Haplotype of JAK2 Confers Susceptibility to JAK2 Exon 12 Mutation-Positive Polycythemia Vera

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Letters to the Editor 1924 The ‘GGCC’ haplotype of JAK2 confers susceptibility to JAK2 exon 12 mutation-positive polycythemia vera

Leukemia (2009) 23, 1924–1926; doi:10.1038/leu.2009.110; distinct clinical MPN entities. To examine the contribution of published online 14 May 2009 interindividual genetic differences on MPN disease phenotypes, Pardanani et al. studied genetic variation in the erythropoietin Acquired somatic mutations in the Janus kinase 2 (JAK2) and receptor (EPOR), granulocyte colony-stimulating factor receptor 3 thrombopoietin receptor (MPL) genes have been described as (GCSFR), MPL as well as in the JAK2 gene. In a cohort of 179 major contributors to the pathogenesis and clinical character- MPN patients, an intragenic JAK2 haplotype showed significant istics of myeloproliferative neoplasms (MPN).1 Among these, the association with the diagnosis of polycythemia vera, but not G4T single nucleotide transversion in exon 14 of JAK2 (JAK2- essential thrombocythemia (ET) or primary myelofibrosis (PMF) V617F) is the most frequent in MPN.1 Point mutations and (Figure 1). deletions in exon 12 of the JAK2 gene are the most common Two independent studies have recently offered further insights genetic aberrations associated with JAK2-V617F-negative into the effect of hereditary factors on the pathogenesis of 6,7 polycythemia vera.2,3 Despite numerous investigations on the MPN. Genotyping single-nucleotide polymorphisms in MPN impact of JAK2 mutations on MPN phenotype, the genetic patients with uniparental disomy on chromosome 9p revealed a mechanisms facilitating the somatic acquisition of JAK2-V617F haplotype that includes the JAK2 gene and predisposes to the 7 and its influence on phenotypic variation among the three main development of JAK2-V617F-positive MPN. In a study addres- MPN entities remain unclear. sing the incidence of multiple acquisitions of the JAK2-V617F 6 Bone marrow transplant models have shown that the expres- mutation, we obtained evidence that JAK2-V617F is preferen- sion of JAK2-V617F in murine bone marrow hematopoietic cells tially acquired on one of the two common JAK2 haplotypes, induces phenotypic features resembling human MPN.4,5 Most which we refer to as the GGCC haplotype (Figure 1). A total interestingly, JAK2-V617F-transformed Balb/c and C57Bl/6 mice of 88% of all JAK2-V617F mutations were acquired on the exhibited strain-specific differences in JAK2-V617F-associated chromosome carrying the GGCC haplotype of JAK2. Association disease phenotype.5 This finding gave rise to the hypothesis that analysis showed that MPN patients heterozygous or homo- individual genetic variation influences the clinical manifestation zygous for the GGCC haplotype were at higher risk of acquiring of JAK2-V617F-positive MPN and provided a first clue as to how the JAK2-V617F mutation. The same conclusions were reached a single point mutation in the JAK2 gene can constitute three in an independent study with a single-nucleotide polymorphism

Figure 1 Association between the GGCC haplotype and JAK2 mutations. (a) Allelic association results of single-nucleotide polymorphisms (SNPs) in the JAK2 genomic region on chromosome 9p. Dots represent P-values of association analysis between SNP genotypes and polycythemia vera (PV) (blue dots, Pardanani et al.3), JAK2-V617F-positive and -negative myeloproliferative neoplasms (green dots, Olcaydu et al.6) and JAK2 exon 12 mutation-positive PV (red dot, this study). The x axis represents chromosomal position in mega base pairs. HapMap recombination rates (combined phase I and II, release 21) are shown on the right y axis (black line). Horizontal arrows delineate the RefSeq gene position of JAK2, INSL6 and INSL4 genes. Dashed grey bar indicates the SNP-genotyped region tagging the GGCC haplotype (red horizontal bar) and containing JAK2-V617F and JAK2 exon 12 mutations. (b) Detailed view of the genomic region susceptible to JAK2 mutations. JAK2 introns (in) 11–15 and exons (ex) 12–15 are shown (not in scale). The JAK2 exon 12 mutation locus and the SNP used for association analysis in this study (rs10974944) are depicted. Exon 14 and the locus of the JAK2-V617F mutation is shown with the SNP in intron 14 (rs12343867) used for the earlier association study.6

