Mutations in a delta 8-delta 7 sterol isomerase in the tattered mouse and X-linked dominant chondrodysplasia punctata. jderry@immunex.com.

Tattered (Td) is an X-linked, semi-dominant mouse mutation associated with prenatal male lethality. Heterozygous females are small and at 4-5 days of age develop patches of hyperkeratotic skin where no hair grows, resulting in a striping of the coat in adults. Craniofacial anomalies and twisted toes have also been observed in some affected females. A potential second allele of Td has also been described. The phenotype of Td is similar to that seen in heterozygous females with human X-linked dominant chondrodysplasia punctata (CDPX2, alternatively known as X-linked dominant Conradi-Hünermann-Happle syndrome) as well as another X-linked, semi-dominant mouse mutation, bare patches (Bpa). The Bpa gene has recently been identified and encodes a protein with homology to 3beta-hydroxysteroid dehydrogenases that functions in one of the later steps of cholesterol biosynthesis. CDPX2 patients display skin defects including linear or whorled atrophic and pigmentary lesions, striated hyperkeratosis, coarse lusterless hair and alopecia, cataracts and skeletal abnormalities including short stature, rhizomelic shortening of the limbs, epiphyseal stippling and craniofacial defects (MIM 302960). We have now identified the defect in Td mice as a single amino acid substitution in the delta8-delta7 sterol isomerase emopamil binding protein (Ebp; encoded by Ebp in mouse) and identified alterations in human EBP in seven unrelated CDPX2 patients.

Chromosomal localization of GABAA receptor subunit genes: relationship to human genetic disease.

Hybridization of GABAA receptor probes to human chromosomes in situ and to DNA from sorted human chromosomes has localized the genes encoding a beta subunit and three isoforms of the alpha subunit. The alpha 2 and beta genes are both located on chromosome 4 in bands p12-p13 and may be adjacent. The alpha 1 gene is on chromosome 5 (bands q34-q35) and the alpha 3 gene is on the X chromosome. The alpha 3 locus was mapped also on the mouse X chromosome using genetic break-point analysis in an interspecies pedigree. The combined results locate the human alpha 3 gene within band Xq28, in a location that makes it a candidate gene for the X-linked form of manic depression.

Sox5 regulates beta-cell phenotype and is reduced in type 2 diabetes.

Type 2 diabetes (T2D) is characterized by insulin resistance and impaired insulin secretion, but the mechanisms underlying insulin secretion failure are not completely understood. Here, we show that a set of co-expressed genes, which is enriched for genes with islet-selective open chromatin, is associated with T2D. These genes are perturbed in T2D and have a similar expression pattern to that of dedifferentiated islets. We identify Sox5 as a regulator of the module. Sox5 knockdown induces gene expression changes similar to those observed in T2D and diabetic animals and has profound effects on insulin secretion, including reduced depolarization-evoked Ca2+-influx and ß-cell exocytosis. SOX5 overexpression reverses the expression perturbations observed in a mouse model of T2D, increases the expression of key ß-cell genes and improves glucose-stimulated insulin secretion in human islets from donors with T2D. We suggest that human islets in T2D display changes reminiscent of dedifferentiation and highlight SOX5 as a regulator of ß-cell phenotype and function.

Mapping of the glycine receptor alpha 2-subunit gene and the GABAA alpha 3-subunit gene on the mouse X chromosome.

We have mapped the gene for the alpha 2-subunit of the inhibitory glycine receptor (Glra2) to the telomeric end of the mouse X chromosome by backcross analysis of a Mus musculus/Mus spretus interspecific cross. In addition, we have extended the mapping of the GABAA alpha 3-subunit receptor gene (Gabra3). A deduced gene order of cen-Cybb-Hprt-DXPas6-Gabra3-Rsvp-Gdx/Cf-8- Dmd-Pgk-1-DXPas2-Plp-DXPas1-Glra2-tel places Gabra3 proximal to the visual pigment gene Rsvp and Glra2 in the region of loci for hypophosphatemia (Hyp), steroid sulfatase (Sts), and the E1 alpha-subunit of pyruvate dehydrogenase (Pdha1). This establishes the XF region of the mouse X chromosome as homologous with the Xp22.1-p22.3 region of the human X chromosome and indicates the presence of an evolutionary breakpoint in the region of Xp21.3.

