Clinical Manifestations

The patient was born at 33 + 2 weeks of gestation via vaginal delivery due to premature rupture of membranes, with a birth weight of 1720 g (P10–25). The Apgar score was 9 at 1 min, and the blood glucose level at birth was 4.8 mmol/L. At 1 week of age, echocardiography demonstrated a small patent ductus arteriosus (1.1 mm) and a patent foramen ovale (2.3 mm). By 1 month of age, the ductus arteriosus had closed spontaneously, while the foramen ovale persisted; cranial MRI showed only mild prematurity-related changes. The infant was clinically stable, tolerated full oral feeding with adequate weight gain, and was discharged in good condition.

From 6 months of age, the patient developed recurrent upper respiratory tract infections and bacterial enteritis, for which he received multiple courses of symptomatic and antibiotic therapy. At 18 months of age, the patient contracted varicella, which initially presented with rash on the legs and subsequently spread to the back, retroauricular areas, and face, without accompanying fever. He was treated with acyclovir for 5 days, after which the rash resolved.

At 2 years 4 months, the patient was admitted with fever, generalized rash, mucocutaneous symptoms, and cervical lymphadenopathy. Laboratory tests showed elevated inflammatory markers (CRP, IL-6, SAA, and ESR), and chest X-ray suggested bronchopneumonia. Respiratory pathogen panel and blood metagenomic sequencing results were negative, and antibiotics were ineffective. A diagnosis of Kawasaki disease was made. Echocardiography during the acute phase revealed no coronary artery abnormalities but identified a small pericardial effusion and trivial mitral regurgitation. The patient received intravenous immunoglobulin and high-dose aspirin. After 7 days of treatment, his symptoms improved, and he was then discharged. On follow-up echocardiography, the pericardial effusion had resolved and no coronary artery involvement was detected. Aspirin was subsequently tapered to 33.3 mg once daily for antiplatelet therapy.

Conventional Immunological Features

White blood cell, lymphocyte, and neutrophil counts remained within normal ranges. Analysis of lymphocyte subsets revealed a reduced proportion of CD16+CD56+ NK cells, an increased proportion of CD19+ B cells, elevated CD4+ T cells, and decreased CD8+ T cells. Hypogammaglobulinemia was detected at 2 months of age but resolved by 1 year and 5 months. During the episode of Kawasaki disease, serum IL-5, IL-6, IL-8, and IL-12p70 levels were markedly elevated (Table 1).

Table 1 Immunological profile of the patient

Autoantibody testing performed as part of routine clinical evaluation revealed positivity for antinuclear antibodies (ANA) and anti-Ro52 antibodies by immunofluorescence (Fig. 1a). To further characterize the patient’s autoantibody landscape, we performed a comprehensive antigen microarray profiling covering 120 IgG autoantigens. A total of 40 antigens showed elevated reactivity (Z-score > 0) (Fig. 1b), among which 13 were markedly increased (Z-score > 3). These highly enriched autoantibodies spanned multiple antigen categories, including nuclear antigens (Jo-1, Ro/SSA 60 kDa, ssDNA), cytoplasmic or membrane-associated proteins (α-actinine, CD4), cell matrix proteins (Collagen VI), phospholipid-related antigens (phosphatidyl-serine), and several circulating proteins (Calprotectin, Complement C1q, C-reactive protein, Factor H, Insulin, Troponin-I). This broad autoreactive signature highlights a dysregulated humoral immune response in the patient.

Fig. 1

Autoantibody assessments in the patient. (a) Immunofluorescence results showing positive (red) and negative (gray) signals; (b) IgG autoantibody profiling of antigen microarray, displaying antigens with Z-score > 0 as a heatmap

Genetic Analysis Reveals p.I115T MSN Mutation

WES identified a hemizygous MSN c.344T > C (p.I115T) variant in the patient. Sanger sequencing confirmed this variant and showed that it was maternally inherited, with the mother being a heterozygous carrier (Fig. 2a-b). This missense variant is not recorded in major population databases and therefore appears to be novel. Multiple in silico prediction tools suggested a damaging effect, supporting its potential pathogenicity (Fig. 2c).

Fig. 2

Identification of the MSN c.344T > C (p.I115T) mutant. (a) Pedigree illustrating the inheritance pattern of the mutant; (b) Sanger sequencing confirming the c.344T > C (p.I115T) substitution in the patient; (c) Summary of in silico predictions for the functional impact of the mutant

p.I115T MSN Variant Exhibits Reduced Protein Stability

To determine whether the p.I115T substitution affects MSN protein stability, wild-type and p.I115T MSN plasmids were generated and transiently introduced into HEK293T cells. Protein turnover was evaluated using a CHX assay. Quantitative immunoblot analysis demonstrated that the p.I115T mutant exhibited markedly reduced stability, with accelerated degradation relative to the wild-type MSN protein (Fig. 3).