Leukemia Letters to the Editor 1925 located in the intron 12 of the JAK2 gene, which is contained in tion of single-nucleotide polymorphism variants can influence the GGCC haplotype and was confirmed to be significantly genetic repair or replication mechanisms leading to a higher associated with JAK2-V617F-positive MPN.8 susceptibility and accumulation of defects at that particular One interpretation of these data is that the GGCC haplotype genomic region. We still cannot exclude the possibility that the of JAK2 is more susceptible to DNA repair defects or replication described haplotype is in linkage with a yet unidentified infidelity and, therefore, is more likely to acquire somatic functional variant, and thereby influences disease manifestation mutations such as JAK2-V617F. To further explore the hypothesis of differential mutability as a haplotype property, we investigated whether somatic mutagenesis on the GGCC Table 1 List of JAK2 exon 12 mutation-positive PV cases haplotype is restricted to the JAK2-V617F mutation site or involves a larger genomic region. To determine the haplotype Location ID JAK2 exon 12 mutation preference of other acquired mutations of JAK2, we tested whether the various JAK2 exon 12 mutations also preferentially Vienna/Austria PV01 E543-D544del PV02 R541K, E543-D544del arise on the GGCC haplotype. We examined a cohort of 44 Basel/Switzerland PV03 ins33 Caucasian polycythemia vera patients of Western European PV04 I540-E543delinsMK descent who were positive for JAK2 exon 12 mutations (Table 1). PV05 F537-K539delinsL We genotyped all patients for a tagging single-nucleotide PV06 I540-E543delinsMK polymorphism in intron 12 of the JAK2 gene (rs10974944), that PV07 N542-E543del is in linkage disequilibrium with the GGCC haplotype.6 PV08 H538-K539delinsL PV09 N542-E543del Association analysis revealed a significant correlation between PV10 N542-E543del the occurrence of JAK2 exon 12 mutations and the GGCC PV11 N542-E543del haplotype when compared with an Austrian non-MPN control PV12 I540-E543delinsMK population (n ¼ 290; Table 2, Figure 1). According to allelic PV13 F537-K539delinsL association analysis, patients carrying the G variant of PV14 E543-D544del rs10974944 were at higher risk of acquiring a point mutation PV15 E543-D544del PV16 K539L or deletion in exon 12 of JAK2 (P ¼ 0.0016; OR (odds PV17 K539L ratio) ¼ 2.10; 95% CI (confidence interval) ¼ 1.31–3.35). About Pavia/Italy PV18 N542-E543del 64 percent of all JAK2 exon 12 mutation-positive patients PV19 E543-D544del carried the GGCC haplotype in a heterozygous or homozygous PV20 N542-E543del state. The minor allele frequencies did not differ significantly in PV21 R541-E543delinsK the Austrian non-MPN control cohort and in the publicly PV22 E543-D544del PV23 V536-I546dup11 available genotype data of the Caucasian HapMap CEU and TSI PV24 N542-E543del populations (0.24, 0.25 and 0.30, respectively), excluding the PV25 F537-I546dup10, F547L influence of population-specific differences in allelic distribu- PV26 N542-E543del tion on the computed association analysis. Our data provide PV27 R541-E543delinsK evidence that the increased susceptibility to somatic mutagen- Dijon/France PV28 H538QK539L esis exhibited by the JAK2 GGCC haplotype is not restricted to PV29 R541-E543delinsK PV30 F537-I546dup10, F547L JAK2-V617F, but also includes the less frequent but more PV31 K539L diverse JAK2 exon 12 mutations. PV32 F537-I546dup10, F547L There are two alternative hypotheses that could explain the PV33 F537-I546dup10, F547L observed associations: (i) JAK2 mutations occur randomly on Bordeaux/France PV34 N542-E543del both haplotypes, but the GGCC haplotype holds certain features PV35 N542-E543del necessary to propagate MPN disease phenotype, or (ii) the PV36 K539L GGCC haplotype is more susceptible to somatic mutagenesis PV37 F537IK539L Salt Lake City/USA PV38 K539L and therefore the V617F mutation and exon 12 mutations of the PV39 N542-E543del JAK2 gene preferentially arise on this particular DNA sequence PV40 F537-K539delinsK variant. Several possible mechanisms have been suggested to PV41 I540S, R541-E543delinsK influence the disease susceptibility through haplotype variants. Florence/Italy PV42 F537-K539 Yet, ‘differential mutability’, as a hypothetical haplotype PV43 K539L property, has not been considered in investigations of cancer PV44 F547V susceptibilities so far. Our data suggest that a certain combina- Abbreviations: JAK2, janus kinase 2; PV, polycythemia vera.

