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KIT GNNK splice variants: Expression in systemic mastocytosis and influence on the activating potential of the D816V mutation in mast cells

2013; Elsevier BV; Volume: 41; Issue: 10 Linguagem: Inglês

10.1016/j.exphem.2013.05.005

1873-2399

Eunice C. Chan, Yun Bai, Geethani Bandara, Olga Šimáková, Erica Brittain, Linda M. Scott, Kimberly D. Dyer, Amy D. Klion, Irina Marić, Alasdair M. Gilfillan, Dean D. Metcalfe, Todd M. Wilson,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Stem cell factor–dependent KIT activation is an essential process for mast cell homeostasis. The two major splice variants of KIT differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain. We hypothesized that the expression pattern of these variants differs in systemic mastocytosis and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK– and GNNK+ transcripts from bone marrow mononuclear cells was developed. The GNNK−/GNNK+ copy number ratio showed a trend toward a positive correlation with the percentage of neoplastic mast cell involvement, and KIT D816V containing transcripts displayed a significantly elevated GNNK−/GNNK+ copy number ratio. Relative expression of only the GNNK− variant correlated with increasing percentage of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK− isoform of wild type KIT was associated with increased granule formation, histamine content, and growth. When accompanying the KIT D816V mutation, the GNNK– isoform enhanced cytokine-free metabolism and moderately reduced sensitivity to the tyrosine kinase inhibitor, PKC412. These data suggest that neoplastic mast cells favor a GNNK− variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus could influence therapeutic sensitivity in systemic mastocytosis. Stem cell factor–dependent KIT activation is an essential process for mast cell homeostasis. The two major splice variants of KIT differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain. We hypothesized that the expression pattern of these variants differs in systemic mastocytosis and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK– and GNNK+ transcripts from bone marrow mononuclear cells was developed. The GNNK−/GNNK+ copy number ratio showed a trend toward a positive correlation with the percentage of neoplastic mast cell involvement, and KIT D816V containing transcripts displayed a significantly elevated GNNK−/GNNK+ copy number ratio. Relative expression of only the GNNK− variant correlated with increasing percentage of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK− isoform of wild type KIT was associated with increased granule formation, histamine content, and growth. When accompanying the KIT D816V mutation, the GNNK– isoform enhanced cytokine-free metabolism and moderately reduced sensitivity to the tyrosine kinase inhibitor, PKC412. These data suggest that neoplastic mast cells favor a GNNK− variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus could influence therapeutic sensitivity in systemic mastocytosis. KIT, the receptor for stem cell factor (SCF), is expressed on the surface of various hematopoietic progenitor cells and on mature mast cells [1Escribano L. Ocqueteau M. Almeida J. Orfao A. San Miguel J.F. Expression of the c-kit (CD117) molecule in normal and malignant hematopoiesis.Leuk Lymphoma. 1998; 30: 459-466Crossref PubMed Scopus (106) Google Scholar]. Binding of SCF induces KIT dimerization, inherent tyrosine kinase activation, and resulting activation of downstream signaling pathways, including the PI3-kinase, MAPK, and Ras/ERK pathways [2Gilfillan A.M. Rivera J. The tyrosine kinase network regulating mast cell activation.Immunol Rev. 2009; 228: 149-169Crossref PubMed Scopus (322) Google Scholar]. These KIT-mediated signaling cascades are critical for proper mast cell proliferation, activation, and differentiation [3Jensen B.M. Akin C. Gilfillan A.M. Pharmacological targeting of the KIT growth factor receptor: a therapeutic consideration for mast cell disorders.Br J Pharmacol. 