Genetic analysis of type 1 diabetes using whole genome approaches.

Whole genome linkage analysis of type 1 diabetes using affected sib pair families and semi-automated genotyping and data capture procedures has shown how type 1 diabetes is inherited. A major proportion of clustering of the disease in families can be accounted for by sharing of alleles at susceptibility loci in the major histocompatibility complex on chromosome 6 (IDDM1) and at a minimum of 11 other loci on nine chromosomes. Primary etiological components of IDDM1, the HLA-DQB1 and -DRB1 class II immune response genes, and of IDDM2, the minisatellite repeat sequence in the 5' regulatory region of the insulin gene on chromosome 11p15, have been identified. Identification of the other loci will involve linkage disequilibrium mapping and sequencing of candidate genes in regions of linkage.

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of ≈40 nitr genes are contiguous in the genome and span ≈0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Susceptibility to the biological effects of polyaromatic hydrocarbons is influenced by genes of the major histocompatibility complex

Polyaromatic hydrocarbons are ubiquitous environmental chemicals that are important mutagens and carcinogens. The purpose of this study was to determine whether genes within the major histocompatibility complex (MHC) influence their biological activities. Cell-mediated immunity to dimethylbenz(a)anthracene (DMBA) was investigated in congenic strains of mice. On three different backgrounds, H-2k and H-2a haplotype mice developed significantly greater contact-hypersensitivity responses to DMBA than H-2b, H-2d, and H-2s mice. In B10.A(R1) mice, which are Kk and Id, a vigorous contact-hypersensitivity response was present, indicating that the response was governed by class I, rather than class II, MHC genes. C3H/HeN (H-2k) and C3H.SW (H-2s) strains were also compared for the development of skin tumors and the persistence of DMBA–DNA adducts. When subjected to a DMBA initiation, phorbol 12-tetradecanoate 13-acetate (TPA)-promotion skin-tumorigenesis protocol, C3H/HeN mice, (which develop cell-mediated immunity to DMBA) were found to have significantly fewer tumors than C3H.SW mice (a strain that failed to develop a cell-mediated immune response to DMBA). DMBA–DNA adducts were removed more rapidly in C3H/HeN than in C3H.SW mice. The results indicate that genes within the MHC play an important role in several of the biological activities of carcinogenic polyaromatic hydrocarbons. The observations are consistent with the hypothesis that cell-mediated immunity to chemical carcinogens serves to protect individuals by removing mutant cells before they can evolve into clinically apparent neoplasms.

Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of γ-interferon (γIFN), i.e., ds polynucleotides increase class I much more than class II, whereas γIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-κB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from γIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.

Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin

Coelomocytes, the heterogeneous population of sea urchin putative immune cells, were found to express a complex set of transcripts featuring scavenger receptor cysteine-rich (SRCR) repeats. SRCR domains define a metazoan superfamily of proteins, many of which are implicated in development and regulation of the immune system of vertebrates. Coelomocytes transcribe multiple SRCR genes from among a multigene family encoding an estimated number of 1,200 SRCR domains in specific patterns particular to each individual. Transcription levels for given SRCR genes may range from pronounced to undetectable, yet all tested animals harbor the genomic loci encoding these genes. Analysis of several SRCR genes revealed multiple loci corresponding to each type. In the case of one SRCR type, a cluster of at least three genes was detected within a 133-kb bacterial artificial chromosome insert, and conserved as well as unique regions were identified in sequences of three genomic clones derived from a single animal. Array hybridizations with repeated samples of coelomocyte messages revealed substantial alterations in levels of expression of many SRCR genes, with fluctuations of up to 10-fold in 1 week and up to 30-fold over a period of 3 months. This report is the first demonstration of genomic and transcriptional complexity in molecules expressed by invertebrate coelomocytes. The mechanisms controlling SRCR gene expression and the functional significance of this dynamic system await elucidation.

Evolution by the birth-and-death process in multigene families of the vertebrate immune system

Concerted evolution is often invoked to explain the diversity and evolution of the multigene families of major histocompatibility complex (MHC) genes and immunoglobulin (Ig) genes. However, this hypothesis has been controversial because the member genes of these families from the same species are not necessarily more closely related to one another than to the genes from different species. To resolve this controversy, we conducted phylogenetic analyses of several multigene families of the MHC and Ig systems. The results show that the evolutionary pattern of these families is quite different from that of concerted evolution but is in agreement with the birth-and-death model of evolution in which new genes are created by repeated gene duplication and some duplicate genes are maintained in the genome for a long time but others are deleted or become nonfunctional by deleterious mutations. We found little evidence that interlocus gene conversion plays an important role in the evolution of MHC and Ig multigene families.

