Bone Marrow Proliferative Diseases And Mast Cell Disease, The Receptor Tyrosine Kinase Gene Mutation Detection And Analysis

Objective: Myeloproliferative disorders (MPD) are clonal disorders characterized by excess proliferation of cells from one or more myeloid lineages. It is consist of a heterogenous spectrum conditions mainly including chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), chronic idiopathic myelofibrosis (IMF), chronic neutrophilic leukemia (CNL) , chronic eosinophilic leukemia (CEL) , chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) . CML is the most common type in MPD and 90%-95% of it is characterized by the presence of the BCR/ABL fusion gene which results in the production of constitutively active tyrosine kinase fusion protein that is believed to be the primary and possibly the only driving force behind the disease. The chimeric protein lead to an increase in activating multiple signalling pathways including RAS/RAF/MAPK, PI3K and STAT and has been found to be response very well to signal transduction inhibitor (STI) imatinib mesylate. The BCR/ABL negative CML and other MPD are rare and heterogeneous diseases of which the molecular pathogenesis is poorly understood. While the most of these cases have a normal karyotype theminority of them present an acquired reciprocal chromosomal translocation that disrupts specific receptor tyrosine kinase genes including PDGFRA, PDGFRB and FGFRl and produce constitutively active fusion proteins which are functionally and structurally analogous to BCR/ABL fusion protein. Signal transduction pathways have a wide spectrum of functions including regulations of cell proliferation and differentiation and have been reported to be involved in tumourgenesis. Constitutive activation of the signal transduction pathway may be due to the activating mutations at the receptor level (RTK) or downstream mediators (RAS or RAF) or protein tyrosine phosphatase (PTP).Receptor tyrosine kinase (RTK) and their downstream effectors (RAS, RAF) have emerged as significant components in the pathogenesis of haematological malignancies. Class III RTKs which include KIT, FMS, FLT3, PDGFRA and PDGFRB comprise a similar structure: a five immunoglobin-like domains in the extracellular ligand-binding region, a single hydrophobic transmembrane domain (TM) and two intracellular catalytic (TK1, TK2) domain which is separated by a kinase insert region (KI).The PTPN11 gene encodes the non-receptor-type protein tyrosine phosphatase SHP-2. SHP-2 is a member of the PTP family which is known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle and oncogenic transformation. Recently mutations in exon 3 and exon 13 which encode the N-SH2 domain and PTP domain respectively were described in patients with NS and JMML causing a gain of function of this gene.With the advent of targeted signal transduction therapy, an accurate clinical and molecular diagnosis of MPD has become increasingly important. Therefore in this study, we undertook extensively mutation analysis using PCR, DHPLC, TOPO TA cloning and direct sequencing method in 42 MPDpatients for the KIT mutations in JM (exonll) and AL (exonl7) regions and the analogous region of PDGFRs and FMS, the latter also including exon 7, exon 9 and exon 21/22 for the previously reported mutations. For FLT3 gene we screened FLT3/ITD in exon 14 and exon 15 and D835 point mutation in exon 20 and we also screened exon 3 and exon 13 of PTPN11 gene.Methods Genomic DNA from 42 BCR/ABL negative MPD patients were abstracted from bone marrow or peripheral blood using phenol/chloroform method.Genomic DNA amplification The entire coding sequence with exon-intron boundary was amplified. PCR was performed with AmpliTaq Gold DNA polymerase (Applied Biosystem) and 25ng genomic DNA in standard conditions. PCR products were resolved by electrophoresis on 1.5% agarose gel and visualised after staining with ethidium bromide.DHPLC analysis and sequencing Denaturing high-performance liquid chromatography (DHPLC) analysis was performed on a WAVE? DNA Fragment Analysis System (Transgenomic). Each PCR product was mixed with an equal quantity of amplified human placental DNA (sigma) and was denatured and reannealed to allow heteroduplexes formation. All the conditions for the DHPLC analysis, including melting temperature and buffer gradients were determined using the Transgenomic software Navigator VI.0. Samples with extra peaks or with different peak appearance were direct sequenced using the BigDye? Terminator V3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystem) and ABI 3100 Genetics Analyser.Detection of FLT3/ITD PCR products which yielded an extra band on agarose gel were cloned into plasmid vector pCR4-TOPO using TOPO TA Cloning Kit (Invitrogen) and transformed to TOP 10 chemically competent E.coli cells for amplification. Plasmid DNA was abstracted using QIAprep Spin Miniprep Kit (Qiagen) and then direct sequenced.ARMS assay was used to screen known nucleotide change in normal controls.Results DHPLC and direct sequencing detected all the previously reported SNPs in screened exons of KIT and FMS as well as some novel silent changes in PDGFRs. We found two patients had the G413S change in exon 9 of FMS however we also found this change in 3 of 43 normal controls using ARMS assay suggesting that this change is a SNP. Two patients were found to have both a normal band and an aberrant band in agarose gel when using primers for exonl4 and exonl5 of FLT3 to amplify genomic DNA. Sequence analysis confirmed a 39 bp duplication with 9 bp insertion and a 57 bp duplication with 9 bp insertion in exon 14 of FLT3 resulting in an addition of 16 and 22 amino acids in these two patients respectively. In both cases the duplications and insertions maintained the integrity of the reading frame. No mutations were detected in PTPN11 gene. The previously reported mutations in KIT exonU and exonl7, PDGFRA exonl2 and exonl8, L301 and Y969 in FMS and D835 in FLT3 were not found in this cohort of patients. In addition, five samples were found to harbour mutations at codon 12 and codon 13 in NRAS gene in our lab's previous work, no mutation was revealed in KRAS, HRAS and BRAF.Conclusions In our study, we detected all the previously reported SNPs as well as some novel silent changes suggesting that DHPLC is a reliable and sensitive method for mutation detection. The silent single base pair substitutions found in PDGFRB and FMS gene may cause structure alternations to mRNA which will require further study to confirm. Two patients were found to have FLT3/ITD in exon 14 of FLT3 and we believe this mutation is crucial to the pathogenesis of the disease. We concluded that the RAS signal transduction pathway may play a very important role in molecular...