Gene Mutations Associated With Human Skeletal Disorders Occur FGFR3
Transcription
Gene Mutations Associated With Human Skeletal Disorders Occur FGFR3
From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 1998 92: 2987-2989 FGFR3 Gene Mutations Associated With Human Skeletal Disorders Occur Rarely in Multiple Myeloma Nicola Stefano Fracchiolla, Stefano Luminari, Luca Baldini, Luigia Lombardi, Anna Teresa Maiolo and Antonino Neri Updated information and services can be found at: http://www.bloodjournal.org/content/92/8/2987.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on October 28, 2014. For personal use only. CORRESPONDENCE 2987 2. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson K, Peto J, Smith PG: Design and analysis of randomized clinical trials requiring prolonged observation of each patient. I. Introduction and design. Br J Cancer 34:585, 1976 3. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson K, Peto J, Smith PG: Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br J Cancer 35:1, 1977 4. The Italian Cooperative Study Group on Chronic Myeloid Leukemia: Interferon alfa-2a as compared with conventional chemotherapy for the treatment of chronic myeloid leukemia. N Engl J Med 330:820, 1994 5. Hasford J, Pfirrmann M, Hehlmann R, Allan NC, Kluin-Nelemans JC, Alimena G, Steegmann JL, Ansari H: A new prognostic score for survival of patients with chronic myeloid leukemia treated with interferon alfa. J Nat Cancer Inst 90:850, 1998 6. Mahon F, Montastruc M, Faberes C, Reiffers J: Predicting complete cytogenetic response in chronic myelogenous leukemia patients treated in chronic myelogenous leukemia patients treated with recombinant interferon-a. Blood 84:3592, 1994 7. Guilhot F, Chastang C, Michallet M, Guerci A, Harousseau JL, Maloisel F, Bouabdallah R, Guyotat D, Cheron N, Nicolini F, Abgrall JF, Tanzer J: Interferon alpha2b (IFN) and cytarabine (Ara-C) increase survival and cytogenetic response in chronic myeloid leukemia (CML). Results of a randomized trial. N Engl J Med 337:223, 1997 8. Allan NC, Richards SM, Shepherd PCA: Interferon-a therapy with busulphan or hydroxyurea compared with either BU or HU alone in treatment of chronic phase CML. Results from the MRC III trial. Int J Hematol 64:S68, 1996 (abstr 258, suppl 1) 9. Shepherd PCA, Richards SM, Allan NC: Progress with interferon in CML—Results of the MRC UK CML III study. Bone Marrow Transplant 17:S15, 1996 (suppl 3) 10. CML Trialists’ Collaborative Group: Interferon alfa versus chemotherapy for chronic myeloid leukemia: A meta-analysis of seven randomized trials. J Natl Cancer Inst 89:1616, 1997 11. Hehlmann R, Heimpel H, Hasford J, Kolb HJ, Pralle H, Hossfeld DK, Queisser W, Lo¨ffler H, Hochhaus A, Heinze B, Georgii A, Bartram CR, Griesshammer M, Bergmann L, Essers U, Falge C, Queisser U, Meyer P, Schmitz N, Eimermacher H, Walther F, Fett W, Kleeberg UR, Ka¨bisch A: Randomized comparison of interferon-a with busulfan and hydroxyurea in chronic myelogenous leukemia. Blood 84:4064, 1994 FGFR3 Gene Mutations Associated With Human Skeletal Disorders Occur Rarely in Multiple Myeloma To the Editor: Fibroblast growth factor receptor 3 (FGFR3) is one of four distinct tyrosine-kinase receptors (FGFR1-4) that are capable of binding a repertoire of at least nine related mitogenic fibroblast growth factors (FGFs). FGFRs encode proteins that all contain three glycosylated extracellular Ig-like domains, a transmembrane domain (TM), and a split cytoplasmic tyrosine-kinase domain. Point mutations in distinct domains of the FGFR3 gene are associated with autosomal dominant human skeletal disorders, such as achondroplasia, thanatophoric dysplasia types I and II, and hypochondroplasia.1,2 Recent reports indicate that the point mutations associated with these disorders produce constitutively activated FGFR3, which shows autophosphorylation in the absence of ligand and is no longer regulated by FGF binding.3-6 We and others have recently provided the first evidence of FGFR3 gene involvement in human cancer.7,8 In particular, the FGFR3 gene located at 4p16.3 is translocated to chromosome 14q32 as a result of a novel and karyotypically undetectable t(4;14)(p16.3;q32) chromosomal translocation in multiple myeloma (MM), a malignant proliferation of plasma cells. Molecular studies have shown this lesion in five MMderived cell lines and in four primary tumors. Although the breakpoints on 4p16.3 are located approximately 50 to 120 kb centromeric to FGFR3, the gene is overexpressed in these cases, but absent or barely detectable in cell lines without the translocation. Interestingly, FGFR3 gene mutations associated with distinct human skeletal