A targeted association study in systemic lupus erythematosus identifies multiple susceptibility alleles.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Multiple genetic and environmental factors contribute to the pathogenesis of this disease. Recent genome-wide association studies have added substantially to the number of genes associated with SLE. To replicate some of these susceptibility loci, single-nucleotide polymorphisms reported to be associated to SLE were evaluated in a cohort of 245 well-phenotyped Canadian SLE trios. Our results replicate previously reported associations to alleles of interferon regulatory factor 5 (IRF5), major histocompatibility complex (MHC), tumor necrosis factor (ligand) superfamily member 4 (TNFSF4), Kell blood group complex subunit-related family member 6 (XKR6), B-cell scaffold protein with ankyrin repeats 1 (BANK1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), ubiquitin-conjugating enzyme E2L 3 (UBE2L3) and islet cell autoantigen 1 (ICA1). We also identify putative associations to cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a gene associated with several autoimmune disorders, and ERBB3, a locus on 12q13 that was previously reported to be associated with type 1 diabetes. This study confirms the existence of multiple genetic risk factors for SLE, and supports the notion that some risk factors for SLE are shared with other inflammatory disorders.

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part II).

Down syndrome (DS) is the most common genetic cause of mental retardation. To explain the impact of extra chromosome 21 in the pathology of DS, gene dosage effect hypothesis has been proposed, but several investigators including our group have challenged this hypothesis. Although analysis of the sequence of chromosome 21 has been essentially completed, the molecular and biochemical mechanisms underlying the pathology are still unknown. We therefore investigated expression levels of six proteins encoded on chromosome 21 (HACS1, DYRK1A, alphaA-crystallin, FTCD, GARS-AIRS-GART, and CBS) in fetal cerebral cortex from DS and controls at 18-19 weeks of gestational age using Western blot analysis. Protein expression of HACS1 was significantly and remarkably decreased in DS, and the expression levels of five proteins were comparable between DS and controls suggesting that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are continuing to quantify proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level.

HACS1 encodes a novel SH3-SAM adaptor protein differentially expressed in normal and malignant hematopoietic cells.

SH3 and SAM domains are protein interaction motifs that are predominantly seen in signaling molecules, adaptors, and scaffold proteins. We have identified a novel family of putative adaptor genes that includes HACS1. HACS1 encodes a 441 amino acid protein that is differentially expressed in hematopoietic cells and has restricted expression in human tissues. Its SH3 domain is most similar to the same motif in Crk and its SAM domain shares homology with a family of uncharacterized putative scaffold and adaptor proteins. HACS1 maps to human chromosome 21q11.2 in a region that is frequently disrupted by translocation events in hematopoietic malignancies. Polyclonal antibodies against HACS1 recognized a 49.5 kDa protein whose mRNA is expressed in human immune tissues, bone marrow, heart, lung, placenta and brain. Cell lines and primary cells from acute myeloid leukemias and multiple myeloma patients express HACS1. Immunostaining and cellular fractionation studies localized the HACS1 protein predominantly to the cytoplasm.

Cloning and expression analysis of a novel WD repeat gene, WDR3, mapping to 1p12-p13.

WD repeat proteins are components of multiprotein complexes that are involved in a wide spectrum of cellular activities, such as cell cycle progression, signal transduction, apoptosis, and gene regulation. These proteins are characterized by repeat units bracketed by Gly-His and Trp-Asp (GH-WD). We report here the isolation of a new member of the WD repeat gene family, WDR3, which encodes a putative 943-amino-acid nuclear protein consisting of 10 WD repeat modules. WDR3 is widely expressed in hematopoietic cell lines and in nonhematopoietic tissues. Fluorescence in situ hybridization mapped WDR3 to human chromosome 1p12-p13, a region that is affected by chromosomal rearrangements in a number of hematologic malignancies and solid tumors.

AKAP350, a multiply spliced protein kinase A-anchoring protein associated with centrosomes.

