Background
Autoimmune polyendocrinopathy syndrome type 1 (APS-1), also called autoimmune polyendocrinopathy-candidiasis–ectodermal dystrophy/dysplasia (APECED), is a rare autosomal recessive syndrome (OMIM 240300) with a small female preponderance [
1,
2]. It occurs more frequently in certain populations, including Iranian Jews (1/9000), Finns (1/25,000), and Sardinians (1/14,400) [
3]. APS-1 is characterized by immune dysregulation which leads to some endocrine and nonendocrine manifestations [
2].
To define this syndrome, patients must have at least two of the three major components, i.e. a chronic mucocutaneous candidiasis (CMC), hypoparathyroidism, and autoimmune adrenal insufficiency [
4,
5]. Other components of APS-1 include type 1 diabetes, autoimmune hepatitis, hypothyroidism, primary hypogonadism, pernicious anemia, alopecia, ectodermal dysplasia, malabsorption, and vitiligo [
6].
The disease’s inheritance is in the Mendelian fashion [
7]. The underlying genetic abnormality which causes APS-1 is a mutation in the Autoimmune Regulator (AIRE) gene mapped to chromosome 21q22.3 [
8]. Fourteen exons of this gene encode a proline-rich protein with 545 amino acids [
7]. This protein plays a role in the regulation of transcription [
7]. There are distinctive subdomains in the AIRE protein structure, including a highly-conserved N-terminal homogeneously staining region (HSR) domain (exons 1 & 2), the nuclear localization signal (NLS) (exon 3), the putative DNA binding SAND domain (exons 5, 6 & 7), two plant homeodomain (PHD) type zinc fingers (PHD1: exons 8 & 9, PHD2: exons 11 & 12), the proline-rich region (PRR) (exon 10), and four LXXLL motifs [
7,
9,
10]. To date, many different mutations (more than 115 mutations) [
9] including small insertions, deletions, and single nucleotide substitutions [
11] have been reported with the predominant gene mutation varying across different ethnic groups [
12,
13]. It has been suggested that mutation in different regions of the AIRE gene has various results on the function of the AIRE protein with a currently poorly-described mechanism [
7,
9,
14]. In this article, we report three AIRE variations in a patient with APS-1 from an Iranian Muslim family.
Discussion and conclusions
To date, many APS-1-causing mutations have been identified in the AIRE gene across different ethnic groups [
9]. The Iranian Jewish population is well-known for carrying a founder AIRE gene mutation (Y85C) which causes a tyrosine to a cysteine change in the HSR domain [
14]. Other reported AIRE mutations in the Iranian population have been R139X, R257X, K50NfsX168, and L323SfsX51 [
12].
In line with the recessive mode of the inheritance of APS-1, most mutations of the AIRE gene are homozygous or compound heterozygous [
7,
9,
19,
20]. Pathogenic mutations spread over the entire coding sequence of the AIRE gene where at least four mutational hot spots including exons 2, 6, 8, and 10 have been reported [
11]. The proband in our study had one silent polymorphism in the hot spot exon 10 and one silent polymorphism in exon 14. Moreover, the promoter region or the intronic sequence mutation with an impact on the transcription and/or RNA splicing is possible [
11,
12]. Our proband had one intronic mutation with a probable RNA splicing effect.
Two variants, c.1197 T > C and c.1578 T > C, which were observed in our patient, were reported for the first time in 1998 [
21‐
23]. These two synonymous variants, one in exon 10 (proline-rich region) and the other in exon 14 (near one of the four interspersed LXXLL motifs) do not change the amino acid type at the protein level and are unlikely to have functional implications. In other words, the substitution of the T nucleotide in position 1197 to C (c.1197 T > C) does not change alanine amino acid in position 399 (p.Ala399=); similarly, the substitution of the T nucleotide in position 1578 to C (c.1578 T > C) does not alter aspartate amino acid in position 526 of the AIRE protein (p.Asp526=). Cetani et al. reported a 38-year-old Italian APECED woman who carried a G228 W mutation and three common silent heterozygous polymorphisms (588 C/T, S196S; 1197 T/C, A399A; and 1578 T/C D526D) [
11]. Sun et al. also presented a 15-year-old Chinese APS-1 patient with c.57 T > C, c.588C > T, c.834C > G, c.1197 T > C, and c.1578 T > C SNPs [
24]. These findings which are in line with our results highlight the fact that the two SNPs c.1197 T > C and c.1578 T > C are possible to occur at the same time.
