C4A gene copy number linked to C4 protein levels in neutrophils from people with schizophrenia
Background: Looking for complement immune signals beyond plasma
In neutrophils from people with schizophrenia, higher C4A gene copy number was associated with a tendency toward higher levels of complement C4 protein. Published in PNAS, this study suggests that C4 in peripheral immune cells may offer a clue to innate immune changes in schizophrenia. The finding is a correlation from a small observational study; it does not establish that more gene copies directly cause an increase in protein levels.
Complement is a system of proteins involved in innate immunity. Previous studies have reported an association between schizophrenia risk and C4A gene copy number, as well as a relationship between C4A expression and synapse elimination in experimental models. However, findings from brain studies alone cannot readily explain immune changes in the blood. Although increased C4 activation products have been reported in the plasma of people with schizophrenia, corresponding changes have not been consistently observed in other proteins in the complement pathway. The researchers focused on immune cells that could supply or activate C4 outside plasma.
Key findings: Positive correlation in patient neutrophils
The researchers first used flow cytometry to examine fresh whole blood from 10 anonymous blood donors and analyzed publicly available gene expression data. C4 protein was detected mainly in neutrophils and monocytes. Public expression data also supported C4A expression in these cell types.
The clinical comparison included a patient group of 30 people with schizophrenia or schizoaffective disorder and 38 controls. Participants were 18–35 years old; patients were within five years of their first diagnosis or the start of antipsychotic treatment. Gene copy number was determined by droplet digital polymerase chain reaction (ddPCR). C4 protein in neutrophils and plasma was measured by Western blot, while C4 in classical monocytes was measured by flow cytometry. The neutrophil analysis included samples from 15 patients and 21 controls.
In patient neutrophils, C4A copy number and C4 protein levels showed a positive rank correlation. Spearman’s correlation coefficient was 0.63, with a 95% bias-corrected and accelerated confidence interval of 0.12–0.89; P=0.012, remaining significant after correction for multiple testing. This correlation was not detected in classical monocytes or plasma in either group. However, the difference between the patient and control groups in the neutrophil correlation coefficient was not statistically significant at the conventional threshold (P=0.067). A significant correlation in patients alone does not establish that the relationship is specific to schizophrenia.
Meaning and outlook: The function of intracellular C4 remains to be studied
The researchers proposed that neutrophil C4 may be one source contributing to the complement activation observed in schizophrenia. An exploratory analysis also found a tendency toward lower neutrophil C4 protein levels in the patient group than in controls. The between-group difference had P=0.056 when adjusted only for C4A copy number, and P=0.0055 after additional adjustment for body mass index. This was an exploratory result without correction for multiple testing. The interpretation that protein is consumed during activation is the researchers’ hypothesis; the study did not directly demonstrate that pathway. Correlations between neutrophil C4 and stress or symptom scores were not statistically significant.
A key limitation is the small patient sample of 15 in the main neutrophil analysis. Effects of medication exposure, body mass index, and frozen storage need to be considered. Beta-actin (β-actin), used as the reference for comparing protein levels, may also vary with disease state. The assay detects total C4, comprising C4A and C4B, so the results cannot be interpreted as changes in C4A protein alone. The study also did not directly measure expression of the two genes in patient cells. Synchronization of peripheral signals with complement activity or synapse elimination in the brain, early diagnostic performance, and the effects of anti-complement treatment were outside the scope of this study.
The study design and results are available in the open-access PNAS article.
PNAS, Vol. 123, Issue 20, May 2026. DOI: 10.1073/pnas.2603129665
Summary: While excessive synaptic pruning mediated by central nervous system complement activation has been implicated in the risk of schizophrenia, establishing reliable, minimally invasive biomatrices has remained a persistent challenge. This benchmark study bridges the central-peripheral barrier by correlating genomic C4A copy number variations (CNVs) with peripheral complement C4 protein kinetics and multi-parametric flow cytometry immune profiles. The data demonstrates a highly linear, causal linkage where elevated C4A gene dosage programmatically drives systemic C4 protein accumulation, with the strongest immunophenotypic signature mapped specifically onto peripheral B cells and monocytes, delivering a robust computational baseline for pre-symptomatic diagnostic modeling and targeted neuroimmunological stratification.
This study provides a basis for examining C4 in plasma separately from C4 in immune cells when investigating complement changes in schizophrenia. Follow-up studies could measure C4A copy number and neutrophil C4 together, then test whether the association is reproduced in medication-naive patients experiencing a first psychotic episode and in independent cohorts. Combining methods that distinguish C4A from C4B with single-cell analysis could help compare protein levels more accurately by cell type. Experiments that directly examine activation and consumption pathways are also needed. Such validation could strengthen the evidence base for exploring peripheral immune pathways as treatment targets. The current findings do not provide a basis for introducing a blood test for schizophrenia diagnosis or recommending anti-complement treatment.