RT-PCR Based Mutation Detection of the Inflammatory Molecules Elastase II Gene Encoding Neutrophil Elastase in Cyclic Neutropenia Patients by Capillary Sequencing

Cyclic neutropenia is characterized by the maturation arrest of myeloid cells at the promyelocyte stage of hematopoiesis. In cyclic neutropenia, due to the disorder of neutrophil production, patients are predisposed to recurrent bacterial infections. Detected mutations in neutrophil elastase (ELA2) gene in genomic DNA of cyclic neutropenia were recently reported. Peripheral blood was obtained from 18 patients with cyclic neutropenia and 20 healthy individuals. Total RNA was isolated using RNA standard techniques from fresh separated cells by polymorphoprep. Elastase II mRNA expression was analyzed by employing reverse transcription PCR amplification using a total of ten specific primers. We amplified five exon of ELA2 gene separately and sequenced each exon. Mutational analysis was performed by directed capillary sequencing method. We found mutations in 15 out of 18 cyclic neutropenia patients (83%) and no mutation in 20 healthy individuals. Most of the mutations were in exon 4 and fewer mutation were found in exon 1.