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Autoimmune Regulator-1 Messenger Ribonucleic Acid Analysis in a Novel Intronic Mutation and Two Additional Novel AIRE Gene Mutations in a Cohort of Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients

Katarina Trebuak Podkrajek, Nina Bratani, Ciril Krinik and Tadej Battelino

Centre for Medical Genetics (K.T.P., T.B.) and Department of Pediatric Endocrinology, Diabetes, and Metabolism (N.B., C.K., T.B.), University Children’s Hospital, SI-1000 Ljubljana, Slovenia

Address all correspondence and requests for reprints to: Prof. Tadej Battelino, M.D., Ph.D., University Children’s Hospital, Ljubljana, Vrazov trg 1, SI-1000 Ljubljana, Slovenia. E-mail: tadej.battelino{at}mf.uni-lj.si.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disease associated with mutations in the AIRE gene.

Objective: Our objective was to investigate clinical and mutational characteristics of 12 Slovenian patients from 10 families with APECED.

Methods: Direct sequencing, restriction fragment length polymorphism, and amplification refractory mutation system analyses were used to identify AIRE gene mutations. Autoimmune regulator (AIRE)-1 mRNA analysis was used to confirm pathogenicity of the intronic mutation.

Results: The prevalence of APECED in Slovenian population was estimated to be 1 in 43,000, which is significantly higher compared with the neighboring populations. Three novel mutations were identified among six different mutations detected in the AIRE gene. The first novel mutation was an intronic mutation (653-7_-5delCTC) affecting proper splicing by using a nearby new acceptor splice site as demonstrated by AIRE-1 mRNA analyses. The second and third novel mutations were frame-shift mutations located in exon 5 (540delG) and exon 9 (1064-1068dupCCCGG), both leading to premature truncation of the AIRE protein. The Finnish R257X mutation was the most frequent AIRE gene mutation in Slovenian patients with APECED (16 of 24 alleles).

Conclusions: Three novel AIRE gene mutations were identified. For the first time, a novel intronic mutation was investigated on the mRNA level in APECED. This could be particularly important for APECED patients where no or only heterozygous mutation on the genomic DNA level is detected.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
AUTOIMMUNE POLYENDOCRINOPATHY-candidiasis-ectodermal dystrophy (APECED; Online Mendelian Inheritance in Man no. 240300) or autoimmune polyglandular syndrome type 1 (APS-1) is a rare autosomal recessive disorder, characterized by the presence of two of three major diseases: hypoparathyroidism, Addison’s disease, and chronic mucocutaneous candidiasis. Several associated clinical symptoms include other autoimmune endocrinopathies (hypergonadotropic hypogonadism, insulin-dependent diabetes mellitus, and autoimmune thyroid disease) and nonendocrine disorders (ectodermal dystrophy, vitiligo, alopecia, pernicious anemia, chronic atrophic gastritis, malabsorption, and chronic active hepatitis) (1). The incidence of the disease is higher in some genetically isolated populations (Finland, 1 in 25,000; Iranian Jews, 1 in 9000; and Sardinia, 1 in 14,400). APECED is associated with mutations in the AIRE (autoimmune regulator) gene (2, 3). In accordance with its structural motifs [two plant homeo domain (PHD) zinc-finger motifs, a proline-rich region, four LXXLL motifs, a Spn100, autoimmune regulator (AIRE)-1, NucP41/75, deformed epidermal autoregulatory factor 1 (DEAF-1)/suppresin (SAND) domain, and a homogenously stained region (HSR) domain] (Fig. 1) (2, 3, 4), AIRE is shown to act as a transcriptional activator (5). Only five intronic mutations were identified among at least 54 different mutations in the AIRE gene spread all over the coding region (reviewed in Refs.6 and 7).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Schematic diagram of the AIRE gene, AIRE protein domains, and mutations detected in the present study. Novel mutations are in boxes.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

Thirteen APECED patients from 11 unrelated Slovenian families were identified at the Department of Pediatric Endocrinology Diabetes, and Metabolism, University Children’s Hospital Ljubljana, Slovenia. The study protocol was approved by the Ethical Committee of the Slovenian Ministry of Health, and written informed consent was obtained by all participants before the study.

Mutational analyses of the AIRE gene

All 14 exons and the exon/intron boundaries of the AIRE gene were individually PCR amplified (3, 8), directly sequenced using the Big Dye terminator cycle sequencing kit and ABI PRISM 310 automated sequencer (PE Applied Biosystems, Norwalk, CT) and compared with the normal AIRE gene sequence (GenBank access no. AB006684). R257X was alternatively confirmed by TaqI restriction fragment length polymorphism analysis. Novel 540delG and 653-7_-5delCTC mutations were verified in 50 unrelated healthy Slovenian controls by an amplification refractory mutation system (ARMS), using a flanking ß-globin fragment for coamplification (Table 1). The 1064-1068dupCCCGG mutation was screened in the healthy control group on 13% polyacrylamide gel.

