At the molecular level, ALPS is primarily linked to abnormalities in the FAS-mediated apoptotic pathway. The FAS gene, also known as TNFRSF6 or CD95, encodes a cell-surface death receptor that plays a key role in activation-induced cell death, a fundamental mechanism for maintaining peripheral immune tolerance. Germline or somatic mutations in FAS account for most genetically defined ALPS cases. These alterations impair the elimination of autoreactive or excess lymphocytes, thereby promoting lymphoproliferation and autoimmunity. CASP10 variants were historically included in the genetic classification of ALPS, but their causal role has been challenged by recent functional evidence showing that the investigated missense variants, as well as a homozygous frameshift variant abolishing CASPASE-10 protein expression, did not impair FAS-mediated apoptosis.

Despite this well-established pathogenic framework, the translation of molecular insights into practical diagnostic tools remains limited. Current diagnostic algorithms for ALPS rely on a combination of clinical features, immunophenotyping, particularly the expansion of TCRαβ⁺ CD4⁻CD8⁻ double-negative T cells, functional apoptosis assays, and genetic testing. However, many of these investigations are not readily available in routine clinical practice, particularly outside specialized referral centers. Consequently, ALPS may remain underdiagnosed or be misclassified as isolated immune cytopenia, especially at disease onset. In this context, transcriptional analysis of genes involved in the apoptotic pathway, including FAS and FASL, may represent a potential diagnostic adjunct.

The study measured FAS and FAS ligand messenger RNA expression by quantitative real-time polymerase chain reaction in whole-blood samples from patients with pediatric-onset ALPS (n = 24), patients with chronic ITP (n = 27), and healthy controls (n = 22). Expression levels were compared among groups, and their diagnostic performance was assessed using receiver operating characteristic curve analysis. Total RNA was isolated from whole-blood samples using an automated extraction system, DNase treatment was included to remove contaminating genomic DNA, and relative gene expression was calculated using the comparative threshold cycle method normalized to GAPDH as the endogenous reference gene.

The results showed that FAS expression was significantly higher in patients with ALPS than in healthy controls (p < 0.01), but no significant difference was observed between ALPS and ITP. FAS expression showed moderate discrimination between ALPS and healthy controls but poor discrimination between ALPS and ITP. FAS ligand expression did not differ significantly among groups and showed no meaningful diagnostic performance. Neither FAS nor FAS ligand expression correlated with double-negative T-cell percentages or differed according to treatment status in patients with ALPS.

Receiver operating characteristic curve analysis confirmed these findings. FAS expression showed moderate discriminative ability between ALPS patients and healthy controls, with an area under the curve (AUC) of 0.742 (95% CI: 0.600–0.885; p = 0.0049). In contrast, FAS expression demonstrated poor performance in distinguishing ALPS from ITP patients (AUC = 0.568; 95% CI: 0.405–0.731; p = 0.406), indicating limited diagnostic utility in a clinically relevant setting. FASL expression did not demonstrate meaningful discriminative ability in either comparison, showing no significant performance in distinguishing ALPS from ITP patients (AUC = 0.517) or from healthy controls (AUC = 0.534).

These findings highlight a key distinction: transcript abundance does not necessarily reflect protein function or apoptotic pathway activity. In ALPS, pathogenic FAS variants can impair receptor function and downstream signaling without necessarily reducing transcript levels. Preserved or increased FAS messenger RNA levels may therefore coexist with markedly impaired apoptotic responses. Compensatory transcriptional responses or chronic lymphocyte activation could contribute to the observed increase, although these mechanisms were not investigated in this study. The recent reassessment of CASP10 further illustrates the need for functional evidence when interpreting alterations in apoptosis-related genes.

FASL expression did not differ significantly among patients with ALPS, patients with ITP, and healthy controls. FAS ligand activity is regulated at post-transcriptional and post-translational levels, including membrane trafficking, proteolytic cleavage into soluble forms, and spatial regulation at the immune synapse. These mechanisms are not captured by bulk messenger RNA measurements and provide a biological rationale for distinguishing FASL transcript abundance from ligand activity. This distinction is also relevant to other inborn errors of immunity, in which qualitative signaling defects cannot necessarily be inferred from messenger RNA expression alone.

Several methodological limitations should be acknowledged. The relatively small sample size, particularly in the ALPS group, reflects the rarity of the disease and the single-center design. This may have limited the statistical power to detect moderate differences between ALPS and ITP; therefore, the findings should be considered exploratory and require confirmation in larger, multicenter cohorts. The present study focused on transcriptional measurements and did not include an integrated analysis of functional and genetic findings. Gene expression was measured in whole blood, which contains a heterogeneous mixture of cell populations; consequently, cell-specific transcriptional changes may have been masked. Finally, seven patients with ALPS were receiving immunomodulatory treatment at the time of sampling, which may have influenced gene expression, although FAS and FASL expression did not differ significantly between treated and untreated patients.

Taken together, these findings have clear implications for clinical practice. In patients presenting with autoimmune cytopenias, FAS and FASL expression should not be used as standalone biomarkers to support or exclude a diagnosis of ALPS. If considered, transcriptional data should be interpreted only within a comprehensive diagnostic framework that prioritizes clinical features, immunophenotypic markers such as double-negative T-cell expansion, functional apoptosis assays, and targeted genetic testing. From a translational perspective, the present study underscores the distinction between pathogenic relevance and biomarker utility. Although disruption of the FAS–FASL pathway is central to ALPS pathogenesis, this defect is primarily functional and cannot be reliably inferred from transcript abundance alone. Diagnostic strategies should therefore prioritize integrated approaches combining clinical, immunophenotypic, functional, and genetic data rather than relying on isolated transcriptional measurements.

Subject of Research: Immunology

Article Title: FAS and FASL expression in autoimmune lymphoproliferative syndrome and immune thrombocytopenia: limited diagnostic utility as transcriptional biomarkers

Article References: Montanari, P., Rondot, F., Pau, A., Clemente, A., Calvi, C., Galliano, I., Gambarino, S., & Bergallo, M. (2026). FAS and FASL expression in autoimmune lymphoproliferative syndrome and immune thrombocytopenia: limited diagnostic utility as transcriptional biomarkers. Molecular Biology Reports, 53(1), Article 1730. https://doi.org/10.1007/s11033-026-12950-0

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12950-0

Keywords: autoimmune lymphoproliferative syndrome, immune thrombocytopenia, FAS, FAS ligand, apoptosis, biomarkers, double-negative T cells, inborn errors of immunity, FASL, expression, autoimmune, lymphoproliferative