Systemic autoinflammatory diseases (SAIDs) are characterized by unprovoked inflammatory episodes such as recurrent/periodic fever, serositis, skin lesions, abdominal symptoms, arthritis/arthralgia, and central nervous system involvement [1-4]. In this expanding and still ongoing disease category, genetic diagnosis has been challenging because disease manifestations overlap among themselves and with other immunological disease categories, such as infection and autoimmune diseases. Differential diagnosis with laboratory findings of this disease category has not been so helpful because their underlying pathophysiology shares a common immunological defect [5]. Prior to the dissemination of next-generation sequencing (NGS) technologies, the genetic diagnosis was limited to genes associated with prototypic recurrent fevers (MEFV, MVK, TNFRSF1A, and NLRP3 genes). In the era of NGS, we can incorporate the expanded SAID-associated genes into our targeted panel gene list, and we are now hoping to adopt the routine genetic diagnosis in this challenging category of diseases and finally, provide a genetic characterization of many previously undiagnosed patients, and give an insight to the biological mechanisms of the innate immunity and autoinflammation. Recent diagnostic approaches of SAIDs can shorten the diagnostic odyssey and provide early access to the optimal treatment adapted to the underlying disease pathophysiology of the immune system. Here, we discuss the clinical usefulness of NGS targeted sequencing for SAIDs and recent advance of understandings for this heterogeneous disease group as for underlying primary immunodeficiency.

Genetic testing for 4 prototypic hereditary recurrent fevers (HRFs).

Genetic testing using Sanger sequencing as a first-line diagnostic tool was recommended for the patients with a clear clinical diagnosis of 4 prototypic HRFs. Genetic testing guidelines were suggested for two recessively inherited diseases [6]: Familial Mediterranean fever (FMF) associated with MEFV gene (MIM *608107) and mevalonate kinase deficiency (MKD) associated with MVK gene (MIM *251170), and two dominantly inherited diseases: tumor necrosis factor (TNF) receptor-associated periodic syndrome associated with TNFRSF1A gene (MIM *191190) and cryopyrin-associated periodic syndrome (CAPS) associated with NLRP3 gene (MIM *606416). This guideline recommended to screen mutational hot spots (exons 2, 3, 5, 10 of the MEFV gene; exons 2-11 of the MKV gene; exons 2-4 of the TNFRSF1A; exon 3 of the NLRP3). However, such a screening approach should always consider the possibility of the presence of a causal mutation outside the defined regions of interest. We can find genotype-phenotype information of these 4 prototypic HRFs in the well-organized registry for autoinflammatory diseases (Eurofever, https://www.printo.it/eurofever/index) [7]. Genetic diagnosis of these 4 prototypic HRFs could guide the therapy according to the specific defect in immunological process. Colchicine is the first-line therapeutic modality for FMF [8]. However, IL-1 or IL-6 blockade for MKD, and IL-1 blockade for CAPS are recommended [9-13].

Targeted sequencing using NGS technologies is the current method of choice for SAIDs with ambiguous phenotypes and locus heterogeneity. NGS targeted gene panels usually analyze coding sequences of hundreds of genes. Therefore, we can incorporate other recently described SAID-associated genes, such as adenosine deaminase 2 (ADA2, MIM *607575), nucleotide-binding oligomerization domain protein 2 (NOD2, MIM *605956), proline/serine/threonine phosphatase-interacting protein 1 (PSTPIP1, MIM *606347), and tumor necrosis factor-alpha-induced protein 3 (TNFAIP3, MIM *191163) genes. Deficiency of plasma adenosine deaminase 2 caused by homozygous or compound heterozygous mutations of the ADA2 gene has been identified in various systemic inflammatory diseases and immune deficiencies, manifesting fever, vasculitis, polyarteritis nodosa, recurrent stroke, and pure red cell aplasia [14-16]. Blau syndrome is a representative autoinflammatory granulomatous disease resulting from mutations of the recognition receptor NOD2, presenting with granulomatous polyarthritis, dermatitis and uveitis [17,18]. Pyogenic sterile arthritis, pyoderma gangrenosum and acne syndrome is a pleiotropic autosomal dominant autoinflammatory disease caused by mutations of CD2-binding protein PSTPIP1, which interacts with pyrin [19,20]. Haploinsufficiency of nuclear factor kappa B (NF-κB) regulatory protein A20 encoded by TNFAIP3, mediates a familial Behcet-like autoinflammatory syndrome, characterized by painful and recurrent mucosal ulceration affecting the oral mucosa, gastrointestinal tract, and genital areas. Several non-truncating TNFAIP3 mutations may provoke autoimmune conditions such as rheumatoid arthritis, SLE, and Sjogren associated non-Hodgkin lymphoma [21-23]. Therefore, expert practice guidelines from the International Society of Systemic Autoinflammatory Disease recommended including these 4 recently identified genes (ADA2, NOD2, PSTPIP1, and TNFAIP3) in its diagnostic scheme in addition to the 4 previously annotated HRF associated genes (MEFV, MVK, NLRP3, and TNFRSF1A) [22].

