Atypical Hemolytic Uremic Syndrome (aHUS), C3 glomerulopathy (C3G) and diseases linked to abnormalities of the complement system

In patients with thrombotic microangiopathy (TMA) or glomerulonephritis in whom a complement-mediated disease is suspected, studies performed at Secugen provide a detailed genetic and functional characterisation of the complement system. This information supports the diagnosis of complement-mediated aHUS and C3G, helps to estimate the risk of post-transplant recurrence and provides relevant data for therapeutic management and counselling of family members. The results are integrated into interpretative reports oriented towards clinical practice and must always be evaluated in conjunction with the available clinical, laboratory and histological information.

Atypical haemolytic uraemic syndrome (aHUS) is a thrombotic microangiopathy (TMA) mediated by complement, within a group of clinical entities that share microangiopathic haemolytic anaemia, thrombocytopenia and variable organ damage, mainly renal. Classical TMAs include thrombotic thrombocytopenic purpura (TTP) and typical haemolytic uraemic syndrome (HUS-STEC); aHUS represents the form of TMA associated with dysregulated activation of the alternative complement pathway, while other diseases such as C3 glomerulopathy (C3G) and paroxysmal nocturnal haemoglobinuria (PNH) also fall within the broad spectrum of complement-mediated disorders, often with pathophysiological and clinical overlap.

aHUS is a rare disease with an incidence that has increased over recent years and has become one of the most frequent causes of acute kidney injury in certain age groups, particularly in children without other obvious risk factors. Unlike typical HUS, which is associated with Shiga-toxin-producing Escherichia coli infection, aHUS is not related to this type of infection and is considered an atypical form of haemolytic uraemic syndrome, with a more guarded prognosis and significantly higher rates of recurrence and progression to end-stage renal disease (ESRD).

C3 glomerulopathy (C3G) is a form of glomerulonephritis characterised by intense and predominant C3 deposits on immunofluorescence, with little or no immunoglobulin staining, and a membranoproliferative-type histology or related proliferative patterns. Clinically, it presents with proteinuria, macroscopic or microscopic haematuria, oedema and a decline in renal function, and may progress to chronic kidney disease. Its pathogenesis is linked to abnormal activation of the complement system in the fluid phase, with accumulation of C3 breakdown products in glomerular capillaries, and a documented role for mutations in complement genes (CFH, CFI, MCP, CFB, C3) and regulatory autoantibodies (such as anti-factor H) in a subset of patients.

There may be a pathophysiological continuum between aHUS and C3G, with some patients carrying complement gene mutations who can develop, at different times, both TMA episodes and C3G-type lesions. This highlights the need for accurate differential diagnosis and for an integrated approach that includes both genetic and functional assessment of the complement system.

Genetics, complement and pathophysiology of aHUS and C3G

aHUS is a polygenic and multifactorial disease with a clear genetic component. Mutations and polymorphisms in genes encoding regulators of the alternative complement pathway – such as factor H (CFH), membrane cofactor protein (MCP or CD46), factor I (CFI) and factor B (CFB) – predispose to uncontrolled complement activation on the endothelium of the renal microvasculature. This activation leads to endothelial injury, formation of microthrombi in arterioles and capillaries and, ultimately, the acute renal dysfunction that typifies the syndrome.

In addition to these genetic risk factors, there are environmental triggers (bacterial or viral infections, certain drugs such as immunosuppressants, oral contraceptives or antineoplastic agents) that can precipitate disease in genetically predisposed individuals. Genetic protective factors have also been described, and their assessment enables a more accurate estimation of an individual’s risk of developing aHUS.

Age at onset and clinical course are highly heterogeneous, reflecting the underlying genetic diversity. Patients with CFH mutations tend to present earlier and with more severe disease, whereas those with MCP mutations often have somewhat milder courses, and those with CFI or CFB mutations frequently display severe forms with a high rate of progression to ESRD. This genetic profile also influences post-transplant prognosis, as the probability of aHUS recurrence is very high in patients with CFH, CFI or CFB mutations, while it is much lower in those with MCP mutations.

In C3G, genetic studies show that a proportion of patients carry alterations in the same complement genes implicated in aHUS (CFH, CFI, MCP, CFB, C3), as well as anti-factor H autoantibodies and variants in the CFH/CFHR1-5 cluster, which modulate the stability of regulatory proteins and activation of the alternative pathway. This genetic overlap supports the concept of a shared “complementopathy” spectrum, in which the same genetic alteration may manifest clinically as aHUS, C3G or even mixed forms, depending on the interaction with other factors (immunological, environmental, thrombotic).

Secugen: reference laboratory for complement-mediated diseases

Secugen S.L. is a diagnostic laboratory specialised in molecular genetics of the complement system, with accumulated experience since 2008 and the use of next-generation sequencing (NGS) technologies since 2010. We have analysed more than 2,000 samples related to complement-mediated diseases for national and international hospitals and have provided our services to leading pharmaceutical companies working in the field of complement-targeted therapies.