CML66, a broadly immunogenic tumor antigen, elicits a humoral immune response associated with remission of chronic myelogenous leukemia

This report describes a tumor-associated antigen, termed CML66, initially cloned from a chronic myelogenous leukemia (CML) cDNA expression library. CML66 encodes a 583-aa protein with a molecular mass of 66 kDa and no significant homology to other known genes. CML66 gene is localized to human chromosome 8q23, but the function of this gene is unknown. CML66 is expressed in leukemias and a variety of solid tumor cell lines. When examined by Northern blot, expression in normal tissues was restricted to testis and heart, and no expression was found in hematopoietic tissues. When examined by quantitative reverse transcription–PCR, expression in CML cells was 1.5-fold higher than in normal peripheral blood mononuclear cells. The presence of CML66-specific antibody in patient serum was confirmed by Western blot and the development of high titer IgG antibody specific for CML66 correlated with immune induced remission of CML in a patient who received infusion of normal donor lymphocytes for treatment of relapse. CML66 antibody also was found in sera from 18–38% of patients with lung cancer, melanoma, and prostate cancer. These findings suggest that CML66 may be immunogenic in a wide variety of malignancies and may be a target for antigen-specific immunotherapy.

Ancestral major histocompatibility complex DRB genes beget conserved patterns of localized polymorphisms.

Genes within the major histocompatibility complex (MHC) are characterized by extensive polymorphism within species and also by a remarkable conservation of contemporary human allelic sequences in evolutionarily distant primates. Mechanisms proposed to account for strict nucleotide conservation in the context of highly variable genes include the suggestion that intergenic exchange generates repeated sets of MHC DRB polymorphisms [Gyllensten, U. B., Sundvall, M. & Erlich, H. A. (1991) Proc. Natl. Acad. Sci. USA 88, 3686-3690; Lundberg, A. S. & McDevitt, H. 0. (1992) Proc. Natl. Acad. Sci. USA 89, 6545-6549]. We analyzed over 50 primate MHC DRB sequences, and identified nucleotide elements within macaque and baboon DRB6-like sequences with deletions corresponding to specific exon 2 hypervariable regions, which encode a discrete alpha helical segment of the MHC antigen combining site. This precisely localized deletion provides direct evidence implicating segmental exchange of MHC-encoded DRB gene fragments as one of the evolutionary mechanisms both generating and maintaining MHC diversity. Intergenic exchange at this site may be fundamental to the diversification of immune protection in populations by permitting alteration in the specificity of the MHC that determines the repertoire of antigens bound.

Immune response of HLA-DQ8 transgenic mice to peptides from the third hypervariable region of HLA-DRB1 correlates with predisposition to rheumatoid arthritis.

The major histocompatibility complex class II genes play an important role in the genetic predisposition to many autoimmune diseases. In the case of rheumatoid arthritis (RA), the human leukocyte antigen (HLA)-DRB1 locus has been implicated in the disease predisposition. The "shared epitope" hypothesis predicts that similar motifs within the third hypervariable (HV3) regions of some HLA-DRB1 alleles are responsible for the class II-associated predisposition to RA. Using a line of transgenic mice expressing the DQB1*0302/DQA1*0301 (DQ8) genes in the absence of endogenous mouse class II molecules, we have analyzed the antigenicity of peptides covering the HV3 regions of RA-associated and nonassociated DRB1 molecules. Our results show that a correlation exists between proliferative response to peptides derived from the HV3 regions of DRB1 chains and nonassociation of the corresponding alleles with RA predisposition. While HV3 peptides derived from nonassociated DRB1 molecules are highly immunogenic in DQ8 transgenic mice, all the HV3 peptides derived from RA-associated DRB1 alleles fail to induce a DQ8-restricted T-cell response. These data suggest that the role of the "shared epitope" in RA predisposition may be through the shaping of the T-cell repertoire.

Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells.

The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound.

A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense.

In this paper we report a recessive mutation, immune deficiency (imd), that impairs the inducibility of all genes encoding antibacterial peptides during the immune response of Drosophila. When challenged with bacteria, flies carrying this mutation show a lower survival rate than wild-type flies. We also report that, in contrast to the antibacterial peptides, the antifungal peptide drosomycin remains inducible in a homozygous imd mutant background. These results point to the existence of two different pathways leading to the expression of two types of target genes, encoding either the antibacterial peptides or the antifungal peptide drosomycin.