RESUMO
In the present study, we used exome sequencing to analyze PRF1, UNC13D, STX11, and STXBP2, as well as genes associated with primary immunodeficiency disease (RAB27A, LYST, AP3B1, SH2D1A, ITK, CD27, XIAP, and MAGT1) in Thai children with hemophagocytic lymphohistiocytosis (HLH). We performed mutation analysis of HLH-associated genes in 25 Thai children using an exome sequencing method. Genetic variations found within these target genes were compared to exome sequencing data from 133 healthy individuals. Variants identified with minor allele frequencies <5% and novel mutations were confirmed using Sanger sequencing. Exome sequencing data revealed 101 non-synonymous single nucleotide polymorphisms (SNPs) in all subjects. These SNPs were classified as pathogenic (n = 1), likely pathogenic (n = 16), variant of unknown significance (n = 12), or benign variant (n = 72). Homozygous, compound heterozygous, and double-gene heterozygous variants, involving mutations in PRF1 (n = 3), UNC13D (n = 2), STXBP2 (n = 3), LYST (n = 3), XIAP (n = 2), AP3B1 (n = 1), RAB27A (n = 1), and MAGT1 (n = 1), were demonstrated in 12 patients. Novel mutations were found in most patients in this study. In conclusion, exome sequencing demonstrated the ability to identify rare genetic variants in HLH patients. This method is useful in the detection of mutations in multi-gene associated diseases.
Assuntos
Análise Mutacional de DNA/métodos , Exoma/genética , Estudos de Associação Genética , Testes Genéticos/métodos , Linfo-Histiocitose Hemofagocítica/diagnóstico , Mutação , Análise de Sequência de DNA/métodos , Criança , Humanos , Linfo-Histiocitose Hemofagocítica/genética , Proteínas de Membrana/genética , Técnicas de Diagnóstico Molecular , Família Multigênica/genética , Perforina/genética , Polimorfismo de Nucleotídeo Único , Proteínas Qa-SNARE/genética , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPRESUMO
Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human hematologic malignancies. Mutations in fms-like tyrosine kinase 3 (FLT3) gene including internal tandem duplication (ITD) and point mutation in the tyrosine kinase domain (TKD) as well as in nucleoplasmin (NPM1) gene are associated with pathogenesis of acute myeloblastic leukemia (AML). Several reports have demonstrated high incidences of the FLT3 and NPM1 mutations in adult AML patients. Since the pathogenesis of pediatric AML is different from that of adult and the FLT3 and NPM1 mutations have not been well characterized in childhood AML. Therefore, the objective of this study was to determine the frequencies of FLT3 and NPM1 mutations in 64 newly diagnosed childhood AML patients. All blood and bone marrow samples were previously diagnosed with AML by using flow cytometry and/or cytochemistry. FLT3-ITD and FLT3-TKD were detected by PCR and PCR-RFLP methods, respectively. The NPM1 mutation was analyzed by PCR and direct DNA sequencing. The FLT3 mutations were detected in 7 of 64 (11.1%), including FLT3-ITD in 4 of 64 (6.3%) and FLT-TKD in 3 of 62 (4.8%). The NPM1 mutation was not detected in this cohort. By multivariate analysis, white blood cell counts, peripheral blood and bone marrow blast cell counts at diagnosis were significantly higher in children with FLT3-ITD (P<0.05). In addition, the median percentage of CD117 was significantly higher in leukemic blast cells with FLT3-ITD than those with wild type (P=0.01). We did not find any FLT3 mutations in children aged less than 5 years. The AML M3 cell type was most frequently associated with FLT3 gene mutations (50%). In conclusion, the FLT3 mutations was found in 11.1% but none of NPM1 mutation was detected in Thai children with AML. These data support the hypothesis of different biology and pathogenesis between adult and childhood AML.