2008; 154: 1572-1582Crossref PubMed Scopus (55) Google Scholar].As a result of alternative messenger RNA splicing, two major isoforms of KIT are expressed, characterized by the presence or absence of four amino acids (GNNK) in the juxta-membrane region of the extracellular domain [4Hayashi S. Kunisada T. Ogawa M. Yamaguchi K. Nishikawa S. Exon skipping by mutation of an authentic splice site of c-kit gene in W/W mouse.Nucleic acids research. 1991; 19: 1267-1271Crossref PubMed Scopus (107) Google Scholar, 5Crosier P.S. Ricciardi S.T. Hall L.R. Vitas M.R. Clark S.C. Crosier K.E. Expression of isoforms of the human receptor tyrosine kinase c-kit in leukemic cell lines and acute myeloid leukemia.Blood. 1993; 82: 1151-1158Crossref PubMed Google Scholar, 6Piao X. Curtis J.E. Minkin S. Minden M.D. Bernstein A. Expression of the Kit and KitA receptor isoforms in human acute myelogenous leukemia.Blood. 1994; 83: 476-481Crossref PubMed Google Scholar]. These isoforms are generally coexpressed, often with the GNNK− variant as the predominant transcript. Biological differences between the two GNNK isoforms have been described. The GNNK− isoform generally exhibits stronger signal transduction [7Montero J.C. Lopez-Perez R. San Miguel J.F. Pandiella A. Expression of c-Kit isoforms in multiple myeloma: differences in signaling and drug sensitivity.Haematologica. 2008; 93: 851-859Crossref PubMed Scopus (30) Google Scholar, 8Young S.M. Cambareri A.C. Odell A. Geary S.M. Ashman L.K. Early myeloid cells expressing c-KIT isoforms differ in signal transduction, survival and chemotactic responses to stem cell factor.Cell Signal. 2007; 19: 2572-2581Crossref PubMed Scopus (12) Google Scholar] and potential tumorigenicity [9Caruana G. Cambareri A.C. Ashman L.K. Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts.Oncogene. 1999; 18: 5573-5581Crossref PubMed Scopus (81) Google Scholar]. Expression differences of the two variants in malignant cell lines, solid tumors, and hematologic malignancies have suggested a possible prognostic utility [5Crosier P.S. Ricciardi S.T. Hall L.R. Vitas M.R. Clark S.C. Crosier K.E. Expression of isoforms of the human receptor tyrosine kinase c-kit in leukemic cell lines and acute myeloid leukemia.Blood. 1993; 82: 1151-1158Crossref PubMed Google Scholar, 10Guerrini F. Galimberti S. Ciabatti E. et al.Molecular detection of GNNK- and GNNK+ c-kit isoforms: a new tool for risk stratification in adult acute myeloid leukaemia.Leukemia. 2007; 21: 2056-2058Crossref PubMed Scopus (4) Google Scholar, 11Theou N. Tabone S. Saffroy R. et al.High expression of both mutant and wild-type alleles of c-kit in gastrointestinal stromal tumors.Biochim Biophys Acta. 2004; 1688: 250-256Crossref PubMed Scopus (27) Google Scholar, 12Sakuma Y. Sakurai S. Oguni S. Hironaka M. Saito K. Alterations of the c-kit gene in testicular germ cell tumors.Cancer science. 2003; 94: 486-491Crossref PubMed Scopus (93) Google Scholar].Systemic mastocytosis is a myeloproliferative neoplasm characterized by the clonal expansion of neoplastic mast cells [13Metcalfe D.D. Mast cells and mastocytosis.Blood. 2008; 112: 946-956Crossref PubMed Scopus (415) Google Scholar]. The KIT D816V activating mutation, located in the intracellular tyrosine kinase domain, is observed in more than 90% of adult patients with systemic mastocytosis [14Nagata H. Worobec A.S. Semere T. Metcalfe D.D. Elevated expression of the proto-oncogene c-kit in patients with mastocytosis.Leukemia. 1998; 12: 175-181Crossref PubMed Scopus (27) Google Scholar], and early acquisition of this mutation during hematopoiesis contributes to disease severity [15Garcia-Montero A.C. Jara-Acevedo M. Teodosio C. et al.KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: A prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients.Blood. 2006; 108: 2366-2372Crossref PubMed Scopus (392) Google Scholar]. Alterations in KIT messenger RNA processing can also have a critical role in disease pathogenesis, as novel KIT transcripts have been detected in aggressive mast cell malignancies [16Ozer O. Zhao Y.D. Ostler K.R. et al.The identification and characterisation of novel KIT transcripts in aggressive mast cell malignancies and normal CD34+ cells.Leuk Lymphoma. 