DNA methylation profile in patients with indolent systemic mastocytosis

Abstract Background Mastocytosis is a clinically heterogeneous, usually acquired disease of the mast cells with a survival time that depends on the onset of the disease and ranges from skin‐limited to systemic disease, including indolent and more aggressive variants. The crucial element in pathogenesis is the presence of oncogenic KIT somatic mutation D816V. Further epigenetic alterations are responsible for regulating the expression of genes. It is essential to identify indicators of disease progression, and the specific clinical picture to establish an appropriate therapeutic strategy. Objective The aim of this study was to analyze the relation of mastocytosis symptoms and epigenetic changes, and to identify epigenetic predictors of the disease. Methods Global DNA methylation profile analysis was performed in peripheral blood collected from 73 patients with indolent systemic mastocytosis (ISM) and 43 healthy adult volunteers. Levels of 5‐methylcytosine (5‐mC) and 5‐hydroxymethylcytosine (5‐hmC) were determined using an ELISA‐based method, while the methylation of the Alu and LINE‐1 repeats were assayed with the quantitative methylation‐specific PCR technique. A questionnaire interview was conducted among the study participants to collect data on possible epigenetic modifiers. Additionally, the methylation profile was compared between three human mast cell lines: ROSA KIT D816V, ROSA KIT WT, and HMC‐1.1 KIT V560G, in order to assess the association between KIT mutations and methylation profile. Results A significantly lower level of DNA hydroxymethylation (5‐hmC) in the blood was found in patients with ISM as compared to the controls (0.022% vs. 0.042%, p = 0.0001). Differences in the markers of global DNA methylation (5‐mC, Alu, LINE‐1) were not statistically significant, although they did indicate generally higher DNA methylation in patients with mastocytosis. The 5‐hmC level was significantly associated with allergy (p = 0.011) in patients with ISM, showing a higher level of 5‐hmC in patients with allergy as compared to patients without allergy. The in vitro study revealed significant differences between the studied cell lines at the level of 5‐mC, Alu, and LINE‐1. Conclusions This study confirms that epigenetic changes are involved in mastocytosis, and suggests that allergy may be an important epigenetic modifier of the disease. A possible association between KIT mutations and methylation status observed in human mast cell lines requires further investigation in human studies. Clinical Implications Epigenetic alterations are involved in mastocytosis pathology. The possible role of allergy as an important epigenetic modifier suggests the more impaired function of mast cells in ISM patients without allergy. Capsule summary Decreased DNA demethylation in the blood DNA of patients with ISM confirms that epigenetic alterations are involved in mastocytosis pathology. We observed a possible role of allergy as an important epigenetic modifier. There is a possible association between KIT mutations and the methylation status observed in human mast cell lines.

higher level of 5-hmC in patients with allergy as compared to patients without allergy. The in vitro study revealed significant differences between the studied cell lines at the level of 5-mC, Alu, and LINE-1.

Conclusions:
This study confirms that epigenetic changes are involved in mastocytosis, and suggests that allergy may be an important epigenetic modifier of the disease. A possible association between KIT mutations and methylation status observed in human mast cell lines requires further investigation in human studies.
Clinical Implications: Epigenetic alterations are involved in mastocytosis pathology.
The possible role of allergy as an important epigenetic modifier suggests the more impaired function of mast cells in ISM patients without allergy.
Capsule summary: Decreased DNA demethylation in the blood DNA of patients with ISM confirms that epigenetic alterations are involved in mastocytosis pathology. We observed a possible role of allergy as an important epigenetic modifier.
There is a possible association between KIT mutations and the methylation status observed in human mast cell lines. The presence of oncogenic KIT mutation D816V is an important element in pathogenesis of mastocytosis as it has been detected in more than 80% of adult cases with SM. 3  specific microRNA expression, loss of suppressor gene function, activation of specific oncogenes (tyrosine kinases, signal transduction proteins), impaired DNA replication and repair processes, apoptosis, and by causing instability of the MC genome. 5-7 A number of apoptosis-associated genes, including FAS and several TSGs, including p16 and p21, were found to be hypermethylated in mast cell lines HMC-1 but not in normal bone marrow cells. Moreover, p16 and FAS were found to be hypermethylated in both HMC-1 subclones, whereas p21 was found to be hypermethylated only in HMC-1.2 cells harboring KIT D816V, but not in HMC-1.1 cells lacking KIT D816V. 8 Aberrant expression of micro RNAs is detected in mast cells with a KIT mutation. It has been indicated that cells with KIT mutations have lower expression of miR −539 and miR −381. 6 These miRNAs are involved in the inhibition of microphthalmia-associated transcription factor (MITF) expression which regulates the development of MC and melanocyte, and the synthesis of tryptase and melanin. [5][6][7] Epigenetic alterations are responsible for regulating the expression of genes that do not modify the DNA sequence. The bestknown epigenetic marker is methylation of DNA which predominantly occurs in cytosines that precede guanines; these are called dinucleotide CpGs and are one of the epigenetic modifications frequently observed in a variety of biological and pathological processes. 9 DNA methyltransferases (DNMTs) are involved in the process of DNA methylation by catalyzing the transfer of a methyl group to the 5-position of the cytosine in DNA and the generation of 5methylcytosine (5-mC). 10 In the process of DNA demethylation, 5-mC can be converted into 5-hydroxymethylcytosine (5-hmC) by TET hydroxylases, which play a key role in the active demethylation of DNA. Therefore, global DNA hypomethylation leads to chromosomal instability which may result in cell proliferation and cancer. 11 There are known non-LTR retrotransposons-including long interspersed elements-LINE-1 and short interspersed elements-Aluwhich affect the human genome in many different ways: generating insertion mutations, genomic instability, alterations in gene expression and also contributing to genetic innovation. 12  KIT gene was performed using the qPCR in bone marrow aspirate. 16 Our local registry is part of the European Competence Network on Mastocytosis (ECNM) Registry which was described previously. 17 The control group included 43 healthy adult volunteers who were not diagnosed with any chronic diseases including allergy. Peripheral blood samples were collected from patients with ISM during diagnosis or during the follow-up visit in the Allergology Department, or from healthy volunteers recruited in the University of Lodz. Informed consent was obtained from all study participants. The database of the ECNM registry, data storage, and data distribution comply with the rules and regulations of data protection laws, with local ethics committee regulations of each participating center, and with the declaration of Helsinki. 17 The enrolment to the study was performed between January and December 2019.

