Federica Incani

Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

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.

DAXX is a new AIRE-interacting protein.

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.

AIRE acetylation and deacetylation: effect on protein stability and transactivation activity.

BackgroundThe 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 AIRE gene cause the autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population.AIRE protein is primarily known as a transcriptional regulator and is capable of interacting with numerous proteins. The first characterized partner of AIRE is the ubiquitous transcription factor CREB-binding protein (CBP), which regulates DNA transcription through the acetylation and deacetylation of histones. More recently, the role of p300 in AIRE acetylation, which could influence the selection of AIRE activated genes, has been described.ResultsIn this study, we have precisely mapped, by mass spectrometry experiments, the sites of protein acetylation and, by mutagenesis assays, we have described a set of acetylated lysines as being crucial in influencing the subcellular localization of AIRE. Furthermore, we have also determined that the de-acetyltransferase enzymes HDAC1-2 are involved in the lysine de-acetylation of AIRE.ConclusionsOn the basis of our results and those reported in literature, we propose a model in which lysines acetylation increases the stability of AIRE in the nucleus. In addition, we observed that the interaction of AIRE with deacetylases complexes inhibits its transcriptional activity and is probably responsible for the instability of AIRE, which becomes more susceptible to degradation in the proteasome.

New mutations in DYNC2H1 and WDR60 genes revealed by whole-exome sequencing in two unrelated Sardinian families with Jeune asphyxiating thoracic dystrophy

Jeune asphyxiating thoracic dystrophy (JATD; Jeune syndrome, MIM 208500) is a rare autosomal recessive chondrodysplasia, phenotypically overlapping with short-rib polydactyly syndromes (SRPS). JATD typical hallmarks include skeletal abnormalities such as narrow chest, shortened ribs, limbs shortened bones, extra fingers and toes (polydactyly), as well as extraskeletal manifestations (renal, liver and retinal disease). To date, disease-causing mutations have been found in several genes, highlighting a marked genetic heterogeneity that prevents a molecular diagnosis of the disease in most families. Here, we report the results of whole-exome sequencing (WES) carried out in four JATD cases, belonging to three unrelated families of Sardinian origin. The exome analysis allowed to identify mutations not previously reported in the DYNC2H1 (MIM 603297) and WDR60 (MIM 615462) genes, both codifying for ciliary intraflagellar transport components whose mutations are known to cause Jeune syndrome.

β-defensin CNV is not associated with susceptibility to Candida albicans infections in Sardinian APS I patients.

OBJECTIVE The aim of this study was to investigate whether a variation in the genomic copy number (CNV) of the β-defensin cluster could be associated with the pre-disposition to chronic mucocutaneous candidiasis (CMC) in Sardinian APECED patients. SUBJECTS AND METHODS The β-defensin copy number variation was determined by MLPA analysis in 18 Sardinian APECED patients with CMC and in 21 Sardinian controls. Statistical analyses were performed with one-way ANOVA test. RESULTS No statistically significant results were observed between the patients and controls groups. CONCLUSIONS According to the results we have obtained, it appears that either β-defensin genomic CNV is not a modifier locus for CMC susceptibility in APECED patients, or any effect is too small for it to be detected using such sample size. An extensive study on APECED patients from different geographical areas might reveal the real implication of the β-defensin CNV in the susceptibility to Candida albicans infections.