We report two novel mutations, c.230T>C (p.F77S) and c.64_69del (p.V22_D23del) within the HSR domain of the AIRE protein in two patients of Italian descent affected by APECED. Both mutations were found in the compound heterozygous state respectively with c.994+5G>T and c.232T>A (p.W78R). With the two-hybrid assay in the yeast system we found that constructs containing the two mutations fail to interact with the wild-type protein. These findings indicate that both mutations negatively affected the homodimerization properties of the AIRE protein, thereby leading to a defective function.

No abstract

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5-and 3-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.Cystic fibrosis (CF 2 ; MIM entry #219700) is the most frequent severe autosomic recessive disorder in the European population (1). Indeed, CF affects ϳ1 in 2500 births, and ϳ1 in 25 individuals are heterozygotes, with marked regional variations (2). CF is caused by mutations of the CF transmembrane conductance regulator (CFTR or ABCC7; MIM #602421) gene, which is also involved in a broad spectrum of phenotypes, including male infertility due to congenital bilateral absence of the vas deferens (3, 4), disseminated bronchiectasis (5, 6), and chronic pancreatitis (7,8). The mutational spectrum of this disease is made up of Ͼ1600 different mutations, 98% of which consist of point mutations or microdeletions/insertions (Cystic Fibrosis Mutation Database). A significant fraction (ϳ13%) of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined.Splicing mutations act by directly disrupting splice sites or by creating new ones. Varied levels of alternative splicing have been detected for some of the splicing mutations (9 -11). These differences are most likely due to different utilization of intragenic splicing elements by trans-acting splicing factors (12, 13). cis-Acting elements required for splicing in mammalian cells include short and poorly conserved consensus sequences, which specify the 5Ј-splice site, branch site, and 3Ј-splice site. The 3Ј-splice site is preceded by a polypyrimidine tract of variable length.The branch point is typically located 18 -40 nucleotides upstream of the polypyrimidine tract. These splicing signals are es...

PAGE 17257:Fig. 2, line 9 should read: F, a projection through a 350 nm thick section of longitudinally sectioned P3H1 null tendon from which the tilt series (supplemental Fig. S1) was collected. ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

The AIRE protein plays a remarkable role as a regulator of central tolerance by controlling the promiscuous expression of tissue-specific antigens in thymic medullary epithelial cells. Defects in the AIRE gene cause the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population. To this day, the precise function of the AIRE protein in regulating transcription and its interacting proteins has yet to be entirely clarified. The knowledge of novel AIRE interactors and their precise role will improve our knowledge of its biological activity and address some of the foremost autoimmunity-related questions. In this study, we have used a yeast two-hybrid system to identify AIRE-interacting proteins. This approach led us to the discovery of a new AIRE-interacting protein called DAXX. The protein is known to be a multifunctional adaptor with functions both in apoptosis and in transcription regulation pathways. The interaction between AIRE and DAXX has been validated by in vivo coimmunoprecipitation analysis and colocalization study in mammalian cells. The interaction has been further confirmed by showing in transactivation assays that DAXX exerts a strong repressive role on the transcriptional activity of AIRE.