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Immunogenetics ; 69(10): 653-659, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28597365


Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency, which is characterized by the dysfunction and/or absence of T lymphocytes. Early diagnosis of SCID is crucial for overall survival, and if it remains untreated, SCID is often fatal. Next-generation sequencing (NGS) has become a rapid, high-throughput technology, and has already been proven to be beneficial in medical diagnostics. In this study, a targeted NGS panel was developed to identify the genetic variations of SCID by using SmartChip-TE technology, and a novel pathogenic frameshift variant was found in the CD3E gene. Sanger sequencing has confirmed the segregation of the variant among patients. We found a novel deletion in the CD3E gene (NM000733.3:p.L58Hfs*9) in two T-B+ NK+ patients. The variant was not found in the databases of dbSNP, ExAC, and 1000G. One sibling in family I was homozygous and the rest of the family members were heterozygous for this variant. T cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) analyses were performed for T and B cell maturation. TRECs were not detected in both patients and the KREC copy numbers were similar to the other family members. In addition, heterozygous family members showed decreased TREC levels when compared with the wild-type sibling, indicating that carrying this variant in one allele does not cause immunodeficiency, but does effect T cell proliferation. Here, we report a novel pathogenic frameshift variant in CD3E gene by using targeted NGS panel.

Sequência de Bases , Complexo CD3/genética , Mutação da Fase de Leitura , Deleção de Sequência , Imunodeficiência Combinada Severa/genética , Linfócitos B/imunologia , Linfócitos B/patologia , Complexo CD3/imunologia , Proliferação de Células , Consanguinidade , Feminino , Expressão Gênica , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Lactente , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Masculino , Linhagem , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/patologia , Irmãos , Linfócitos T/imunologia , Linfócitos T/patologia , Turquia
Methods Mol Biol ; 506: 13-21, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19110616


Hematopoietic stem cells (HSC) are rare with estimated frequencies of 1 in 10,000 bone marrow cells and 1 in every 100,000 blood cells. The most important characteristic of HSC is their capacity to provide complete restoration of all blood cell lineages after bone marrow ablation. Therefore they are considered as the ideal targets for various clinical applications including stem cell transplantation and gene therapy. In adult mice and men, the main stem cell source is the bone marrow. For clinical applications HSC derived from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (PB) have been demonstrated to have several advantages compared to bone marrow; therefore, they are slowly replacing BM as alternative source of stem cells. The mouse is the model organism of choice for immunological and hematological research; therefore, studies of murine HSC are an important research topic. Here we described the most often used protocols and methods to isolate human and mouse HSC to high purity.

Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/imunologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Humanos , Separação Imunomagnética , Camundongos
J Leukoc Biol ; 75(2): 314-23, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14634063


The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34(+) cells and PBSC significantly differ from BM CD34(+) cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34(+) cells, and CD34(+) PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34(+) cells contained the highest frequency of CAFC(wk6) (3.6- to tenfold higher than BM CD34(+) cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34(+), with a higher proportion of CD45(+) cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34(+) SC and 64 genes between BM CD34(+) cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34(+) HSC and leads to a better understanding of the discrepancy among the SC sources.

Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Animais , Antígenos CD34 , Apoptose/genética , Células Sanguíneas/citologia , Células da Medula Óssea/citologia , Ciclo Celular/genética , Ensaio de Unidades Formadoras de Colônias , Sangue Fetal/citologia , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Análise de Sequência com Séries de Oligonucleotídeos
Br J Haematol ; 117(1): 226-37, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11918560


Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34+CD38- cells from purified CD34+ cells upon stimulation with Flt3-ligand, stem cell factor and thrombopoietin. Over a 100-fold (range 80 to 128-fold) expansion of CD34+CD38- cells was observed with bone marrow and cord blood (CB). The expanded CD34+CD38- cells remained negative for lineage-specific markers and could be induced to differentiate into granulocytes, monocytes, megakaryocytes, erythrocytes, and T and B-lymphocytes in vitro. Lineage differentiation assays with single CD34+CD38- cells showed no loss of multilineage potential of expanded cells after ex vivo culture. We also demonstrated that the increase in frequency of CD34+CD38- cells was not as a result of the downregulation of CD38 expression during the culture. Quantitative analysis showed that the number of 6 week cobblestone area forming cells (CAFCwk6), a measure of proliferating HSC, in cytokine-stimulated CD34+ cells were increased by 20-fold. Expanded CD34+CD38- cells could be transduced efficiently with retroviruses encoding the low affinity nerve growth factor receptor (LNGFR) marker gene (17% to 44%, mean 27%), resulting in long-lasting expression of retroviral-encoded genes in progeny HSC and differentiated progenitors. We conclude that the combination Flt3-ligand (FL), stem cell factor and thrombopoietin (TPO) induced strong ex vivo proliferation of CD34+CD38- cells and that the absolute number of expanded cells with stem cell activity increased substantially in this population.

Antígenos CD34 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Adenoviridae/genética , Células da Medula Óssea/imunologia , Contagem de Células , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Sangue Fetal , Vetores Genéticos/administração & dosagem , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Proteínas de Membrana/farmacologia , Receptor de Fator de Crescimento Neural/genética , Fator de Células-Tronco/farmacologia , Estimulação Química , Trombopoetina/farmacologia , Transdução Genética