The substitution of the T nucleotide in the second nucleotide of intron 9 to A (c.1095 + 2 T > A) is located in a region between PHD1 and PRR. This mutation occurs in the conserved splice donor sequence and leads to a change in the wild type donor site, affecting splicing [
12]. Recently, Seifi-Alan et al. reported an Iranian Muslim family with the same mutation in intron 9 (c.1095 + 2 T > A) in which the parents were both heterozygous for this mutation. The 9-year-old proband in this report suffered from APS-1 (CMC, Addison’s disease, hypoparathyroidism, and hypothyroidism) [
12]. The proband in our study had the same phenotype (with the exception of late onset CMC and the lack of Addison’s disease in our case), suggesting that c.1095 + 2 T > A may be regarded as a founder mutation in Iranian population, which cause APS-1 development. The late onset CMC and the lack of adrenal insufficiency in our case may be due to the fact that even patients with the same AIRE mutations present with a wide range of clinical manifestations and different courses of the disease [
13]. According to Perheentupa et al., who followed up 91 APS1 patients up to 50 years, the initial manifestation of APS1 appeared at age 0.2–18 years. Among the main signs of APS1, CMC, hypoparathyroidism, and adrenal insufficiency were the presenting features in 60, 32, and 5% of patients, respectively. The CMC, hypoparathyroidism, and adrenal insufficiency affected 100, 87, and 81% of APS1 patients respectively by the time they were 40 years old [
25]. The presenting feature in our case was hypoparathyroidism (age of 6) and later at age of 14, CMC was detected which is congruent with some cases of Perheentupa report. Our patient did not have adrenal insufficiency. Nevertheless, as we did not check anti-adrenal autoantibodies, the adrenal autoimmunity was not excluded. The vertebral fragility fractures in our case was justified by a combination of GH and estrogen deficiency.
Although the main expression of AIRE occurs in the medullary tissue of thymus [
23], the dendritic cells of the blood also express this gene [
26,
27]. Therefore, we examined the mRNA level of this gene in the PBMCs to test the effect of this splicing site mutation on the expression level. Our study indicated an up-regulation of AIRE expression in the patient with a homozygous c.1095 + 2 T > A mutation in comparison to patient’s parents with heterozygous mutation and other siblings (Fig.
3). The AIRE mRNA level was not reported in Seifi-Alan et al.’s report [
12], and as a result, we could not compare this finding to pervious results. However, Björses et al. reported 13 Iranian Jews with APS-1 who had an A374G (Y85C) mutation in exon 2 with a transcriptional activity of 115% of the wild-type AIRE. Transcriptional activity of the Y85C mutation was in contrast to other mutations with low or no transcriptional activity reported by Björses et al. [
7]. The mechanism suggested for this finding is that while the Y85C mutant AIRE protein has a correct length, it has a significantly shorter half-life and as a result, it rapidly deteriorates [
14]. A similar mechanism may explain a higher mutant AIRE mRNA expression level we found here. Further studies are required to examine the effect of this intronic mutation (c.1095 + 2 T > A) on the function and stability of the AIRE protein and its cellular consequences. The expression level of the AIRE mRNA was assessed in PBMCs in our study, not at the site of its actual transcriptional activity located at thymus. Therefore, further investigations in the thymic tissue are required to confirm our results.
In conclusion, we identified three previously reported AIRE gene variations in an Iranian Muslim APS-1 patient. Furthermore, the association between c.1095 + 2 T > A mutation and APS-1 disease was confirmed in this report. Finally, AIRE gene expression analysis was performed, which indicated the functional impact of this variation for the first time. This study expands the diversity of variants that could cause APS-1. In addition to diagnostic utility, this information will help us better understand the pathophysiology of APS-1. More genetic studies are required to determine the frequency of these variants and their diagnostic credibility.
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