Adult-onset Still’s disease (AOSD) is a rare systemic inflammatory disease characterized by intermittent fever, arthritis, and evanescent skin rash in the absence of infection, malignancy, or rheumatological disease (1–4). It usually occurs in young adults with a bimodal age distribution, the first peak between 15 and 25 years and the second between 36 and 46 years. The clinical course varies from one individual to another, ranging from benign and self-limited forms to severe complications such as macrophage activation syndrome (1–4).

Although the etiopathology of AOSD remains unknown, growing evidence supports that AOSD is an autoinflammatory disorder where the innate immune system plays a pivotal role (1). The main hypothesis states that, in patients with some genetic predisposition, several external factors, such as infectious agents and other environmental factors, activate innate immune cells through Toll-like receptors (TLRs), leading to an abnormal response in both innate and adaptive immunity with an overproduction of cytokines, mainly IL-1b, IL-18, and IL-6 (1–4).

AOSD is considered a polygenic disease in contrast to other systemic autoinflammatory diseases (SAIDs) that are related to specific genetic mutations, such as the familial Mediterranean fever syndrome (FMF), TNF receptor-associated periodic fever syndrome (TRAPS), cryopyrin-associated periodic syndrome (CAPS), hyper IgD syndrome (HIDS), or Yao syndrome. Although AOSD shares common clinical and pathogenic features with these SAIDs, its genetic profile seems to be different. The presence of polymorphisms in genes encoding innate immunity-associated factors has been reported in AOSD patients (5–9). However, a unique association with HLA genes (10–13), not observed in other SAIDs, suggests the involvement of the adaptative immune system.

Molecular genetic analysis has become an excellent diagnostic tool for most SAIDs (14–19). However, it is not routinely used in patients with AOSD suspicion despite the fact that it could be very useful in differentiating it from other SAIDs (20, 21). In this regard, experts in the field call for a revision of the current classification criteria for AOSD that include the exclusion of other periodic fever syndromes with the aid of molecular genetics testing (22). Given that AOSD is considered a polygenic disease, the use of molecular genetic techniques targeting specific genes, such as Sanger sequencing, has been more restricted compared to its application in monogenic SAIDs. In this regard, next-generation sequencing (NGS) represents a particularly valuable tool for AOSD. NGS markedly outperforms Sanger sequencing in terms of scalability, speed, cost efficiency, sensitivity, versatility, and clinical applicability. Furthermore, NGS facilitates the development of disease-specific gene panels. Its high sensitivity enables the detection of low-frequency variants, including somatic mutations and mosaicisms, which are often challenging to identify with Sanger sequencing. Additionally, the capability for deep coverage with NGS enhances the precision of variant detection, which is crucial for investigating complex diseases and identifying rare or novel sequence variants.

Scarce studies have focused on the role of molecular genetics analysis in AOSD (22). Our study aimed to analyze the results of NGS in our cohort of patients with AOSD and to conduct a literature review on the potential value of molecular genetic techniques to improve diagnosis and differentiate AOSD from other SAIDs.

We identified 24 patients with AOSD diagnosis in whom NGS analysis was performed ( Figure 1 ). The main demographic, clinical, and laboratory features are summarized in Table 1 .

Molecular genetic techniques are becoming increasingly essential tools for the diagnosis of SAIDs. However, their role in the diagnostic algorithm of AOSD still remains limited. Previous studies assessing the potential genetic variants in AOSD have only assessed single coding regions of a few genes using Sanger sequencing (5–9). However, NGS panels are becoming the genetic technique of choice because they allow rapid and simultaneous analysis of the complete coding sequence of several SAID-related genes (15). In addition, increasing the NGS gene panel has been related to a higher diagnostic rate in autoinflammatory diseases (26). To the best of our knowledge, our study is the first to assess the role of NGS panels in AOSD patients.

We found that 20.8% of our 24 AOSD patients were carriers of genetic VUS in genes related to other SAIDs, including NOD2, TNFRSF1A, TNFAIP3, and SCN9A. No pathogenic genetic variants were identified. Table 3 summarizes previous reported data of genetic studies in AOSD patients. The prevalence of VUS varies widely across populations. Our results are in line with those reported in a Caucasian population ranging from 15% to 27.8% (9). However, Asian studies found a higher frequency of VUS in MEFV. In this regard, a prevalence of 53.1% and 63.3% of VUS in MEFV was reported in Korean and Japanese AOSD patients, respectively (5, 8). It is worth mentioning that none of our patients carried VUS in MEFV.

The clinical implications of carrying genetic VUS on severity and therapeutic response in AOSD patients are still under investigation. Our two patients with NOD2 variants did not present atypical manifestations but were refractory to conventional DMARDs and required biologic therapy to control disease activity. The patient carrying a genetic VUS in TNFRSF1A experienced testicular pain, which is not frequent in AOSD, and was refractory to different lines of DMARDs requiring canakinumab to achieve complete remission. The patient with SCN9A VUS had atypical severe neurologic manifestations and responded poorly to different cDMARDs and biological therapy until anakinra was initiated. In contrast, the patient with the TNFAIP variant did not show atypical symptoms and achieved a complete response with glucocorticoids alone ( Table 2 ; Figure 2 ).

Taking into account our whole cohort of 24 AOSD patients, 4 out of 5 patients with genetic variants required biologic therapy, while only 5 of 18 patients without genetic variants needed biologics due to cDMARD refractoriness. These findings suggest that AOSD patients with genetic variants might be prone to a more severe and refractory course of the disease. In this regard, Nonaka et al. (8) found that MEFV VUS carriers were more likely to present with a polycyclic phenotype and require biological therapy to control inflammatory disease activity. Along the same lines, Sighart et al. (9) reported that two-thirds of patients with VUS in MEFV experienced a severe course of the disease and two patients in whom TNFRSF1A genetic variants were detected required bDMARDs.

Recent studies have identified and validated four distinct patient clusters in AOSD, as well as three disease-related endotypes, each characterized by different immune cell profiles (27–29). In our study, among the five patients carrying genetic VUS, one patient (with a TNFRSF1A VUS) could be classified into cluster 1 or the “juvenile/transitional” group, one patient (with a TNFAIP3 VUS) into cluster 2 or the “uncomplicated” group, two patients (with NOD2 VUS) into cluster 3 or the “hyperferritinemic” group, and one patient (with SCN9A VUS) into cluster 4 or the “catastrophic” group. Taking this into account, future studies focused on the distribution of VUS across the different clusters would be highly valuable, as it may help elucidate their role in driving variations in clinical presentation, prognosis, and treatment response.

This study has potential limitations, including a small sample size and regional specificity (northern Spain). Additionally, we recognize that the use of a targeted gene panel may not capture all relevant variants, and the VUS identified were not classified as pathogenic, which complicates their interpretation. Despite these limitations, we consider that the use of NGS in our cohort of patients allowed us to detect genetic VUS that were associated with atypical clinical manifestations and refractoriness to conventional therapy. Based on our experience, we advocate for the use of NGS in all AOSD patients to exclude other SAIDs, facilitate the identification of those who may present with atypical manifestations, and improve the detection of patients likely to exhibit poor treatment response. Future collaborative international studies are particularly needed to enlarge the patient sample and encompass broader regional areas. Whole-exome sequencing will also help to identify more coding variants.

In conclusion, our findings support that NGS could be of value in AOSD patients. NGS was useful to rule out the presence of pathogenic genetic variants related to other SAIDs and to detect VUS that might be associated with a more severe and refractory course of the disease. Further studies are needed to establish stronger associations. The detection of VUS may help to identify patients at risk for atypical and severe manifestations and poor response to conventional therapy.