Slc15a4, AP-3, and Hermansky-Pudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells

Despite their low frequency, plasmacytoid dendritic cells (pDCs) produce most of the type I IFN that is detectable in the blood following viral infection. The endosomal Toll-like receptors (TLRs) TLR7 and TLR9 are required for pDCs, as well as other cell types, to sense viral nucleic acids, but the mechanism by which signaling through these shared receptors results in the prodigious production of type I IFN by pDCs is not understood. We designed a genetic screen to identify proteins required for the development and specialized function of pDCs. One phenovariant, which we named feeble, showed abrogation of both TLR-induced type I IFN and proinflammatory cytokine production by pDCs, while leaving TLR responses intact in other cells. The feeble phenotype was mapped to a mutation in Slc15a4, which encodes the peptide/histidine transporter 1 (PHT1) and has not previously been implicated in pDC function. The identification of the feeble mutation led to our subsequent observations that AP-3, as well as the BLOC-1 and BLOC-2 Hermansky-Pudlak syndrome proteins are essential for pDC signaling through TLR7 and TLR9. These proteins are not necessary for TLR7 or TLR9 signaling in conventional DCs and thus comprise a membrane trafficking pathway uniquely required for endosomal TLR signaling in pDCs.     

Biogenesis of lysosome-related organelles complex 3 (BLOC-3): a complex containing the Hermansky-Pudlak syndrome (HPS) proteins HPS1 and HPS4

Hermansky-Pudlak syndrome (HPS) defines a group of autosomal recessive disorders characterized by deficiencies in lysosome-related organelles such as melanosomes and platelet-dense granules. Several HPS genes encode proteins of unknown function including HPS1, HPS3, and HPS4. Here we have identified and characterized endogenous HPS3 and HPS4 proteins from HeLa cells. Both proteins were found in soluble and membrane-associated forms. Sedimentation-velocity and coimmunoprecipitation experiments revealed that HPS4 but not HPS3 associates with HPS1 in a complex, which we term biogenesis of lysosome-related organelles complex 3 (BLOC-3). Mutant fibroblasts deficient in either HPS1 or HPS4 displayed abnormal localization of lysosomes and late endosomes, which were less concentrated at the juxtanuclear region in mutant cells than in control fibroblasts. The coat-color phenotype of young homozygous double-mutant mice deficient in subunits of BLOC-3 (HPS1) and BLOC-1 (pallidin) was indistinguishable from that of BLOC-1 single mutants. Taken together, these observations suggest that HPS1 and HPS4 are components of a protein complex that regulates the intracellular localization of lysosomes and late endosomes and may function in a BLOC-1-dependent pathway for melanosome biogenesis.     

A human serotonin 1D receptor variant (5HT1D beta) encoded by an intronless gene on chromosome 6.

An intronless gene encoding a serotonin receptor (5HT1D beta) has been cloned and functionally expressed in mammalian fibroblast cultures. Based on the deduced amino acid sequence, the gene encodes a 390-amino acid protein displaying considerable homology, within putative transmembrane domains (approximately 75% identity) to the canine and human 5HT1D receptors. Membranes prepared from CHO cells stably expressing the receptor bound [3H]serotonin with high affinity (Kd 4 nM) and displayed a pharmacological profile consistent, but not identical, with that of the characterized serotonin 5HT1D receptor. Most notably, metergoline and serotonergic piperazine derivatives, as a group, display 3- to 8-fold lower affinity for the 5HT1D beta receptor than for the 5HT1D receptor, whereas both receptors display similar affinities for tryptamine derivatives, including the antimigraine drug sumatriptan. Northern blot analysis revealed an mRNA of approximately 5.5 kilobases expressed in human and monkey frontal cortex, medulla, striatum, hippocampus and amygdala but not in cerebellum, olfactory tubercle, and pituitary. The 5HT1D beta gene maps to human chromosome 6. The existence of multiple neuronal 5HT1D-like receptors may help account for some of the complexities associated with [3H]serotonin binding patterns in native membranes.     