Our approach combines targeted NGS by hybridisation capture on Illumina MiSeq platforms, with read lengths of at least 250 nucleotides, which allows more accurate alignment, particularly in genetically complex regions such as the CFH/CFHR cluster. The achieved coverage is 20× in 99.9% of positions, with an average depth greater than 400×, ensuring high sensitivity and reliability for the detection of variants, including copy-number variants (CNVs) derived from NGS data.

We complement this strategy with MLPA (Multiplex Ligation-dependent Probe Amplification), using two probe sets: one commercial and one specifically designed in-house for the CFH/CFHR1-5 cluster. This combination increases the accuracy in detecting rearrangements and CNVs in a critical region where conventional methods often have limitations.

Our panel is specifically optimised for complement genes and comprises a total of 54 genes, including CFH, CFI, MCP (CD46), CFB, C3, THBD, DGKE, CFP and CFHR1-5, as well as ADAMTS13, covering key intronic and intergenic regions and avoiding coverage gaps that can occur in clinical exome or whole-exome panels. We complement NGS with Sanger sequencing of critical regions, such as exons 9 and 23 of CFH and exon 6 of CFHR1, to ensure maximum accuracy. In addition, all variants identified by NGS are independently confirmed by Sanger sequencing, and results are reported together with CFH and MCP (CD46) risk/protective haplotypes, which are clinically relevant.

Diagnostic application in aHUS, C3G and other complement-mediated diseases

Secugen offers an integrated approach covering aHUS, C3G, paroxysmal nocturnal haemoglobinuria (PNH) and other complement-related glomerulopathies. The analyses included in the study of complement-mediated diseases include:

• Detection of mutations and CNVs by NGS in the 54-gene complement panel associated with complement-mediated TMA.
• Genotyping of risk/protective haplotypes in CFH and MCP (CD46).
• Analysis of rearrangements in the CFH/CFHR1-5 region.
• Biochemical and functional studies of the complement system, including:
  • Quantification by ELISA of factor H, factor I and factor B levels.
  • Quantification by flow cytometry of MCP (CD46) and DAF (CD55).
  • Functional assessment of factor H.
  • Detection of anti-factor H autoantibodies, which is particularly relevant in patients with homozygous CFHR3-1 deletion and in certain C3G cases with marked dysregulation of the alternative pathway.

This approach allows:

• Comprehensive genetic and functional characterisation of the patient, integrating risk and protective factors within the spectrum of complement-mediated diseases.
• Provision of evidence in favour of a complement-mediated aetiology (such as aHUS or C3G) and contribution to its distinction from other TMAs and glomerulopathies, always in the context of global clinical assessment and other specific investigations (including ADAMTS13 testing, STEC infection studies and histological findings).
• Guidance on the use of complement-targeted therapies, as well as assessment of the risk of post-transplant recurrence and the risk of HUS/aHUS in related living kidney donors.

Quality, accreditation and research experience

Secugen is licensed by the Community of Madrid to operate as a diagnostic centre with a Genetics Unit and is certified according to ISO 9001:2015, reflecting our commitment to quality and analytical traceability. We are also the only company in Spain with EQA certification as a developer of an R&D project in the complement field, with the project “SECUGEN016 – Platform for the diagnosis of complement-mediated diseases in the context of precision medicine”, funded by the Ministry of Economy, Industry and Competitiveness.

We have led the PIDMECOMP project (Integrated platform for the molecular diagnosis of complement-related diseases) in collaboration with the ISCIII and the CIB-CSIC, supported by the Spanish Government, consolidating our position as a reference centre in translational research in this field.

The laboratory has analysed more than 2,000 cases of complement-mediated diseases, including aHUS, C3G and PNH, and has developed its own mutation database that allows more robust interpretation of findings. We also benefit from the scientific advice of Professor Santiago Rodríguez de Córdoba, an international authority in complement genetics and its involvement in human disease.

Publications and clinical experience

Secugen’s technical expertise has been reflected in scientific publications in the field, such as the book chapter “Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequencing and MLPA” (Methods Mol Biol. 2021;2227:159-178) and the article “Novel immunochromatographic test for rapid detection of anti-factor H autoantibodies with an assessment of its clinical relevance” (Front. Immunol., 2025).

The Secugen team is composed of highly qualified personnel with extensive experience in molecular diagnostics and in the development of innovative methodologies in the complement field, enabling us to offer nephrologists, haematologists, immunologists and genetics services rigorous diagnostic support aligned with precision-medicine principles and oriented towards clinical decision-making in thrombotic microangiopathies, C3 glomerulonephritis and other complement-mediated diseases.