disorders2 have also been identified in some MM tumors carrying the t(4;14)(p16.3; q32): in particular, the Y373C mutation in the KMS-11 cell line,7,8 the K650E mutation in the OPM2 cell line,7 and the K650M mutation in a primary MM tumor.7 These findings prompted us to look for FGFR3 mutations known to be associated with skeletal disorders in a representative panel of MM, including 80 primary cases (60 patients at first diagnosis, 12 at relapse, and 8 affected by plasma cell leukemia) and 10 MM-derived cell lines (including the KMS-11 and OPM2 cell lines). The analysis was performed by means of the polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) direct sequencing of genomic DNA. We amplified five distinct genomic FGFR3 fragments containing codons affected by mutations: codon 248, the entire TM domain Fig 1. Schematic representation of the primers from the human FGFR3 gene used in the study. The FGFR3 exons are indicated by white boxes, and the introns are indicated by lines. The 38 untranslated region is indicated by the dashed box. The approximate locations of the primers, the length of the amplified fragments, and the approximate positions of codons 248, 540, 650, and 807 are indicated. The nucleotide sequence of FGFR3 cDNA and the intron-exon organization of the gene have been previously reported.12,13 The sequences of the primers are as follows: 248F (intron 6), 58-CCTGAGCGTCATCTGCC-38, and 248R (exon 7), 58-CCATTGCATCCCACACGG-38; TD5 (exon 10), 58-AGGAGCTGGTGGAGGCTGA-38, and TD3 (exon 10), 58-GGAGATCTTGTGCACGGTGG-3814; 540F (exon 13), 58-ACTGACAAGGACCTGTCGGAC-38, and 540R (exon 13), 58GCCCTGCGTGCAGGCGCC-38; 650F (exon 15), 58-GCATCCACAGGGACCTGG-38, and 650R (intron 15), 58-AGGCGGTGTTGGCGCCAG-38; 14S (exon 15), 58-GTGCACAACCTCGACTAC-38 (this primer was used with 650R to obtain a DNA fragment suitable for the restriction enzyme analysis of codon 650); 807F (exon 19), 58-CCTGTCGGCGCCTTTCGAGCAGTAC-38, and 807R (exon 19), 58-CACCAGCAGCAGGGTGGGCTGCTAG-38.15 From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 2988 CORRESPONDENCE constitutive oncogenic signal for the growth and/or survival of malignant plasma cells. This possibility is supported by the evidence that the bone marrow environment and, in particular, the stromal cells with which the plasma cells interact10 are able to produce FGFs.11 The FGFR3 mutations reported in MM probably represent somatic events, suggesting that FGFR3 gene may be deregulated by different mechanisms. However, we were unable to detect FGFR3 mutations associated with skeletal disorders in our series of samples, except in the cell lines previously reported.7,8 This finding suggests that such mutations represent rare events in MM and support the hypothesis that they may occur after the translocation and deregulation of the FGFR3 gene, thus contributing to tumor progression by means of ligand-independent activation. ACKNOWLEDGMENT We are grateful to Dr T. Otsuki, Dr F. Malavasi, and Dr A. Solomon for providing us with the some of the MM-derived cell lines (KMM1, KMS-11, KMS-12, LP-1, and UTMC-2) used in this study and to G Ciceri for technical assistance. The cell lines U266, Sultan, ARH-77, and RPMI 8226 were obtained from ATCC and the OPM2 cell line was obtained from DSMZ. This work was supported by a grant from the Associazione Italiana Ricerca sul Cancro (AIRC) to A.N. and a grant ‘‘Ricerca Corrente 1994’’ from the Ministero Italiano della Sanita` to Ospedale Maggiore IRCCS. Nicola Stefano Fracchiolla Stefano Luminari Luca Baldini Luigia Lombardi Anna Teresa Maiolo Antonino Neri Servizio di Ematologia Istituto di Scienze Mediche Universita` di Milano Ospedale Maggiore IRCCS Milan, Italy Fig 2. PCR-SSCP analysis of the FGFR3 gene. N, normal control; migrating fragments different from the normal control are indicated by arrows. (codons 371, 373, 375, and 380), codon 540, codon 650, and codon 807 (Fig 1). The mutations at codon 650 were also investigated by means of a restriction enzyme analysis of the PCR-amplified fragment using Mbo II and Bbs I enzymes, as previously described.9 We detected allelic variations of the FGFR3 gene only in the fragment specific for the TM domain. An abnormal fragment with the same pattern of migration was observed in 2 cases (the LP-1 cell line and a primary tumor; Fig 2); in both cases, a novel single basepair mutation involving codon 384 in the form of a T to C transition (TTC-CTC) led to a conservative Phe = Leu amino acid substitution (data not shown). Interestingly, this mutation abrogates a Mbo II restriction site and creates a new Mnl I site that allows restriction enzyme analysis of the PCR-amplified fragment. The apparently similar