Mpl Baltimore: a Thrombopoietin Receptor Polymorphism Associated with Thrombocytosis

Home , Thrombopoietin, Thrombopoietin receptor

Mpl Baltimore: A thrombopoietin receptor polymorphism associated with thrombocytosis

Alison R. Moliterno*, Donna M. Williams, Laura I. Gutierrez-Alamillo, Roberto Salvatori, Roxann G. Ingersoll, and Jerry L. Spivak

Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

Communicated by Victor A. McKusick, The Johns Hopkins University School of Medicine, Baltimore, MD, June 17, 2004 (received for review March 26, 2004) The chronic myeloproliferative disorders (MPD) are clonal hema- was established according to Polycythemia Vera Study Group topoietic stem cell disorders of unknown etiology. We have re- criteria (1). Platelet counts were obtained during routine out- ported defective thrombopoietin receptor (Mpl) protein expres- patient clinic visits by using an automated cell counter. Periph- sion in MPD patients. To determine whether the basis of abnormal eral blood genomic DNA samples from racially identified Mpl protein expression was due to mutations in the Mpl gene, we healthy controls were kindly provided by Josef Prchal (Baylor sequenced Mpl cDNA from MPD patients. We found a single College of Medicine, Houston). nucleotide substitution (G1238T) that results in a change from lysine to asparagine at amino acid 39 (K39N) in three African- Mpl Cloning and Sequencing. Platelets were harvested from pe- American women referred for an evaluation of an MPD. We ripheral blood as described in ref. 4, and RNA was extracted with subsequently screened more than 400 patients and controls and phenol and chloroform (5). cDNA was generated with the found that the K39N substitution is a polymorphism restricted to SuperScript II RT kit (GIBCO͞BRL) by using a gene-specific African Americans and that Ϸ7% of African Americans are het- primer (5Ј-GGAATGGTTGGCAATGTGGG-3Ј) complemen- erozygous for K39N. African Americans with the K39N polymor- tary to the distal 3Ј end of Mpl-P. Mpl cDNA was amplified with phism had a signiﬁcantly higher platelet count than controls forward (5Ј–GATGGGCTAAGGCAGGCACA-3Ј) and reverse without the polymorphism (P < 0.001) and reduced platelet protein (5Ј-GGAATGGTTGGCAATGTGGG-3Ј) primers under the Mpl expression. Expression of an Mpl cDNA containing the K39N following conditions: 40 cycles of denaturation at 94°C for 30 s, substitution in cell lines was associated with incomplete processing annealing at 58°C for 30 s, and extension at 72°C for 2 min. PCR and a reduction in Mpl protein, recapitulating the Mpl protein products were excised and purified from agarose gels and cloned defect observed in platelets from individuals with K39N. K39N with Topo II by using the direct PCR Topo TA cloning system represents an identiﬁed functional Mpl polymorphism and is as- (Invitrogen). Plasmids isolated from transformed clones sociated with altered protein expression of Mpl and a clinical were fully sequenced with four sequencing primers (T7, 5Ј- phenotype of thrombocytosis. CAGCCAGGGGAACTTCAG-3Ј located in exon 4, 5Ј-AAG- CTTCTTCTACCACAG-3Ј located in exon 6, and SP6). DNA he chronic myeloproliferative disorders (MPD), polycythe- templates were sequenced by using the fluorescent dideoxy Tmia vera, essential thrombocytosis, and idiopathic myelofi- terminator method of cycle sequencing on an Applied Biosys- brosis (IMF), are clonal hematopoietic stem cell disorders tems Division 3700 DNA analyzer by following the manufactur- characterized by increases in one or more of the formed elements er’s protocols. of the blood and a tendency to develop extramedullary hematopoiesis and myelofibrosis. As the causative molecular lesions of Denaturing Gradient Gel Electrophoresis. DNA was extracted from these disorders are unknown, diagnosis relies on broad clinical peripheral blood or buccal smears by using a commercially and laboratory criteria (1). Thrombocytosis is an integral feature available kit according to the manufacturer’s instructions (Gen- of the MPD, yet it is also a feature of many benign and malignant tra, Minneapolis). Mpl exon 2 was amplified from 500 ng of conditions. peripheral blood genomic DNA by using a modified forward Thrombopoietin (TPO) and the TPO receptor (Mpl) are the primer containing a 40-bp GC-rich tail (5Ј-CGCCCGCCGC- key regulators of platelet mass and are required for both stem GCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCACATA- cell proliferation and for the terminal differentiation of platelets AACATGCCTGGGAG-3Ј) to increase the sensitivity of muta- (2). We have demonstrated that the majority of polycythemia tion detection during denaturing gel electrophoresis and an vera and IMF patients have a reduction in platelet Mpl protein unmodified reverse primer (5Ј-CAGATACATGGGGAGTGG- that is associated with incomplete Mpl glycosylation (3, 4). The 3Ј) with the following PCR conditions: 35 cycles of denaturation molecular basis of this defect is unknown, but it is not a at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C consequence of Mpl gene disruption or transcriptional repres- for 2 min. Denaturing gel electrophoresis of PCR products was sion. To define the molecular basis for these observed Mpl performed at constant voltage (80 V) for 16 h at 60°C by using protein defects, we sequenced Mpl cDNA from six MPD patients a 7.5% acrylamide gel containing a linear gradient of 30–90% of and found a missense mutation in one of these patients. We the denaturants urea and formamide. Abnormally migrating report herein that the missense mutation represents a single- bands were excised from the gel and sequenced. nucleotide polymorphism in the Mpl gene that is restricted to African Americans. We show that this Mpl polymorphism was Expression of K39N in 32D Cells. Retroviral expression vectors were associated with mild thrombocytosis in African Americans het- constructed by replacement of the green fluorescent protein erozygous for the polymorphism and with chronic, extreme cDNA with the full-length Mpl cDNA (normal or K39N variant) thrombocytosis in homozygous individuals.

