Johanna Aaltonen

Mutations in the AIRE Gene: Effects on Subcellular Location and Transactivation Function of the Autoimmune Polyendocrinopathy-Candidiasis–Ectodermal Dystrophy Protein

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal disease with recessive inheritance. It is characterized by multiple autoimmune endocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The defective gene responsible for this disease was recently isolated, and several different mutations in the novel gene, AIRE, have been identified, by us and by others, in patients with APECED. We have shown that the APECED protein is mainly localized, both in vitro and in vivo, to the cell nucleus, where it forms distinct speckles. This accords with the predicted structural features of the protein, which suggest involvement of AIRE in the regulation of gene transcription. Here, we report the results of mutational analyses of a series of 112 patients with APECED who were from various ethnic backgrounds. A total of 16 different mutations, covering 91% of disease alleles, were observed; of these, 8 were novel. The mutations are spread throughout the coding region of AIRE, yet four evident mutational hotspots were observed. In vitro expression of four different naturally occurring nonsense and missense mutations revealed a dramatically altered subcellular location of the protein in cultured cells. Interestingly, the wild-type APECED protein tethered to the Gal4 DNA-binding domain acted as a strong transcriptional activator of reporter genes in mammalian cells, whereas most of the analyzed mutant polypeptides had lost this capacity.

A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis.

Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131-136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R. , Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531-535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-b superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-b family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.

Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B

Although targeted gene disruption of GDF-9, an oocyte derived growth factor, leads to an arrest of folliculogenesis and causes infertility in female mice, little is known on the expression of GDF-9 protein in the ovary. We show that GDF-9 protein is expressed in rat oocytes during folliculogenesis from the early primary follicle stage onwards but the most intensive immunostaining was seen in primary and preantral follicles. Northern blot analyses of the ontogeny of GDF-9 gene expression in postnatal rat ovaries showed that the GDF-9 transcript levels are clearly increased on the second postnatal day concomitant with the appearance of primary follicles. Interestingly, Northern blot and in situ hybridization analyses indicate a similar expression pattern for GDF-9B, the rat ortholog of a mouse GDF-9 like factor for which we recently reported the partial amino acid sequence. The polypeptide sequences deduced from isolated ovarian cDNAs indicate that the rat GDF-9 prepropeptide is 440 amino acids (aa) in length and the putative mature peptide is 135 aa whereas rat GDF-9B is 391 aa long and the mature region is 125 aa. We conclude that (1) the GDF-9 protein is highly expressed in the oocytes of primary follicles of rat ovaries suggesting that it plays a role mainly in early folliculogenesis and that (2) the full-length polypeptide sequence of GDF-9B suggests that this novel TGF-beta family member is likely to be a secreted growth factor that may regulate folliculogenesis at similar developmental stages as GDF-9.

Cloning of the APECED gene provides new insight into human autoimmunity

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is the only autoimmune disease characterized so far that is caused by a defect in a single gene. We have recently isolated the defective gene in this disease by positional cloning and have identified several different mutations in APECED patients. This novel gene, AIRE, contains two plant homeodomain (PHD)-type zinc finger motifs and a newly described putative DNA-binding domain SAND. We have further shown that the protein encoded by the AIRE gene is localized to the nuclear body-like structures of cell nuclei. Similar discrete speckles within the nucleus have been suggested to be involved in the regulation of transcription, oncogenesis and differentiation of cells. Together with the predicted structural features of the APECED protein the new data obtained both in vitro and ex vivo suggest that this protein participates in the regulation of gene expression in a restricted set of tissues and cells.

Autoimmune polyglandular disease type I. Exclusion map using amplifiable multiallelic markers in a microtiter well format.

The pathogenetic background of human autoimmunity is only partially understood. By discovering the defective gene causing the autosomal recessive polyglandular autoimmune disease type I (PGDI, APECED) we hope to provide new insights into autoimmune responses in general. Here we have taken advantage of newly developed amplifiable multiallelic microsatellite markers and performed the analyses using the microtiter well format of the polymerase chain reaction. This rapid semiautomated protocol was applied to analyze 62 assigned highly polymorphic loci. The linkage analyses coupled with the EXCLUDE analysis resulted in an exclusion map of this polyglandular autoimmune disease and in the preliminary assignment of the APECED locus to chromosome 22. The method proved to be an effective and economical tool for gene mapping compared with standard blotting and hybridization.

Assignment of ACVR2 and ACVR2B the human activin receptor type II and IIB genes to chromosome bands 2q22.2-->q23.3 and 3p22 and the human follistatin gene (FST) to chromosome 5q11.2 by FISH.

Supported by The Academy of Finland, Helsinki University Central Hospital Research Funds, the Duodecim Society, Medical Association of Finland (Finska Läkaresällskapet i Finland) and the Maud Kuistila Foundation. We thank Dr. Pieter de Jong, Roswell Memorial Institute, Buffalo, NY, USA, for providing the PAC library. Ms. Ritva Javanainen and Ms. Maritta Putkiranta are thanked for excellent technical assistance.

LINKAGE ANALYSES TO ASSIGN THE LOCUS FOR AUTOIMMUNE POLYGLANDULAR DISEASE TYPE I

The autoimmune phenomena associated with many human diseases are still only partially understood. Unravelling the molecular palhogenesis of inherited diseases with a strong autoimmune component in their clinical expression could help to dissect the molecular background of abnormal immune response. One such genetic disorder is autosomal resessive autoimmune polyglandular disease type I (PGD I), also known as autoimmune polyendocrinopathy-candidiasis - ectodermal dystrophy (APECED, MIM 240300). This disease is especially enriched in the genetically isolated population of Finland. Here we have taken advantage of newly developed amplifiable multiallelic microsatellite markers coupled with the microtiter well format of the polymerase chain reaction and linkage analyses to establish the most probable chromosomal location for the APECED locus. The rapid “semiautomated” protocol was here aplied to analyze 100 chromosomally assigned polymorphic loci. The method proved to be an effective and economical tool for gene mapping compared with standard blotting and hybridization and resulted in the preliminary assignment of the APECED locus.