Protein kinase A-anchoring proteins (AKAPs) localize the second messenger response to particular subcellular domains by sequestration of the type II protein kinase A. Previously, AKAP120 was identified from a rabbit gastric parietal cell cDNA library; however, a monoclonal antibody raised against AKAP120 labeled a 350-kDa band in Western blots of parietal cell cytosol. Recloning has now revealed that AKAP120 is a segment of a larger protein, AKAP350. We have now obtained a complete sequence of human gastric AKAP350 as well as partial cDNA sequences from human lung and rabbit parietal cells. The genomic region containing AKAP350 is found on chromosome 7q21 and is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, a protein associated with the N-methyl-D-aspartate receptor. Rabbit parietal cell AKAP350 is missing a sequence corresponding to a single exon in the middle of the molecule located just after the yotiao homology region. Two carboxyl-terminal splice variants were also identified. Both of the major splice variants showed tissue- and cell-specific expression patterns. Immunofluorescence microscopy demonstrated that AKAP350 was associated with centrosomes in many cell types. In polarized Madin-Darby canine kidney cells, AKAP350 localized asymmetrically to one pole of the centrosome, and nocodazole did not alter its localization. During the cell cycle, AKAP350 was associated with the centrosomes as well as with the cleavage furrow during anaphase and telophase. Several epithelial cell types also demonstrated noncentrosomal pools of AKAP350, especially parietal cells, which contained multiple cytosolic immunoreactive foci throughout the cells. The localization of AKAP350 suggests that it may regulate centrosomal and noncentrosomal cytoskeletal systems in many different cell types.

Identification of sequence-tagged transcripts differentially expressed within the human hematopoietic hierarchy.

Hematopoiesis is regulated by a complex gene expression program. To gain further insight into the molecular mechanisms underlying this process in humans, we sampled the transcriptional activity of the CD34+ hematopoietic progenitor line KG1a by single-pass sequencing the 5' ends of 1018 clones from a unidirectional cDNA library. Searches of public databases with the resulting expressed sequence tags (ESTs) identified 101 clones that showed no sequence similarity to any of the existing entries and that were therefore considered to derive from previously undescribed genes. Of the remaining 917 ESTs, 553 (a total of 485 distinct transcripts) corresponded to known genes. A further 279 KG1a ESTs matched or exhibited sequence similarity to ESTs or genomic sequences from humans and other species. Among the latter were putative human orthologs of developmental and cell cycle control genes from Caenorhabditis elegans, Drosophila, and yeast, as well as genes whose predicted amino acid sequences showed similarity to mammalian transcription factors. Hybridization of selected novel KG1a ESTs to globally amplified cDNAs prepared from single primary human hematopoietic precursors and homogeneous populations of terminally maturing hematopoietic cells revealed transcripts that are expressed preferentially at a specific stage or in a particular lineage within the hematopoietic hierarchy. Thus, included in the KG1a EST dataset are candidates for new human genes that may play roles in hematopoietic differentiative progression and lineage commitment.

Expression of schwannomin in lens and Schwann cells.

Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder characterized by the development of bilateral vestibular schwannomas, meningiomas, ependymomas and juvenile lens opacities. The NF2 gene encodes a tumor suppressor protein, schwannomin (or merlin), with sequence homology to erythrocyte band 4.1, talin, ezrin, moesin and radixin. Using an antibody that recognizes the carboxy-terminal epitope of isoform 1 of schwannomin, we looked at its expression in lens and Schwann cells, two cell-types affected by the NF2 phenotype. Schwannomin was detected as an approximately 80 kDa protein in both cytoplasmic and cytoskeleton fractions. Indirect immunofluorescence localized schwannomin to the cytoplasm and was frequently observed in dynamic cellular regions such as leading edges and ruffling membranes. Its level of expression in the lens inversely correlates with the degree of lens cell differentiation suggesting a role for schwannomin in differentiation-specific events.

Type of mutation in the neurofibromatosis type 2 gene (NF2) frequently determines severity of disease.

The gene predisposing to neurofibromatosis type 2 (NF2) on human chromosome 22 has revealed a wide variety of different mutations in NF2 individuals. These patients display a marked variability in clinical presentation, ranging from very severe disease with numerous tumors at a young age to a relatively mild condition much later in life. To investigate whether this phenotypic heterogeneity is determined by the type of mutation in NF2, we have collected clinical information on 111 NF2 cases from 73 different families on whom we have performed mutation screening in this gene. Sixty-seven individuals (56.2%) from 41 of these kindreds revealed 36 different putative disease-causing mutations. These include 26 proposed protein-truncating alterations (frameshift deletions/insertions and nonsense mutations), 6 splice-site mutations, 2 missense mutations, 1 base substitution in the 3' UTR of the NF2 cDNA, and a single 3-bp in-frame insertion. Seventeen of these mutations are novel, whereas the remaining 19 have been described previously in other NF2 individuals or sporadic tumors. When individuals harboring protein-truncating mutations are compared with cases with single codon alterations, a significant correlation (P < .001) with clinical outcome is observed. Twenty-four of 28 patients with mutations that cause premature truncation of the NF2 protein, schwannomin, present with severe phenotypes. In contrast, all 16 cases from three families with mutations that affect only a single amino acid have mild NF2. These data provide conclusive evidence that a phenotype/genotype correlation exists for certain NF2 mutations.

