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- Design and methods
Background: Mastocytosis is a heterogenous disease involving mast cells (MC) and their progenitors. Cutaneous and systemic variants of the disease have been reported. In contrast to cutaneous mastocytosis (CM), patients with systemic mastocytosis (SM) are at risk to develop disease progression or a nonMC-lineage haematopoietic neoplasm. Little is known, however, about factors predisposing for the development of SM. One factor may be cytokine regulation of MC progenitors.
Methods: We examined the role of the interleukin-13 (IL-13) promoter gene polymorphism -1112C/T, known to be associated with increased transcription, in mastocytosis using allele-specific polymerase chain reaction method. Serum tryptase and IL-13 levels were determined by immunoassay, and expression of the IL-13 receptor in neoplastic MC by reverse transcription-polymerase chain reaction and flow cytometry.
Results: The frequency of the -1112T allele of the IL-13 promoter was significantly higher in patients with SM compared with CM (P < 0.008) and in mastocytosis patients compared with healthy controls (P < 0.0001). Correspondingly, the polymorphism was found to correlate with an elevated serum tryptase level (P = 0.004) and with adult-onset of the disease (P < 0.0015), both of which are almost invariably associated with SM. Serum IL-13 levels were also higher in SM patients compared with CM (P = 0.011), and higher in CT- than in CC carriers (P < 0.05). Finally, we were able to show that neoplastic human MC display IL-13 receptors and grow better in IL-13-containing medium.
Conclusions: The -1112C/T IL-13 gene polymorphism and the resulting ‘hypertranscription’ may predispose for the development of SM.
Mastocytosis is a myeloid stem cell disease characterized by a pathologic increase in mast cells (MC) in various organs, including the skin, bone marrow, liver, spleen or/and lymph nodes (1–4). The clinical presentation and course of mastocytosis are variable, ranging from pure cutaneous involvement, termed cutaneous mastocytosis (CM), to different forms of systemic mastocytosis (SM), and in rare cases, mast cell leukemia (MCL). According to the WHO classification, the following variants of SM have been described: indolent systemic mastocytosis (ISM), mastocytosis with an associated clonal haematologic nonMC-lineage disease (SM-AHNMD), aggressive systemic mastocytosis (ASM) and MCL (5, 6).
Several mechanisms contributing to MC growth and survival are considered to be involved in the pathogenesis of mastocytosis. A number of previous and more recent data suggest that stem cell factor (SCF) and its transmembrane tyrosine kinase receptor, KIT, play a key role in abnormal growth and survival of MC in SM (5, 6). In particular, it has been described that point mutations in the KIT gene are frequently detected in these patients (5–11). The most commonly detected mutation is KIT D816V, which is found in more than 80% of all SM cases (5–11). This KIT mutation is considered to lead to ligand-independent (auto)phosphorylation of KIT and thus to uncontrolled growth of MC (5, 6, 11, 12). However, although KIT D816V is a well recognized ‘pro-oncogenic hit’ in SM and considered critical for survival and differentiation of neoplastic MC, several lines of evidence suggest that the mutation per se is neither sufficient to induce malignant proliferation of MC nor to even cause SM. Rather, the mutant is also detectable in cutaneous MC in CM (9, 13, 14) as well as in bone marrow MC in ISM (5–11), a disease-variant with a completely stable clinical course and no signs of MC proliferation or disease progression, even when recording these patients over decades (5, 6). Based on these observations, it has been hypothesized that additional factors, apart from KIT mutations and SCF, may be responsible for disease evolution and disease progression in SM.
A number of previous data suggest that growth of MC is not only regulated by SCF (and KIT), but also by other cytokines. These cytokines include IL-4, IL-6, IL-10 and IL-13 (15–20). One exciting new hypothesis is that distinct polymorphisms in cytokine genes or cytokine receptor genes are associated with the systemic, indolent or aggressive variants of SM. Likewise, Daley and colleagues described that the gain of function Q576R-polymorphism of the IL-4 receptor gene is more frequently detectable in patients with CM than in SM (21).
