PMC3540864_01
Pedigree of the two patients with APS-1 and detection of the c.463G>A AIRE mutation.. (A) Pedigree of the Chinese family with APS-1. Numbered family members were subjected to mutational analysis. Genotypes were shown as wild-type (G/G), heterozygous (G/A) and homozygous (A/A) of the c.463G>A AIRE mutation. Arrow indicates the proband. (B) Direct sequence analysis of the AIRE gene identified homozygous carriers of c.463G>A mutation in patients, while heterozygous carriers were found in unaffected members (III-1, III-2, IV-2, V-1) of the family. Control and IV-4 carried wild-type AIRE. Arrow indicates the homozygous G>A mutation at the end of exon 3.
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They were born from healthy consanguineous parents of third cousins (Figure 1A). Direct sequencing of the AIRE gene revealed that the affected patients were homozygote for a missense mutation c.463G>A at the end of exon 3, which belongs to the conserved splice donor sequence (Figure 1B).
Alternative splicing of AIRE by the c.463G>A mutation.. (A) Schematic diagram of the AIRE minigene constructs. Complete genomic DNA spanning from exon 2 to exon 5 of AIRE gene was amplified from wild-type control and the APS-1 patient (homozygous for the c.463G>A mutation), respectively, for minigene analysis. P1 and P2 were exonic primers to evaluate the results of RT-PCR. (B) Minigene constructs were individually transfected into HeLa cells. RT-PCR products amplified with the primers (P1 and P2) showed an aberrant splicing pattern in mutant minigene compared with wild-type minigene. The predicted cDNA transcripts are shown, which was confirmed by DNA sequence analysis. * indicates the premature termination codon in intron 3. (C) RT-PCR analysis of AIRE mRNA from lymph node needle biopsy of a heterozygous carrier (III-1; G/A) and a healthy control (G/G) using the same primers of minigene analysis. A cDNA fragment of 295-bp, which corresponded to predicted wild-type transcript, was identified in both control and the heterozygous carrier of the c.463G>A mutation. In addition, a longer 678-bp transcript was identified in the heterozygous carrier. DNA sequence analysis of the 678-bp transcript revealed the retention of exon 3. (D) In vitro translation assay. Total cell lysates of HEK293T cells transfected with wild-type or intron 3-retaining AIRE were immunoblotted with FLAG antibody. Compared with the wild-type, the intron 3-retaining AIRE translated a shorted product. beta-actin was used as an internal control.
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Two minigene constructs, harboring the wild-type and mutant AIRE fragments (1902-bp genomic DNA, spanning from AIRE exon 2 to exon 5) (Figure 2A), respectively, were generated. Twenty-four hours after transfection, total RNA was extracted, reverse transcribed and amplified with primers (P1 and P2) located in exon 2 and exon 5 (Figure 2A). Differential splicing pattern was found between the cells transfected with wild-type and mutant minigenes (Figure 2B). Sequencing analysis of the 678-bp amplicons revealed that this transcript was an alternatively spliced product which retained intron 3 (Figure 2B). While a 295-bp transcript was detected in both the control and heterozygous subjects, consistent with the minigene analysis result in cell lines, we specifically detected an additional aberrant 678-bp fragment in the heterozygous subject (Figure 2C). cDNA sequence analysis confirmed the 295-bp and 678-bp transcripts as wild-type and intron 3-retaining transcript, respectively (Fig. 2C). The intron 3-retaining AIRE translated an abnormal protein band corresponding to the expected truncated AIRE (Figure 2D).