This study was approved by the Ethics in Research Committee of the First Affiliated Hospital, Zhejiang University (approval number: 2021IIT757). Informed consent was obtained from all individuals involved in the study.

Serum samples were serially diluted (1:200; 1:2,000; 1:20,000, and 1:200,000) and incubated with recombinant human IFN-α2 or IFN-ω at a final concentration of 150 pg/ml or 200 pg/ml for 3 h at room temperature. IFN-α2 or IFN-ω levels were determined using a human IFN-α2 or IFN-ω ELISA kit (TSZ, United States) according to the manufacturer’s instructions. Inhibition assays were performed in duplicate for all samples.

Blood samples were harvested from all participants and genomic DNA was extracted from peripheral blood leukocytes using RelaxGene Blood DNA System (Tiangen, Beijing, China) in accordance with the manufacturer’s instructions. DNA was dissolved in sterilized double-distilled water and kept at -20 °C until assayed. DNA degradation and contamination were monitored on 1% agarose gel. All DNA samples were examined for protein contamination (as indicated by the A260/A280 ratio) and reagent contamination (indicated by the A260/A230 ratio) with NanoDrop ND 1000 spectrophotometer (NanoDrop, Wilmington, DE).

Sanger sequencing was performed for the proband and other family members. 13 pairs of primers (Supplementary Table S1) covering the whole exons of AIRE gene were designed upon request. The touch-down PCR was performed in a 30 μl reaction mixture containing 1 μl DNA template, 2 μl forward primer (2.5 μM), 2 μl reverse primer (2.5 μM), 15 μl TaqMix/Taq DNA polymerase and 10 μl double-distilled water. The PCR conditions were: initial denaturation for 5 min at 95°C, 35 cycles of amplification (at 95°C for 30 s, at 65–50°C for 30 s starting from 65°C with decreasing by 1°C every cycle for 15 cycles until remaining at 50°C for 20 cycles, and at 72°C for 30 s), and final elongation at 72°C for 10 min. The amplification products were tested by 2% agarose gel electrophoresis and further analyzed by Sanger sequencing with automated DNA capillary sequencer (3730XL, Applied Biosystems). The sequencing results were analyzed by Mutation Surveyor V3.00.

AIRE cDNA PCR amplification product was sub-cloned into pEGFP-C1 vector using ClonExpress^®II (Vazyme, Nanjing, China) to construct recombined plasmid pEGFP-AIRE. Site-directed mutagenesis PCR was used to construct the AIRE mutants. Primers used in site-directed mutagenesis were shown in Supplementary Table S2. Each mutant was achieved by two-step PCRs using pEGFP-AIRE as the template.

For c.1024C>T, two pairs of pEGFP-C1-AIRE-F and pEGFP-C1-AIRE-M1024-R and pEGFP-C1-AIRE-R and pEGFP-C1-AIRE-M1024-F were used in the first PCR step. pEGFP-C1-AIRE-F and pEGFP-C1-AIRE-R were used in the second step. For each mutation, amplification products in the first step were cleaned (Axygen, CA, United States), mixed and used as the template in the second PCR reaction. All final PCR amplifications were recombined into the digested pEGFP-C1 vector using ClonExpress^®II. Recombinant plasmids pEGFP-AIRE and pEGFP-AIRE-M1024 were transformed into Escherichia coli DH5α cells. DNA was prepared using a plasmid DNA purification kit (NucleoBond Xtra midi kit, MN, Germany) according to the manufacturer’s instructions. Sanger sequencing was used to verify sequences. pEGFP-C1-AIRE was cloned into pmCherry-C1 vector by double digestion (BglII and SalⅠ).

HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂. 1 µg plasmids DNA with different ratios of WT and mutant AIRE reconstructed vectors were co-transfected into HEK293T cells using PolyJet Transfection reagent (SignaGen Laboratories, MD, United States) according to the manufacturer’s instructions.

After incubation for 24 h, transfected cells were fixed using 4% paraformaldehyde for 15 min at room temperature. After fixation, cells were washed three times with PBS. Thereafter, they were incubated with 0.2% Triton X-100 for permeabilization. After washing with PBS, nuclei were stained with DAPI (1 mg/ml). Confocal images were taken with the microscope (Olympus FV3000, Japan).

Transfected cells were harvested and lysed with RIPA buffer for 20 min on ice, followed by centrifugation at 12,000 rpm for 20 min at 4°C. Afterwards, protein extracts were separated on a 10% SDS-polyacrylamide gel and electroblotted onto PVDF membranes. After blocking with 5% skimmed milk, the membranes were incubated with GFP-Tag monoclonal antibody (1:2500, ImmunoWay Biotechnology Company, United States) overnight at 4°C. The membranes were then incubated with HRP^* goat anti-mouse IgG (H + L) (1:200,000, ImmunoWay Biotechnology Company, United States) for 2 h at room temperature. Finally, the chemiluminescence kit (Biosharp, Anhui) was used to detect the protein signals according to the manufacturer’s protocol.