Physical linkage of the A-raf-1, properdin, synapsin I, and TIMP genes on the human and mouse X chromosomes.

Genes encoding the neuron-specific phosphoprotein synapsin I (SYN1), the glycoprotein tissue inhibitor of metalloproteinases (TIMP), the proto-oncogene A-raf-1 (ARAF1), and properdin (PFC), a positive regulator of the alternative pathway of human complement, lie within a conserved synteny encompassing the proximal short arm of the human X chromosome (Xp21.1-p11) and the centromeric end of the mouse X chromosome (A1-A5). We have used a mouse interspecific cross to demonstrate genetic linkage of Syn-1, Timp, and Araf and also show physical linkage, with Timp lying only 10 kb from Araf, within an intron of the Syn-1 gene. Detailed restriction mapping shows that Timp is transcribed in the same direction as Araf but in the opposite direction to the Syn-1 gene. Analysis of the corresponding region of the human X chromosome indicates a similar arrangement and in addition shows that the properdin gene lies within 5 kb of the 5' end of the synapsin I gene.

RBM3, a novel human gene in Xp11.23 with a putative RNA-binding domain.

Using cDNA selection with a YAC from the Xp11.2 region, we have identified a novel gene (RBM3) that encodes a polypeptide with high sequence similarity to a group of proteins that bind to RNA. On a YAC contig map, RBM3 is located between OATL1 and GATA1/TFE3 in sub-band Xp11.23, and gives rise to alternatively spliced transcripts in a variety of human tissues. The longest open reading frame encodes a 157 amino acid protein with a predicted molecular weight of 17 kDa. Its putative RNA-binding domain most closely resembles that of two previously characterized human RNA-binding proteins, YRRM, the gene for which has been implicated in azoospermia, and hnRNP G, a glycoprotein, also identified as an auto-antigen. The homology of RMB3 in both sequence and size to an RNA binding protein from maize, AAIP , suggests that it functions in a fundamental pathway conserved from plants to mammals.

Isolation of a novel gene mutated in Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency characterized by eczema, thrombocytopenia, and recurrent infections. Linkage studies have placed the gene at Xp11.22-p11.23. We have isolated from this interval a novel gene, WASP, which is expressed in lymphocytes, spleen, and thymus. The gene is not expressed in two unrelated WAS patients, one of whom has a single base deletion that produces a frame shift and premature termination of translation. Two additional patients have been identified with point mutations that change the same arginine residue to either a histidine or a leucine. WASP encodes a 501 amino acid proline-rich protein that is likely to be a key regulator of lymphocyte and platelet function.

Dystroglycan: brain localisation and chromosome mapping in the mouse.

Duchenne muscular dystrophy (DMD) is accompanied by varying degrees of mental retardation. The molecular basis for this is unknown, although at least four dystrophin transcripts regulated by specific promoters and undergoing elaborate splicing control are present in brain areas associated with cognitive function. In muscle the absence of dystrophin causes instability of a dystrophin-associated protein complex (DAPC) linking the cytoskeleton to the extracellular matrix; this disruption is accompanied by muscle necrosis. The laminin-binding component of DAPC, dystroglycan, in contrast to other components of DAPC, has been found in brain homogenates. This suggests that the link between the membrane cytoskeleton and extracellular matrix mediated by dystrophin-dystroglycan may play a functional role in brain. We have cloned a mouse dystroglycan partial cDNA and have mapped this gene in the mouse to chromosome 9. Further, in situ hybridisation to mouse brain sections shows that the dystroglycan gene is expressed in relatively few structures and co-localises with dystrophin mRNA in hippocampus, dentate gyrus, olfactory bulb and Purkinje neurons but, surprisingly, not in the cortex. Dystroglycan is also expressed in those brain areas where the dystrophin-related protein (utrophin) is present. Our results provide a basis for a future characterisation of the role of dystrophin-dystroglycan association in the brain.