intensity of the normal and mutated bands in both cases, as well as the detection of the mutation in 2 of 100 normal individuals by means of restriction enzyme analysis, suggest that it may represent a rare genetic polymorphism. Finally, the FGFR3 gene was apparently not expressed in the LP-1 cell line; it remains to be seen whether this particular variant may affect FGFR3 biological activity. Although no specific genetic lesions have been found to be associated with MM (unlike other types of lymphoid neoplasms), cytogenetic and more recent molecular analyses suggest that chromosomal translocations involving the Ig locus on chromosome 14q32 may play an important role in gene deregulation.7,8 In this context, the recent identification of the t(4;14)(p16.3;q32) in MM, associated with an apparent deregulation of the FGFR3 gene, may provide some insights into the pathogenesis of this neoplasia. Although more work is needed to assess the role and frequency of the t(4;14) in MM, it can be suggested that deregulation of FGFR3 gene expression may lead to a REFERENCES 1. Muenke M, Schell U: Fibroblast-growth-factor receptor mutations in human skeletal disorders. Trends Genet 11:308, 1995 2. Webster MK, Donoghue DJ: FGFR activation in skeletal disorders: Too much of a good thing. Trends Genet 13:178, 1997 3. Naski MC, Wang Q, Xu J, Ornitz DM: Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia. Nat Genet 13:233, 1996 4. Webster MK, Donoghue DJ: Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J 15:520, 1996 5. Webster MK, d’Avis PY, Robertson SC, Donoghue DJ: Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II. Mol Cell Biol 16:4081, 1996 6. d’Avis PY, Robertson SC, Meyer AP, Bardwell WM, Webster MK, Donoghue DJ: Constitutive activation of fibroblast growth factor receptor 3 by mutations responsible for the lethal skeletal dysplasia thanatophoric dysplasia type I. Cell Growth Differ 9:71, 1998 7. Chesi M, Nardini E, Brents LA, Schroch E, Ried T, Kuehl WM, Bergsagel PL: Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet 16:260, 1997 8. Richelda R, Ronchetti D, Baldini L, Cro L, Viggiano L, Marzella R, Rocchi M, Otsuki T, Lombardi L, Maiolo AT, Neri A: A novel From www.bloodjournal.org by guest on October 28, 2014. For personal use only. CORRESPONDENCE chromosomal translocation t(4;14)(p16.3;q32) in multiple myeloma involves the fibroblast growth-factor receptor 3 gene. Blood 10:4062, 1997 9. Tavormina PL, Shiang R, Thompson LM, Zhu YZ, Wilkin DJ, Lachman RS, Wilcox WR, Rimoin DL, Cohn DH, Wasmuth JJ: Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nat Genet 9:321, 1995 10. Caligaris-Cappio F, Bergui L, Gregoretti MG, Gaidano G, Gaboli M, Schena M, Zallone AZ, Marchisio PC: Role of bone marrow stromal cells in the growth of human multiple myeloma. Blood 77:2688, 1991 11. Allouche M: Basic fibroblast growth factor and hematopoiesis. Leukemia 9:937, 1995 12. Keegan K, Johnson DE, Williams LT, Hayman MJ: Isolation of 2989 an additional member of the fibroblast growth factor receptor family, FGFR-3. Proc Natl Acad Sci USA 88:1095, 1991 13. Perez-Castro AV, Wilson J, Altherr MR: Genomic organization of the human fibroblast growth factor receptor 3 (FGFR3) gene and comparative sequence analysis with the mouse fgfr3 gene. Genomics 41:10, 1997 14. Shiang R, Thompson LM, Zhu YZ, Church DM, Fielder TJ, Bocian M, Winckur ST, Wasmuth JJ: Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 78:335, 1994 15. Rousseau F, Saugier P, Le Merrer M, Munnich A, Delezoide A-L, Maroteaux P, Bonaventure J, Narcy F, Sanak M: Stop codon FGFR3 mutations in thanatophoric dwarfism type 1. Nat Genet 10:11, 1995 Interleukin-2 Receptor Subunit Expression and Function on Human Peripheral T Cells Is Not Dependent on the Anticoagulant To the Editor: In a recent report, David et al1 addressed the ongoing controversy on expression levels of interleukin-2 receptor (IL-2R) a, b, and g chains on various mononuclear cells from the peripheral blood. Using freshly isolated peripheral blood mononuclear cells (PBMCs) from (sodium) heparinized blood and fluorescein isothiocyanate (FITC)-labeled commercial monoclonal antibodies, they showed that all three IL-2R chains usually are hardly detectable on either CD4 or CD8 T cells from healthy donors and from hemochromatosis patients. These results are in contrast with the much higher levels of IL-2R subunits on T cells observed by several investigators, including ourselves.2-9 David et al1 tentatively explain the discrepancy by invoking effects of anticoagulant and of storage. Indeed, if Ca21 chelators were used instead of heparin, the levels of all three IL-2R chains on T cells apparently increased, and