The IL-13 gene has been mapped to the cytokine cluster on chromosome 5q31-33 (22). Recently, several different single nucleotide polymorphisms (SNPs) in the IL-13 promoter region have been described (23–27). The best described SNP, namely the transition of cytosine (allele C) to thymine (allele T) at the -1112 site in the promoter region, leads to a change in the binding rate of nuclear proteins to this region and to overproduction of IL-13 in Th2 lymphocytes, which may play a role in allergic and chronic inflammatory diseases (23–27). Indeed, IL-13 gene polymorphisms have been associated with inflammatory and atopic disorders (23–27). One potential target cell bearing receptors for IL-13 in inflamed tissues are MC, which may grow better when exposed to this cytokine (20). Therefore, it may also be of interest to learn whether the IL-13 gene exhibits distinct polymorphisms in disorders associated with an enhanced growth and survival of MC, such as mastocytosis. However, no data on the frequency and role of IL-13 gene polymorphisms in mastocytosis have been presented so far.
The aims of the present study were to analyze the frequency of the IL-13 gene polymorphism at position -1112 in patients with mastocytosis, and to define whether the polymorphism is associated with a distinct variant of the disease.
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- Design and methods
Several different factors may contribute to the pathogenesis of mastocytosis. Recent data suggest that the genetic background may also play a potential role in disease manifestation and evolution (21, 35, 36). However, so far, little is known about involved genes and their exact role in mastocytosis. The results of our study show that the systemic variant of mastocytosis is associated with the -1112T polymorphism (genotypes CT + TT) of the IL-13 gene, known to lead to a high transcription rate. In particular, this polymorphism was detected at higher frequency in SM than in CM or in healthy controls. In addition, patients with SM were found to have higher serum IL-13 levels compared with CM patients or controls, suggesting that the polymorphism is of functional significance. Finally, we provide evidence that neoplastic MC display the IL-13 R and grow better in IL-13-containing medium. All in all, these data strongly suggest that the IL-13 promoter polymorphism -1112T may predispose for the development of SM.
The notion that the IL-13 promoter polymorphism -1112C/T is associated with systemic disease manifestation was supported by several different observations. First, the polymorphism was detected with high frequency in adult-onset mastocytosis, usually resembling SM, but less frequently in childhood mastocytosis, where CM is the predominant subtype (5, 6). Moreover, we found a relationship between the polymorphism and the serum tryptase level. In fact, the frequency of the polymorphism was high in patients with elevated serum tryptase, indicating the presence of SM (5, 6), and low in those with normal tryptase (usually in the CM group).
A number of different cytokines have been described to regulate the growth of normal or/and neoplastic MC (15–20). One factor regulating the growth and function of normal MC is IL-13 (20, 37, 38). Based on our data, it is tempting to speculate that IL-13 acts on (pre)neoplastic MC or/and their progenitors to stimulate their growth and survival in patients with SM, which would be an explanation for the SM-predisposing function of the polymorphism. However, this hypothesis must be discussed in light of the KIT mutation D816V that has been discussed as a major pathogenetic factor in SM. One possible scenario would be that KIT D816V-positive clones can only grow to overt SM when the respective (pre/neoplastic) MC progenitors are exposed to high levels of IL-13. An alternative possibility would be that the polymorphism predisposes for the occurrence of KIT mutations in MC progenitors. Finally, the IL-13 polymorphism may be linked to other (unknown) defects that are responsible for the higher susceptibility to develop an MC proliferative disorder.
An interesting observation was that the -1112T polymorphism C/T of the IL-13 promoter was also detected in the two patients with MMAS, a condition that is associated with a low burden of MC and the presence of only two minor SM criteria [subdiagnostic as SM requires three minor SM criteria (5, 6)]. This observation suggests that the presence of the polymorphism is not sufficient for the full manifestation of SM in all patients, and that additional factors (hits) may be required to lead to SM in these cases. Indeed, the polymorphism C/T of the IL-13 promoter may not be the only predisposing factor for SM-development (21). Rather, it may well be that multiple hits and predisposing (genetic) factors are necessary for full manifestation of SM.