Materials and Methods Abbreviations: MPD, myeloproliferative disorders; TPO, thrombopoietin. Study Design. Patients and control subjects from a general Data deposition: The sequence reported in this paper has been deposited in the National hematology clinic were enrolled in this Institutional Review Center for Biotechnology Information’s dbSNP database (accession no. 24779593). Board-approved study and gave written informed consent. The *To whom correspondence should be addressed. E-mail: [email protected]. diagnosis of polycythemia vera, essential thrombocytosis, or IMF © 2004 by The National Academy of Sciences of the USA

11444–11447 ͉ PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0404241101 Downloaded by guest on September 26, 2021 in the murine stem cell virus-based bicistronic retrovirus vector MGIN kindly provided by Linzhao Cheng (The Johns Hopkins University) (6). Retroviruses were generated by transient co- transfection into the human kidney cell line 293T of the Mpl expression vector plus a ⌿Ϫ ecotropic packaging vector (7), then used to infect 32D cells within 1 week. Stable, polyclonal 32D cell lines expressing the wild-type or K39N Mpl were obtained by G418 selection. Mpl mRNA expression was measured with real-time RT-PCR by using the Applied Biosystems Prism 7700 sequence detection system with primers and a probe designed to bind to the exon 11 and 12 region of the Mpl gene to target full-length transcripts (forward, TCCTGCACACTACAG- GAGACTGA; reverse, TCCACTTCTTCACAGGTATCT- GAGACT; probe, 6FAM-TCCTAGGCCAGTACCTTAGG- GACACTGCA-TAMRA). Mpl threshold cycle values were normalized to 18S rRNA as an internal standard by using TaqMan control reagents, and transcript levels were calibrated relative to standard curves generated with 32D-Mpl cells expressing the normal Mpl cDNA.

