Positional cloning of the APECED gene.

Autoimmune polyglandular syndrome type I (APS 1, also called APECED) is an autosomal-recessive disorder that maps to human chromosome 21q22.3 between markers D21S49 and D21S171 by linkage studies. We have isolated a novel gene from this region, AIRE (autoimmune regulator), which encodes a protein containing motifs suggestive of a transcription factor including two zinc-finger (PHD-finger) motifs, a proline-rich region and three LXXLL motifs. Two mutations, a C-->T substitution that changes the Arg 257 (CGA) to a stop codon (TGA) and an A-->G substitution that changes the Lys 83 (AAG) to a Glu codon (GAG), were found in this novel gene in Swiss and Finnish APECED patients. The Arg257stop (R257X) is the predominant mutation in Finnish APECED patients, accounting for 10/12 alleles studied. These results indicate that this gene is responsible for the pathogenesis of APECED. The identification of the gene defective in APECED should facilitate the genetic diagnosis and potential treatment of the disease and further enhance our general understanding of the mechanisms underlying autoimmune diseases.

Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness.

Approximately 50% of childhood deafness is caused by mutations in specific genes. Autosomal recessive loci account for approximately 80% of nonsyndromic genetic deafness. Here we report the identification of a new transmembrane serine protease (TMPRSS3; also known as ECHOS1) expressed in many tissues, including fetal cochlea, which is mutated in the families used to describe both the DFNB10 and DFNB8 loci. An 8-bp deletion and insertion of 18 monomeric (approximately 68-bp) beta-satellite repeat units, normally present in tandem arrays of up to several hundred kilobases on the short arms of acrocentric chromosomes, causes congenital deafness (DFNB10). A mutation in a splice-acceptor site, resulting in a 4-bp insertion in the mRNA and a frameshift, was detected in childhood onset deafness (DFNB8). This is the first description of beta-satellite insertion into an active gene resulting in a pathogenic state, and the first description of a protease involved in hearing loss.

Cholesterol 25-hydroxylase is a metabolic switch to constrain T cell-mediated inflammation in the skin.

Interleukin-27 (IL-27) is an immunoregulatory cytokine whose essential function is to limit immune responses. We found that the gene encoding cholesterol 25-hydroxylase (Ch25h) was induced in CD4+ T cells by IL-27, enhanced by transforming growth factor­ß (TGF-ß), and antagonized by T-bet. Ch25h catalyzes cholesterol to generate 25-hydroxycholesterol (25OHC), which was subsequently released to the cellular milieu, functioning as a modulator of T cell response. Extracellular 25OHC suppressed cholesterol biosynthesis in T cells, inhibited cell growth, and induced nutrient deprivation cell death without releasing high-mobility group box 1 (HMGB1). This growth inhibitory effect was specific to actively proliferating cells with high cholesterol demand and was reversed when extracellular cholesterol was replenished. Ch25h-expressing CD4+ T cells that received IL-27 and TGF-ß signals became refractory to 25OHC-mediated growth inhibition in vitro. Nonetheless, IL-27­treated T cells negatively affected viability of bystander cells in a paracrine manner, but only if the bystander cells were in the early phases of activation. In mouse models of skin inflammation due to autoreactive T cells or chemically induced hypersensitivity, genetic deletion of Ch25h or Il27ra led to worse outcomes. Thus, Ch25h is an immunoregulatory metabolic switch induced by IL-27 and dampens excess bystander T effector expansion in tissues through its metabolite derivative, 25OHC. This study reveals regulation of cholesterol metabolism as a modality for controlling tissue inflammation and thus represents a mechanism underlying T cell immunoregulatory functions.

