Functional analysis of SAND mutations in AIRE supports dominant inheritance of the G228W mutation

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a rare disorder caused by mutations in the autoimmune regulator gene (AIRE) and characterized by a variable combination of organ-specific autoimmune diseases. Studies on AIRE-deficient mice suggest that AIRE is an important factor in the establishment and maintenance of self-tolerance. The AIRE protein contains several structural domains often found in transcriptional regulators and functions as a transcriptional transactivator in vitro. To date, more than 50 patient mutations have been identified in the coding region of the AIRE gene. So far, APECED has been reported to be inherited in an autosomal recessive manner. However, in contrast to all other AIRE mutations, a novel mutation c.682T>G (p.G228W) in the DNA-binding and/or multimerization domain SAND was recently described to be inherited in a dominant fashion. We analyzed the effects of mutant AIRE proteins containing the patient mutations c.682T>G (p.G228W) and c.755C>T (p.P252L) located in the SAND domain on the properties of the wild-type AIRE in a heterozygous situation in vitro. In addition to the patient mutations, we analyzed the effects of a double mutation [c.727A>G;c.728A>C;c.739C>G;c740G>C] (p.K243A;R247A) of positively charged Amino acids in the SAND domain. Of the mutants studied, only c.682T>G (p.G228W) mutant changed the subcellular localization and in addition severely disrupted the transactivating capacity of the wild-type AIRE. Our results indicate that the c.682T>G (p.G228W) mutant AIRE protein acts with a dominant negative effect by binding to the wild-type AIRE, thus preventing the protein from forming the complexes needed for transactivation.