SAT-101 TWO CASES OF ATYPICAL HEMOLYTIC UREMIC SYNDROME ASSOCIATED WITH HYBRID GENES IN CFH/CFHR GENE CLUSTER CAUSED BY NOVEL GENOMIC RECOMBINATION

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a rare disease resulting from dysregulation of complement pathway. Variants in the gene encoding complement factor H (CFH), the key regulator of complement pathway, are most frequently reported as the causes of aHUS. So far, six kinds of hybrid genes in CFH/CFHR gene cluster have been identified in aHUS and all the hybrid genes reportedly contain some part of CFH. We have previously established the hemolytic assay by using sheep blood cells, which can detect complement abnormalities associated with CFH (Yoshida Y, et al. 2017 PLOS ONE). Through this assay, we have identified two patients whose plasma lysed sheep blood cells, but having neither anti-CFH antibodies nor causative variants in seven complement-related genes (CFH, CFI, CFB, C3, MCP, THBD and DGKE). Based on these findings, we speculate that these two patients have hybrid genes in CFH/CFHR gene cluster. Therefore, in this study, we aimed to analyze these patients focusing on CFH/CFHR gene cluster. Patient-1 presented with acute kidney failure requiring long-term hemodialysis at the age of 32. She received a kidney transplant at the age of 36 and developed severe thrombotic microangiopathy (TMA) attack on POD 2, however, eculizumab started on POD 36 improved her kidney function markedly. Patient-2 presented with typical features of TMA at the age of 55. He recovered gradually after the treatment of plasma exchange. For these two patients, Sanger sequencing and whole-exome sequencing were performed for the investigation of causative variants in seven complement genes (CFH, CFI, CFB, C3, MCP, THBD and DGKE). We also performed multiplex ligation-dependent probe analysis (MLPA), aiming to detect copy number variations in CFH/CFHR gene cluster. According to the result of MLPA, breakpoint analysis was thereafter performed to reveal hybrid genes. For these two patients, whose plasma lysed sheep blood cells, having neither anti-CFH antibodies nor causative variants in seven complement genes, we found unusual copy number variations. Patient-1 showed two types of unusual copy number variations: heterozygous deletion extending from CFH exon 23 to its downstream sequence and heterozygous duplication extending from CFHR1 exon 5 to 6. Patient-2 showed an unusual heterozygous deletion extending from CFH exon 23 to CFHR1 exon 5, which included the whole sequence of CFHR3. Breakpoint analyses revealed the site of rearrangements and both cases had hybrid genes made up of CFH and CFHR1. Genomic breakpoints we revealed in this study were novel. In this study, we have newly identified two cases of aHUS having hybrid genes in CFH/CFHR gene cluster. The hemolytic assay played an important role in screening aHUS patients with CFH-related abnormalities, which include hybrid genes in CFH/CFHR gene cluster. MLPA could detect this type of genetic abnormalities, while Sanger sequencing and whole-exome sequencing could not. Functional analysis, such as the hemolytic assay, and copy number variation analysis are proven to be useful methods for the diagnosis of aHUS associated with hybrid genes in CFH/CFHR gene cluster.