Anti-complement factor H (CFH) antibodies and a novel CFH gene mutation in an atypical hemolytic uremic syndrome patient with complement activation of the classical pathway

Abstract Atypical hemolytic uremic syndrome (aHUS) is a rare disease caused by overactivation of the complement alternative pathway. aHUS involves the presence of antibodies against complement factor H and its mutations in the complement genes. A 2-month-old boy presented with discoid rash, hemolytic anemia, thrombocytopenia, multiple antibodies, and hypocomplementemia with a very low level of C4 (< 3 mg/dL), indicating activation of the complement pathway, together fulfilling the systemic lupus erythematosus (SLE) criteria of the American College of Rheumatology at 5 months of age. However, most of these findings normalized spontaneously without any intervention. Further investigations revealed a high level of anti-complement factor H antibodies and a novel heterozygous missense mutation (p.Glu1172Ala, located in exon 22) in a complement gene, CFH. At 2 years of age, his SLE-like symptoms have not recurred, but hematuria and schistocytes were persistent. Eventually, aHUS was diagnosed rather than SLE. Our findings suggest that multiple antibody complex, including anti-complement factor H antibody, may temporarily activate the classical pathway, resulting in SLE-like findings.


Introduction
Atypical hemolytic uremic syndrome (aHUS) is a type of thrombotic microangiopathy (TMA) caused by overactivation of the complement alternative pathway. aHUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal dysfunction [1]. Approximately 10% of aHUS cases had antibodies against complement factor H (CFH) that regulates the alternative pathway [2]. Recent studies have shown that mutations of complement genes (such as CFHR1 and CFHR3) are strongly associated with the generation of anti-CFH antibodies [3,4]. However, the mechanism of action between complement gene mutation and the generation of anti-CFH antibodies has been poorly understood. Here we describe a patient with symptoms of aHUS, anti-CFH antibodies, and a novel missense CFH mutation.

Case report
The male patient was born after an uneventful pregnancy of 38 weeks, weighing 2,578 g. The family history was unremarkable and the parents were non-consanguineous. He was born in Japan. His mother was Thai, and his father was half Chinese and half Filipino. He had an aneurysmal malformation in the vein of Galen and received therapeutic embolization at 18 days of age. Symptomatic epilepsy after embolization was treated with carbamazepine for 2 months.
Complement genes of genomic DNA were analyzed by the Miseq Sequencing System (Illumina, San Diego, CA) at The Japanese Association for Complement Research. Genetic variants with rare allele frequency (< 0.005) were identified using the Exome Aggregation Consortium. The sequencing revealed a novel heterozygous missense mutation (p.Glu1172Ala in exon 22) in the CFH gene. Although this mutation was not listed in HGVD, p.Glu1172Ala was predicted by in silico analysis using PolyPhen-2, PROVEAN, and SIFT analyses as 'probably damaging', 'deleterious', and 'damaging',  respectively. Complement C4 gene copy number variation was measured using QuantStudio 3D Digital PCR System (Thermo Fisher Scientific). He had five copies of total C4 (reference range 2-8), two copies of C4A, and three copies of C4B, suggesting that his genetic status of the C4 gene did not reduce the C4 level. Based on the combination of microangiopathic hemolytic anemia, thrombocytopenia, acute kidney injury, high level of anti-CFH IgG, and mutation in the CFH gene, his condition was diagnosed as aHUS, rather than SLE. Thereafter, symptoms of aHUS or SLE have not recurred for 2 years, except for persistent hematuria and schistocytes.

Discussion
It is often difficult to clarify the etiology of TMA. This case presented aHUS symptoms with anti-CFH antibodies and a novel CFH mutation. Typically, the alternative pathway is activated in aHUS patients. In this case, multiple autoantibodies may have been involved in the activation of the classical pathway and the SLE-like symptoms.
In this case, symptoms of TMA were speculated to occur secondarily to SLE at first. However, his condition recovered without any intervention. Complement levels that were low at 5 months old normalized at 11 months old, accompanying a decrease in multiple autoantibody levels. In contrast, hematuria and schistocytes persisted. This clinical course was atypical for SLE. Further studies revealed an exceedingly high level of anti-CFH antibodies and a heterozygous mutation in the CFH gene. CFH is a major regulatory protein of the complement alternative pathway and is composed of 20 short consensus repeats (SCRs). Two major functional regions are located at the N-terminal SCRs 1-4 and C-terminal SCRs 19-20. N-terminal SCRs mediates regulatory activities of decay of the C3 convertase and cofactor. C-terminal SCRs includes binding sites for ligands such as C3b, C3d, and glycosaminoglycan, and mediates surface recognition leading inhibition of complement activation. Acquired autoantibodies against CFH have been described in about 10% of aHUS cases [4][5][6]. Anti-CFH antibodies induce functional CFH deficiency by binding to its C-terminal region and thereby reducing its regulatory function [2][3][4].
In a different pattern from typical aHUS cases, this case showed the activation of the classical pathway and SLE-like symptoms, which may be caused by immune complexes of multiple autoantibodies that bind the C1q, leading to type 3 hypersensitivity, and consequently SLE-like symptoms, similar to serum disease or type 3 allergy. In fact, the levels of anti-CFH antibody in this patient were higher than those in the previous cases [7,8]. Although we did not measure C1q level and immune complexes binding to C1q, his C4 and C3 levels decreased together and he had multiple autoantibodies, suggesting the activation of the classical pathway rather than the alternative or lectin pathways. The cause of antibodies generated in this patient was unclear. Although, to our knowledge, no studies have examined the association of specific pathogen and anti-CFH antibody, respiratory syncytial virus infection might have induced to generate multiple autoantibodies, and consequently activated the classical pathway. There is also a possibility that therapeutic embolization causes the autoimmune-like phenomenon. However, to our knowledge, there is no report of this phenomenon in patients with aneurysmal malformation in the vein of Galen. Besides, the same phenomenon did not occur again even though he underwent therapeutic embolization seven times.
Several causative genetic variants for aHUS have been identified. Approximately 50% of aHUS patients have loss-of-function variants in complement regulatory genes (CFH, MCP, CFI) or gain-offunction variants of complement factors (C3, CFB). In our patient, a single novel missense mutation, p.Glu1172Ala in the CFH gene, was found in a heterozygous state. No other mutations were found in complement genes. The majority of the reported CFH mutations are heterozygous likely pathogenic variants [9]. Previous reports have shown that only 9.2% of aHUS patients with CFH mutation carried abnormalities in other complement genes, suggesting that a mutation in the CFH gene alone may be sufficient to cause aHUS [9]. In addition, in silico analysis algorithms suggested the pathogenicity of the mutation. This mutation (exon 22 of CFH gene) is located in the major functional region C-terminal SCR 20, which is the recognition region and binding sites for the surface of endothelial cells. The exon 20-22 of the CFH gene is known as a hotspot for mutations in aHUS [10]. Therefore, the heterozygous CFH mutation in our case may be a cause of aHUS. Further studies are needed to elucidate this association.

Conclusions
We found anti-CFH antibodies and a novel CFH mutation (p.Glu1172Ala) in an aHUS patient with temporary activation of the classical complement pathway. Our findings suggest that high levels of anti-CFH antibodies may activate the classical pathway, resulting in SLE-like symptoms.