overnight storage of heparinized blood also seemed to upregulate IL-2R subunit expression. These observations are very important, because they not only seem to settle a long-standing controversy on IL-2R expression, but they also imply that the use of Ca21 chelators as anticoagulant instead of heparin could dramatically influence the sensitivity of the T cells to IL-2. Because IL-2 and other common g-chain triggering cytokines are central to almost any T-cell function, EDTA or citrate anticoagulants should be avoided if subsequent functional testing is envisioned. A lot of immunological research is based on buffy coats, which routinely are anticoagulated with citrate. In our own studies of T-cell function during human immunodeficiency virus (HIV) infection, we have systematically used EDTA blood as starting material, because it is readily available and because we did not find a functional difference between lymphocytes derived from blood anticoagulated with heparin, citrate, or EDTA in preliminary experiments. In view of the findings of David et al,1 we felt obliged to carefully control the effect of Ca21 chelators on IL-2R expression and function and we did not observe any significant influence of the anticoagulant. In three separate experiments, blood from five healthy control subjects (all lab personnel) was drawn at 10 AM in three different tubes from Sarstedt containing either sodium heparin (final concentration, 0.3 mg/mL), potassium-EDTA (final concentration, 1.6 mg/mL), or sodiumcitrate (final concentration, 10.6 mmol/L). The largest part of each tube was immediately processed for mononuclear cell (PBMC) separation, using Histopaque 1077 (Sigma, Bornem, Belgium), whereas the rest was kept at room temperature. At 2 PM, 50 µL of whole blood and 50 µL of PBMCs (containing 200,000 cells), derived from each of the three anticoagulant tubes, were incubated for 20 minutes at 4°C with 0.1 µg of the nonconjugated reference monoclonals anti-Tac (IL-2Ra–specific; obtained from Dr Thomas Waldman, National Institutes of Health, Bethesda, MD) and with 2R-B (IL-2Rb–specific; from Dr Takashi Uchiyama, Institute for Virus Research, Kyoto University, Kyoto, Japan). As an isotypic (IgG1) control, we used purified 56D3 directed against an irrelevant parasitic antigen (provided by Dr J. Brandt, Institute of Tropical Medicine, Antwerpen, Belgium). After washing with phosphate-buffered saline (PBS), containing 0.5% bovine serum albumin, 1 µL of FITC-labeled F(ab8) 2 goat antimouse IgG (Tago, Burlingame, CA) was added for another 20 minutes. After washing again, the remaining binding sites on the FITC-conjugate were blocked with 5 µL of mouse serum. Next, 5 µL of phycoerythrin (PE)-labeled anti-CD4 and 5 µL of peridinin-chlorophyll A protein (PercP)-labeled anti-CD3 (both from Becton Dickinson, Erembodegem, Belgium) were added for the last 20 minutes. The tubes with whole blood were then subjected to the Becton Dickinson lysing solution. All preparations were washed once and fixed with 1% paraformaldehyde. The samples were analyzed on a FACScan (Becton Dickinson) using the LYSYS I software. Based on the scatter and the CD3/CD4 expression, the CD41 and CD42 T lymphocytes were gated separately and the distribution of the first fluorescence was represented in a histogram for each subset. An example of this analysis is shown in Fig 1. It is evident that, within both the CD41 and CD42 T-cell populations, the expression profile of IL-2Ra is rather broad and tends to be bimodal (a negative and a positive subpopulation), whereas the curve of IL-2Rb is unimodal and shows a shift to the right, which is most evident in the CD42 subset. We chose to express the results for both chains as percentage of positive cells, after establishing a narrow threshold at a relative fluorescence intensity of 10, based on the background of the control monoclonal. A summary of the results is shown in Table 1. No significant difference was observed in the level of IL-2R a and b chains on CD41 or CD42 T cells, according to the anticoagulant used and regardless of whether the cells were stained in the context of whole blood or PBMCs. Comparing the mean fluorescence intensity of all gated cells (instead of the percentage of positive cells) showed similar results and confirmed that the low level of IL-2Rb expression on CD41 T cells significantly differed from background (data not shown). We next wanted to know whether the anticoagulant influences the sensitivity to IL-2. To this end, we cultured the three preparations of PBMCs at a final concentration of 106/mL in RPMI, supplemented with antibiotics (GIBCO, Paisley, UK) and 10% bovine calf serum (Hyclone,