Thrombotic microangiopathies (TMAs) are a heterogenous group of disorders characterized by thrombocytopenia, microangiopathic hemolytic anemia (MAHA), and end-organ damage. Atypical hemolytic uremic syndrome (aHUS), more precisely termed complement-mediated hemolytic uremic syndrome (CM-HUS), is a rare, incidence ranging between 0.23 – 1.9 per million population per year, but potentially life-threatening condition (1). Incidence and prevalence vary based on region and age (1).Unlike Shiga toxin-associated HUS and immune thrombotic thrombocytopenic purpura (iTTP), CM-HUS is primarily driven by dysregulation of the alternative complement pathway, resulting in uncontrolled activation, excessive inflammation, and endothelial injury.

In this review, we summarize current knowledge on the role of complement proteins and autoantibodies in the pathogenesis, diagnosis, and treatment of CM-HUS.

The cases demonstrate two patients with a shared underlying disease process but distinct clinical presentation and age at onset.

Patients with CM-HUS typically present with MAHA, thrombocytopenia, and end-organ damage, often manifesting as acute kidney injury (AKI). Compared to other forms of TMA, CM-HUS is characterized by more severe renal involvement, often progressing to end-stage kidney disease (ESKD) if untreated. Unlike iTTP, CM-HUS is less responsive to plasma exchange, and renal deterioration may continue despite intervention (2, 3). Hypertension is a common clinical feature, and distinguishing CM-HUS from hypertensive emergency can be challenging.

Extra-renal manifestations, including neurological symptoms such as seizures, confusion, and encephalopathy, as well as gastrointestinal involvement such as hepatotoxicity, pancreatitis, and gastrointestinal bleeding, are less common but have been reported (3).

In both cases presented, patients had thrombocytopenia, MAHA, and end-organ damage in the form of kidney injury. Case 2 also exhibited extra-renal manifestations, including altered mental status.

The diagnosis of CM-HUS is one of exclusion. The initial step involves ruling out other potential causes of TMA, including Shiga toxin-associated HUS, TTP (ruled out by demonstrating ADAMTS13 activity >10%), and TMAs secondary to conditions such as medications, malignant hypertension, autoimmune disease, transplant, or pregnancy. If no alternative etiology is identified, a working diagnosis of CM-HUS should be pursued and treatment with complement inhibitor therapy should be initiated. Laboratory evaluation aims to detect complement abnormalities that support the clinical suspicion of CM-HUS. Testing options include complement serology, autoantibody assessment, evaluation of complement activation on cell membranes, and complement genetic analysis (4).

Below we describe the complement abnormalities associated with CM-HUS and outline how laboratory tests evaluate these abnormalities to support the clinical diagnosis.

The pathophysiologic mechanism underlying CM-HUS involves the dysregulation of the amplification loop of the alternative pathway leading to unregulated formation of the the membrane attack complex, and subsequent tissue injury (Figure 1). CM-HUS is a genetic disorder that may occur in either familial or sporadic forms. The first gene linked to CM-HUS, CFH, was identified in 1998. Since then, variants in several additional complement-related genes have been implicated. Most pathogenic variants are heterozygous, and approximately 20% of patients have a combination of mutations (5). A key challenge in CM-HUS diagnosis is that only 40-50% of affected individuals have identifiable pathogenic variants in complement-regulating proteins.

Complement gene variants implicated in CM-HUS include gain-of-function variants in complement activators or loss-of-function variants in complement regulators (Figure 2). More than 120 variants in CFH, CFI, and CD46 (MCP) have been reported among patients with CM-HUS (6).

In addition to pathogenic complement gene variants, other genetic features that can modify risk are haplotypes, which are a specific combination of alleles at adjacent loci along the same chromosome that are inherited together as a unit (7). In CM-HUS, haplotypes on their own are not sufficiently pathogenic to cause disease on their own, but when they occur alongside pathogenic variants they can increase the risk of developing the disease or manifestations of the disease (ie. end stage renal disease) (8, 9).

