A De Novo Deletion in the Regulators of Complement

BRIEF COMMUNICATION www.jasn.org A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H–Related 3 Gene in Atypical Hemolytic Uremic Syndrome † Rachel C. Challis,* Geisilaine S.R. Araujo,* Edwin K.S. Wong,* Holly E. Anderson,* Atif Awan, ‡ † Anthony M. Dorman, Mary Waldron, Valerie Wilson,* Vicky Brocklebank,* Lisa Strain,* | B. Paul Morgan,§ Claire L. Harris,§ Kevin J. Marchbank, Timothy H.J. Goodship,* and David Kavanagh* *Institutes of Genetic Medicine and |Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom; †Department of Nephrology, Our Lady’s Children’s Hospital, Crumlin, Dublin; ‡Department of Renal Pathology, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin, Ireland; and §Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, United Kingdom ABSTRACT The regulators of complement activation cluster at chromosome 1q32 contains the to arise through microhomology– complement factor H (CFH)andfive complement factor H–related (CFHR)genes. mediated end joining (MMEJ)11 (Supple- This area of the genome arose from several large genomic duplications, and these mental Figure 1). These genetic variants low-copy repeats can cause genome instability in this region. Genomic disorders have all been shown to impair C3b/host affecting these genes have been described in atypical hemolytic uremic syndrome, polyanion binding on host cells, thus im- arising commonly through nonallelic homologous recombination. We describe a pairing local complement regulation.5,8,11 novel CFH/CFHR3 hybrid gene secondary to a de novo 6.3-kb deletion that arose Recently, a reverse CFHR1/CFH through microhomology–mediated end joining rather than nonallelic homologous hybrid gene arising through nonallelic recombination. We confirmed a transcript from this hybrid gene and showed a se- homologous recombination has been creted protein product that lacks the recognitiondomainoffactorHandexhibits described in aHUS. This encodes an impaired cell surface complement regulation. The fact that the formation of this FHR1, where the C-terminal CCPs are hybrid gene arose as a de novo event suggests that this cluster is a dynamic area of replaced by the C-terminal CCPs of the genome in which additional genomic disorders may arise. FH.12,13 Unlike previously reported hy- J Am Soc Nephrol 27: 1617–1624, 2016. doi: 10.1681/ASN.2015010100 brid proteins, this does not impair FH cell surface binding but instead, acts as a competitive inhibitor of FH.13 The complement factor H (CFH)and majority of mutations in CFH occur at the complement factor H–related (CFHR1– C-terminal end responsible for host poly- CFHR5) genes reside in a 360-kb region anion binding.5–7 Several of these CFH in the regulators of complement activa- Received January 27, 2015. Accepted September mutations (e.g., S1191L and V1197A) 7, 2015. tion (RCA) cluster on chromosome have been shown to be gene conversion 1q32 (Figure 1A).1 This is an area of events between CFH and CFHR1.8 Non- R.C.C., G.S.R.A., and E.K.S.W. contributed equally to this work. the genome that arose from several large allelic homologous recombination involv- genomic duplications,2,3 and these low- ing CFH and CFHR1 can result in CFH/ Published online ahead of print. Publication date available at www.jasn.org. copy repeats can cause genome instabil- CFHR1 hybrid genes (Supplemental Fig- ity in this region. ure 1).9,10 A single family with a hybrid Correspondence: Dr.DavidKavanagh,Instituteof Genetic Medicine, International Centre for Life, Mutations in CFH are the most com- CFH/CFHR3 gene (factor H [FH] protein Central Parkway, Newcastle upon Tyne, NE1 3BZ, mon genetic predispositions to the complement control protein modules UK. Email: [email protected] – – thrombotic microangiopathy: atypical he- [CCPs] 1 19 and factor H related 3 Copyright © 2016 by the American Society of molytic uremic syndrome (aHUS).4 The [FHR3] CCP1–CCP5) has been reported Nephrology J Am Soc Nephrol 27: 1617–1624, 2016 ISSN : 1046-6673/2706-1617 1617 BRIEF COMMUNICATION www.jasn.org Figure 1. A 6.3-kb deletion in the RCA cluster results in a novel CFH/CFHR3 hybrid gene transcript. (A) Genomic organization of the RCA cluster region containing the CFH and CFHR genes. (B) MLPA of CFH, CFHR3, CFHR1, CFHR2,andCFHR5 showing the copy number ratio. The dotted lines represent ratios considered within the normal range. (C) Identification of breakpoint. Genomic DNA was amplified 1618 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 1617–1624, 2016 www.jasn.org BRIEF COMMUNICATION In this study, we report a novel CFH/ and DGKE) did not reveal any muta- degradation product. No abnormal bands CFHR3 hybrid gene arising through tions, although heterozygosity was found were detected in either parent (Supple- MMEJ that impairs cell surface comple- for the common Y402H polymorphism mental Figure 4A). An FH C terminus– ment regulation. (rs1061170) in