Cloning and characterization of a novel zinc finger gene in Xp11.2.

During a systematic search for open reading frames in chromosome band Xp11.2, a novel gene (ZNF157) that encodes a putative 506-amino-acid protein with the sequence characteristics of a zinc-finger-containing transcription factor was isolated. ZNF157 is encoded by four exons distributed over > 20 kb of genomic DNA. The second and third exons contain sequences similar to those of the previously described KRAB-A and KRAB-B domains, motifs that have been shown to mediate transcriptional repression in other members of the protein family. A fourth exon contains 12 zinc finger DNA binding motifs and finger linking regions characteristic of ZNF proteins of the Krüppel family. ZNF157 maps to the telomeric end of a cluster of ZNF genes that includes ZNF21, ZNF41, and ZNF81.

Characterization of the interaction of zopiclone with gamma-aminobutyric acid type A receptors.

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.

A transcript map of a 2-Mb BAC contig in the proximal portion of the mouse X chromosome and regional mapping of the scurfy mutation.

A physical clone contig has been constructed, spanning 2 Mb on the proximal mouse X chromosome containing the mouse scurfy (sf) and tattered (Td) mutations. Extensive transcript mapping in this interval has identified 37 potential transcription units, including a number of novel genes, and 4 pseudogenes. These genes have been ordered by STS content and restriction mapping. Comparison of the transcript map to the corresponding region in human Xp11.23-p11.22 shows extensive homology, with complete conservation of gene order for loci in common between the two maps. Further, using a novel method to identify simple sequence length polymorphisms, we have developed a number of genetic markers, which has enabled the region containing the sf mutation to be narrowed to <300 kb. This contig has already allowed the cloning of the Td gene using a candidate gene approach and now serves as a starting point for the cloning of the sf mutation.

Mapping of the phosphorylase kinase alpha subunit gene on the mouse X chromosome.

Phosphorylase kinase is a glycogenolytic enzyme in several animal tissues. Within the last few years all four subunits of the enzyme have been cloned. The beta, gamma, and delta subunits are known to be autosomal. We have mapped the alpha subunit of phosphorylase kinase, recently cloned by Zander et al. (1988), in an interspecific mouse pedigree and localized it on the X chromosome, where it maps between the X-linked zinc finger protein and phosphoglycerate kinase genes, close to the latter. In man and mouse several X-linked disorders of this enzyme have been described. Although the X-linked phosphorylase kinase deficiency in mice may be caused by a mutation in the structural gene for the alpha subunit, mapped here, the existence of a separate regulatory locus, important in the normal expression or function of the enzyme in muscle, still remains a possibility.

Expression of four alternative dystrophin transcripts in brain regions regulated by different promoters.

Cognitive impairment occurs in one-third of patients with Duchenne muscular dystrophy, a lethal X-linked, recessive disease caused by mutations in the dystrophin gene which is expressed in both brain and muscle, the two transcripts having alternative first exons. Previous reports have indicated that the 'brain-type' dystrophin transcript predominates in brain. Using in situ hybridisation with antisense oligonucleotides, expression of four distinct mRNAs in specific brain areas is demonstrated here; the 14 kb muscle-type and brain-type transcripts were found to coexist in cortical and hippocampal neurons and two new transcripts have been identified in dentate gyrus and cerebellar Purkinje neurons, respectively. The latter has a novel first exon which was isolated and sequenced from mouse and human, and which would encode a protein with a different amino-terminus from the known muscle- and brain-type isoforms. Mapping in human located this exon in a large intron between the muscle-type promoter and second exon of the dystrophin gene. This finding of four alternative transcripts regulated by different promoters in brain reveals a new complexity to dystrophin expression that may have important insights for mental retardation mechanisms.

Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization.

