RESUMO
Leukocyte adhesion deficiency II (LAD II) is characterized by the lack of fucosylated glycoconjugates, including selectin ligands, causing immunodeficiency and severe mental and growth retardation. No deficiency in fucosyltransferase activities or in the activities of enzymes involved in GDP-fucose biosynthesis has been found. Instead, the transport of GDP-fucose into isolated Golgi vesicles of LAD II cells appeared to be reduced. To identify the gene mutated in LAD II, we cloned 12 cDNAs from Caenorhabditis elegans, encoding multi-spanning transmembrane proteins with homology to known nucleotide sugar transporters, and transfected them into fibroblasts from an LAD II patient. One of these clones re-established expression of fucosylated glycoconjugates with high efficiency and allowed us to identify a human homolog with 55% identity, which also directed re-expression of fucosylated glycoconjugates. Both proteins were localized to the Golgi. The corresponding endogenous protein in LAD II cells had an R147C amino acid change in the conserved fourth transmembrane region. Overexpression of this mutant protein in cells from a patient with LAD II did not rescue fucosylation, demonstrating that the point mutation affected the activity of the protein. Thus, we have identified the first putative GDP-fucose transporter, which has been highly conserved throughout evolution. A point mutation in its gene is responsible for the disease in this patient with LAD II.
Assuntos
Proteínas de Transporte/genética , Guanosina Difosfato Fucose/metabolismo , Síndrome da Aderência Leucocítica Deficitária/genética , Proteínas de Transporte de Monossacarídeos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Transporte/metabolismo , Compartimento Celular , Teste de Complementação Genética , Glicosilação , Complexo de Golgi/química , Humanos , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/etiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Homologia de Sequência de AminoácidosRESUMO
Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, mutations are found in ITGB2, the gene that encodes the ß subunit of the ß(2) integrins. This syndrome is characterized directly after birth by delayed separation of the umbilical cord. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Le(a) and Le(b) blood group antigens. Finally, in LAD-III (also called LAD-I/variant) the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells that is involved in the regulation of ß integrin conformation.
Assuntos
Antígenos CD18/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Leucócitos/metabolismo , Proteínas de Membrana/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Neoplasias/genética , Antígenos CD18/sangue , Adesão Celular/genética , Pré-Escolar , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Recém-Nascido , Síndrome da Aderência Leucocítica Deficitária/sangue , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/imunologia , Leucócitos/imunologia , Proteínas de Membrana/sangue , Proteínas de Transporte de Monossacarídeos/sangue , Proteínas de Neoplasias/sangue , Neutrófilos/imunologia , Neutrófilos/metabolismo , Conformação ProteicaRESUMO
We have previously reported a newly discovered congenital disorder of neutrophil adhesion, leukocyte adhesion deficiency syndrome type 2 (LAD II). The clinical manifestations of this syndrome are similar to those seen in the classic leukocyte adhesion deficiency syndrome, now designated type 1 (LAD I), but the two syndromes differ in the molecular basis of their adhesion defects. LAD I is caused by a deficiency in the CD18 integrin adhesion molecules while LAD II patients are deficient in expression of sialyl-Lewis X (SLeX), a carbohydrate ligand for selectins. In this report we demonstrate that neutrophils from a LAD II patient bind minimally or not at all to recombinant E-selectin, purified platelet P-selectin, or P-selectin expressed on histamine-activated human umbilical vein endothelial cells, but have normal levels of L-selectin and CD11b/CD18 integrin, and adhere to and migrate across endothelium when CD11b/CD18 is activated. We compare LAD I and LAD II patient neutrophil function in vitro, demonstrating that integrin and selectin adhesion molecules have distinct but interdependent roles in neutrophil adhesion during an inflammatory response.
Assuntos
Síndrome da Aderência Leucocítica Deficitária/sangue , Neutrófilos/fisiologia , Antígenos CD11/fisiologia , Antígenos CD18/fisiologia , Adesão Celular , Células Cultivadas , Quimiotaxia de Leucócito , Selectina E/fisiologia , Endotélio Vascular/fisiologia , Feminino , Citometria de Fluxo , Humanos , Técnicas In Vitro , Síndrome da Aderência Leucocítica Deficitária/classificação , Masculino , Selectina-P/fisiologia , Probabilidade , Valores de Referência , Veias UmbilicaisRESUMO
Rare hereditary deficiencies have been described which affect each functional stage of polymorphonuclear neutrophils. They almost invariably lead to recurrent acute infection. Among the abnormalities involving adhesion and motility, the following can be noted: the Buckley syndrome; and leucocyte type 1 and 2 adhesion deficiencies, respectively caused by a deficiency in membrane expression of beta 2 integrin CD11/CD18, and sialyl lewis X. Granulation system abnormalities include relatively non-symptomatic myeloperoxidase deficiency, specific granulation deficiency or the Chediak-Higashi syndrome with the presence of giant lysosomal granulations. Chronic or familial septic granulomatosis constitutes the main disease described due to the oxidative PMN burst connected with the functional impairment of one of the constituents of NADPH oxidase (with an incidence of one in 5.10(6) to one in 10(6) births) The transmission is X-linked, or autosomal recessive depending on the mutation. The antenatal detection of the X-linked component, gp91 phox, can be made in suspected carrier mothers. In addition to the standard treatment (Bactrim and Itraconazole), bone marrow transplantation may also be carried out, and in future gene therapy may be introduced.
