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.

Recurrent herpes simplex virus infection in a patient with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy associated with L29P and IVS9-1G>C compound heterozygous autoimmune regulator gene mutations.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) has characteristic clinical features with organ-specific autoimmune polyendocrine diseases and candidiasis, caused by the mutations of autoimmune regulator (AIRE) gene. Although almost all patients are complicated with mucocutaneous candidiasis, no apparent susceptibility to other infections has yet been reported. We herein report that a patient with APECED suffered from recurrent herpes simplex virus type 1 (HSV-1) infection after severe primary herpetic stomatitis, associated with sequential HSV-1 isolates of the same genomic profile, consistent with endogeneous recurrence. Thus, not only candidiasis but also HSV infection should receive more attention in patients with APECED, with treatment being administered accordingly.

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.

Fukutin-related protein gene mutated in the original kindred limb-girdle MD 2I.

The authors mapped an autosomal recessive form of limb-girdle MD on chromosome 19q13.3 (LGMD2I), further narrowed down the candidate region to 1.1 Mb, and identified one new homozygous mutation in the fukutin-related protein (FKRP) gene on patients of the original Tunisian family. Immunohistochemical and immunoblot analysis showed abnormal expression of alpha-dystroglycan and laminin-alpha2 supporting the hypothesis that FKRP has a role in the interaction between the extracellular matrix components.

Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency.

Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.

Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.

The human ABCG1 gene encodes a member of the ATP-binding cassette (ABC) superfamily of transporter proteins and is highly induced when macrophages are incubated with oxysterols. Using mRNA from oxysterol-treated human THP-1 cells together with 5'-rapid amplification of cDNA ends and polymerase chain reaction, we identified a novel ABCG1 transcript that encodes a putative protein of 786 residues containing a new amino terminus of 203 amino acids. Characterization of the genomic organization and structure of the human ABCG1 gene demonstrates that: (i) the gene consists of 23 exons spanning 98 kilobase pairs (kb) on chromosome 21q22.3, (ii) the 203 amino acids are encoded on three previously unidentified exons, 8-10, and (iii) a promoter, containing a TATA box and two liver X receptor (LXR) alpha response elements (LXREs), is located upstream of exon 8. Northern analysis using exon-specific probes confirms that oxysterol treatment results in >10-fold induction of ABCG1 transcripts that are derived from either exons 8-23 or exons 5, 7, and 11-23. Electromobility shift assays demonstrate that LXRalpha and retinoid X receptor alpha bind to the two LXREs in intron 7. Cells were transiently transfected with reporter luciferase constructs under the control of either (i) 9 kb of genomic DNA corresponding to intron 7 and part of exon 8 and containing either wild-type or mutant LXREs or (ii) two copies of the wild-type or mutant LXRE. In all cases, the wild-type construct was regulated in an LXR- and oxysterol-dependent manner, and this regulation was attenuated when the LXREs were mutated. In conclusion, the human ABCG1 gene contains multiple promoters, spans more than 98 kb and comprises 23 exons that give rise to alternative transcripts encoding proteins with different amino-terminal sequences. Elucidation of the various roles of different ABCG1 isoforms will be important for our understanding of mammalian cholesterol homeostasis.

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.

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.

Isolation and characterization of the UBASH3A gene on 21q22.3 encoding a potential nuclear protein with a novel combination of domains.

In order to identify candidate genes for Down syndrome phenotypes or monogenic disorders that map to human chromosome 21q22.3, we have used genomic sequence and expressed sequence tags mapping to an autosomal recessive deafness (DFNB10) critical region to isolate a novel 2.5-kb cDNA that maps between TFF1 and D21S49. A semi-quantitative reverse transcription/polymerase chain reaction method revealed that UBASH3A gene expression is limited to only a few tissues, with its highest expression in spleen, peripheral blood leukocytes, and bone marrow. The putative 661-amino-acid protein shows considerable homology to a hypothetical protein from Drosophila melanogaster but only domain homologies to other organisms. Both the human and D. melanogaster proteins contain protein-protein interaction domains, viz., SH3 and ubiquitin-associated (UBA) domains, in addition to a novel domain also containing a nuclear localization signal. This is the first protein described containing both UBA and SH3 domains. The gene, thus called UBASH3A, spans 40 kb and is divided into 15 exons. Mutation analysis excluded UBASH3A as being responsible for DFNB10.

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.

Cloning and characterization of a putative human glycerol 3-phosphate permease gene (SLC37A1 or G3PP) on 21q22.3: mutation analysis in two candidate phenotypes, DFNB10 and a glycerol kinase deficiency.

