Clinical relevance of murine double minute 2 single nucleotide polymorphisms 309 in familial myeloproliferative neoplasm
Authors: Elisa Rumi, Ilaria Casetti, Daniela Pietra, Chiara Elena, Ilaria Ambaglio, Cristiana Pascutto, Francesco Passamonti, Mario Cazzola
Journal: American Journal of Hematology (2011)

The genetic variants predisposing to familial myeloproliferative neoplasms (MPN) are still to be defined. Germline TP53 mutations are involved in genetic predisposition to cancer. Inactivation of the p53 pathway may occur by up regulation of the MDM2 protein. A single nucleotide polymorphisms (SNP 309 T/G) in the MDM2 promoter was shown to increase the affinity of the transcriptional factor Sp1, resulting in higher levels of MDM2 RNA and protein and subsequent attenuation of the p53 pathway. We investigated the distribution of MDM2 SNP 309 in 71 familial MPN to evaluate its influence on disease susceptibility and age of disease onset, the association with the JAK2 (V617F) mutation and the impact on disease progression. The distribution of the high risk MDM2 SNP 309 (G allele) did not differ between patients with familial MPN and healthy relatives belonging to the same pedigree or to a control population. MDM2 SNP 309 did not affect age of MPN onset and was not associated with the JAK2 (V617F) mutation. Disease progression-free survival did not significantly differ between patients carrying the MDM2 SNP 309 G allele and those carrying the MDM2 SNP 309 T allele. MDM2 SNP 309 shows no clinical relevance in familial MPN. Myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are chronic myeloid disorders characterized by clonal expansion of hematopoietic stem cells, over-production of mature blood elements, and a variable risk of progression to acute myeloid leukemia [ 1]. Although MPN have in most instances a sporadic occurrence, familial clustering has been previously reported [ 2, 3]. Common mutations involved in the pathogenesis of MPN such as JAK2, MPL, and TET2 mutations arenot inherited, but somatically acquired also in familial cases [4]. Genetic variants contributing to MPN susceptibility are still to be defined. The p53 pathway is one of the best-characterized tumor suppressor pathway involved in hereditary cancer predisposition. TP53 is activated upon cellular stresses and initiates a transcriptional program leading to cell cycle arrest, cellular senescence, and apoptosis, thus ensuring the fidelity of DNA duplication process [ 5]. Germline TP53 mutations are involved in genetic predisposition to cancer [6, 7]. Inactivation of the p53 pathway may also occur by up regulation of the human homologue of the Murine Double Minute 2 protein (MDM2). The MDM2 protein acts as a direct negative regulator of p53 by inhibiting its activity and promoting its degradation [5]. Overexpression of MDM2 in mice favors tumorigenesis [8], while reduction of MDM2 levels in mice enhances tumor resistance [9]. A single nucleotide polymorphisms (SNP 309 T/G, rs2279744) in the MDM2 promoter region was identified and was shown to increase the affinity of the transcriptional factor Sp1, resulting in higher levels of MDM2 RNA and protein and the subsequent attenuation of the p53 pathway [10]. In humans, the G variant of MDM2 SNP309 is associated with accelerated tumor formation in both hereditary and sporadic cancers [5, 11]. The effects of MDM2 SNP309 G allele in hematological disorders are not well clarified, with some studies reporting an association between SNP309 G allele and the risk of myeloid or lymphoid disorders [12, 13] and other studies excluding its clinical relevance [14, 15]. In this study, we investigated the distribution of MDM2 SNP 309 in familial MPN to evaluate its influence on disease susceptibility and age of disease onset, the association with the JAK2 (V617F) mutation and the impact on disease progression. We identified in our database 71 consecutive MPN families with two or more affected individuals evaluated at the Division of Hematology, Fondazione IRCCS Policilinico S. Matteo, Pavia, Italy from 1981 to 2010. In 25 of these 71 families, we had the possibility to evaluate also one or more healthy relatives belonging to the same pedigree. As a whole, DNA for MDM2 SNP309 analysis was available in 116 MPN patients and 84 healthy relatives. The study was approved by the Local Ethics Committee. The procedures followed were in accordance with the Declaration of Helsinki of 1975, as revised in 2000, and samples were obtained after patients provided written informed consent. We assumed heterozygosity and homozygosity for the G variant of MDM2 SNP 309 as “high risk condition” because associated with a major inhibition of the p53 pathway, while homozygosity for the T variant was considered as “low risk condition”. To test whether the MDM2 SNP309 G contributes to familial occurrence of MPN, two different statistical analyses were carried out. First, we analyzed the subgroup of 25 pedigrees for which both patients and healthy subjects within the same pedigree were available and we compared the distribution of the high-risk SNP309 G variant between patients and healthy relatives. Among 45 patients, 28 (62.2%) carried the high-risk G allele, whereas 40 of 84 healthy relatives (47.6%) displayed the G variant. Although the high risk G variant seemed to be more represented in patients than in healthy relatives (62.2 vs. 47.6%) the association was not statistically