2008; 49: 1567-1577Crossref PubMed Scopus (11) Google Scholar, 17McArthur G.A. Splicing the way to leukemia with KIT.Leuk Lymphoma. 2008; 49: 1431-1432Crossref PubMed Scopus (1) Google Scholar]. We hypothesized that alterations in the expression pattern of the GNNK variants exist in systemic mastocytosis; therefore, we developed a novel real-time PCR assay to examine the GNNK transcripts and their relationship to the KIT D816V mutation.In this study, we report that KIT D816V containing transcripts in mastocytosis displayed an elevated GNNK−/GNNK+ copy number ratio. Furthermore, the GNNK− isoform, in association with the KIT D816V mutation, enhanced cytokine-free metabolism and reduced sensitivity to the tyrosine kinase inhibitor, PKC412. This study suggests that normal mast cell homeostasis is dependent on the relative levels of the KIT GNNK isoforms expressed and, furthermore, preferential expression might influence the molecular pathogenesis and therapeutic responses in KIT D816V systemic mastocytosis.MethodsStudy subjectsFollowing informed consent, 25 patients with systemic mastocytosis (11 men, 14 women, ages 24–74 years) and 16 healthy subjects (10 men, 6 women, ages 29–62 years) underwent bone marrow biopsies as part of research protocols approved by the National Institute of Allergy and Infectious Diseases Institutional Review Board (NCT00044122, NCT00806364, NCT00090662). Systemic mastocytosis was diagnosed and classified according to the World Health Organization criteria [18Horny H.-P. Metcalfe D.D. Bennett J.M. et al.Mastocytosis.in: Swerdlow S.H. Campo E. Harris N.L. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. International Agency for Research on Cancer, Lyon, France2008Google Scholar] (Table 1). All bone marrow microscopic examinations were performed in a blinded manner by a hematopathologist. Complementary DNA (cDNA) was prepared from isolated bone marrow mononuclear cells as described previously [19Wilson T.M. Maric I. Simakova O. et al.Clonal analysis of NRAS activating mutations in KIT-D816V systemic mastocytosis.Haematologica. 2011; 96: 459-463Crossref PubMed Scopus (66) Google Scholar]. As a clinical diagnostic, the presence of the KIT D816V mutation was determined by PCR/restriction fragment length polymorphism (RFLP) as described previously [20Shah N.P. Lee F.Y. Luo R. Jiang Y. Donker M. Akin C. Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis.Blood. 2006; 108: 286-291Crossref PubMed Scopus (235) Google Scholar].Table 1Characteristics of study subjects with systemic mastocytosisCMML = chronic myelomonocytic leukemia; ET = essential thrombocytosis; F = female; Indolent (B) = indolent with one B finding; M = male; MDS = myelodysplastic syndrome; SM = systemic mastocytosis; WHO = World Health Organization.Light gray shading (patients 1–10) indicates the indolent disease group; medium grey shading (patients 11–18) indicates the indolent (B)/smoldering disease group; dark grey shading (patients 19–25) indicates the advanced disease group.Normal serum tryptase <11.5 ng/mL. Open table in a new tab Generation of control plasmids to validate real-time quantitative PCR assayTo validate the KIT GNNK quantitative (qPCR) assay, plasmids containing either the GNNK+ or GNNK− sequences were generated. The GNNK variants were amplified from cultured human mast cell (HuMC) cDNA using the following primer sets: the GNNK+ primer set, GNNK+ F1 5ʹ-GCA TTT AAA GGT AAC AAC AAA GAG C-3ʹ and GNNK+ R1 5ʹ-ATC TGC ATC CCA GCA AGT CT-3ʹ; the GNNK− primer set, GNNK− F1 5ʹ-CCT ATT TTA ACT TTG CAT TTA AAG AGC-3ʹ and GNNK−R1 5ʹ-CCC AGC AAG TCT TCA TTA TGT C-3ʹ. The PCR products were cloned into the pCR 2.1-TOPO plasmid using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. These plasmids were used as positive, negative, and interfering template controls to validate the qPCR assay. In addition, the molecular weight of the plasmids was calculated, and a serial dilution of plasmid DNA was prepared to produce standard curves from which transcript copy numbers could be derived in the samples.Real-time qPCR assayQuantitative