CM-HUS is not a monogenic disorder as patients may carry multiple disease-associated variants or risk haplotypes. Although CM-HUS is considered a genetic disease, due to incomplete penetrance, the presence of genetic variants alone is insufficient to cause clinical TMA. Instead, at least two “hits” are generally required. The second hit typically involves an environmental trigger. In a cohort of 272 patients, a triggering event was identified in 70% of cases, most commonly diarrheal or gastrointestinal illnesses and upper respiratory tract infections (6). Pregnancy is another, though less common, trigger.

Depending on the genetic variant(s) identified, the risk of developing CM-HUS, as well as clinical manifestations and outcome, may vary (10, 11). For instance, individuals with CFH or CFI variants are more likely to present before one year of age (12). Below (Table 1), we summarize the current understanding of the most commonly implicated genes and proteins in CM-HUS.

Complement genetic testing utilizes next-generation sequencing (NGS) to perform full gene sequencing and copy number analysis of multiple genes related to the complement system. Sanger sequencing and multiplex ligation–dependent probe amplification (MLPA) may also be performed to confirm findings and evaluate for large deletions (2, 53).

Several centers offer comprehensive complement genetic testing, and the gene panels and turnaround times vary. Multi-gene NGS panels commonly include CFH, CFI, CD46 (also known as MCP), CFB, C3, and CFHR1-5. Often, panels may also include non-complement genes, such as DGKE and coagulation-related genes. Additionally, ADAMTS13 may be included due to its association with TTP, which is often in the differential diagnosis. Some panels include the specific C5 variants that have been associated with eculizumab resistance (30). The most well characterized of these variants are p.Arg885His and p.Arg885Cys polymorphisms. These variants alter the eculizumab binding site on C5 which then prevents complement inhibition with eculizumab necessitating the use of medications that target alternative epitopes of C5 or other complement targets (30).

Laboratories typically use the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines for variant classification. Clinical classification of genetic variants in CM-HUS remains challenging from a laboratory standpoint for various reasons. The ACMG/AMP criteria were not intended to be used to interpret variants in genes associated with non-Mendelian or multigenic disorders; however, due to a lack of alternative guidelines, the ACMG/AMP has typically been adopted for variant classification in CM-HUS (54). The low penetrance of CM-HUS makes it difficult to establish a minor allele frequency cut-off to identify variants more common in the general population than expected for the disease. Assessment of segregation in family members is not reliable because not all individuals may be exposed to the second trigger. In addition, an individual may have a pathogenic variant but might never develop CM-HUS (55). Many patients will not have pathogenic variants and will present with variants of uncertain significance (VUS). For rare disorders with low penetrance, such as aHUS, it can be difficult to find sufficient evidence to reach a classification of benign/likely benign or pathogenic/likely pathogenic. In a research setting, the variant protein corresponding to genetic variants identified in patients can be expressed in vitro, followed by functional studies to characterize the variant protein’s impact on the alternative pathway (16, 56–58). For example, if a complement variant leads to the non-expression of the carrying allele, this would represent strong evidence of pathogenicity. Other useful information to help guide variant classification includes prevalence data of the respective variant (ie demonstrating that the prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls) (54). When a functional study is performed after a variant has been classified, or when other information emerges (e.g. additional reports of affected individuals with the same variant), a variant classification can be updated, such as to re-classify a VUS as likely benign or likely pathogenic.

Given the nuances of variant classification and genetic test interpretation, it is important for clinical laboratories performing this test to have a deep understanding of this disorder. Paired clinical and genetic data are essential for accurate variant interpretation and meaningful clinical correlation. As the tools available for genetic testing and interpretation become more robust and more data regarding complement variants in CM-HUS emerges previously published incidences of pathogenic variants in CM-HUS will need to be reassessed (16, 22). Due to the complexity of complement genetics and variable penetrance, genetic counseling should be offered to individuals with CM-HUS.