Immunoblotting. Equal amounts of protein were applied to a 10% Criterion gel (Bio-Rad), with electrophoresis, membrane trans- Fig. 1. Sequence and mutation detection analysis of G1238T. (A) Sequence fer, and immunoblotting with antibodies against Mpl or glyco- electropherograms of wild-type Mpl (Upper) and G1238T mutation (Lower, protein IIb performed as described in ref. 4. Antiserum to ␤ arrow) from cDNA clones. (B) Denaturing gradient gel electrophoresis of Mpl -actin was purchased from Sigma. Preparation of platelet and exon 2 PCR products. Lane 1, no template PCR control; lane 2, K39N- cell lysates was performed as described in ref. 4. heterozygous individual; lane 3, K39N-homozygous individual; lane 4, K39N- negative individual. Lower arrow indicates mobility of wild-type allele; upper Statistical Analysis. Statistical significance of sets of values was arrow indicates K39N allele, and the two faint upper bands are heterodu- analyzed with Student’s t test. plexes of wild-type and K39N double-stranded DNA products generated during PCR. Results We cloned and sequenced Mpl-P cDNAs from the platelet RNA were heterozygous for K39N. To avoid any bias that might have of six MPD and two control subjects. In an African-American woman with a 25-year history of isolated thrombocytosis and been introduced by only studying patients referred to a hema- erythrocytosis, we found a single base change (guanosine to tology clinic, we screened an additional 61 healthy, unrelated thymidine at nucleotide 1,238 in exon 2, National Center for African Americans; five (8%) were heterozygous for K39N. Biotechnology Information’s dbSNP database accession number 24779593) that predicts a lysine to asparagine substitution at Association of K39N and Thrombocytosis. Thrombocytosis was the amino acid 39 (K39N) (Fig. 1A). To determine the frequency of common phenotype of the three original K39N positive African- K39N in other MPD patients, we devised a PCR-based dena- American MPD patients. To determine whether K39N was turing gel electrophoresis assay by using peripheral blood as the associated with thrombocytosis in control subjects, we compared source of genomic DNA (8). The single nucleotide base change the platelet counts of the seven K39N heterozygous control resulting in K39N produced a unique signature of the hetero- subjects (five females, two males) to the 93 K39N-negative control individuals (61 females and 32 males) genotyped in the duplex products compared with that of the wild-type (Fig. 1B) MEDICAL SCIENCES hematology clinic study. Six of the seven K39N heterozygotes sequence. The K39N pattern was absent in 108 Caucasian MPD Ͼ patients. However, the K39N mutation was present in two other had an elevated platelet count ( 350,000 platelets per microli- African-American patients referred for an evaluation of an ter). The mean platelet count of the seven subjects with the MPD: one female with a 20-year history of thrombocytosis K39N polymorphism was significantly higher than the mean Ͻ (chronically between 560,000 and 719,000 platelets per microli- platelet count of 93 individuals without the polymorphism (P ter) and another female with thrombocytosis of unknown dura- 0.001), even when those controls with thrombocytopenia (15 of tion (a count of 1,450,000 single platelets per microliter). There the controls were thrombocytopenic) were excluded from the was no history of arterial or venous thrombosis, hemorrhage, or analysis (Fig. 2A). The referring diagnoses of the seven K39N erythromelalgia in these three patients. K39N was present in the heterozygous subjects were anemia (two), monoclonal gam- heterozygous state in these three African American patients of mopathy (two), von Willebrand disease (one), sickle cell anemia a total of 18 African Americans referred for MPD evaluations. (one), and HIV infection (one). K39N was present in DNA obtained from buccal smears from Two additional patients, a sister and brother, both with sickle two of these patients available for study. Platelet Mpl protein cell anemia, were referred to our study for chronic, extreme expression was markedly reduced in two of these patients thrombocytosis. Both siblings had counts between 800,000 and available for study (data not shown). 1,000,000 platelets per microliter chronically in the absence of infection or iron deficiency. Both siblings had a history of acute K39N Frequency in Control Subjects. Because the three K39N chest syndrome. Both were genotyped and found to be homozy- positive MPD patients were African American, we sought to gous for K39N. Studies of their mother and father (healthy determine whether K39N represented a rare, sporadic mutation individuals with sickle cell trait) revealed both to be K39N specific to MPD patients or whether K39N represented a genetic heterozygotes, with counts of 285,000 and 313,000 platelets per variation (polymorphism) specific to African Americans. microliter, respectively. Because patients with sickle cell disease K39N was not detected in 250 Caucasian, 40 Hispanic, or 39 have chronic elevations in platelet count because of the asplenic Asian individuals. Screening 100 randomly selected African state, we compared the mean platelet count of these two Americans referred to a general hematology clinic yielded 7 that homozygotes with the platelet counts of 29 sickle cell disease