Common mutations in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients of different origins.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; OMIM *240300, also called APS 1,) is a rare autosomal recessive disorder that is more frequent in certain isolated populations. It is generally characterized by two of the three major clinical symptoms that may be present, Addison's disease and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. Patients may also have a number of other clinical symptoms including chronic gastritis, gonadal failure, and rarely, autoimmune thyroid disease and insulin-dependent diabetes mellitus. We and others have recently identified the gene for APECED, which we termed AIRE (for autoimmune regulator). AIRE is expressed in thymus, lymph nodes, and fetal liver and encodes a protein containing motifs suggestive of a transcriptional regulator, including two zinc finger motifs (PHD finger), a proline-rich region, and three LXXLL motifs. Six mutations, in cluding R257X, the predominant Finnish APECED allele, have been defined. R257X was also observed in non-Finnish APECED patients occurring on different chromosomal haplotypes suggesting different mutational origins. Here we present mutation analyses in an extended series of patients, mainly of Northern Italian origin. We have detected 12 polymorphisms, including one amino acid substitution, and two additional mutations, R203X and X546C, in addition to the previously described mutations, R257X, 1096-1097insCCTG, and a 13-bp deletion (1094-1106del). R257X was also the common mutation in the Northern Italian patients (10 of 18 alleles), and 1094-1106del accounted for 5 of 18 Northern Italian alleles. Both R257X and 1094-1106del were both observed in patients of four different geo-ethnic origins, and both were associated with multiple different haplotypes using closely flanking polymorphic markers showing likely multiple mutation events (six and four, respectively). The identification of common AIRE mutations in different APECED patient groups will facilitate its genetic diagnosis. In addition, the polymorphisms presented provide the tools for investigation of the involvement of AIRE in other autoimmune diseases, particularly those affecting the endocrine system.

APECED mutations in the autoimmune regulator (AIRE) gene.

Autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy (APECED) is a rare recessively inherited disorder caused by mutations in the AIRE (autoimmune regulator) gene. APECED is characterized by variable combinations of endocrine autoimmune diseases such as Addison's disease, hypoparathyroidism, and type 1 diabetes. The AIRE protein contains motifs suggestive of a transcription regulator and can activate transcription of a reporter gene when fused to a heterologous DNA biding domain. In this article, mutation analyses of over 200 APECED patients published by several laboratories are summarized. To date 42 different mutations have been identified. These mutations include nonsense and missense mutations, small insertions and deletions leading into frame shifts, and splice site mutations. Although mutations are spread throughout the coding region of the gene some hotspots emerge, including the more common and recurrent mutations R257X and 967-979del13bp. Some of the identified mutations have been shown to affect subcellular localization or transactivation properties of the protein, thus providing insights into the functional properties of the predicted protein motifs.

Novel mutations in the myocilin gene in Japanese glaucoma patients.

Myocilin is a gene responsible for juvenile onset primary open angle glaucoma (POAG) mapped as the GLC1A locus and, many mutations have been reported worldwide. Some mutations were found not only in patients with juvenile onset POAG, but also in patients with late onset POAG and in patients with normal tension glaucoma. To investigate the mutation prevalence in Japan, we performed a mutation analysis in 140 unrelated Japanese patients. We have identified the 10 sequence variants, of which four were highly probable for disease-causing mutations (Arg46ter, Arg158Gln, Ile360Asn, and Ala363Thr), and six polymorphisms (Gln19His, Arg76Lys, Asp208Glu, Val439Val, Arg470His, and Ala488Ala). Thus, myocilin mutations were found at the rate of 4/140 (2.9%) probands, similar to previous reports with other ethnic populations.

Detection of the 170-kDa bullous pemphigoid antigen by immunoprecipitation.

There has been controversy concerning the nature of the bullous pemphigoid (BP) antigen: immunoprecipitation identified BP antigen as a single, unique 230-kDa protein, whereas immunoblot analysis showed multiple antigen molecules, mainly 230- and 170-kDa proteins. In this study, to further characterize the 170-kDa protein, we have examined whether the 170-kDa protein is detected by immunoprecipitation. Extracts of human squamous cell carcinoma cells revealed the 170-kDa protein with immunoblot analysis. Although the conventional immunoprecipitation detected only the 230-kDa protein, some BP sera that detected the 170-kDa protein with immunoblotting also precipitated the 170-kDa protein with our modified immunoprecipitation, in which the cells were extracted with 1% sodium dodecylsulfate (SDS) buffer and reacted with the sera under reduced SDS concentration. The 170-kDa protein-specific BP sera clearly showed hemidesmosomal plaque staining with immunofluorescence of cultured cells. These results indicate that the 170-kDa protein is indeed one of the BP antigens and that the 230- and 170-kDa BP antigens are integrated in different ways in hemidesmosomes.

