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Blood ; 132(22): 2362-2374, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128


ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

J Clin Immunol ; 35(5): 501-11, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25975970


Familial Hemophagocytic Lymphohistiocytosis type 2 (FHL2) results from mutations in PRF1. We described two unrelated individuals who presented with FHL, in whom severely impaired NK cytotoxicity and decrease perforin expression was observed. DNA sequencing of PRF1 demonstrated that both were not only heterozygous for the p.54R > C/91A > V haplotype but also presented with the novel variant p.47G > V at the perforin protein. Perforin mRNA was found to be increased in a individual with that genotype. A carrier of the novel variant also demonstrated altered perforin mRNA and protein expression. Phylogenetic analysis and multiple alignments with perforin orthologous demonstrated a high level of conservation at Gly47. PolyPhen-2 and PROVEAN predicted p.47G > V to be "probably damaging" and "deleterious", respectively. A thermodynamic analysis showed that this variant was highly stabilizing, decreasing the protein internal energy. The ab initio perforin molecular modeling indicated that Gly47 is buried inside the hydrophobic core of the MACPF domain, which is crucial for the lytic pore formation and protein oligomerization. After the in silico induction of the p.47G > V mutation, Val47 increased the interactions with the surrounding amino acids due to its size and physical properties, avoiding a proper conformational change of the domain. To our knowledge, this is the first description supporting that p.47G > V is a pathogenic variant that in conjunction with p.54R > C/91A > V might result in the clinical phenotype of FHL2.

Citotoxicidade Imunológica , Células Matadoras Naturais/fisiologia , Linfo-Histiocitose Hemofagocítica/diagnóstico , Perforina/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Biologia Computacional , Citotoxicidade Imunológica/genética , Regulação para Baixo , Feminino , Humanos , Lactente , Linfo-Histiocitose Hemofagocítica/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Perforina/genética , Conformação Proteica , Relação Estrutura-Atividade , Adulto Jovem
J Clin Immunol ; 32(4): 670-80, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22437823


Familial hemophagocytic lymphohistiocytosis (FHL), is a rare autosomal recessive disorder characterized by an impairment of cytotoxic cells and uncontrolled activation of macrophages. This study presents the first description of four patients with FHL type 2 in Latin America. Patient 1 fulfilled the disease diagnostic criteria since 2 months of age, whereas patients 2, 3 and 4 exhibited the typical manifestations of the disease only later in their childhood. The PRF1 genetic analysis in these patients revealed two previously reported mutations: L17fsx50 and R54C. Interestingly, seven out of the 8 alleles evaluated here in patients carried the haplotype R54C/A91V, suggesting that this is a highly frequent FHL type 2 allele in Colombia. This haplotype confers residual cytotoxic function leading to late onset disease. Therefore, this report highlights the remarkable complexity of FHL diagnostic, emphasizing the importance of the genetic characterization of the disease.

Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Idade de Início , Criança , Pré-Escolar , Colômbia , Feminino , Predisposição Genética para Doença , Haplótipos , Humanos , Lactente , Células Matadoras Naturais/imunologia , Masculino , Proteínas de Membrana/genética , Mutação , Perforina