Widespread but cell type-specific expression of the mouse neurofibromatosis type 2 gene.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease in which loss of function mutations of the NF2 gene lead to the development of schwannomas, meningiomas and juvenile cataracts. We studied the mouse NF2 homologue (Nf2) to determine its precise pattern of mRNA and protein expression. In situ hybridization showed that Nf2 is expressed in neuronal cells as well as in epithelial and fibre cells of the lens. The Nf2 protein, schwannomin, is expressed as a single protein isoform of approximately 80 kDa in neuronal and non-neuronal tissues. In Purkinje cells of the cerebellum and motor neurones of the spinal cord, the protein is in the cytoplasm. In non-neuronal tissues immunostaining showed expression in cells of the tunica intima of blood vessels. We conclude that there is a widespread but cell type-specific expression of schwannomin.

Schwannomin: new insights into this member of the band 4.1 superfamily.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease characterized by the development of central nervous system tumours. The NF2 gene was recently cloned and found to encode a protein, schwannomin (or merlin), with homology to the band 4.1 superfamily. This superfamily of proteins includes ezrin, moesin, radixin, and talin, as well as several protein tyrosine phosphatases. How does a cytoskeleton-associated protein act as a tumour suppressor? While this fundamental question remains unanswered, recent studies have begun to address key questions regarding the function of schwannomin. In this review, we examine what is known about the band 4.1 superfamily and how this information pertains to schwannomin. In addition, we summarize recent studies of schwannomin itself.

The mouse neurofibromatosis type 2 gene maps to chromosome 11.

Neurofibromatosis type 2 (NF2) is a dominantly inherited disease characterized by the development of bilateral vestibular schwannomas and meningiomas, which together represent 30% of primary brain tumors. The NF2 gene, which has recently been isolated, maps to the long arm of human chromosome 22. Using recombinant inbred mice, we have determined the chromosomal position of the mouse homologue of the NF2 gene. Analysis of the allele distribution in AKXD recombinant inbred strains using a simple sequence repeat polymorphism (D11Mcg1) in the 3' untranslated region of the mouse cDNA maps the mouse NF2 gene to the proximal region of chromosome 11, closely linked to Pmv-2. This region also contains the genes for leukemia inhibitory factor and neurofilament heavy-chain polypeptide and thus represents a region of conserved synteny between human chromosome 22 and mouse chromosome 11. Using additional polymorphic markers, we established the following locus order from the centromere: D11Mit1/D11Mit72/D11Mcg1-D11Mit74-Pmv-2-D11Mi t2-D11Mit77/D11Mit78/D11Mit63.

The mouse homologue of the neurofibromatosis type 2 gene is highly conserved.

Neurofibromatosis type 2 (NF2) is a complex nervous system disorder characterized by the development of schwannomas (especially vestibular), meningiomas, ependymomas and juvenile lens opacities. Mutation in the NF2 gene, which encodes for the schwannomin protein (SCH), a member of the band 4.1 superfamily of genes, predisposes carriers to these central nervous system tumors. We have isolated a mouse cDNA from a brain library which contains the complete open reading frame of the mouse homologue of the NF2 gene. This cDNA encodes for a 596 amino acid protein with 98% identity to the human SCH. Cross species hybridization experiments predict that the NF2 gene is highly conserved in other vertebrates. Northern analysis detects a 4.5 kb transcript in mouse brain, kidney, cardiac muscle, skin and lung suggesting ubiquitous expression. The predicted secondary structure of SCH, which is shared by all members of the band 4.1 superfamily, includes a highly conserved amino-terminal domain which is believed to bind to proteins in the plasma membrane and a large highly charged alpha-helix domain proposed to associate with the cytoskeleton. The NF2 gene is the first example of a tumor suppressor gene whose protein product appears to act as a membrane cytoskeleton-linker. These results show that the NF2 gene is highly conserved and suggests that the analysis of the mouse NF2 gene might yield insights into the function of the human gene.