The extracellular aminoterminal domain of bovine desmoglein 1 (Dsg1) is recognized only by certain pemphigus foliaceus sera, whereas its intracellular domain is recognized by both pemphigus vulgaris and pemphigus foliaceus sera.

The major antibody binding regions of desmoglein 1 (Dsg1) in pemphigus foliaceus and pemphigus vulgaris were examined using cDNA-encoded fusion proteins combining glutathione S-transferase with various domains of bovine Dsg1, namely, the extracellular regions EC1-2, EC3-5, EC1-5, and the entire intracellular region IC. In immunoblot analyses using these fusion proteins, EC1-2, as well as EC1-5, which comprises EC1-2, were recognized by 50% of the sporadic pemphigus foliaceus sera and 45% of Brazilian pemphigus foliaceus sera that reacted with Dsg1 in immunoblotting of bovine desmosome preparations. None of these fusion proteins reacted with any sera of pemphigus vulgaris. None of these sera showed reactivity with EC3-5. In contrast, the IC domain was recognized by 91% of pemphigus vulgaris sera reactive with Dsg1 in bovine desmosome preparations, and by certain pemphigus foliaceus and Brazilian pemphigus foliaceus sera. These results indicate that major epitopes of Dsg1 recognized by pemphigus foliaceus and Brazilian pemphigus foliaceus sera are located in the extracellular amino-terminal domain EC1-2, and that sera of the Dsg1-positive pemphigus vulgaris contain antibodies against the intracellular domain, which may not play a pathogenic role. Possible reasons for this selectivity of antigen binding site are discussed.

Localization of 16 exons to a 450-kb region involved in the autoimmune polyglandular disease type I (APECED) on human chromosome 21q22.3.

As a step toward identifying the pathogenic genes for autoimmune polyglandular disease type I (APECED) and other disorders mapped to the PFKL locus on chromosome 21q22.3, we have constructed a cosmid/BAC (bacterial artificial chromosome) contig of 450 kb covering markers D21S1460-D21S25-PFKL-D21S154 and performed exon trapping. We isolated 22 distinct exons including 6 exons derived from two known genes (PFKL and EHOC-1). Among 16 novel exons, 2 exons matched with human expressed sequence tags (EST) and 7 exons showed homology at predicted amino acid sequence level with proteins from other species. These 16 exons were mapped back to the cosmid contigs, 12 of which were confirmed for their expression by polymerase chain reaction (PCR) screening of human cDNA libraries of various tissues. These exon sequences and a transcript map will aid for isolation of corresponding genes which will be identified as candidate genes involved in the pathogenesis of disorders mapped to the 21q22.3 region.

Molecular mechanisms of human single-minded 2 (SIM2) gene expression: identification of a promoter site in the SIM2 genomic sequence.

We previously postulated that the single-minded 2 (SIM2) gene identified on the human chromosome 21q22.2 is a good candidate gene for the pathogenesis of mental retardation in Down syndrome because its mouse homolog exhibits preferential expression in the mouse diencephalon during early embryogenesis. We analyzed the genomic sequence of the entire SIM2 gene which consists of 11 exons and spans over 50 kb. As a step toward understanding the molecular mechanisms of SIM2 gene expression, we have analyzed the human SIM2 gene expression in nine established human cell lines. Three transcripts of 3.6, 4.4, and 6.0 kb were detected in the glioblastoma cell line, T98G, neuroblastoma cell line, TGW, and transformed embryonic kidney cell line, 293. The RACE analysis using SIM2-expressing human cell line T98G provided evidence for the transcription start site at approximately 1.2 kb upstream of the translation initiation site. The transfection assay using various deletion constructs with reporter gene suggested the presence of a presumptive promoter region. Transient transfection assay in T98G cell line revealed a significant promoter activity located in the 60 bp sequence between nt -1385 and -1325 upstream region of the translation initiation site. This 60 bp sequence contains cis-elements for c-myb, E47 and E2F transcription factors. Moreover, the gel retardation assay using oligo-DNA of various cis-element sequences indicated the presence of protein factor(s) which bind to the cis-element for c-myb. These results suggested that binding of a protein transcription factor(s) such as c-myb or that alike regulates transcription of the SIM2 gene by binding to a small upstream region.