CFH. Multiplex ligation– specific antibody (L20/3) did not reveal An 8-month-old boy presented with a dependent probe amplification (MLPA) additional aberrant bands, indicating that diarrheal illness; on admission, creati- of CFH and the CFHRs revealed a dele- these protein species lacked FH CCP20 nine was 52 mmol/L, and urea was tion in the CFH gene (Figure 1B). To de- (Figure 3C). Genotyping showed the pa- 11.1 mmol/L. A blood film showed mi- fine the extent of the deletion and the tient to be heterozygous for the Y402H croangiopathic hemolytic anemia, and breakpoint, Sanger sequencing of geno- polymorphism in FH. This allowed the lactate dehydrogenase was 5747 units/L. mic DNAwas undertaken. This showed a use of mAbs specific for histidine or Stool culture was positive for Escherichia 6.3-kb deletion extending from CFH in- tyrosine in CCP7 of FH.14 The normal coli O157:H7, and a diagnosis of Shiga tron 20 to the CFH 39 intergenic region allele’s product reacted to the histidine- toxin–producing E. coli (STEC) hemo- incorporating exons 21–23 of CFH (Fig- specific mAb (MBI7) and gave a single lyticuremicsyndrome(HUS)was ure 1C). Directly flanking the breakpoint band of approximately 150 kD, consis- made. He did not require RRT and did was a 7-bp region of microhomology tent with FH. The hybrid allele reacted not receive plasma exchange. He was (Figure 1D). The deletion was not de- to the tyrosine-specificmAb(MBI6), discharged 2 weeks later with a creati- tected in either parent, suggesting that showingadoubletatapproximately nine of 107 mmol/L. He was readmitted this was a de novo event. 160 kD (Figure 3D). FHR3 is known to 2 weeks later with an upper respira- We hypothesized that the loss of CFH be alternately glycosylated,15–17 and we tory tract infection. Creatinine on read- exons 21–23 and the 39 UTR of CFH hypothesize that the doublet is caused mission was 141 mmol/L, urea was would lead to aberrant splicing. In silico by an alternately glycosylated hybrid pro- 20.6 mmol/L, lactate dehydrogenase analysis showed that the next acceptor tein. Additionally, it can be seen that the was 2398 units/L, and platelets were splice site following the CFH exon 20 presumed degradation product at 120 kD 1283109/L, with evidence of microan- donor site was 59 of CFHR3 exon 2, is only seen using the tyrosine-specific giopathic hemolytic anemia on blood thus potentially producing a transcript mAb, consistent with this only arising film. This relapse suggested a diagnosis for a hybrid CFH/CFHR3 gene (Supple- from the hybrid protein. of aHUS rather than STEC HUS. Serum mental Figure 3). mRNA for this puta- To confirm these findings, FH species complement levels were within the nor- tive hybrid CFH/CFHR3genewas were purified from serum using affinity mal range: C3, 1.05 g/L (0.68–1.80 g/L); confirmed by amplifying patient and chromatography with an OX24 mAb. C4, 0.30 g/L (0.18–0.60 g/L); and FH, control cDNA with CFH– and CFHR3– These were separated bya 6% SDS-PAGE 0.51 g/L (0.35–0.59 g/L). He com- specific primers. This showed a product before trypsin digestion. Mass spec- menced plasma exchange and required in the patient and not in healthy controls trometrywasthencarriedoutonthese three sessions of hemofiltration. A renal (Figure 1E). three purified bands (Figure 4A). Pep- biopsy confirmed a thrombotic micro- To confirm that this hybrid transcript tide species identified from the approx- angiopathy (Figure 2). Creatinine re- led to the synthesis and secretion of a imately 160-kD band confirmed that turned to a baseline of approximately hybrid protein, Western blotting was this was a hybrid FH/FHR3 protein. 100 mmol/L. He was treated with weekly performed using a series of epitope– Protein fragments from the 150-kD plasma exchange for 3.5 years before defined anti–FH mAbs (Figure 3A). An band were consistent with FH. Frag- starting Eculizumab, on which he has initial blot probed with OX24, an FH ments from CCP5, CCP6, CCP8, been maintained for 3 years. Creatinine CCP5–specific antibody, showed, in ad- CCP9, and CCP14 of FH were seen in is currently 200 mmol/L, and there have dition to FH, two additional bands only the band at approximately 120 kD. Cov- been no additional episodes of aHUS in the patient (Figure 3B). The species at erage at CCP7 was insufficient to deter- while on treatment with Eculizumab approximately 160 kD was consistent mine whether there was a tyrosine or (Supplemental Figure 2). with the predicted intact hybrid histidine at position 402. The Western Sanger sequencing of aHUS-associated FH/FHR3. A species at approximately blot analysis and mass spectrometry genes (CFH, CFI, CFB, CD46, C3, THBD, 120kDwashypothesizedtobea data together show that breakdown using a forward primer specificforCFH exon 20 (1f) and a reverse primer in the CFH 39 region intergenic region (1r) and sequenced.

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