A novel hybrid CFHR1/CFH gene causes atypical hemolytic uremic syndrome

Abstract

Background: Mutations in complement factor H (CFH) are associated with complement dysregulation and the development of an aggressive form of atypical hemolytic uremic syndrome (aHUS) that progresses to end-stage renal disease (ESRD) and in most patients has a high rate of recurrence following transplantation. Sequence analysis of CFH and its downstream complement factor H-related genes (CFHR1-5) reveals several macrohomologous blocks caused by large genomic duplications. This high degree of sequence identity renders this area susceptible to nonallelic homologous recombination (NAHR) events, resulting in large-scale deletions, duplications, and the generation of hybrid CFH genes.

Case-diagnosis: Here, we report the finding of a novel CFHR1/CFH hybrid gene created by a de novo NAHR event in a 14-year-old girl with aHUS. The resulting fusion protein contains the first three short consensus repeats (SCRs) of CFHR1 and the terminal two SCRs of CFH.

Conclusions: This finding demonstrates a novel pathogenic mechanism for the development of aHUS. Additionally, since standard Sanger sequencing is unable to detect such rearrangements, all aHUS patients should receive comprehensive genetic screening that includes analysis of copy number variation in order to identify patients with poor clinical prognoses.

Figure 1. A novel hybrid CFHR1/CFH gene

a) MLPA determination of copy number over the CFH-CFHR region identified a duplication beginning with CFH exon 21 (CFH SCR 19) and ending after CFHR1 exon 4 (CFHR1 SCR 3). MLPA probes are represented by arrows and SCRs by ovals. Red arrows indicate duplicated probes. The breakpoint sequence was localized to a 29 bp sequence between intron 4 of CFHR1 and intron 20 of CFH. SNP differences (circled) between CFH and CFHR1 defined the exact location of the breakpoint. SCRs are represented by ovals. b) The breakpoint sequence was localized to a 29 bp sequence between intron 4 of CFHR1 and intron 20 of CFH. SNP differences (circled) between CFH and CFHR1 defined the exact location of the breakpoint. c) The novel hybrid CFHR1/CFH gene originates from NAHR between homologous blocks B and B′. The resulting fusion protein comprises SCRs 1-3 of CFHR1 and SCRs 19-20 of CFH. The direction of the recombination is indicated by black arrows. d) Diagnostic PCR utilizing a common forward primer (F) in CFHR1 intron 4, a reverse primer (R) in CFHR1 intron 4, and a reverse primer in CFH intron 20 (R′) in the same reaction resulted in both a 593-bp breakpoint and a 821-bp wild-type (WT) product in our patient. The 593-bp breakpoint fragment was not seen in the mother (M), father (F), or in a control patient (Ctrl). e) Western blot using a polyclonal rabbit anti-CFHR1 antibody against sera from the patient (P) and controls (C0-C3) with known genetic copy number variations in CFHR1 (C0 = zero copies of CFHR1, C1 = one copy, C2 = two copies, and C3 = three copies). The presence of an additional CFHR1/CFH fusion protein (*) accounts for the increased band density seen in the patient. Differential glycosylation of CFHR1 SCRs 2 and 3 accounts for the two different band sizes at 37 and 43 kDa. CFH (~150 kDa) was used as a loading control.