The Rho family of GTPases control diverse biological processes, including cell morphology and mitogenesis. We have identified WASP, the protein that is defective in Wiskott-Aldrich syndrome (WAS), as a novel effector for CDC42Hs, but not for the other Rho family members, Rac and Rho. This interaction is dependent on the presence of the G protein-binding domain. Cellular expression of epitope-tagged WASP produces clusters of WASP that are highly enriched in polymerized actin. This clustering is not observed with a C-terminally deleted WASP and is inhibited by coexpression with dominant negative CDC42Hs-N17, but not with dominant negative forms of Rac or Rho. Thus, WASP provides a novel link between CDC42Hs and the actin cytoskeleton, which suggests a molecular mechanism for many of the cellular abnormalities in WAS. The WASP sequence contains two novel domains that are homologous to other proteins involved in action organization.

Mapping of a liver phosphorylase kinase alpha-subunit gene on the mouse X chromosome.

Phosphorylase kinase (PHK) is a regulatory enzyme of the glycogenolytic pathway composed of a complex of four subunits. We recently mapped the muscle alpha-subunit gene (Phka) to the mouse X chromosome in a region syntenic with the proximal long arm of the human X chromosome and containing the human homologue of this gene, PHKA. We now report the mapping of the liver alpha-subunit gene to the telomeric end of the mouse X chromosome. This mapping position would suggest a location for the human liver alpha-subunit gene on the proximal short arm of the X chromosome, a region recently implicated in X-linked liver glycogenosis (XLG).

WASP gene mutations in Wiskott-Aldrich syndrome and X-linked thrombocytopenia.

The WASP gene has been recently cloned from Xp11.23 and shown to be mutated in three patients with the Wiskott-Aldrich syndrome (WAS). We have developed a screening protocol for identifying WASP gene alterations in genomic DNA and have identified a spectrum of novel mutations in 12 additional unrelated families. These missense, nonsense and frameshift mutations involve eight of the 12 exons of the gene. Two mutations creating premature termination codons were associated with lack of detectable mRNA on Northern blots. Four amino acid substitutions, Leu27Phe, Thr48Ile, Val75Met and Arg477Lys, were found in patients with congenital thrombocytopenia and no clinically evident immune defect indicating that the WASP gene is the site for mutations in X-linked thrombocytopenia as well as in WAS. A T-cell line from a WAS patient contained two independent DNA alterations, a constitutional frameshift mutation, also present in peripheral blood leukocytes from the patient, and compensatory splice site mutation unique to the cell line. The distribution of eight missense mutations provides valuable information on amino acids which are essential for normal protein function, and suggests that sites in the first two exons are hot-spots for mutation.

The Wiskott-Aldrich syndrome and X-linked congenital thrombocytopenia are caused by mutations of the same gene.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, small platelets, eczema, recurrent infections, and immunodeficiency. Besides the classic WAS phenotype, there is a group of patients with congenital X-linked thrombocytopenia (XLT) who have small platelets but only transient eczema, if any, and minimal immune deficiency. Because the gene responsible for WAS has been sequenced, it was possible to correlate the WAS phenotypes with WAS gene mutations. Using a fingerprinting screening technique, we determined the approximate location of the mutation in 13 unrelated WAS patients with mild to severe clinical symptoms. Direct sequence analysis of cDNA and genomic DNA obtained from patient-derived cell lines showed 12 unique mutations distributed throughout the WAS gene, including insertions, deletions, and point mutations resulting in amino acid substitutions, termination, exon skipping, or splicing defects. Of 4 unrelated patients with the XLT phenotype, 3 had missense mutations affecting exon 2 and 1 had a splice-site mutation affecting exon 9. Patients with classic WAS had more complex mutations, resulting in termination codons, frameshift, and early termination. These findings provide direct evidence that XLT and WAS are caused by mutations of the same gene and suggest that severe clinical phenotypes are associated with complex mutations.

The mouse homolog of the Wiskott-Aldrich syndrome protein (WASP) gene is highly conserved and maps near the scurfy (sf) mutation on the X chromosome.