PCR was performed using RT² SYBR Green Master Mixes (SABiosciences, Valencia, CA, USA) according to the manufacturer's instructions with modified PCR cycles on the ABI PRISM 7500 SDS system (Applied Biosystems, Carlsbad, CA, USA). To distinguish GNNK+ and GNNK− splice variants in the samples, it was critical to develop and validate highly specific primers selective for the alternatively spliced variants. This process is described in detail in Supplementary Figure 1A (online only, available at www.exphem.org). The primer sets used for each specific transcript are as follows: GNNK+ F1 5ʹ-GCA TTT AAA GGT AAC AAC AAA GAG C-3ʹ and GNNK+ R2 5ʹ-CTG TAC TTC ATA CAT GGG TTT CTG-3ʹ; GNNK− F1 5ʹ CCT ATT TTA ACT TTG CAT TTA AAG AGC-3ʹ and GNNK− R2 5ʹ-ATA ATG CAC ATC ATG CCA GCT A-3ʹ. The amplification cycles were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 65°C for 1 min 30 sec. All reactions were performed in triplicate, and the specificity of the reactions was confirmed by dissociation curve analysis. The GNNK variant transcript copy numbers were determined using standard curves generated with the control plasmids, and the GNNK−/GNNK+ copy number ratio was calculated for each sample. In addition, the 2−ΔΔCt method was used to calculate the expression level of each GNNK variant relative to that of a healthy control sample. GAPDH Ct values were consistent between samples, and GAPDH was used as the reference transcript to normalize for technical variations.Detection of the KIT D816V mutation in the GNNK isoformsGNNK+ F1/R1 and GNNK− F1/R1 primer sets were used to amplify the GNNK variant-specific transcripts, including the D816V region. The amplification cycles were as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 45 sec, 63°C for 1 min and 72°C for 90 sec, and the final extension of 72°C for 7 min. The PCR products were run on 2% agarose gels. DNA was recovered from the agarose gels using the QIAEX II Gel Extraction Kit (Qiagen, Valencia, CA, USA) according to manufacturer's instructions and eluted in 15 μL of water. The presence of the KIT D816V mutation was determined by PCR/RFLP [20Shah N.P. Lee F.Y. Luo R. Jiang Y. Donker M. Akin C. Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis.Blood. 2006; 108: 286-291Crossref PubMed Scopus (235) Google Scholar] using HinfI restriction enzyme digestion, which generates a confirmatory 188-bp band.Amplification of KIT D816V transcriptsKIT D816V containing transcripts were amplified using the following allele-specific primers: D816V F 5ʹ-AGT GCA TTC AAG CAC AAT GG-3ʹ and D816V R 5ʹ-TTA GAA TCA TTC TTG ATG A-3ʹ. As part of the primer validation, the KIT D816V mutation was readily detected in the cDNA of the neoplastic HMC1.2 (KIT D816V positive) cell line. In contrast, no amplification product was detected in normal human mast cells and the HMC1.1 (KIT D816V negative) cell line under various annealing temperatures (Supplementary Figure 1B, online only, available at www.exphem.org). The optimized amplification cycles were as follows: 94°C for 5 min, followed by 36 cycles of 94°C for 45 sec, 55°C for 90 sec and 72°C for 90 sec, and the final extension of 72°C for 7 min. The PCR products were run on 2% agarose gels. DNA was recovered from the agarose gels using the QIAEX II Gel Extraction Kit (Qiagen) according to the manufacturer's instructions and eluted in 15 μL of water. The eluted PCR products were diluted, and the GNNK variant transcript copy number quantified by qPCR.Expression constructs and mutagenesisHuman wild type KIT GNNK+ (WT+) or GNNK− (WT−) open reading frames were cloned into the pcDNA 3.1 expression vector (Invitrogen) using standard molecular biology techniques. The D816V mutation associated with either the GNNK+ (D816V+) or the GNNK− (D816V−) variant was generated using the QuickChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. The KIT variants (designated as WT−, WT+, D816V−, and D816V+; Fig. 1) were confirmed by Sanger sequencing and subsequently subcloned into the pMX-puro retroviral expression vector (Cell BioLabs, San Diego, CA, USA) using standard molecular biology techniques.Mast cell culture and stable expression of KIT GNNK isoformsThe immature murine mast cell line, IC2, was a gift from Tatsuki Kataoka (Department of Diagnostic Pathology, Kyoto University Hospital, Kyoto, Japan). IC2 cells express FcεRI, lack surface KIT expression, and exhibit minimal granule formation [21Lunderius C. Xiang Z. Nilsson G. Hellman L. Murine mast cell lines as indicators of early events in mast cell and basophil development.Eur J Immunol. 