The first published report of Factor H antibodies in CM-HUS in 2005 was a description of three pediatric patients (59). Since then, FH antibodies have become the best-studied acquired drivers of CM- HUS, detected in approximately 24% of pediatric and 19% of adult CM-HUS cases (60, 61). FH antibodies are primarily of the IgG class, and the majority bind to the C-terminal region of FH, impairing its function (59, 62, 63). The C-terminus of FH is needed for FH to be docked onto C3b-covered cells or surfaces, which is a critical step in complement regulation.

FH antibodies are strongly associated with deficiency of FH-related protein 1 (FHR1). Ninety percent of patients with FH antibodies have complete deficiency of the FH-related proteins FHR1 and FHR3 due to a homozygous combined CFHR3-CFHR1 deletion. However, in about 15% of patients, FHR1 deficiency is caused by compound heterozygous deletion of CFHR3-CFHR1 and CFHR1-CFHR4 (64). Homozygous CFHR1-CFHR4 deletion has also been rarely reported (65). In some patients, a heterozygous deletion of CFHR1, with or without accompanying deletions in CFHR3 or CFHR4, may be paired with a nonsense or frameshift variant on the other allele. If this combination results in complete absence of CFHR1 protein production, the functional effect may mimic a homozygous deletion (Mayo Clinic, unpublished data). However, this scenario remains poorly characterized, and its clinical significance is not yet fully understood (To date, there have been no reports of Factor H antibodies with this rare clinical situation).

Not all patients with homozygous CFHR1 and CFHR3 gene deletion have FH antibodies. In fact, the relative risk for a patient with CFHR3-CFHR1 deletion developing an FH antibody is small, as homozygous CFHR3-CFHR1 deletion is common and present in about 3% of Americans of European descent (66). Rates of CFHR3-CFHR1 deletions vary among populations, as do rates of FH antibodies. European CM-HUS cohorts report FH antibody frequencies of 5-25%, while cohorts in children in India report frequencies in the 50% range (67). Interestingly, the population frequency of the CFHR1 deletion in healthy cohorts in India was similar to those reported in other studies, suggesting unrecognized mechanisms for the high prevalence of FH-antibody-mediated CM-HUS in Indian children.

Ten percent of patients with FH antibodies do not have a deletion in CFHR1 (65). In some instances, likely pathogenic variants in CFH, THBD, and C3 are enriched in patients who develop FH antibodies (64). A meta-analysis of 19 studies including 384 patients demonstrated a pooled prevalence of pathogenic or likely pathogenic variants of 3% in anti-factor H CM-HUS (68).

Patients with anti-FH CM-HUS are typically younger than those with non-anti-FH CM-HUS (2, 61, 64).

The course of disease presentation is generally more acute with diarrhea and/or fever reported in >50% of cases suggesting that antibody-mediated HUS may be triggered by infection (68, 69). Individuals with high antibody titers at presentation (>8000 U/ml, normal threshold 150 AU/mL) are associated with higher mortality and long-term risk of renal disease (61).

Autoantibodies for Factor I have been described in the setting of CM-HUS but not in C3G. Currently there is no consensus on whether antibodies to FI are definitely implicated in the pathogenesis of CM-HUS. They are exceedingly rare, and thought to form complexes with Factor I, however this seems to have limited impact on complement function. It is still poorly understood how antibodies to FI contribute to disease development, and other susceptibility genetic variants may need to be present (e.g. CFH) (70).

The patient in case 1 had subsequent blood antibody testing that was positive for Salmonella typhi, suggesting an infectious trigger to her acute episode.

Effective management of CM-HUS requires a multidisciplinary approach, with coordinated input from hematologists, nephrologists, pathologists, and geneticists to guide diagnosis and treatment.