After transfection for 24 h, the cells were lysed with RNAisoTM Plus (Takara, Japan). Chloroform (0.2 ml) was added to the homogenate. The tube was then spun and top aqueous layer was transferred to a new tube. Isopropyl alcohol was added and centrifuged. The pellet was washed with 1 ml of 75% ethanol and vortexed briefly. The tube was then centrifuged again, and the RNA pellet was dried. Afterward, the pellet was dissolved in 100 μl of ultra-pure water and quantified using the NanoDrop 2000 Spectrophotometer. Total RNA (1 μg) was then reverse-transcribed into cDNA in a 20 μl reaction mix using a PrimeScript RTTM reagent Kit (Takara, Japan). Quantitative PCR was performed using PowerUp^TM SYBR Green Master Mix (Applied Biosystems, United States). Each sample was tested in triplicate. The primer sequences were listed in Supplementary Table S3.

Data were analyzed by ordinary one-way ANOVA using GraphPad Prism v8.0.2. p < 0.05 was considered statistically significant.

The patient was found to have low blood pressure during physical examination 12 years prior and was not further examined at that time. He was admitted to a local hospital for numbness and convulsions in his limbs 8 years prior and was diagnosed with primary hypoparathyroidism. He was then diagnosed with Addison’s disease in our hospital due to weakness, darkening of the skin and mucous membranes, poor appetite, and low blood pressure 7 years prior. After his diagnosis, he received supplemental therapy with calcium, calcitriol and hydrocortisone. During treatment, he sometimes felt fatigued in his hands and feet. After this hospitalization, he underwent a series of tests, and the results were shown in Table 1. The dose of hydrocortisone was adjusted according to the patient’s clinical symptoms and laboratory results, with the final dose being 20 mg at 8:00 and 10 mg at 16:00. The level of calcium was 2.03 mmol/L, but the patient had no signs or symptoms of hypocalcaemia. Therefore, the doses of calcium and calcitriol were not adjusted (Brandi et al., 2016). In addition, Abs against IFN-α2 and IFN-ω in the proband were tested. The results showed that IFN-α2 Ab was positive, whereas IFN-ω Ab was negative.

He was diagnosed with APS-1 according to the clinical diagnostic criteria of APS-1. However, he did not suffer from chronic mucocutaneous candidiasis and other autoimmune diseases, including autoimmune thyroiditis, type I diabetes, autoimmune hepatitis, and vitiligo.

Next, sequencing was performed to identify the AIRE mutation sites. The results revealed a novel homozygous mutation of the AIRE gene (c.1024C>T) in the proband (Figure 1). Additionally, his parents were found to carry the same heterozygous variant of the AIRE gene. However, his older brother was wildtype. Of note, the proband’s parents were in a consanguineous marriage. Unfortunately, the proband’s parents did not undergo further testing for special reasons, so it was unclear whether they had organ-specific autoimmune diseases.

Oftedal et al. reported that several missense PHD1 mutations suppressed gene expression driven by WT AIRE in a dominant-negative manner (Oftedal et al., 2015). Given that p.Q342X mutant is close to PHD1 domain, we speculate whether this mutation would also exert a similar dominant-negative effect. To this end, HEK293T cells transfected with either WT-AIRE and/or p.Q342X mutant expression vectors were used. We then tested the mRNA expression of a panel of AIRE - dependent (keratin 14- KRT14 and S100 calcium binding protein A8-S100A8) and - independent (protein arginine methyltransferase 3, PRMT3 and cyclin H-CCNH) genes. As expected, p.Q342X mutant diminished the expression of AIRE-dependent genes (Figure 2). Surprisingly, when HEK293T cells were co-transfected with different ratios of WT AIRE and p.Q342X mutant, p.Q342X mutant significantly diminished the ability of WT AIRE and p.Q342X mutant. p.Q342X mutant significantly abrogated the ability of WT AIRE to induce the expression of KRT14 and S100A8 (Figure 2). These results indicate that p.Q342X mutant exerted a dominant-negative effect on WT AIRE function.

The c.1024C>T mutation of the AIRE gene was predicted to generate a truncation protein. To confirm this prediction, HEK293T cells were transfected with WT-AIRE or p.Q342X mutant expression vectors. Western blot analysis showed that the molecular weight of WT-AIRE protein was 58 kDa. Since the detected band contained the GFP protein (27 kDa), its molecular weight was shown to be 85 kDa. The molecular weight of p.Q342X mutant was 63 kDa in Figure 3. The actual molecular weight was 36kD after removing GFP protein, in agreement with the calculated weight of the mutant. It demonstrated that the c.1024C>T mutation leaded to the truncation of the AIRE protein.

To further understand the property of p.Q342X mutant, immunofluorescent staining was performed to analyze the nuclear localization pattern. HEK293T cells were co-transfected with Cherry-tagged WT AIRE plasmids together with expression vectors encoding p.Q342X mutant tagged with EGFP. Our results revealed that p.Q342X mutant localized in nuclear speckles. Moreover, it co-localized with WT AIRE protein (Figure 4), suggesting that its nuclear localization pattern is not disrupted.