The locus specific database for SAIDs (Infevers, https://infevers.umai-montpellier.fr/web/index.php) deposited 43 additional genes into an expanding list of SAID-associated genes: ADAM17, ALPK1, AP1S3. CARD14, CDC42, CEBPE, COPA, ELF4, F12, IKBKG, IL1RN, IL36RN, LACC1, LPIN2, NCSTN, NLRC4, NLRP1, NLRP12, NLRP7, OTULIN, PLCG2, POMP, PSMA3, PSMB4, PSMB8, PSMB9, PSMB10, PSMG2, PSTPI1, RBCK1, RELA, RIPK1, SAMD9L, SH3BP2, SLC29A3, STING1, TNFAIP3, TNFRSF1A, TNFRSF11A, TRAP1, TRNT1, UBA1, WDR1. Most of them are associated with the players of the innate immune system, such as inflammasomes and IL-1β production (MVK, NLRP1, NLRP3, NLRC4, PSTPIP1), NF-κB signaling (NOD2, TNFAIP3, TNFRSF1A, TNFRSF11A, TRAP1), ubiquitination (UBA1), and type I interferon production (COPA, POMP, STING1) [5].

Type I interferonopathy has been denoted and classified within the broader disease entity of SAIDs [5,24]. Aicardi-Goutieres syndrome (AGS) is the first defined Mendelian disease associated with type I interferon upregulation, characterized by inflammation and tissue damage in the central nervous system, brain calcification, chronic cerebrospinal fluid (CSF) lymphocytosis, increased CSF alpha-interferon. Mutations of the ADAR, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and TREX1 can cause dysfunction of nucleases involved in the immune system, resulting in inflammatory damage to brain, skin and other body systems that lead to the characteristic features of AGS [25]. Type I interferon (IFN-I) and their cognitive receptors and signaling pathways are the essential components of the innate immune system. Type I interferonopathy-associated genes can be grouped according to their functions as nucleic acid sensing (IFIH1, DDK58), nucleic acid signaling (STING1, COPA), nuclear acid metabolism (RNASEH2A, RNASEH2B, RNASEH2C, POLA1, BLM, ATM, DCLRE1C, SAMHD1, TREX1, DNASE2, ADAR1, SKIV2L PTPN1, LSM11, RNU7-1), proteasome (PSMB4, PSMB8, PSMB9, PSMB10, PSMD12, PSMA3, PSMG2, POMP), mitochondrial integrity (NGLY1, ATAD3A), and post interferon receptor signaling pathways (ISG15, JAK1, STAT1, STAT2, USP18).

SAID is a rapidly expanding disease category that is associated with the innate immune dysregulation, encompassing hereditary recurrent/periodic fevers, other recently defined SAIDs, and type I interferonopathies. Since they share the immunological pathways and interactions, genetic diagnostic approaches are challenging. Therefore, adoption of NGS technology in this complicated disease category, having locus heterogeneity and various expressivity, is mandatory. NGS panel testings, including primary immunodeficiency panel and hemophagocytic lymphohistiocytosis panel, are now routinely performed in Korea. There were some retrospective studies and case reports to reveal the genetic cause of SAIDs [26,27]. So far, SAID NGS panel testing is not widely performed. As for SAIDs, the list of panel genes should be periodically updated because they are still expanding rapidly. The SAID panel should include classical 4 HRF-associated genes and additional SAIDs-associated genes. The inclusion of type I interferonopathy-associated genes in the panel is also recommended. Table 1 shows the representative SAID gene panel in Korea. After the adoption of this NGS panel for 18 months, they could identify the causative mutations of classical HRF-associated genes and other SAID-associated genes in the patients with recurrent fever and/or systemic inflammatory diseases (Table 2).

Genetic diagnosis of autoinflammatory diseases has been challenging so far. However, the advent of NGS technologies and expanding knowledge about the innate immunity and inflammation have made the routine genetic diagnosis of SAIDs possible. Here we reviewed the classical recurrent hereditary fevers and recently defined SAIDs, and type I inteferonopathies with their corresponding molecular mechanisms of the innate immunity. Now is the time to adopt the targeted NGS panel testing in the routine genetic diagnosis of SAIDs in Korea.

I declare that I do not have any conflicts of interests.

This study was supported by a 2021 research grant from Pusan National University Yangsan Hospital.