Table 2 Association analysis of rs10974944 and JAK2 exon 12 mutation-positive PV

Model Allele/Genotype w2 P-value Odds ratio (95% CI)

Allelic C G 9.908 0.001646 1 2.10 (1.313–3.348) Trend C G 10.130 0.001456 1 2.10 (1.313–3.348) Dominant CC CG/GG 6.538 0.01056 1 2.32 (1.20–4.48) Recessive CC/CG GG 7.763 0.01494 1 3.68 (1.39–9.70) Genotypic CC CG GG 11.160 0.00378 1 1.96 (0.98–3.93) 5.09 (1.80–14.45) Abbreviations: CI, conﬁdence interval; JAK2, janus kinase 2; PV, polycythemia vera; Trend, Cochran–Armitage trend test. Association analysis was computed using the PLINK statistical program http://pngu.mgh.harvard.edu/purcell/plink and SNPStats statistical web-tool http://bioinfo.iconcologia.net/SNPStats.

Leukemia Letters to the Editor 1926 and outcome. Investigations on other oncogenic mutations in 8ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA; MPN patients, such as MPL mutations, and their association 9 with certain gene variants might reveal further insights into the Division of Hematology, University of Utah School of mechanism underlying the relationship between haplotype Medicine, Salt Lake City, Utah, USA and 10Department of Internal Medicine I, Division of Hematology variants and somatic mutability. Furthermore, earlier identiﬁed and Blood Coagulation, Medical University of Vienna, cancer susceptibility loci might represent genomic regions of Vienna, Austria divergent mutagenesis and thus, may carry somatic mutations of E-mail: [email protected] potential relevance for cancer pathogenesis.

