A novel AIRE mutation leads to autoimmune polyendocrine syndrome type-1

Abstract

Autoimmune polyendocrine syndrome type-1 (APS-1) is a rare inherited monogenic autoimmune disease characterized by the presence of at least two of three following major clinical features: chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency. Mutations in autoimmune regulator (AIRE) gene have been found to contribute to APS-1. In the present study, we reported a 36-years-old male APS-1 patient who presented with hypoparathyroidism and Addison's disease. The proband underwent complete clinical examinations and mutation screening was performed by Sanger sequencing on AIRE gene. A novel homozygous mutation in exon 9 of the AIRE gene (c.1024C>T) was identified. Based on sequencing findings, HEK293T cell-based assays were conducted to analyze the subcellular localization and mutant transcript processing. Our results revealed that p.Q342X mutant localized in nuclear speckles and exerted a dominant-negative effect on wildtype AIRE function. We reported the c.1024C>T mutation of AIRE gene for the first time, which enriched the AIRE mutation database and contributed to further understanding of APS-1.

Keywords: autoimmune polyendocrine syndrome type-1 (APS-1); autoimmune regulator (AIRE); exon; homozygous mutation; pedigree.

FIGURE 1

Genetic analysis of AIRE mutation. (A) Pedigree of the APS-1 family. Solid symbol indicates the affected individuals, open symbol belongs to the unaffected individuals, square represents male and circle represents female. Arrow indicates the proband (Ⅱ2). The half-shaded icons denote the mutation carriers. (B) Sequencing profiles of the pedigree. The sequencing result of the proband (Ⅱ2) shows the homozygous mutation (c.1024C>T) in exon 9 of the AIRE gene (GenBank Accession: NM_000383.3) indicated by the arrow. The proband’s parents carry the heterozygous variant of the AIRE gene and his older brother is wildtype.

FIGURE 2

The AIRE- dependent (KRT14 and S100A8) and -independent (PRMT3 and CCNH) genes were tested. Cells were transfected with various amounts of WT AIRE and p.Q342X mutant, alone or in combinations. Thereafter, the mRNA expressions of KRT14, S100A8, PRMT3, and CCNH were examined using qRT-PCR. The results were shown as relative mRNA expression compared to cells transfected only with WT AIRE. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Western blot analysis of HEK293T cells transfected with WT-AIRE or p.Q342X mutant constructs. The bands were blotted with anti-GFP antibody. Tubulin was used as an internal control. Compared with WT AIRE, the p.Q342X mutant translated a shortened product. M: protein marker.

FIGURE 4

Subcellular localization of p.Q342X mutant. Confocal images showed the subcellular localization of WT AIRE and p.Q342X mutant. Overlay images showed the degree of co-localization (yellow). Nuclei were visualized with DAPI counterstain (blue). The scale bar is 10 μm.