Detailed mapping of the ERG-ETS2 interval of human chromosome 21 and comparison with the region of conserved synteny on mouse chromosome 16.

We have carried out a detailed annotation of 550 kb of genomic DNA on human chromosome 21 containing the ERG and ETS2 genes. Comparative genomic analysis between this region and the interval of conserved synteny on mouse chromosome 16 indicated that the order and orientation of the ERG and ETS2 genes were conserved and revealed several regions containing potential conserved noncoding sequences. Four pseudogenes including those for small protein G, laminin receptor, human transposase protein and meningioma-expressed antigen were identified. A potentially novel gene (C21orf24) with alternative mRNA transcripts, consensus splice donor and acceptor sites, but no coding potential nor murine orthologue, was identified and found to be expressed in a range of human cell lines. We have identified four novel splice variants that arise from a previously undescribed 5' exon of the human ERG gene. Comparison of the cDNA sequences enabled us to determine the complete exon-intron structure of the ERG gene. We have also identified the presence of noncoding RNAs in the first and second introns of the ETS2 gene. Our studies have important implications for Down syndrome as this region contains multiple mRNA transcripts, both coding and potentially noncoding, that may play as yet undescribed roles in the pathogenesis of this disorder.

Human BAC library: construction and rapid screening.

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.

Criteria for gene identification and features of genome organization: analysis of 6.5 Mb of DNA sequence from human chromosome 21.

To establish criteria for and the limitations of novel gene identification, to identify novel genes of potential relevance to Down Syndrome and to investigate features of genome organization, 6. 550kb. In total, 41 novel gene models were predicted, and for a subset of these, RT-PCR experiments helped to verify and refine the models, and were used to assess expression in early development and in adult brain regions of potential relevance to Down syndrome. Results suggest generally low and/or restricted patterns of expression, and also reveal examples of complex alternative processing, especially in brain, that may have important implications for regulation of protein function. Analysis of complete gene structures of the known genes identified a number of very large introns, a number of very short intergenic distances, and at least one potentially bi-directional promoter. At least 3/4 of known genes and 1/2 of predicted genes are associated with CpG islands. For novel genes, three cases of overlapping genes are predicted. Results of these analyses illustrate some of the complexities inherent in mammalian genome organization and some of the limitations of current sequence analysis technologies. They also doubled the number of potential genes within the region.

Quantitative determination of heteroplasmy in Leber's hereditary optic neuropathy by single-strand conformation polymorphism.

PURPOSE: The maternal inheritance of Leber's hereditary optic neuropathy (LHON) is caused by defects in the genes of mitochondrial DNA (mtDNA). The most prevalent mtDNA mutation, present in 40% to 90% of families with this disease, is a G to A substitution at nucleotide position 11778. The rapid and accurate quantification of heteroplasmy of this mutation will help determine the relative risk for disease expression. METHODS: The authors conducted screening tests for heteroplasmy in 44 visually affected patients with the 11778 mutation and 34 unaffected members of 36 Japanese families with LHON using the single-strand conformation polymorphism analysis. This method can detect even a single base difference between the sequences of wild type and mutant DNA strands. The percentage of mutant mtDNA was calculated using an image analyzer. RESULTS: Single-strand conformation polymorphism analysis allowed the detection of heteroplasmy ranging from 5% to 95%. Five (14%) of the 36 families showed heteroplasmy, and 14 (18%) of the 78 persons tested had heteroplasmy ranging from 10% to 94%. Seven patients with heteroplasmy with visual loss had mutant mtDNA ranging from 62% to 94%. CONCLUSIONS: Single-strand conformation polymorphism analysis is rapid, efficient, and accurate for detecting point mutations and quantifying heteroplasmy in mtDNA. Individuals with heteroplasmy with less than 60% of mutant mtDNA in circulating leukocytes are probably at lesser risk for developing optic atrophy.