2000; 30: 3396-3402Crossref PubMed Scopus (28) Google Scholar]. The IC2 cells were cultured in Alpha MEM (Mediatech, Manassas, VA, USA) medium containing 10% FBS (Gibco, Carlsbad, CA), 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 ng/mL mouse interleukin (IL) 3 (Peprotech, Rocky Hill, NJ, USA). Transfection of the pMX-puro/KIT variants was performed using the Platinum Retroviral Expression System (Cell BioLabs) according to the manufacturer's instructions. Transduction of the virus into IC2 cells was performed using the ViraDuctin Retrovirus Transduction Kit (Cell BioLabs) according to the manufacturer's instructions and followed by positive selection with 1.8 μg/mL puromycin for 1 week. Expression was confirmed with flow cytometry using PE-conjugated anti-human CD117 (BioLegend, San Diego, CA, USA) and PE-conjugated isotype control (BD Biosciences, San Jose, CA, USA) on a LSRII flow cytometer (BD Biosciences) and using FlowJo software (Tree Star, Ashland, OR). Aliquots of the stable transduced cell lines were frozen and revived as needed.Mast cell growth and metabolism assaysFollowing puromycin selection, the KIT-transduced IC2 cells were washed free of IL-3 and cultured in the presence or absence of human SCF (hSCF; 100 ng/mL; PeproTech) for 1 week. To assess cell proliferation differences, 1 × 105 cells/mL were subsequently seeded in a six-well plate, and viable cells were counted daily using the Cellometer Auto T4 cell counter (Nexcelom Bioscience, Lawrence, MA, USA). An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess cell metabolism by measuring the activity of living cells via mitochondrial dehydrogenase activity (Sigma-Aldrich, St. Louis, MO, USA). Cells (2 × 104) in 100 μL medium were incubated in a 96-well plate for 24 hours. Medium was used as the blank control sample. After 21 hours, 10 μL of MTT solution was added into each well and incubated for 3 hours. The cells were centrifuged at 3000 rpm for 10 min; 75 μL supernatant was removed from the samples and controls. MTT solvent (100 μL) was added into each well and resuspended thoroughly to dissolve the crystals. Absorbance was measured at 570 nm within 1 hour.Mast cell maturation studiesThe KIT transduced IC2 cells were cultured in the presence or absence of hSCF 100 ng/mL (PeproTech) for 4 weeks. To assess granule formation, cytospin preparations were prepared and toluidine blue staining was performed [22Radinger M. Jensen B.M. Kuehn H.S. Kirshenbaum A. Gilfillan A.M. Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood.Curr ProtocImmunol. 2010; (Chapter 7:Unit 7.37)Google Scholar]. In addition, 1 × 105 cells were lysed in 100 μL of H2O, and the histamine content was measured by ELISA (Cell-cult/Urine kit, Immuno-Biological Laboratories, Minneapolis, MN, USA) according to the manufacturer's instructions.ImmunoblottingCell lysates were prepared using 1 × 106 cells and 25 μL aliquots were loaded on to 4–12% NuPAGE Bis-Tris gels (Invitrogen) for electrophoretic separation and immunoblotting as described previously [23Iwaki S. Tkaczyk C. Satterthwaite A.B. et al.Btk plays a crucial role in the amplification of Fc epsilonRI-mediated mast cell activation by kit.J Biol Chem. 2005; 280: 40261-40270Crossref PubMed Scopus (89) Google Scholar]. The following antibodies were used for immunoblotting: anti-KIT mAb (Cell Signaling, Danvers, MA, USA), anti–phospho-KIT pAb (pY936), anti-Akt pAb, anti–phospho-Akt pAb (Ser473), anti-Erk pAb, anti–phospho-Erk pAb (Thr202/Tyr204) pAb (Cell Signaling), and anti–β-actin mAb (Sigma). Immunoreactive proteins were visualized with enhanced chemiluminescence ECL (Thermo Scientific, Rockford, IL, USA).Mast cell drug inhibition assaysThe KIT D816V transduced IC2 cells were plated at 4 × 105 cells/mL with PKC412 (1–1000 nmol/L) in a final concentration of 0.1% DMSO [24Bai Y. Bandara G. Ching Chan E. et al.Targeting the KIT activating switch control pocket: A novel mechanism to inhibit neoplastic mast cell proliferation and mast cell activation.Leukemia. 