To demonstrate the effect of the novel intron 5 mutation, total RNA of a patient, 11/I, and two unrelated healthy control subjects (one male, one female) were purified from whole blood using TRI reagent (MRC, Cincinnati, OH). RT was performed using specific reverse primer (8BR; Table 1) and GeneAmp RNA PCR kit (Applied Biosystems, Piscataway, NJ). Nested amplification of specific AIRE-1 cDNA was performed in the first round using primers 4F and 8BR amplifying exons 4–8 and in a second round using primers 4–5F and 8AR amplifying exons 5–8 (Table 1). Amplicons were directly sequenced and compared with the normal AIRE-1 mRNA sequence (GenBank accession no. AB006682).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patient characteristics

Twelve Slovenian patients from 10 families were included in the study (Table 2); one refused participation. None of the kindred was consanguineous or related. Clinical characteristics are summarized in Table 2. The number of all clinical manifestations ranged from three to six; two of three major clinical components were present in all 12 patients, whereas all three major clinical components were present in five patients. Ten patients had at least one additional minor manifestation. Mucocutaneous candidiasis was present in 12 (100%), hypoparathyroidism in 10 (83%) and Addison’s disease in seven (58%) of 12 patients. Four patients with recurrent mucocutaneous candidiasis had also onychomycosis (1/A, 4/C, 8/F, and 9/G), and one had fungal esophagitis (10/H). Additionally, during follow-up, growth failure was documented in seven patients (3/B, 4/C, 5/D, 6/E, 7/E, 10/H, and 12/J), pancreatic exocrine insufficiency in two patients (6/E and 12/J), and fungal intestinal overgrowth in two patients (3/B and 10/H). Gonadal failure was present only in one 21-yr-old male patient.

Mutational analyses

Six different mutations of the AIRE gene were detected, three of which were novel and so far not reported (Table 2). Eleven of 12 patients were either homozygous or heterozygous for the R257X mutation. Three novel mutations were identified and named starting numbering from the AUG codon (GenBank accession no. AB006682) and as recommended by den Dunnen and Antonarakis (9). None of them was detected in 50 healthy Slovenian controls.

A novel 653-7_-5delCTC intron 5 mutation was detected in a heterozygous state of maternal origin in patient 11/I (Fig. 2A). This deletion was located at the acceptor site of intron 5 and was further shown to affect proper splicing. The second novel mutation was a deletion, 540delG, in exon 5 identified in a heterozygous state in patient 9/G (Fig. 2B). The 540delG mutation was a frame-shift mutation, allowing the synthesis of 180 amino acids, followed by 196 unrelated amino acids and then a premature stop codon in exon 10. The third novel mutation was the 1064-1068dupCCCGG mutation in exon 9, identified in patient 12/J (Fig. 2C). It led to a frame-shift at position 358 of the AIRE protein and to premature truncation in exon 10 after a track of 21 unrelated amino acids.

View larger version (40K): [in this window] [in a new window]   FIG. 2. Novel AIRE gene mutations. A, Heterozygous 653-7_-5delCTC mutation, normal intron 5 sequence, and ARMS detection [allele-specific fragments amplified by normal primer set N (189 bp) and primer set M specific for 653-7_-5delCTC (185 bp); 269 bp is ß-globin gene control amplicon]; B, heterozygous 540delG mutation, normal exon 5 sequence, and ARMS detection (387 bp is allele-specific fragment amplified by normal primer set N or primer set M specific for 540delG; 269 bp is ß-globin gene control amplicon); C, 1064-1068dupCCCGG mutation, normal exon 9 sequence, and electrophoretic screening for 1064-1068dupCCCGG (normal amplicon is 5 bp shorter than the mutated one). 9/G, 11/I, and 12/J are patients; F, father; Mo, mother; S, sibling; C, healthy control; m1, 50-bp stepladder; m2, X174 HaeIII digest (the latter two from Sigma Chemical Co., St. Louis, MO).

Nested amplification and sequencing of AIRE-1 cDNA in patient 11/I revealed 43 bp of intron 5 (cDNA 8311–8356, GenBank accession no. AB006684) inserted between exons 5 and 6 in AIRE-1 mRNA (Fig. 3). The insertion included the 653-7_-5delCTC mutation detected on the genomic DNA level and was leading to insertion of 15 amino acids, followed by a frame shift resulting in 51 unrelated amino acids and a premature stop codon in exon 7. Two healthy control subjects had normal AIRE-1 mRNA amplicons.