Assuntos
Síndrome da Aderência Leucocítica Deficitária/genética , Neutrófilos/fisiologia , Antígenos CD/sangue , Antígenos CD18/sangue , Humanos , Síndrome da Aderência Leucocítica Deficitária/sangue , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/diagnóstico , Antígeno de Macrófago 1/sangue , Cromossomo XAssuntos
Erros Inatos do Metabolismo dos Carboidratos/genética , Proteínas de Transporte/genética , Fucose/metabolismo , Complexo de Golgi/metabolismo , Guanosina Difosfato Fucose/metabolismo , Síndrome da Aderência Leucocítica Deficitária/genética , Proteínas de Transporte de Monossacarídeos , Açúcares de Nucleosídeo Difosfato/metabolismo , Processamento de Proteína Pós-Traducional/genética , Substituição de Aminoácidos , Animais , Árabes/genética , Transporte Biológico , Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Dimerização , Cães , Etnicidade/genética , Células Eucarióticas/metabolismo , Retardo do Crescimento Fetal/genética , Fucose/uso terapêutico , Heterogeneidade Genética , Predisposição Genética para Doença , Glicoproteínas/biossíntese , Glicosilação , Humanos , Infecções/etiologia , Deficiência Intelectual/genética , Invertebrados , Lectinas/toxicidade , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/tratamento farmacológico , Síndrome da Aderência Leucocítica Deficitária/metabolismo , Masculino , Mamíferos , Fenótipo , Mutação Puntual , Recidiva , Seleção Genética , TurquiaRESUMO
Up to now three distinct syndromes affecting several steps in the leukocyte adhesion cascade have been described. In LAD I the firm adhesion of leukocyte to the endothelium is defective, because of mutations in the gene encoding the beta(2)-integrin. Recent works both in human and animal models shed light on various mutations and their physiological importance. Furthermore, the beneficial effect of gene therapy is also becoming clear. LAD II which involved the first phase of the cascade, the rolling phase, is caused by mutations in the specific fucose transporter to the Golgi apparatus. Gene targeted mice were able to demonstrate indeed the role of this transporter in the adhesion process and long-term follow-up of patients showed that while in childhood immunodeficiency is the prominent feature, later on in life the metabolic consequences govern the clinical pictures. LAD III is the last syndrome to be described and a primary activation defect in all three beta-integrins 1, 2, and 3 is detected. Just recently mutations in Kindlin 3, a newly recognized component, which binds the cytoplasmic tail of integrin, and is important in integrin activation, the second phase of the adhesion cascade, were found.
Assuntos
Antígenos CD18/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Proteínas de Membrana/genética , Mutação , Proteínas de Neoplasias/genética , Animais , Antígenos CD18/metabolismo , Humanos , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/patologia , Proteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
OBJECTIVE: To describe the application of preimplantion genetic diagnosis (PF to a carrier couple for leukocyte adhesion deficiency type I disease (LAD-1), to achieve a healthy pregnancy. DESIGN: Case report. SETTING: Reproductive center and university hospital. PATIENT(S): A couple in which both partners were carriers for LAD-1; the female partner carried a G400A substitution in exon 4, and the male partner carried a C562T substitution in exon 5 in the CD18 gene. INTERVENTION(S): Day-3 cleavage-stage biopsy after standard in vitro fertilization (IVF) and genetic analysis of blastomeres for two mutations, along with a marker from chromosome 21. MAIN OUTCOME MEASURE(S): Birth of a child unaffected with LAD-1. RESULT(S): Fifteen oocytes were retrieved, of which 10 were fertilized; eight embryos were suitable for embryo biopsy. After genetic analysis, three embryos were found to be unaffected. According to embryo morphology, two embryos were transferred, resulting in the birth of an unaffected child. CONCLUSION(S): This is the first report of preimplantion genetic diagnosis for LAD-1. The successful birth of a healthy child provides evidence that for carrier couples of diseases for which traditional prenatal diagnosis and the decision of whether to terminate a pregnancy might not be acceptable, the application of PGD provides an alternative.
Assuntos
Síndrome da Aderência Leucocítica Deficitária/genética , Diagnóstico Pré-Implantação , Adenina , Adulto , Citosina , Transferência Embrionária , Éxons , Feminino , Fertilização in vitro , Guanina , Humanos , Síndrome da Aderência Leucocítica Deficitária/classificação , Masculino , Gravidez , Resultado da Gravidez , TiminaRESUMO
Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing. In LAD-I, mutations are found in INTG2, the gene that encodes the beta subunit of the beta(2) integrins. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in the gene for a GDP-fucose transporter of the Golgi. This article summarizes all known patient mutations and polymorphisms in these genes.