Human gene mapping and chromosome sorting by laser technology.

More than 30 human genes have been mapped by a combination of the fluorescent in situ hybridization (FISH) and spot blot hybridization in this laboratory for the past five years. Furthermore, the Keio strategy has been established for the molecular analysis of the human genome using flow-sorted human chromosomes.

A novel mutation at codon 124 (R124L) in the BIGH3 gene is associated with a superficial variant of granular corneal dystrophy.

OBJECTIVE: To identify the mutation in a human transforming growth factor beta-induced gene (BIGH3) in a Japanese family with a severe form of granular corneal dystrophy of early onset associated with recurrent corneal erosions. PATIENTS: The tentative clinical diagnosis in this family was Reis-Bücklers corneal dystrophy; 4 persons affected with this disorder have been identified in 4 generations, and 3 of the 4 were examined. The proband underwent keratoplasties in our hospital (Keio University Hospital, Tokyo, Japan). METHODS: The BIGH3 gene was examined for a mutation by the polymerase chain reaction and direct sequencing. Corneal buttons of the proband were stained and examined by electron microscopy. RESULTS: Three affected persons were shown to have a heterozygous G-->T transversion at the second nucleotide position of codon 124 (Arg-->Leu) of the BIGH3 gene. In the proband, corneal deposits between the epithelium and the Bowman layer stained red with Masson trichrome stain. Electron microscopy revealed numerous electron-dense, rod-shaped bodies next to the epithelial basement membrane but no curly fibers suggestive of Thiel-Behnke dystrophy. CONCLUSION: A novel R124L mutation of the BIGH3 gene was associated in this family with a superficial variant of granular corneal dystrophy. CLINICAL RELEVANCE: This mutation causes a severe form of superficial granular corneal dystrophy by producing abnormal keratoepithelin between the epithelium and the Bowman layer and thus clinical similarities to Reis-Bücklers corneal dystrophy.

Expression of APG-2 protein, a member of the heat shock protein 110 family, in developing rat brain.

APG-2 protein is a member of the heat shock protein 110 family, and it is thought to play an important role in the maintenance of neuronal functions under physiological and stress conditions. However, neither the tissue-distribution of APG-2 protein nor developmental change of its expression has been studied at the protein level. Therefore, we generated an antiserum against APG-2 protein and studied expression of this protein in rat brain and other tissues by use of the Western blot method. The results showed a high expression of APG-2 protein in various regions of the central nervous system (cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla pons, and spinal cord) throughout the entire postnatal stage. Similarly, a high level of APG-2 protein was detected in the whole brain of rat embryos and in adult rat tissues such as liver, lung, spleen, and kidney. In contrast, its expression in heart was high at postnatal days 1 and 3, but thereafter drastically decreased to a low level. Furthermore, APG-2 protein was detected in neuronal primary cultures prepared from rat cerebral cortex, and its level did not change notably during neuronal differentiation. These results show that APG-2 protein is constitutively expressed in various tissues and also in neuronal cells throughout the entire embryonic and postnatal period. suggesting that it might play an important role in these tissues under non-stress conditions.

The classic form of granular corneal dystrophy associated with R555W mutation in the BIGH3 gene is rare in Japanese patients.

PURPOSE: To identify the BIGH3 gene mutation in 10 unrelated Japanese individuals with granular corneal dystrophy. METHODS: Genomic DNA was obtained from each patient's leukocytes. Exons 4 and 12 of the BIGH3 gene were amplified by polymerase chain reaction and were directly sequenced. RESULTS: Nine of these patients were found to have the R124H mutation, whereas only one had the R555W mutation. Slit-lamp examination showed that the granular corneal dystrophy associated with each mutation is different. CONCLUSIONS: These results, together with our previous findings, show that the classic form of granular corneal dystrophy associated with the R555W mutation is rare in Japanese patients, whereas granular corneal dystrophy accompanied by amyloid deposits and associated with the R124H mutation, Avellino corneal dystrophy, is more common. Direct examination may be insufficient in the proper diagnosis of corneal dystrophy, and BIGH3 mutation analysis may be required.