A deep intronic splice–altering AIRE variant causes APECED syndrome through antisense oligonucleotide-targetable pseudoexon inclusion
Глубокий интронный вариант AIRE, нарушающий сплайсинг, вызывает синдром APECED посредством включения псдоэкзона, на которое можно воздействовать антисмысловым олигонуклеотидом
2024-09-18
SCID: 54.1/nwcqqhuy
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AIRE variantAPECED syndromeantisense oligonucleotidedeep intronic variantpseudoexon inclusion
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Abstract (AI)
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator ( AIRE ) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE -expressing primary patient monocyte-derived dendric cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109–base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE –expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE -transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.
Key Findings
1
Among 104 clinically diagnosed APECED patients, 17 individuals from 14 kindreds lacked coding or canonical splice-region AIRE variants; 15 had Puerto Rican ancestry.
2
An antisense oligonucleotide reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in patient-derived lymphoblastoid cell lines, demonstrating therapeutic reversibility.
3
Pseudoexon inclusion alters the AIRE protein’s carboxyl terminus and abolishes its function, establishing the molecular mechanism causing APECED.
4
The variant creates a cryptic splice site that causes inclusion of a 109-base-pair frameshifting pseudoexon in AIRE transcripts.
5
Whole-genome sequencing identified the deep intronic AIRE variant c.1504-818 G>A, which cosegregated with APECED in all 17 patients and represents a Puerto Rican founder variant.
Research Object
the deep intronic AIRE variant c.1504-818 G>A and its effects on AIRE expression in APECED patient-derived cells
Research Subject
cryptic splice-site activation, 109-bp frameshifting pseudoexon inclusion, resultant AIRE dysfunction, and antisense-oligonucleotide-mediated correction
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2024-09-18
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