New Splice Site Acceptor Mutation in <i>AIRE</i> Gene in Autoimmune Polyendocrine Syndrome Type 1

<div><p>Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison’s disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this <i>AIRE</i> mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the <i>AIRE</i> gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.</p></div

Clinical features and analytical data.

<p>Abbreviations: IDDM (Insulin-dependent Diabetes Mellitus), T2D (Type 2 Diabetes), PTH (Parathyroid hormone), ACTH (Adrenocorticotropic hormone), GAD (Glutamic acid decarboxylase), IA-2 (Islet Antigen 2) and Ab (antibodies).</p

AIRE overview family pedigree and detection of the c.653-1G>A AIRE mutation.

<p>(A) Schematic AIRE protein representation showing the different protein domains: HSR domain (HSR), conserved bipartite nuclear localization signal (NLS), PHD zinc finger motif (PHD), proline-rich region (PRR), LXXLL motif (L), and SAND domain (SAND). (B) Schematic <i>AIRE</i> gene representation, where rectangles indicate exons and the dashed line the introns. Finally, (C) schematic representation of the consensus sequences for the 5′ splice site donor (SSD), branch site and 3′ splice site acceptor (SSA). The star indicates the mutation. (D) Pedigree of the Spanish consanguineous family. Genotypes were shown as wild-type (G/G), heterozygous (G/A) and homozygous (A/A) of the c.653-1G>A <i>AIRE</i> mutation. The arrow indicates the index case. (E) Above, the schematic representation of the junction between intron 5 and exon 6, and below, the direct sequence analysis of the <i>AIRE</i> gene identified homozygous carriers of the c.653-1G>A mutation in APS-1 patients, while heterozygous carriers were found in unaffected relatives (I-1, I-2, III-2) of the family.</p

Nonsense pre-mRNA mediated decay analysis.

<p>(A) Schematic representation of the non-mutated and mutated allele analysis, with the specifically designed primers positions, including the non-mutated forward (white), the mutated forward (black) and the common reverse (grey). (B) Agarose gel electrophoresis of the qRT-PCR products revealed that the cDNA fragment of 76-bp corresponds to the predicted wild-type transcript, whereas the cDNA large fragment of 129-bp corresponds to the aberrant transcript. (C) Expression results of each allele normalized to that of the control G/G carriers for the individuals studied. The table below includes the mean relative expression and the standard error mean (SEM) for the non-mutated (NM) and mutated (M) alleles.</p

Splice site prediction software output.

<p>The scores of the preprocessing models reflect the splice site strength. Abbreviations: HSF (Human Splice Finder), SP (Splice Port) and ASSP (Alternative Splice Site Predictor).</p

Tertiary schematic representation of the aligned non-mutated and mutated AIRE proteins.

<p>The non-mutated AIRE protein shows the different functional domains: HSR in red, NLS in green, SAND in blue, PHD in orange and PRR in yellow. The mutated AIRE protein, for its part, is represented in black.</p