The mouse WASP gene, the homolog of the gene mutated in Wiskott-Aldrich syndrome, has been isolated and sequenced. the predicted amino acid sequence is 86% identical to the human WASP sequence. A distinct feature of the mouse gene is an expanded polymorphic GGA trinucleotide repeat that codes for polyglycine and varies from 15 to 17 triplets in different Mus musculus strains. The genomic structure of the mouse WASP gene is expressed as an approximately 2.4-kb mRNA in thymus and spleen. Chromosomal mapping in an interspecific M. Musculus/M. spretus backcross placed the Wasp locus near the centromere of the mouse X chromosome, inseparable from Gata1, Tcfe3, and scurfy (sf). This localization makes Wasp a candidate for involvement in scurfy, a T cell-mediated fatal lymphoreticular disease of mice that has previously been proposed as a mouse homolog of Wiskott-Aldrich syndrome. Northern analysis of sf tissue samples indicated the presence of WASP mRNA in liver and skin, presumably as a consequence of lymphocytic infiltration, but non abnormalities in the amount or size of mRNA present.

Molecular characterization of the human interleukin (IL)-17 receptor.

Human interleukin 17 (hIL-17) is a T-cell derived cytokine that exhibits 63% amino acid sequence identity to mouse IL-17 (mIL-17) and 57% identity to a viral protein encoded by the herpesvirus saimiri (HSV) gene 13 (HVS13). The IL-17 family of proteins binds to a unique mouse receptor (mIL-17R). Using nucleic acid hybridization techniques, a cDNA encoding a human homologue of the mIL-17R (hIL-17R) was isolated from a human T cell library. The predicted amino acid sequence of the hIL-17R is 69% identical to the mIL-17R, shares no homology with previously identified cytokine receptor families, and exhibits a broad tissue distribution. The hIL-17R gene was localized to chromosome 22. Monoclonal antibodies (mAbs) generated against the hIL-17R were able to block the IL-17-induced production of cytokine from human foreskin fibroblast (HFF) cells. Binding studies suggest that recombinant hIL-17 binds to the hIL-17R with low affinity.

Interleukin 18 is a primary mediator of the inflammation associated with dextran sulphate sodium induced colitis: blocking interleukin 18 attenuates intestinal damage.

BACKGROUND AND AIMS: Persistent inflammation observed in inflammatory bowel disease may be the consequence of an increased or aberrant immune response to normal gut constituents or an overall immune dysregulation and imbalance. Cytokines play an important role in immune regulation and interleukin 18 (IL-18) is one such cytokine that has emerged as being instrumental in driving CD4+ T cell responses towards a Th1-type. IL-18 can also directly mediate inflammation, moderate interleukin 1 activity, and can act on cell types other than T cells. It has been reported recently that IL-18 mRNA and protein are upregulated in gut tissue from IBD patients. The aim of this study was to understand more about the role of IL-18 in contributing to the pathology of IBD and to assess whether blocking IL-18 activity may be of therapeutic benefit as a treatment regimen for IBD. METHODS: Mice with dextran sulphate sodium (DSS) induced colitis were treated with recombinant IL-18 binding protein (IL-18bp.Fc), a soluble protein that blocks IL-18 bioactivity. Histopathological analysis was performed and RNA from the large intestine was analysed using the RNase protection assay and gene arrays. RESULTS: IL-18 RNA levels increased very early in the colon during DSS colitis. Treatment of mice with IL-18bp.Fc inhibited IBD associated weight loss and significantly inhibited the intestinal inflammation induced by DSS. IL-18bp.Fc treatment also attenuated mRNA upregulation of multiple proinflammatory cytokine genes, chemokine genes, and matrix metalloprotease genes in the large intestine that are commonly elevated during IBD. CONCLUSIONS: IL-18bp treatment attenuated inflammation during DSS induced colitis in mice. Neutralising IL-18 activity may therefore be of benefit for ameliorating the inflammation associated with human intestinal diseases.