Molecular organization of the cytokine gene cluster, involving the human IL-3, IL-4, IL-5, and GM-CSF genes, on human chromosome 5

The human genes for the hematopoietic growth factors interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been mapped to 5q23-31. We present in situ hybridization evidence that the human IL-4 gene is located at 5q23.3-31.2, suggesting that the four cytokine genes may be closely linked. We used pulsed-field gel electrophoresis to prepare subchromosomal restriction maps surrounding these genes to define this possible linkage more precisely. The IL-4 and IL-5 genes are tightly linked, being 90 to 240 kilobases (kb) apart, as has been shown for the IL-3 and GM-CSF genes, which are only 9 kb apart. Possible overlap of the map containing the IL-4 and IL-5 genes with restriction sites 5' to the IL-3 gene suggests that the four cytokine genes may be localized within 500 kb of each other. The endothelial cell growth factor gene (ECGF), which has also been localized to the 5q31 region, did not appear to be close to the cytokine genes. Linkage of the IL-3, IL-4, IL-5, and GM-CSF genes has important implications in the evolutionary origin and regulation of expression of these genes. The four cytokine genes are located in the region of the long arm of chromosome 5, which is deleted in the 5q- anomaly. The present study provides a basis for further investigations of this disorder.     

Identification and genetic mapping of a homeobox gene to the 4p16.1 region of human chromosome 4.

A human craniofacial cDNA library was screened with a degenerate oligonucleotide probe based on the conserved third helix of homeobox genes. From this screening, we identified a homeobox gene, H6, which shared only 57-65% amino acid identity to previously reported homeodomains. H6 was physically mapped to the 4p16.1 region by using somatic cell hybrids containing specific deletions of human chromosome 4. Linkage data from a single-stranded conformational polymorphism derived from the 3' untranslated region of the H6 cDNA placed this homeobox gene more than 20 centimorgans proximal of the previously mapped HOX7 gene on chromosome 4. Identity comparisons of the H6 homeodomain with previously reported homeodomains reveal the highest identities to be with the Nk class of homeobox genes in Drosophila melanogaster.     

Chromosomal mosaicism for inversion of chromosome 3(p25, p26) in a case of idiopathic hypereosinophilic syndrome (IHES)

Cytogenetic analysis and molecular genetic studies help for diagnosis of IHES and differentiation between IHES and other hematologic disorders with eosinophilia. We want to present a case of IHES with chromosomal mosaicism for inversion of chromosome 3(p25, p26), who achieved complete remission after treatment with Imatinib and short course of prednisolone.     

Rat obesity gene fatty (fa) maps to chromosome 5: evidence for homology with the mouse gene diabetes (db).

The autosomal recessive mutations fa (rat) and db (mouse) cause obesity syndromes that develop early and ultimately become severe. Although both fa/fa rats and db/db mice have been studied extensively as models of human obesity and diabetes, the molecular bases of these phenotypes remain unknown. We have mapped fa in 50 fa/fa (obese) offspring of a (13M x Brown Norway) F1 fa/+ intercross relative to two molecular markers, Ifa and Glut-1, which flank db on mouse chromosome 4 and which are located on rat chromosome 5. Ifa and Glut-1 are linked to fa, with a gene order, Ifa-fa-Glut-1, that is identical to that for the region around db in the mouse genome. These results place fa on rat chromosome 5 and suggest that db and fa are mutations in homologous genes.     

Homozygous loss of the cyclin-dependent kinase 4-inhibitor (p16) gene in human leukemias

Recently, it has been shown that the homozygous deletion of the cyclin- dependent kinase-4 inhibitor (CDK4I;p16) gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4I gene is thought to be a putative tumor-suppressor gene. We report here that both alleles of the CDK4I gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4I gene locus by Southern blot analysis. We found that a part or the whole of the CDK4I gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia (ALL), and 13 with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages, whereas all 4 cases showing the homozygous deletion were confined to ALL. It should be noted that 2 of them had no cytogenetic abnormalities of chromosome 9. Our results suggest that loss of the CDK4I function may contribute to immortalization of human leukemia cells and play a causative role at least in development of human lymphocytic leukemias.     

Homologous genes for enolase, phosphogluconate dehydrogenase, phosphoglucomutase, and adenylate kinase are syntenic on mouse chromosome 4 and human chromosome 1p

It is possible to generate interspecific somatic cell hybrids that preferentially segregate mouse chromosomes, thus making possible mapping of mouse genes. Therefore, comparison of the linkage relationships of homologous genes in man and mouse is now possible. Chinese hamster x mouse somatic cell hybrids segregating mouse chromosomes were tested for the expression of mouse enolase (ENO-1; EC 4.2.1.11, McKusick no. 17245), 6-phosphogluconate dehydrogenase [PGD; EC 1.1.1.44, McKusick no. 17220], phosphoglucomutase-2 (PGM-2; EC 2.7.5.1, McKusick no. 17190), and adenylate kinase-2 (AK-2; EC 2.7.4.3, McKusick no. 10302). In man, genes coding for the homologous forms of these enzymes have been assigned to the short arm of human chromosome 1. Analysis of 41 primary, independent, hybrid clones indicated that, in the mouse, ENO-1 and AK-2 are syntenic with PGD and PGM-2 and therefore can be assigned to mouse chromosome 4. In contrast, they were asyntenic with 21 other enzymes including mouse dipeptidase-1 (DIP-1, human PEP-C; EC 3.4.11.(*), McKusick no. 17000) assigned to human chromosome arm 1q and mouse chromosome 1. Karyologic analysis confirmed this assignment. These data demonstrate that a large autosomal region (21 map units in the mouse and 51 map units in the human male) has been conserved in the evolution of mouse chromosome 4 and the short arm of human chromosome 1. Identification of such conserved regions will contribute to our understanding of the evolution of the mammalian genome and could suggest gene location by homology mapping.     

Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism

To investigate the function of the Grb10 adapter protein, we have generated mice in which the Grb10 gene was disrupted by a gene-trap insertion. Our experiments confirm that Grb10 is subject to genomic imprinting with the majority of Grb10 expression arising from the maternally inherited allele. Consistent with this, disruption of the maternal allele results in overgrowth of both the embryo and placenta such that mutant mice are at birth approximately 30% larger than normal. This observation establishes that Grb10 is a potent growth inhibitor. In humans, GRB10 is located at chromosome 7p11.2-p12 and has been associated with Silver-Russell syndrome, in which approximately 10% of those affected inherit both copies of chromosome 7 from their mother. Our results indicate that changes in GRB10 dosage could, in at least some cases, account for the severe growth retardation that is characteristic of Silver-Russell syndrome. Because Grb10 is a signaling protein capable of interacting with tyrosine kinase receptors, we tested genetically whether Grb10 might act downstream of insulin-like growth factor 2, a paternally expressed growth-promoting gene. The result indicates that Grb10 action is essentially independent of insulin-like growth factor 2, providing evidence that imprinting acts on at least two major fetal growth axes in a manner consistent with parent-offspring conflict theory.     

Identification of a homozygous deletion in the AP3B1 gene causing Hermansky-Pudlak syndrome, type 2

We report on the molecular etiology of an unusual clinical phenotype associating congenital neutropenia, thrombocytopenia, developmental delay, and hypopigmentation. Using genetic linkage analysis and targeted gene sequencing, we defined a homozygous genomic deletion in AP3B1, the gene encoding the beta chain of the adaptor protein-3 (AP-3) complex. The mutation leads to in-frame skipping of exon 15 and thus perturbs proper assembly of the heterotetrameric AP-3 complex. Consequently, trafficking of transmembrane lysosomal proteins is aberrant, as shown for CD63. In basal keratinocytes, the incorporated immature melanosomes were rapidly degraded in large phagolysosomes. Despite distinct ultramorphologic changes suggestive of aberrant vesicular maturation, no functional aberrations were detected in neutrophil granulocytes. However, a comprehensive immunologic assessment revealed that natural killer (NK) and NKT-cell numbers were reduced in AP-3-deficient patients. Our findings extend the clinical and molecular phenotype of human AP-3 deficiency (also known as Hermansky-Pudlak syndrome, type 2) and provide further insights into the role of the AP-3 complex for the innate immune system.     

Assignment of the human organic anion transporting polypeptide (OATP) gene to chromosome 12p12 by fluorescence in situ hybridization

Background/Aims: The organic anion transporting polypeptide (OATP) of human liver mediates the basolateral hepatocellular uptake of numerous cholephilic anions and steroidal compounds. The aim of this study was to clone the human OATP gene and to map its chromosomal localization by fluorescence in situ hybridization.Methods: A polymerase chain reaction-amplified fragment of the human OATP gene was used to isolate a genomic OATP clone from a P1-derived artificial chromosome human genomic library. Human metaphase chromosomes were hybridized with digoxigenin-labeled DNA from the genomic OATP clone and incubated in fluoresceinated antidigoxigenin antibodies for in situ detection of specific hybridization signals.Results: Sequence analysis revealed that the isolated P1-derived artificial chromosome clone contained a large portion of the human OATP gene. Fluorescence in situ hybridization of human chromosomes with the genomic OATP clone resulted in the specific labeling of the OATP gene on the short arm of chromosome 12 at band 12p12.Conclusions: Mapping of a genomic OATP clone to chromosome 12p12 represents a first step towards the molecular characterization of the human OATP gene. While no liver disease has so far been associated with cytogenetic abnormalities of the short arm of chromosome 12, the genomic OATP sequence provides the basis for studies on gene structure and on the tissue-specific regulation of OATP gene expression.     

Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

A human cDNA encoding a galactokinase (EC 2.7.1.6) was isolated by complementation of a galactokinase-deficient (gal1-) strain of Saccharomyces cerevisiae. This cDNA encodes a predicted protein of 458 amino acids with 29% identity to galactokinase of Saccharomyces carlsbergensis. Previous studies have mapped a human galactokinase gene (GK1) to chromosome 17q23-25, closely linked to thymidine kinase. The galactokinase gene that we have isolated (GK2) is located on chromosome 15. The relationship between the disease locus for galactokinase deficiency galactosemia, which is responsible for cataracts in newborns and possibly presenile cataracts in adults, and the two galactokinase loci is unknown.     

Genetic analysis of indefinite division in human cells: evidence for a cell senescence-related gene(s) on human chromosome 4.

Earlier studies had demonstrated that fusion of normal with immortal human cells yielded hybrids having limited division potential. This indicated that the phenotype of limited proliferation (cellular senescence) is dominant and that immortal cells result from recessive changes in normal growth-regulatory genes. In additional studies, we exploited the fact that the immortal phenotype is recessive and, by fusing various immortal human cell lines with each other, identified four complementation groups for indefinite division. Assignment of cell lines to specific groups allowed us to take a focused approach to identify the chromosomes and genes involved in growth regulation that have been modified in immortal cells. We report here that introduction of a normal human chromosome 4 into three immortal cell lines (HeLa, J82, T98G) assigned to complementation group B resulted in loss of proliferation and reversal of the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. This result suggests that a gene(s) involved in cellular senescence and normal growth regulation resides on chromosome 4.     

Two-stage candidate gene study of chromosome 3p demonstrates an association between nonsynonymous variants in the MST1R gene and Crohn's disease

BACKGROUND: Genomewide linkage studies identified chromosome 3p21 as an IBD locus. Genomewide association studies have supported this locus and the Wellcome Trust Case Control Consortium (WTCCC) study narrowed it to a 0.6 Mb region. Our objectives were to perform a 2-stage candidate gene association study of the 3p locus and to identify linkage disequilibrium (LD) between significant single-nucleotide polymorphisms (SNPs) and an Oxfordshire subset (n = 282) of the WTCCC as well as the HapMap SNPs. METHODS: A total of 197 SNPs in 53 genes from the 3p locus were genotyped on the Illumina platform in a screening cohort of 469 Crohn's disease (CD) patients and 461 controls. Significant associations were then genotyped on the iPLEX platform in the original as well as a second cohort of 139 CD patients, 670 ulcerative colitis (UC) patients, and 1131 controls. All cases and controls were Caucasian and from the Oxfordshire region of the UK. RESULTS: An intronic SNP rs1128535 in the TRAIP gene was associated with CD in the screening and validation cohorts (combined [n = 608] P = 0.0004 [corrected 0.002], odds ratio [OR] 0.77, 95% confidence interval [CI], 0.67-0.89]). No association was seen for UC. Epistasis was seen with the common CARD15 mutations (P = 0.00003 [corrected 0.0006], OR 0.48, 95% CI, 0.34-0.68). No LD was demonstrated with the WTCCC SNPs. Strong LD was demonstrated with 2 nonsynonymous HapMap SNPs in the MST1R gene in an adjacent LD block to the peak WTCCC association, suggesting a distinct association signal. CONCLUSIONS: The LD with these functional MST1R variants implicate this gene as having a possible role in CD pathogenesis.     

Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

Complementation of the repair defect in hamster xrs mutants has been achieved by transfer of human chromosome 2 using the method of microcell-mediated chromosome transfer. The xrs mutants belong to ionizing radiation complementation group 5, are highly sensitive to ionizing radiation, and have an impaired ability to rejoin radiation-induced DNA double-strand breaks. Both phenotypes were corrected by chromosome 2, although the correction of radiation sensitivity was only partial. Complementation was achieved in two members of this complementation group, xrs6 and XR-V15B, derived independently from the CHO and V79 cell lines, respectively. The presence of human chromosome 2 in complemented clones was examined cytogenetically and by PCR analysis with primers directed at a human-specific long interspersed repetitive sequence or chromosome 2-specific genes. Complementation was observed in 25/27 hybrids, one of which contained only the q arm of chromosome 2. The two noncomplementing hybrids were missing segments of chromosome 2. The use of a back-selection system enabled the isolation of clones that had lost the human chromosome and these regained radiation sensitivity. Transfer of several other human chromosomes did not result in complementation of the repair defect in XR-V15B. These data show that the gene defective in xrs cells, XRCC5, which is involved in double-strand break rejoining, is located on human chromosome 2q.