Conﬂict of interest References

The authors declare no conﬂict of interest. 1 Campbell PJ, Green AR. The myeloproliferative disorders. N Engl J Med 2006; 355: 2452–2466. 2 Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR et al. JAK2 exon 12 mutations in polycythemia Acknowledgements vera and idiopathic erythrocytosis. N Engl J Med 2007; 356: 459– 468. The study was supported by funding from the Austrian Academy 3 Pardanani A, Fridley BL, Lasho TL, Gilliland DG, Tefferi A. Host of Sciences, Austrian Science Fund (FWF, P20033-B11) and the genetic variation contributes to phenotypic diversity in myelo- MPD Foundation. proliferative disorders. Blood 2008; 111: 2785–2789. 4 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C D Olcaydu1, RC Skoda2, R Looser2,SLi2, M Cazzola3, et al. A unique clonal JAK2 mutation leading to constitutive D Pietra3, F Passamonti3, E Lippert4, S Carillo5, F Girodon6, signalling causes polycythaemia vera. Nature 2005; 434: A Vannucchi7, NS Reading8, JT Prchal9,CAy10, I Pabinger10, 1144–1148. H Gisslinger10 and R Kralovics1,10 5 Wernig G, Mercher T, Okabe R, Levine RL, Lee BH, Gilliland DG. 1Center for Molecular Medicine of the Austrian Academy of Expression of Jak2V617F causes a polycythemia vera-like disease Sciences, Vienna, Austria; with associated myeloﬁbrosis in a murine bone marrow transplant 2Department of Biomedicine, University Hospital Basel, Basel, model. Blood 2006; 107: 4274–4281. Switzerland; 6 Olcaydu D, Harutyunyan A, Jager R, Berg T, Gisslinger B, Pabinger I 3 et al. A common JAK2 haplotype confers susceptibility to myelo- Division of Hematology, Fondazione IRCCS Policlinico San proliferative neoplasms. Nat Genet 2009; 41: 450–454. Matteo University of Pavia, Pavia, Italy; 7 Jones AV, Chase A, Silver RT, Oscier D, Zoi K, Wang YL et al. 4Laboratoire d’He´matologie, CHU, Bordeaux, France; 5 JAK2 haplotype is a major risk factor for the development Laboratoire de Cytologie Clinique, CHU CAREMEAU, Nimes, of myeloproliferative neoplasms. Nat Genet 2009; 41: France; 6 446–449. Laboratoire d’He´matologie, Hoˆpital du Bocage, CHU, Dijon, 8 Kilpivaara O, Mukherjee S, Schram AM, Wadleigh M, Mullally A, France; Ebert BL et al. A germline JAK2 SNP is associated with predisposition 7 Department of Hematology, University of Florence, Azienda to the development of JAK2(V617F)-positive myeloproliferative Ospedaliera-Universitaria Careggi, Florence, Italy; neoplasms. Nat Genet 2009; 41: 455–459.

The 1170 A–P single-nucleotide polymorphism (SNP) in the Her-2/neu protein (HER2) as a minor histocompatibility antigen (mHag)

Leukemia (2009) 23, 1926–1929; doi:10.1038/leu.2009.112; samples, we could show that two of them expressed HER2 published online 21 May 2009 (Figure 1a). HER2 comprise five non-synonymous SNPs, two of which have a frequency close to 0.5 (positions 1140 and 1170; An important mechanism of action of allogeneic hematopoietic http://www.genome.utah.edu/genesnps/). By allele-specific RT- cell transplantation (HCT) is the graft versus leukemia (GVL) PCR for the 1170 SNP of a panel of tumor cell lines, we detected effect, and in non-myeloablative conditioning transplantation quite frequent homozygous expression, supporting the 1/1 this immune-mediated effect is the only curative principle.1 It allelic distribution (Figure 1b). By using the SNEP database,7 has been clearly shown that T-cell responses are the main we identified a peptide from the 1170 A (GCC)-P (CCC) effector mechanisms of the GVL reactions,2 and in HLA- polymorphism, which potentially would bind to HLA-A2. By identical donor–recipient transplantations individual targets analyzing the actual binding of the peptides through the recognized by T cells in the graft have been characterized as assembly assay, we showed that the peptides did indeed bind proteins or protein fragments that are not expressed in the donor, to HLA-A2, although with low affinity (data not shown). We and thus represent neo-antigens to the transferred T cells. mHags used peptide-loaded dendritic cell (DC) from a homozygous A/A may be encoded on the Y chromosome (in sex-mismatched donor to stimulate autologous peripheral blood mononuclear HCT)3 or be derived from polymorphismsFthat is, single- cell (PBMC) for cloning and establishment of a cytotoxic nucleotide polymorphisms (SNP) that are disparate between T lymphocyte (CTL) clone specific for the HER2_P peptide as donor and recipient.4 The HER2 protein is a well-characterized described earlier.8 This clone (HER2_P#3) was used to show tumor-associated antigen (TAA), which is overexpressed in specific cytotoxic activity against the HER2_P peptide loaded to many solid cancers.5 Limited data are available regarding the T2 cells, and importantly, absence of recognition of the HER2_A expression in hematological malignancies;6 however, by peptide (Figure 2a). The HLA restriction of the killing was intracellular staining of 14 acute myeloid leukemia (AML) shown by blocking by the HLA class I specific antibody W6/32

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