2012; PubMed Google Scholar]. After 72 hours, an equal volume of 2 × CyQuant direct detection reagent (Invitrogen) was added to the cells in culture. Following a 1-hour incubation at 37°C with detection reagent, sample fluorescence was detected by using 508/527-nm wavelength filter sets.Statistical analysisThe analysis was conducted primarily with a nonparametric approach. Thus, comparisons between groups were made with the Wilcoxon rank sum test, correlations were estimated with the Spearman ρ, and the paired comparison was made with Wilcoxon signed rank test. IC50 values were estimated from a sigmoidal dose-response model, in which differences were assessed with an F test; p < 0.05 was considered statistically significant. Analysis was performed using PRISM software, version 5 (GraphPad, La Jolla, CA, USA).ResultsGNNK−/GNNK+ copy number ratio and GNNK− relative expression increase with percent neoplastic mast cell involvement in systemic mastocytosisWe first examined the cDNA of bone marrow mononuclear cells obtained from normal volunteers and patients with systemic mastocytosis for the absolute expression of the GNNK variants. The absolute copy number of each GNNK variant was derived using standard curves, and the GNNK−/GNNK+ copy number ratio was calculated for each sample. No statistical difference between copy number ratios in normal and mastocytosis patients was observed (p = 0.779; Fig. 2A). However, the copy number ratios increased with percent mast cell involvement, and the correlation approached significance (Spearman ρ = 0.39; p = 0.053; Fig 2B).Figure 2GNNK−/GNNK+ copy number ratio and GNNK− variant relative expression increase with neoplastic mast cell involvement. A real-time qPCR assay was used to analyze the expression of GNNK+ and GNNK− variants in the bone marrow mononuclear cells of healthy control subjects (n = 16) and patients with systemic mastocytosis (n = 25). (A) GNNK−/GNNK+ copy number ratio shown for individuals in each study group. For the patients with mastocytosis, (B) GNNK−/GNNK+ copy number ratio (C) GNNK+ relative expression, and (D) GNNK− relative expression were plotted against the percentage of mast cells in the bone marrow mononuclear cell fraction. All reactions were performed in triplicate, and specificity of the reactions was confirmed by dissociation curve analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether preferential expression of one or both variants contributes to this observation, the relative expression of each variant was examined as compared to a healthy control sample. No correlation between the relative expression of the GNNK+ variant and percent mast cells was observed (Spearman ρ = 0.15; p = 0.477; Fig 2C), whereas GNNK− relative expression directly correlated with the percent mast cells in the bone marrow mononuclear cell fraction (Spearman ρ = 0.51; p = 0.009; Fig 2D). Too few mast cells were present in the healthy control bone marrow mononuclear cells to determine whether this trend observed in patients with mastocytosis was also present in the general population.KIT D816V transcripts have higher GNNK−/GNNK+ copy number ratios in systemic mastocytosisGiven the association of the KIT D816V mutation with mast cell neoplasia, we next investigated whether KIT D816V was preferentially present in either GNNK variant. The KIT D816V PCR/RFLP assay was performed on purified GNNK+ or GNNK− PCR amplification products from patients with mastocytosis and healthy control subjects. Representative gels are shown in Figure 3A. As expected, KIT D816V was not detected in the healthy control subjects or a mastocytosis patient with wild type KIT (Patient 19). A KIT D816V–specific RFLP product (188 bp), however, was detectable in both GNNK+ and GNNK− transcripts from systemic mastocytosis