Using multiple exons trapped from human chromosome 21 (HC21)-specific cosmids with homology to a putative Arabidopsis thaliana glycerol 3-phosphate permease, we have determined the full-length cDNA sequence of a novel HC21 gene encoding a putative sugar-phosphate transporter (HGMW-approved symbol SLC37A1, aka G3PP). The predicted protein has 12 putative transmembrane domains and is also highly homologous to bacterial glpT proteins. The transcript was precisely mapped to 21q22.3 between D21S49 and D21S113. Comparison of the SLC37A1 cDNA to genomic sequence revealed that the gene encompasses 82 kb, and it is split into 19 coding exons and 7 untranslated exons, which are alternatively spliced in a complex and tissue-specific manner. Glycerol 3-phosphate (G3P) is produced by glycerol kinase (GK) and is found in several biochemical pathways in different cellular compartments, such as the glycerol phosphate shuttle and glycerophospholipid synthesis. Thus SLC37A1 mutations may cause a phenotype similar to GK deficiency. Mutational analyses of SLC37A1 in seven patients with no mutations in the GK gene and low GK activity revealed only nonpathogenetic sequence variants, excluding SLC37A1 as the gene for the phenotype in these patients. SLC37A1 maps in the refined critical region of the autosomal recessive deafness locus, DFNB10, on 21q22.3. Mutation analyses also excluded SLC37A1 as the gene for DFNB10.

Myocilin expression in the astrocytes of the optic nerve head.

We investigated the expression of myocilin in the optic nerve head of porcine eyes by Western blotting and immunohistochemical staining. Myocilin was localized in the nucleus, centrosome, glial filament, mitochondria, and some parts of the cell membranes of the astrocytes. Myocilin was also detected at the edge-feet portion of the processes of astrocytes adjacent to the inner limiting membrane and blood vessel wall. The astrocytes are the major cell population in the optic nerve head, contributing to the architecture of the nerve axon and blood vessels. Therefore, myocilin gene mutation and change of myocilin protein are likely to affect the architecture of the optic nerve head and induce various forms of glaucomatous optic nerve damage.

Novel mutation in RP2 gene in two brothers with X-linked retinitis pigmentosa and mtDNA mutation of leber hereditary optic neuropathy who showed marked differences in clinical severity.

PURPOSE: To report the identification of a novel mutation of the RP2 gene in two Japanese brothers with X-linked retinitis pigmentosa of a differing clinical severity. The mother was a carrier of both retinitis pigmentosa and optic atrophy. METHODS: The older brother had a severe form of retinitis pigmentosa associated with macular degeneration and total optic atrophy, whereas the younger brother presented typical X-linked retinitis pigmentosa. RESULTS: Each patient exhibited a novel 2-bp insertion at codon 278 in exon 3 of the RP2 gene as well as a 11778 mutation in mitochondrial DNA. This suggests that the older brother may have developed Leber hereditary optic neuropathy as well as retinitis pigmentosa. CONCLUSION: Molecular testing confirmed the clinical diagnosis in each case. However, such testing did not explain the differences in the severity of the ophthalmoscopic findings between the two brothers.

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.

Refined localization of autosomal recessive nonsyndromic deafness DFNB10 locus using 34 novel microsatellite markers, genomic structure, and exclusion of six known genes in the region.

An autosomal recessive nonsyndromic deafness locus, DFNB10, was previously localized to a 12-cM region near the telomere of chromosome 21 (21q22.3). This locus was discovered in a large, consanguineous Palestinian family. We have identified and ordered a total of 50 polymorphic microsatellite markers in 21q22.3, comprising 16 published and 34 new markers, precisely mapped and ordered on BAC/cosmid contigs. Using these microsatellite markers, the locus for DFNB10 has been refined to an area of less than 1 Mb between markers 1016E7.CA60 and 1151C12.GT45. Six previously published cDNAs were mapped to this critical region, and their genomic structures were determined to facilitate mutation analysis in DFNB10. All six genes in this region (in order from centromere to telomere: White/ABCG1, TFF3, TFF2, TFF1, PDE9A, and NDUVF3) have been screened and eliminated as candidates for DFNB10. The new microsatellite markers and single nucleotide polymorphisms identified in this study should enable the refined mapping of other genetic diseases that map to 21q22.3. In addition, the critical region for DFNB10 has been reduced to a size amenable to an intensive positional cloning effort.

C21orf5, a novel human chromosome 21 gene, has a Caenorhabditis elegans ortholog (pad-1) required for embryonic patterning.

To contribute to the development of the transcription map of human chromosome 21 (HC21), we isolated a new transcript, C21orf5 (chromosome 21 open reading frame 5), encoding a predicted 2298-amino-acid protein. Analysis of the genomic DNA sequence revealed that C21orf5 consists of 37 exons that extend over 130 kb and maps between the CBR3 (carbonyl reductase 3) and the KIAA0136 genes. Northern blot analyses showed a ubiquitously expressed RNA species of 8.5 kb. RNA in situ hybridization on brain sections of normal human embryos revealed a strong labeling in restricted areas of the cerebral cortex. In silico analysis of the deduced C21orf5 protein revealed several highly probable transmembrane segments but no known protein domains or homology with known proteins. However, there were significant homologies to several hypothetical Caenorhabditis elegans proteins and Drosophila melanogaster genomic sequences. To investigate the function of C21orf5, we isolated the cDNA of the C. elegans ortholog and performed double-stranded RNA-mediated genetic interference experiments. The major phenotype observed in the progeny of injected animals was embryonic lethality. Most of the tissues of the embryo failed to undergo proper patterning during gastrulation, and morphogenesis did not occur; thus we termed the ortholog pad-1, for patterning defective 1. These results indicated that pad-1 is essential for the development and the survival of C. elegans. This study provides the first example of the use of C. elegans as a model to study the function of genes on human chromosome 21 that might be involved in Down syndrome.