Moliterno et al. PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11445 Downloaded by guest on September 26, 2021 Fig. 2. Platelet count versus K39N genotype in African Americans. (A) Platelet count versus K39N genotype in 100 African-American subjects from a general hematology clinic. The mean platelet count of the seven African Americans heterozygous for the K39N polymorphism (424,000 platelets per microliter; range 320,000–505,000) was signiﬁcantly higher than the mean platelet count of the 93 African-American controls without the polymorphism (242,000 platelets per microliter; range 5,000–500,000 platelets per microliter) (P Ͻ 0.001). (B) Platelet count versus K39N genotype in 31 African-American sickle cell disease patients. The mean platelet count of the two K39N- homozygous individuals with sickle cell anemia was 858,000 platelets per microliter. The platelet count of the single K39N heterozygous individual with Fig. 3. K39N expression in platelets and cell lines. (A) Immunoblot of platelet sickle cell anemia identiﬁed was 505,000 platelets per microliter. The mean lysates from a K39N-homozygous patient and a K39N-negative control. The platelet count of the 28 K39N-negative individuals with sickle cell disease (25 membranes were probed with Mpl C-terminal antiserum (Upper) and then SS, 3 SC, and 2 S␤ϩ) was 360,000 platelets per microliter with a range of reprobed with antiserum to platelet glycoprotein IIb. (B) Immunoblot of 127,000–584,000 platelets per microliter. 32D-K39N and 32D-Mpl cell lysates probed with Mpl C-terminal antiserum. The membrane was reprobed with actin (Lower) to demonstrate equal protein loading. (C) Quantitative real-time RT-PCR of Mpl mRNA expression in 32D- patients that we genotyped for K39N. The mean platelet count Mpl and 32D-K39N, 32D-vector, and 32D-parental cell lines. Mpl signal was not of the two homozygotes (858,000 platelets per microliter) was detected in either the 32D-vector or 32D-parental cell lines, although 18S significantly higher than the mean platelet count of 28 K39N- signals were detected at high levels in all four cell lines (data not shown). negative sickle cell patients (360,000 platelets per microliter; range 127,000–584,000) (P ϭϽ0.001) (Fig. 2B). scent. Studies of two geographically distinct populations of African Americans indicate that Ϸ7% of African Americans are Expression of K39N-Mpl in Platelets and 32D Cells. We assessed Mpl carriers for K39N. Examination of K39N with respect to platelet protein expression in platelets from K39N-positive individuals count suggests a gene dosage effect with K39N heterozygous and in murine hematopoietic cell lines engineered to express individuals exhibiting mild thrombocytosis, whereas K39N- either K39N Mpl or wild-type Mpl. Platelets from a K39N homozygous individuals exhibit severe thrombocytosis when homozygote expressed a markedly reduced amount of the fully compared with appropriate controls. Studies of platelets from processed Mpl isoform (Fig. 3A). Similarly, expression of K39N patients homozygous for K39N and cell lines engineered to Mpl cDNA in 32D cells, an IL-3-dependent murine cell line that express K39N indicate that this polymorphism dramatically neither expresses Mpl nor responds to TPO, differed both disrupts Mpl protein expression. qualitatively and quantitatively from the wild-type Mpl, with Mutations of the TPO gene and the Mpl gene have been both a reduction in the total amount of protein and the presence described in families with unique clinical phenotypes. Mutations of an isoform with a retarded electrophoretic mobility consistent of the TPO gene that enhance translational efficiency of TPO with incomplete glycosylation (Fig. 3B) in a manner that we mRNA have been demonstrated to be the cause of an autosomal characterized in ref. 4. We examined mRNA expression of K39N dominant hereditary thrombocythemia in three families (9–11), Mpl and wild-type Mpl mRNA in 32D cells by using quantitative whereas more recently, an activating mutation in Mpl was found real-time RT-PCR (Fig. 3C). Mpl transcripts were undetectable to be the basis of thrombocytosis in a single family with in 32D-vector and 32D parental cell lines. Mpl gene expression hereditary thrombocythemia (12). Children with congenital relative to 18S ribosomal RNA in the 32D-K39N cells was 1.81 amegakaryocytic thrombocytopenia, a disorder marked by se- compared with 32D-Mpl cells, supporting the conclusion that the vere congenital thrombocytopenia and eventual aplastic anemia, K39N Mpl protein defects were the result of abnormal post- inherit Mpl alleles from both parents that result in a nonfunc- translational processing and not impaired gene transcription. tioning Mpl protein (13–17). These sporadic human mutations in Taken together, these studies indicate that K39N dramatically TPO and Mpl generate phenotypes in a similar manner to those altered Mpl protein expression. generated in murine models where TPO or Mpl are manipulated. Targeted deletion of either the Mpl or the TPO gene in mice Discussion results in severe thrombocytopenia and a reduction in murine To investigate the possibility that an alteration in Mpl gene bone marrow stem cells (18, 19). expression was responsible for polycythemia vera Mpl protein K39N represents a newly identified allelic variant of Mpl defects, we sequenced Mpl cDNA from MPD patients. During associated with thrombocytosis, yet the in vivo and in vitro this study we identified a single base change that conferred a consequence of K39N expression is a hypofunctional protein. change in amino acid sequence, K39N, in three African- Impaired Mpl function in the setting of thrombocytosis is indeed American patients referred for an evaluation of an MPD. We counterintuitive, especially given the phenotype of marked subsequently found that K39N was a polymorphism of the Mpl thrombocytopenia in the Mpl knockout mice and in individuals gene that is restricted to individuals of African-American de- with congenital amegakaryocytic thrombocytopenia. However,

