Figure 1.
AIRE overview family pedigree and detection of the c.653-1G>A AIRE mutation.
(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 AIRE 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 AIRE 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 AIRE 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.
Table 1.
Clinical features and analytical data.
Figure 2.
Sequencing representation of the cDNA analysis.
(A) Chromatogram and schematic representation of the non-mutated exon 5 and 6 junction. (B) Partial gDNA sequence of exon 5, intron 5 and exon 6. Finally, (C) chromatogram and schematic representation of the mutated allele, including exon 5, part of intron 5 and exon 6. The arrow indicates the mutation.
Table 2.
Splice site prediction software output.
Figure 3.
Nonsense pre-mRNA mediated decay analysis.
(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.
Figure 4.
Tertiary schematic representation of the aligned non-mutated and mutated AIRE proteins.
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.