A major question in this study was how the IL-13 gene polymorphism could contribute to the development of SM. To address this question, we measured serum IL-13 levels in SM patients and examined whether neoplastic MC display IL-13 R and can grow in response to IL-13. Previous studies have already shown that normal MC can express IL-13 R (20). In our experiments, we found that HMC-1 cells, a cell line derived from a patient with MCL, express the IL-13 R as well as the -1112T polymorphism C/T. Both the KIT D816V-positive and the D816V-negative subclone of HMC-1 were found to carry the IL-13 R, suggesting that receptor-expression on MC is independent of the presence of the KIT mutant D816V. An interesting observation was that HMC-1 cells grow significantly better in IL-13-containing medium, which supports the hypothesis that IL-13 is an important factor in SM. Finally, we found that patients with SM indeed exhibit elevated serum IL-13 levels. All these data suggest that IL-13 hypertranscription and elevated IL-13 levels, caused by the polymorphism, may contribute to enhanced MC growth and thus evolution of the disease to SM.
The question why HMC-1 only showed a slight (albeit significant) growth response to IL-13 may have several explanations. One explanation would be that HMC-1 cells produce and secrete IL-13 and utilize IL-13 as autocrine growth regulator, so that the effect of additionally added exogenous IL-13 must be expected to be marginal if at all measurable. A second (additional) possibility would be that the FCS used contained IL-13. Finally, HMC-1 cell growth may be regulated by many different (autocrine) factors, so that the effect of a single cytokine may not be substantial (39).
As mentioned above, it would be of interest to know whether neoplastic MC in SM produce and secrete IL-13 and whether these cells may utilize IL-13 as a potential autocrine growth regulator. This scenario seems likely as normal MC reportedly can express both IL-13 and IL-13 R (20, 37, 38). Whether indeed neoplastic MC express and release IL-13, and can use this cytokine as an autocrine growth regulator is presently under investigation. Based on our data, one could expect that such autocrine regulation is substantially amplified in SM patients through the effect of the gene polymorphism, contrasting the situation in patients with CM. This would then explain the higher burden of MC in SM. An interesting result was the difference in the frequency of the 1112T allele between children and adults with mastocytosis and between children with elevated and normal serum tryptase levels. The children with CM and elevated tryptase levels, in whom the IL-13 polymorphism was found, may have suffered from undiscovered (not proved) SM. In fact, in children, a bone marrow biopsy is not considered as standard as many of these patients enter remission before or during puberty (5, 6). Whether our paediatric patients with the IL-13 polymorphism indeed suffered from SM remains unknown. Thus, the hypothesis that these patients may develop persistent disease and thus SM needs further confirmation. It also remains unknown whether these patients display the KIT mutation D816V. In fact, patients with SM usually display the KIT mutation D816V, whereas many patients with CM lack this mutation. Still, however, some patients with CM present with KIT D816V-positive MC in their skin lesions. In the light of our data, an attractive hypothesis would be that only those CM patients with KIT D816V in whom the IL-13 polymorphism -1112T is present, will develop (evolve to) SM. This may be of clinical importance and may lead to a predictive model that can assist in the estimation of risk to have or to develop SM.
So far, only one study was performed on the role of gene polymorphism in mastocytosis. In particular, Daley et al. analyzed the Q576R polymorphism of the common alpha chain of the IL-4 and IL-13 R (21). In their study, no significant difference in the frequency of the mutation was found when comparing between mastocytosis patients and controls. However, it appeared that the 576R allele of the IL-4Rα of IL-13/IL-4 common receptor is more frequently detected in patients with mastocytosis limited to the skin, and associated with lower tryptase levels and lower soluble KIT receptor serum levels. The authors suggested that the allele 576R may play a protective role in mastocytosis and may predict a better prognosis. The 1112T allele of IL-13 (related to a higher transcription rate) may have an opposite role. In fact, these carriers appear to have an increased risk for the development of a systemic mast cell disease.
In summary, our data show that the -1112C/T polymorphism of the IL-13 promoter is associated with the systemic variant of mastocytosis. This observation may enhance our knowledge concerning the pathophysiology of the disease and may have clinical implications for diagnosis and therapy.