11446 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0404241101 Moliterno et al. Downloaded by guest on September 26, 2021 several lines of experimental evidence indicate both a direct we designate Mpl Baltimore. The polymorphism is autosomal negative regulatory role for Mpl and the presence of platelet and appears to conform to a pattern of autosomal dominance production pathways that are independent of Mpl function. First, with incomplete penetrance in that some heterozygotes have deletion of the distal extracellular domain of Mpl, the site of the normal platelet counts whereas others have sustained elevations K39N mutation, resulted in TPO-independent growth (20). of platelet counts that even satisfy MPD diagnostic criteria. The Second, induction of thrombocytosis has been demonstrated in Ϫ Ϫ Ϫ Ϫ variability in the K39N phenotype that we have observed among Mpl / mice after myelosuppressive therapy (21) and in Mpl / our heterozygotes may be due to the particular genetic and͞or mice who harbored mutations of c-myb (22). In fact, the c-myb Ϫ Ϫ environmental backgrounds in which K39N is expressed. Alter- mutants bred in the Mpl / background mounted a more severe natively, K39N could predispose some individuals to developing ϩ/ϩ thrombocytosis than those bred in a Mpl background, indi- clonal hematopoietic stem cell disorders. These distinctions cating that Mpl was not necessary for this thrombocytosis cannot yet be addressed, as clonal markers specific for the MPDs phenotype and that perhaps loss of Mpl signaling might exag- are lacking. Larger population studies in healthy and disease gerate a predisposition to supraphysiological megakaryocyto- states and family studies will be required to determine the poiesis (22). relationship of K39N to the development of thrombocytosis. Many patients classified clinically as having essential thrombocytosis have polyclonal hematopoiesis, indicating that the We thank Mary Ann Isaacs, Ophelia Rodgers, Michelle Doll, Christo- etiology of essential thrombocytosis is heterogeneous and may pher Garcia, and Dr. Zang Li for technical assistance. This work was not always be related to the transformation of a single hemato- supported by a Doris Duke Clinical Scientist Award (to A.R.M.), the poietic stem cell (23). We have now identified an Mpl polymor- National Organization of Rare Diseases (J.L.S.), and the Bernstein phism unique to individuals of African-American descent, which Family Foundation (A.R.M.).