Fig. 3

HEK293T cells expressing WT or p.I115T MSN were treated with cycloheximide, and MSN protein stability was assessed by Western blot. GAPDH served as a loading control. Representative results of three independent experiments are shown

Reduced MSN Protein Levels in Immune Cells

At baseline, the proportion of MSN + T cells in the patient was 65.6%, which was lower than approximately 80% observed in HCs. After 72 h of ConA stimulation, the proportion of MSN + T cells in the patient decreased to 53.4%, whereas it was slightly increased in HCs, ranging from 80% to 90% (Fig. 4a). These results indicate that T cell activation leads to a more pronounced reduction of MSN expression in the patient compared with HCs, consistent with our HEK293T CHX assay showing reduced stability of the I115T MSN protein. In addition, MSN expression in the patient’s monocytes was lower than in HCs, while expression in B cells was comparable, indicating that the I115T mutation affects MSN protein levels in a cell type-specific manner (Fig. 4b).

Fig. 4

MSN expression in the patient and HCs. (a) Proportion of MSN + T cells at baseline and after 72 h ConA stimulation; (b) MSN expression in B cells and monocytes

Altered T cell Subsets and Th17-skewed Differentiation

The patient exhibited an increased percentage of CD4+ terminal effector memory T cells (TEMRA) at the expense of CD4+ naive and central memory cells (TCM). In the CD8+ compartment, both TCM and TEMRA subsets were markedly elevated compared with HCs, whereas naive CD8+ T cells were reduced (Fig. 5a). The proportion of regulatory T cells (Tregs) and follicular helper T (Tfh) cells in the patient was comparable to that in the controls. However, both the proportion of follicular regulatory T (Tfr) cells and the Tfr/Tfh ratio were decreased, suggesting dysregulation of balance between Tfh and Tfr (Fig. 5b). The proportion of Th1 cells within the patient’s CD4+ T cell population was markedly lower than that in the controls, whereas the proportion of Th17 cells was increased, indicating a shift toward a pro-inflammatory Th17-biased phenotype. CD8+ T cells showed reduced IFN-γ production, suggesting impaired cytotoxic effector function (Fig. 5c).

Fig. 5

Altered T cell subsets in the patient. (a) Proportions of naïve, central memory, effector memory, and TEMRA subsets in CD4+ and CD8+ T cells; (b) Proportions of Treg, Tfh, and Tfr cells; (c) Proportions of Th1 and Th17 in CD4+ T cells, and IFN-γ producing CD8 + T cells

Impaired B cell Maturation and Memory Differentiation

The proportions of memory B cells and marginal zone (MZ) B cells were markedly lower in the patient compared with those in HCs, whereas the proportion of naïve B cells was slightly higher, suggesting that MSN deficiency may preferentially impair B-cell maturation or differentiation toward memory and MZ compartments (Fig. 6a). The proportion of atypical memory CD21−/low B cells was also modestly increased (Fig. 6b). This discordant pattern indicates an abnormal differentiation trajectory of antigen-experienced B cells, potentially limiting the establishment of functionally competent effector and memory B cell pools.

Fig. 6

Altered B cell subsets in the patient. (a) Proportions of naïve, marginal, memory, transitional B cells and plasmablasts in the patient and HCs; (b) Proportion of atypical CD21−/low memory B cells

Hyperactivation and Reduced Apoptosis of Patient CD4+ T Cells

After 36 h of ConA stimulation, the proportion of CD25⁺CD69⁺ CD4⁺ T cells was higher in the patient than in HCs, indicating enhanced CD4⁺ T cell activation (Fig. 7a). In contrast, CD8⁺ T cell activation was comparable between the patient and controls (Fig. 7d). After 72 h of stimulation, CD4⁺ T cell proliferation was slightly increased in the patient (Fig. 7b), whereas CD8⁺ T cell proliferation remained similar to that of HCs (Fig. 7d). The proportion of Annexin V⁺ CD4⁺ T cells was lower in the patient (Fig. 7c), indicating reduced apoptosis, with a slight decrease also observed in CD8⁺ T cells (Fig. 7d).

Fig. 7

T cell activation, proliferation, and apoptosis in the patient. (a) Proportion of CD25+CD69+ CD4+ T cells after 36 h of ConA stimulation; (b) Proliferation of CD4+ T cells after 72 h of stimulation. (c) Proportion of Annexin V+ apoptotic CD4+ T cells. (d) Corresponding activation, proliferation, and apoptosis of CD8+ T cells