significant (Fisher's exact test P = 0.14). Next, the same analysis was conducted considering all familial patients from the 71 families and a control population (n = 151) consisting of subjects with normal hemogram or a hematological reactive condition. The high-risk G variant was observed in 74 of 116 (63.8%) familial MPN patients and in 99 of 151 (65.5%) reactive conditions. The distribution of the high risk MDM2 SNP 309 did not differ between patients with familial MPN and the control population (Fisher's exact test P = 0.79). Taken together these results exclude an influence of MDM2 SNP 309 on susceptibility to familial MPN. Disease anticipation has been reported in familial MPN with a significantly younger age at diagnosis for patients in the second generation than those in the first generation [ 3]. As the MDM2 SNP 309 G allele has been shown to impact on the age of tumor onset in patients with Li-Fraumeni syndrome carrying germline p53 mutation [11], we evaluated whether the G variant might contribute to the disease anticipation observed in familial MPN. To test this hypothesis, we considered the 36 families with two successive generations of affected individuals. The median age at diagnosis of familial MPN was significantly higher in first-generation patients (median 66 years, range 43–85) than in second-generation patients (median 36 years; range 16–57; P < 0.001), as previously reported. However, Mann-Whitney test demonstrated that the median age at MPN onset did not significantly differ between patients carrying the high risk MDM2 SNP 309 G allele and those carrying the low risk SNP 309 T allele, both in the first generation (median 67 years vs. 63 years; P = 0.167) and in the second generation (median 33 vs. 39 years; P = 0.509). When repeating the analysis by studying parent-offspring pairs in the 27 families for whom DNA was available in both generations, disease anticipation was confirmed (P < 0.001) but it was not influenced by the high risk MDM2 SNP 309 (analysis of variance for repeated measurements P = 0.696). These results show that MDM2 SNP 309 does not affect age of onset in familial MPN. A recent report indicated that the JAK2 (V617F) mutation affects p53 response to DNA damage through accumulation of MDM2 protein via enhanced translation [ 16]. To check whether JAK2 (V617F) mutation and MDM2 SNP 309 might synergize in favoring familial MPN onset; we compared the JAK2 mutation rate in patients with high risk and low risk SNP 309. We did not find a higher JAK2 (V617F) mutation rate in patients carrying the MDM2 SNP 309 G allele than in those carrying the MDM2 SNP 309 T allele (71.6 vs. 71.4%, Fisher's exact test P = 1). So, the JAK2 (V617F) mutation is not associated with the MDM2 G variant in familial MPN. Finally, to test whether the MDM2 polymorphisms affecting p53 activity might affect disease progression, we analyzed the occurrence of myelofibrotic or leukemic evolution in patients with high risk and low risk SNP309. Disease progression-free survival did not significantly differ between patients carrying the MDM2 SNP 309 G allele and those carrying the MDM2 SNP 309 T allele (P = 0.116). In conclusion, we analyzed MDM2 SNP 309 in a large cohort of patients with familial MPN and did not find an effect of the polymorphism on disease susceptibility, age at disease onset, disease progression, and association with JAK2 (V617F) mutation. Germline genetic variants other than MDM2 polymorphisms might be involved in the genetic predisposition to familial MPN. Genomic T-lymphocyte DNA was genotyped for MDM2 SNP 306 using the primers and TaqMan probes reported by Onel et al. [ 17]. The allelic discrimination assay was performed on the RotorGene 6000 real-time PCR instrument (Qiagen, Hilden, Germany) in a final volume of 10 μL according to the manufacture's procedure. Granulocyte JAK2 (V617F) mutation burden was assessed using a quantitative real-time polymerase chain reaction-based allelic discrimination assay, as previously reported [ 18]. Descriptive statistics are reported as median and range in the case of continuous variables, while categorical variables are summarized by count and relative frequency (%) in each category. Univariate comparison of continuous variables between groups was performed with nonparametric methods (Wilcoxon/Mann Whitney test for independent groups, and Wilcoxon test for paired samples in the case of repeated measures). Multivariate analysis of disease anticipation was carried out by analysis of variance (ANOVA) for repeated measures. Distribution of categorical variables between groups was tested using the two-tailed Fisher exact test. Progression-free survival was defined as the time between diagnosis and leukemic/myelofibrotic evolution (or last follow-up for patients who did not progress) and was estimated with the Kaplan-Meier method. The Wilcoxon test was applied to the comparison of survival curves. Analyses were performed using Stata SE 11.2 (StataCorp LP, College Station, TX, http://www.stata.com) and Statistica 8 (StatSoft, Inc.-2007, www.statsoft.com) software. Elisa Rumi*, Ilaria Casetti*, Daniela Pietra*, Chiara Elena*, Ilaria Ambaglio*, Cristiana Pascutto*, Francesco Passamonti , Mario Cazzola*, * Department of Hematology Oncology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Pavia, Italy, Division of Hematology, Department Internal Medicine, A.O. Ospedale di Circolo e Fondazione Macchi, Varese, Italy.