Assuntos
Antígenos CD18/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Mutação , Humanos , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/etiologia , Leucócitos/química , Leucócitos/patologia , Dados de Sequência MolecularRESUMO
Leukocyte adhesion deficiency (LAD) type II is the second human disorder identified which involves the adhesion cascade. While in LAD I the integrin family is defective, in LAD II the selectin system is involved. The syndrome has been described in only five patients and is transmitted as an autosomal recessive trait. The infectious episodes and the severity are much milder than those observed in LAD I, and the only persistent clinical symptom is chronic severe periodontitis. Delay separation of the umbilical cord, which is a hallmark for LAD I, was not observed in any of the LAD II patients. The exact defect in the system is absence of the SLeX, which is an important ligand for the selectin on the leukocyte lead ing to a profound defect in leukocyte rolling, the first step in the adhesion cascade. This causes a marked decrease in chemotaxis accompanied by pronounced neutrophilia. Apart from the leukocyte defect, these patients suffer from severe growth and mental retardation and exhibit the rare Bombay blood group type. The primary defect in the syndrome is in fucose metabolism, with the absence of all fucosylated glycans on cell surface membranes. Recently, it is was found that the defect is in a specific transporter of GDP fucose into the Golgi apparatus, and thus no fucosylation process takes place, and no surface expression can be detected. The exact genetic defect in the transporter is still unknown. Four of the patients were of Arabic origin while the fifth was of Turkish origin. It seems that the primary defect is somewhat different and, therefore, fucose administration was effective in the Turkish child, but did not show any beneficial results in the patients of Arabic origin.
Assuntos
Síndrome da Aderência Leucocítica Deficitária/imunologia , Adesão Celular/genética , Movimento Celular/genética , Pré-Escolar , Fucose/metabolismo , Fucose/uso terapêutico , Humanos , Lactente , Recém-Nascido , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/genética , Linfócitos/imunologia , Neutrófilos/imunologiaRESUMO
LAD II/CDG IIc is a rare autosomal recessive disease characterized by a decreased expression of fucosylated antigens on cell surfaces that results in leukocyte adhesion deficiency and severe neurological and developmental abnormalities. Its molecular basis has been identified as a defect in the transporter of GDP-l-fucose into the Golgi lumen, which reduces the availability of the substrate for fucosyltransferases. During metabolic radiolabeling experiments using [3H]fucose, LAD II fibroblasts incorporated significantly less radiolabel compared with control cells. However, fractionation and analysis of the different classes of glycans indicated that the decrease in [3H]fucose incorporation is not generalized and is mainly confined to terminal fucosylation of N-linked oligosaccharides. In contrast, the total levels of protein O-fucosylation, including that observed in Notch protein, were unaffected. This finding demonstrates that the decrease in GDP-l-fucose levels in the fibroblast Golgi caused by the LAD II defect does not impair bulk protein O-fucosylation, but severely affects the bulk addition of fucose as a terminal modification of N-linked glycans. These data suggest that the severe clinical abnormalities including neurological and developmental ones observed in at least some of the LAD II patients may be related to alteration in recognition systems involving terminal fucose modifications of N-glycans and not be due to a defective O-fucosylation of proteins such as Notch.
Assuntos
Fucose/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Síndrome da Aderência Leucocítica Deficitária/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Receptores de Superfície Celular , Fatores de Transcrição , Transporte Biológico Ativo , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fucose/química , Glucosamina/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Guanosina Difosfato Fucose/metabolismo , Humanos , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/genética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Receptor Notch1 , Tunicamicina/farmacologiaRESUMO
Two novel CD18 mutations were identified in a patient who was a compound heterozygote with type 1 leukocyte adhesion deficiency and whose phenotype was typical except that he exhibited hypertrophic scarring. A deletion of 36 nucleotides in exon 12 (1622del36) predicted the net loss of 12 amino acid (aa) residues in the third cysteine-rich repeat of the extracellular stalk region (mut-1). A nonsense mutation in exon 15 (2200G>T), predicted a 36-aa truncation of the cytoplasmic domain (mut-2). Lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen-1 (Mac-1) containing the mut-1 beta(2) subunit were expressed at very low levels compared with wild-type (wt) beta(2). Mac-1 and LFA-1 expression with the mut-2 beta(2) subunit were equivalent to results with wt beta(2). Binding function of Mac-1 with mut-2 beta(2) was equivalent to that with wt beta(2). However, binding function of LFA-1 with the mut-2 beta(2) subunit was reduced by 50% versus wt beta(2). It was concluded that (1) the portion of the CD18 stalk region deleted in mut-1 is critical for beta(2) integrin heterodimer expression but the portion of the cytoplasmic domain truncated in mut-2 is not; and (2) the mut-2 cytoplasmic domain truncation impairs binding function of LFA-1 but not of Mac-1. Studies with the patient's neutrophils (PMNs) were consistent with functional impairment of LFA-1 but not of Mac-1.