patients harboring the KIT D816V mutation (Patients 15, 16, 17, and 20).Figure 3KIT D816V is present in both GNNK variants, and KIT D816V transcripts have higher GNNK−/GNNK+ copy number ratios in systemic mastocytosis. (A) The KIT D816V PCR/RFLP assay was performed on purified GNNK− or GNNK+ PCR amplification products from normal controls (n = 16) and patients with systemic mastocytosis (n = 25). The presence of the KIT D816V was confirmed by the generation of a 188-bp band in the patients known to be positive for the D816V mutation (N = 21; in this representative blot, patients 15, 16, 17, and 20). (B) For individual patients with mastocytosis with the KIT D816V mutation (n = 21), the GNNK−/GNNK+ ratio of KIT D816V specific transcripts was determined, and a paired analysis with the GNNK−/GNNK+ ratio of total KIT transcripts was performed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)With the KIT D816V confirmed in both GNNK variants, we next determined the GNNK−/GNNK+ copy number ratio of KIT D816V specific transcripts. A KIT D816V allele-specific primer was used to amplify KIT D816V containing transcripts from patients with KIT D816V positive systemic mastocytosis (n = 21). These KIT D816V enriched transcripts displayed a significantly higher GNNK−/GNNK+ copy number ratio compared with the total KIT transcript pool of the same sample (p < 0.0001, Wilcoxon signed rank test; Fig 3B). These data thus support our earlier observation that GNNK− predominance is associated with increasing mast cell involvement (Fig 2D).GNNK−/GNNK+ copy number ratio in bone marrow mononuclear cells was unable to distinguish disease severity in systemic mastocytosisWe next attempted to determine a potential association of the GNNK variants with disease severity. The subjects with systemic mastocytosis were divided into three groups: (1) indolent systemic mastocytosis (ISM), (2) indolent with one B finding (ISM-B) or smoldering systemic mastocytosis (SSM), and (3) advanced (ADV) disease as indicated in Table 1 [18Horny H.-P. Metcalfe D.D. Bennett J.M. et al.Mastocytosis.in: Swerdlow S.H. Campo E. Harris N.L. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. International Agency for Research on Cancer, Lyon, France2008Google Scholar]. No statistical difference in GNNK−/GNNK+ copy number ratio (or KIT D816V enriched copy number ratio) was detected between the three disease groups (data not shown). It should be noted, however, that there was no significant difference in the median percentage of mast cells in the bone marrow mononuclear cell fraction between the groups (ISM, 0.07%; ISM-B/SSM, 0.14%; ADV, 0.07%). Other groups have also reported that mast cell involvement within the bone marrow mononuclear cell fraction can be similar in indolent and other forms of systemic mastocytosis [25Sanchez-Munoz L. Morgado J.M. Alvarez-Twose I. et al.Flow cytometry criteria for systemic mastocytosis: bone marrow mast cell counts do not always count.Am J Clin Pathol. 2013; 139: 404-406Crossref PubMed Scopus (5) Google Scholar].Enhanced IC2 mast cell growth and maturation is associated with the GNNK− KIT isoformTo explore the functional effect of these clinical observations, we stably transduced the immature murine mast cell line, IC2, with human WT KIT and KIT D816V in both the GNNK+ and GNNK− isoforms. Comparable surface and total KIT expression was observed between the GNNK isoforms in cells transduced with either WT KIT or KIT D816V (Supplementary Fig 2, online only, available at www.exphem.org). The GNNK− isoform significantly enhanced mast cell proliferation (WT− in Fig 4A; p = 0.004, Wilcoxon rank sum test) and metabolism (WT− in Fig 4C; p < 0.0001, Wilcoxon rank sum test) of WT KIT transduced cells cultured in hSCF at day 3. No difference in the growth rate was observed in cells transduced with KIT D816V in either isoform (D816V+ and D816V− in Fig 4A). These observations were supported by metabolic studies (MTT assay) displaying similar results (Fig 4C). In the absence of hSCF, only KIT D816V transduced cells survived and, although cell proliferation was similar between GNNK varian