View larger version (26K): [in this window] [in a new window]   FIG. 3. Intronic insertion in AIRE-1 mRNA caused by 653-7_-5delCTC mutation. A, Normal AIRE-1 mRNA nucleotide sequence and sequence with 43-bp insertion of intron 5 (cDNA 8311–8356, GenBank accession no. AB006684); B, normal AIRE-1 cDNA amplicon (367 bp) and amplicon with 43-bp insertion (410 bp). 11/I, patient; C, healthy control, m1, 50-bp stepladder (Sigma).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

So far, only five intronic mutations in the AIRE gene are reported (7, 13, 14, 15, 16). The novel 653-7_-5delCTC mutation was located in a polypyrimidine tract in the acceptor site of intron 5 required for efficient spliceosome assembly and splicing of pre-mRNAs (17). The mRNA analyses showed that 43 bp of intron 5 with a CTC deletion detected on the genomic DNA level were inserted between exons 5 end 6. Therefore, the 653-7_-5delCTC mutation in the polypyrimidine tract caused the use of another acceptor site further in intron 5. The insertion caused truncation of a larger AIRE protein segment encompassing the SAND domain and both PHD zinc finger domains. A similar mutant construct with 217 N-terminal amino acids almost completely abolishes AIRE transcriptional properties (18) and therefore affects the expression of proteins, which are under the regulatory influence of AIRE. Therefore, the novel intronic mutation could be responsible for the disease development. The patient with the novel 653-7_-5delCTC mutation had a typical clinical picture with all three major clinical characteristics developed in early childhood before she turned 5. Only 22% of patients with APECED fulfill diagnostics criteria by the fifth year of life (19).

In peripheral blood leukocytes, AIRE mRNA is restrictively expressed in a monocyte/dendritic cell lineage as assessed by RT-PCR analyses in healthy subjects (20). To the best of our knowledge, the present work is the first report of blood leukocytes AIRE-1 mRNA analyses used to elucidate an intronic mutation. AIRE-1 mRNA analyses could point out undetected intronic mutations in APECED patients where establishing the AIRE gene genotype on the genomic DNA level has failed, as was reported for some other diseases (21, 22). So far, nine APECED patients with no mutations (5, 10, 15) and 19 patients with only the heterozygous mutation (5, 8, 10, 12, 13, 15, 23) are described. Additionally, in 12 among 208 chromosomes described in Halonen et al. (24), no mutation was found.

The second novel 540delG mutation is a frame-shift mutation introducing 196 unrelated amino acids and a premature stop codon in exon 10. This leads to the disruption of the SAND domain and the first PHD zinc finger motif and to elimination of a significant part of the AIRE protein. Deletion mutant constructs with only 207 (25) and 209 (26) N-terminal amino acids, resembling the novel 540delG mutation, show a disrupted nuclear staining pattern.

The 540delG deletion is located in gGG trinucleotide, where the first G is lying in intron 4 and was named following the mutation nomenclature rule to count the most 3' nucleotide (9). Because the complete consensus sequence in the 3' splice site (including the polypyrimidine tract, AG site, and also the first G in exon 5) remains unchanged after the deletion of a G, this mutation is unlikely to affect splicing.

The third novel 1064-1068dupCCCGG mutation also leads to the premature truncation of the AIRE protein with the absence of the proline-rich region, two LXXLL motifs, and a second PHD zinc finger motif. The deletion mutant construct of 350 N-terminal amino acids missing the same regions is partly localized in the nucleus, but they fail to form nuclear dots (27). The patient with this mutation presented with mucocutaneous candidiasis in his first year of life and hypothyroidism at the age of 5 yr. Hypothyroidism is an uncommon manifestation in APECED patients, and was described as the first endocrinopathy in only one Finnish patient (19). R257X was the most frequent AIRE gene mutation in Slovenian APECED patients. This is also shown for other Central and Eastern European populations (12).

In conclusion, clinical and mutational characteristics of 12 Slovenian patients from 10 families with APECED were investigated. Three novel mutations in the AIRE gene causing loss of function of the AIRE protein were identified among six different mutations detected. This is the first report of mRNA analyses used to confirm pathogenicity of the AIRE gene intronic mutation. It could be especially important for APECED patients where no or only a heterozygous mutation on the genomic DNA level is detected.

	Acknowledgments

 
We thank Mrs. Jurka Ferran for her expert technical assistance.

	Footnotes

 
This study was supported in part by The Slovenian Ministry of Education, Science, and Sport Grants J3-3096 and J3-6072.

First Published Online May 10, 2005

Abbreviations: AIRE, Autoimmune regulator; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; APS-1, autoimmune polyglandular syndrome type 1; ARMS, amplification refractory mutation system; PHD, plant homeo domain; SAND, Sp100, AIRE-1, NucP41/75, DEAF-1/suppresin.

Received February 28, 2005.

Accepted May 3, 2005.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Trebusak Podkrajsek, K. Articles by Battelino, T. Search for Related Content PubMed PubMed Citation Articles by Trebusak Podkrajsek, K. Articles by Battelino, T. Related Collections Adrenal and Hypertension Thyroid Calcium and Bone Metabolism