Management of CM-HUS is guided by the underlying pathophysiology. In cases of non-antibody-associated CM-HUS treatment is C5 inhibitor therapy (Figure 3) while in FH-antibody-associated- CM-HUS, the goal is antibody elimination. There are two FDA-approved C5 inhibitors for pediatric and adult patients with CM-HUS: eculizumab and ravulizumab. Prior to complement inhibitor therapy, management was supportive care with or without plasma exchange (PEX). Response to PEX is variable and dependent on which complement protein/genetic variant is implicated and, in many instances, can be suboptimal. In patients with MCP variants, plasma-based therapy is not thought to have a great impact on CM-HUS outcome; one potential explanation being that since MCP is a membrane-bound protein, plasma infusion or exchange would not fix the defect. In contrast, patients with CFH variants treated with plasma may have a response, but not consistently (20). In contrast, patients with Factor H antibodies do respond to PEX.

Eculizumab, approved in 2011, is a humanized recombinant monoclonal IgG antibody that binds to and inhibits C5. It effectively treats CM-HUS and has the potential to rescue renal function. Since eculizumab’s approval, real-world data have continued to demonstrate its efficacy. The largest single retrospective comparative cohort study reported efficacy data of patients treated with eculizumab (n=192) compared to a historical control cohort (n=279) from before availability of eculizumab. The 5-year cumulative estimate of ESKD-free survival was 39.5% in the control cohort and 85.5% in the eculizumab-treated cohort with a mutation or Factor H autoantibodies (p=0.000) (23). Prognosis varied according to mutation type in the control cohort. Five-year ESKD-free survival was worse in those with CFH, CFHR1:CFH hybrid, CFI, and C3 variants compared to those with CD46 variants or FHAA. For those with a CFH, CFI, and C3 mutation, ESKD-free survival was significantly better in the eculizumab-treated group, while individuals with a CD46 mutation did not have improved ESKD-free survival in the eculizumab-treated group. There was also no statistically significant difference in ESKD-free survival in individuals with FH-antibody-mediated CM-HUS.

Ravulizumab, FDA approved in 2019, was engineered from eculizumab to have extended complement inhibition with a 4 amino acid difference in its heavy chain. In a phase 3 trial, complete CM-HUS response was observed in 53.5% of patients treated with ravulizumab (median time to response of 86 days (81). There has been no head-to-head comparison between eculizumab and ravulizumab, but comparison studies of patients within the clinical trials have been performed. One study used propensity scoring to compare patient-level data from four eculizumab clinical trials (n=80) and two ravulizumab clinical trials (n=79). At 26 weeks, renal dysfunction, hematologic parameters, and dialysis prevalence were improved with both eculizumab and ravulizumab. Fatigue and quality of life were also improved. There were no statistically significant differences in outcomes between the two different complement inhibitors; however, confidence intervals were wide (82).

Efforts to standardize variant classification in multigenic and incompletely penetrant disorders such as CM-HUS are ongoing and critically needed. While the ACMG/AMP guidelines provide a foundational framework for variant interpretation, they were originally designed for Mendelian disorders and may not fully capture the complexity of oligogenic and multifactorial diseases. Initiatives from different groups have begun to address these gaps. In complement-mediated diseases, statistical modeling and large-scale functional assays have helped reclassify variants in genes such as CFH, moving the field closer to more accurate, consistent, and clinically meaningful genetic interpretation. These developments will help improve genotype-phenotype correlation in CM-HUS (16, 90–92). In addition, as larger data sets have become available it may be possible to begin to understand the interplay among common variants that may modestly impact protein function but together, along with environmental factors, contribute to disease presentation even in the absence of rare pathogenic variants.

CM-HUS reflects the evolving intersection of complement dysregulation, genetic predisposition, and clinical variability. C5 inhibitors have changed the natural history of the disorder, however many questions regarding optimal duration and use in the setting of renal transplant remain. The growing recognition of overlapping genetic variants, incomplete penetrance, and antibody-mediated phenotypes continues to challenge traditional frameworks of diagnosis and treatment and push us toward more individualized strategies. As diagnostics improve and real-world data expand, we are moving towards greater precision, matching the right treatment strategy to each patient, guided by molecular insight and clinical judgement.