1. Berk, P. D., Goldberg, J. D., Donovan, P. B., Fruchtman, S. M., Berlin, N. I. 14. Tonelli, R., Scardovi, A. L., Pession, A., Strippoli, P., Bonsi, L., Vitale, L., & Wasserman, L. R. (1986) Semin. Hematol. 23, 132–143. Prete, A., Locatelli, F., Bagnara, G. P. & Paolucci, G. (2000) Hum. Genet. 107, 2. Kaushansky, K. (1998) N. Engl. J. Med. 339, 746–754. 225–233. 3. Moliterno, A. R., Hankins, W. D. & Spivak, J. L. (1998) N. Engl. J. Med. 338, 15. van den Oudenrijn, S., Bruin, M., Folman, C. C., Peters, M., Faulkner, L. B., 572–580. de Haas, M. & dem Borne, A. E. (2000) Br. J. Haematol. 110, 441–448. 4. Moliterno, A. R. & Spivak, J. L. (1999) Blood 94, 2555–2561. 16. Ballmaier, M., Germeshausen, M., Schulze, H., Cherkaoui, K., Lang, S., 5. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156–159. Gaudig, A., Krukemeier, S., Eilers, M., Strauss, G. & Welte, K. (2001) Blood 6. Cheng, L., Du, C., Murray, D., Tong, X., Zhang, Y. A., Chen, B. P. & Hawley, 97, 139–146. R. G. (1997) Gene Ther. 4, 1013–1022. 17. Ballmaier, M., Germeshausen, M., Krukemeier, S. & Welte, K. (2003) Ann. Proc. Natl. Acad. 7. Pear, W. S., Nolan, G. P., Scott, M. L. & Baltimore, D. (1993) N.Y. Acad. Sci. 996, 17–25. Sci. USA 90, 8392–8396. 18. Alexander, W. S., Roberts, A. W., Nicola, N. A., Li, R. & Metcalf, D. (1996) 8. Miller, K. M., Ming, T. J., Schulze, A. D. & Withler, R. E. (1999) BioTechniques Blood 87, 2162–2170. 27, 1016–1024. 19. Carver-Moore, K., Broxmeyer, H. E., Luoh, S. M., Cooper, S., Peng, J., 9. Wiestner, A., Schlemper, R. J., Van Der Maas, A. P. & Skoda, R. C. (1998) Nat. Blood 88, Genet. 18, 49–52. Burstein, S. A., Moore, M. W. & de Sauvage, F. J. (1996) 803–808. 10. Kondo, T., Okabe, M., Sanada, M., Kurosawa, M., Suzuki, S., Kobayashi, M., 20. Sabath, D. F., Kaushansky, K. & Broudy, V. C. (1999) Blood 94, 365–367. Hosokawa, M. & Asaka, M. (1998) Blood 92, 1091–1096. 21. Levin, J., Cocault, L., Demerens, C., Challier, C., Pauchard, M., Caen, J. & 11. Ghilardi, N., Wiestner, A., Kikuchi, M., Ohsaka, A. & Skoda, R. C. (1999) Br. J. Souyri, M. (2001) Blood 98, 1019–1027. Haematol. 107, 310–316. 22. Carpinelli, M. R., Hilton, D. J., Metcalf, D., Antonchuk, J. L., Hyland, C. D., 12. Ding, J., Komatsu, H., Wakita, A., Kato-Uranishi, M., Ito, M., Satoh, A., Mifsud, S. L., Di Rago, L., Hilton, A. A., Willson, T. A., Roberts, A. W., et al. Tsuboi, K., Nitta, M., Miyazaki, H., Iida, S., et al. (2004) Blood 103, 4198–4200. (2004) Proc. Natl. Acad. Sci. USA 101, 6553–6558. 13. Ihara, K., Ishii, E., Eguchi, M., Takada, H., Suminoe, A., Good, R. A. & Hara, 23. Harrison, C. N., Gale, R. E., Machin, S. J. & Linch, D. C. (1999) Blood 93, T. (1999) Proc. Natl. Acad. Sci. USA 96, 3132–3136. 417–424. MEDICAL SCIENCES

Moliterno et al. PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11447 Downloaded by guest on September 26, 2021