Gupta R, Hauhart R, Meunier V, Loirat C, Dragon-Durey MA, Fridman WH, Janssen BJ, Goodship TH, Atkinson JP: Mutations in complement C3 predispose to Statistical Analyses development of atypical hemolytic uremic syndrome. Blood 112: Data were analyzed using R (version 2.15.1). The Fisher exact test was 4948–4952, 2008 used to compare allelic frequencies in cases and controls. All tests were 13. Sartz L, Olin AI, Kristoffersson AC, Ståhl AL, Johansson ME, Westman K, two-tailed, and P values,0.05 were considered significant. Fremeaux-Bacchi V, Nilsson-Ekdahl K, Karpman D: A novel C3 mutation causing increased formation of the C3 convertase in familial atypical hemolytic uremic syndrome. JImmunol188: 2030–2037, 2012 ACKNOWLEDGMENTS 14. Bresin E, Rurali E, Caprioli J, Sanchez-Corral P, Fremeaux-Bacchi V, Rodriguez de Cordoba S, Pinto S, Goodship TH, Alberti M, Ribes D, This study was supported in part by the Foundation for Children with Valoti E, Remuzzi G, Noris M; European Working Party on Complement Genetics in Renal Diseases: Combined complement gene mutations in Atypical HUS. atypical hemolytic uremic syndrome influence clinical phenotype. JAm Soc Nephrol 24: 475–486, 2013 15. 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55. Jalanko H, Peltonen S, Koskinen A, Puntila J, Isoniemi H, Holmberg C, with the ‘Normandy’ variant of von Willebrand disease. Br J Haematol Pinomaki A, Armstrong E, Koivusalo A, Tukiainen E, Makisalo H, Saland 78: 506–514, 1991 J, Remuzzi G, de Cordoba S, Lassila R, Meri S, Jokiranta TS: Successful liver-kidney transplantation in two children with aHUS caused by a mutation in complement factor H. Am J Transplant 8: 216–221, 2008 56. Gaucher C, Mercier B, Jorieux S, Oufkir D, Mazurier C: Identification of This article contains supplemental material online at http://jasn.asnjournals. two point mutations in the von Willebrand factor gene of three families org/lookup/suppl/doi:10.1681/ASN.2013050453/-/DCSupplemental.

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