| Statistical analysis
Data were analyzed using the Statistica 13.0 software (Stat-Soft Inc).
Group differences were assessed with the Mann-Whitney U test or the Kruskal-Wallis test followed by the post-hoc Dunn's test.
Spearman's rank correlation coefficients were used to describe the correlations between the variables. p values below 0.05 were considered to be statistically significant.

| RESULTS
The study group characteristics are presented in Table 1 Figures 1 and 2), although they did indicate generally higher DNA methylation in patients with mastocytosis.
In the next stage in our research, we analyzed the possible association of methylation/demethylation markers with individual factors in both study groups, as presented in Table 2.
Age, sex, and BMI were not correlated with any of the analyzed markers of DNA methylation or demethylation, apart from significant correlation between Alu and BMI in the control group (r = 0.376, A significant effect of allergy on the 5-mC and 5-hmC levels in the patients with mastocytosis was observed. The trend of lower levels of 5-mC (of borderline significance, p = 0.057) and significantly higher levels of 5-hmC (p = 0.011) were observed in patients with allergic symptoms compared to patients without allergy ( Figure S1).
No significant differences were found at the level of DNA methylation or demethylation with respect to the KIT D816V mutation; however, comparison was limited by the small sample size of the group without mutation (nine subjects). Nevertheless, the trend of a higher level of DNA methylation in KIT D816V carriers was noted.
A statistically significant and positive correlation between 5-mC and 5-hmC (r = 0.315, p = 0.0067, Figure 2B) was found in the patients with ISM. In the control group, the correlation between 5-mC

| DISCUSSION
In this study, we found a significantly lower 5-hmC content in the blood DNA of patients with mastocytosis compared to healthy controls. We also found lower but not significant levels of 5-mC, and of DNA methylation, and may be observed in many diseases, especially in hematologic malignancies. 10,25 The interrelation of 5-mc with 5-hmC was already described in the 90s, and demonstrated in in vitro studies in normal DNA as a response to oxidative stress. 29 5-hmC levels are decreased in various malignancies, including glioblastoma, melanoma, breast, prostate, hepatic, gastric, and renal cancers. 30 Moreover, the loss of 5-hmC or decreased levels of 5-hmC are associated with poorer outcomes in melanoma, and the progressive loss of 5-hmC was revealed in the epigenome from begin nevus to malignant melanoma. 31 Thus, decreased levels of 5-hmC are assumed to be an epigenetic hallmark of melanoma. 31 A low 5-hmC level is also associated with poor prognosis in renal cell carcinoma, gastric, hepatic and ovarian cancers. 26,30,[32][33][34] The significant association between low 5-hmC levels and lymph node metastasis and an advanced stage in TNM classification in various cancers has been revealed in a meta-analysis by Chen et al. 30 Promoter hypermethylation and genomic hypomethylation have frequently been described in hematologic malignancies. 35 We investigated levels of DNA methylation and hydroxymethylation specifically in patients with ISM and found a significantly lower 5-hmC content in patients with mastocytosis compared to healthy controls.
These results are consistent with results previously described by Leoni,26 where overall 5-hmC levels were reduced in all patients with SM. It is also noticeable in that study that the reduction in 5-hmC correlated with the presence of highly detectable levels of KIT D816V mutation. These results may suggest that the initial change in the DNA epigenome correlates with the appearance of the KIT D816V mutation, which may be followed by further genetic and epigenetic alterations that may account for disease variants and progression. 26 In ISM, gene expression profiles were found to be different between patients with a history of IVA and those without. 36 Gene ontology analysis revealed that the differentially expressed genes were involved in several pathways that regulate the balance between proliferation versus terminal differentiation: Wnt signaling pathway, focal and cell adhesion, calcium signaling, extracellular matrix interactions, pathways in cancer, and MAPK signaling. This may suggest that the pattern of gene expression related to neoplastic differentiation is more prevalent in patients without a history of allergy. 36 Therefore, based on this study, it can be assumed that patients without a history of allergy have more pronounced mast cell dysfunction. This is consistent with our study, where we found that patients with ISM without allergies have decreased levels of demethylation markers, and therefore we can assume that the function of their mast cells is more impaired.
It is well-known that atopic diseases develop in genetically predisposed individuals. A dramatic increase in the prevalence of allergy has been noticed over the past few decades. The prevalence of ISM in the adult population is at least 13 cases per 100,000 inhabitants, which was described in the Netherlands. The actual prevalence might be even higher as the diagnosis of ISM can be easily overlooked due to subtle symptoms and a lack of awareness of the disease. 37 The increase in the incidence of allergic disorders may be explained by exposure to negative (risk exacerbating) environmental modifiers, and at participants' current homes significantly increased methylation at the same site. 44 It is well known that DNA hypermethylation in the 5 0 region of FOXP3 is associated with higher exposure of diesel exhaust exposure, and furthermore children with DNA hypermethylation in FOXP3 are assumed to be at higher risk of asthma, persistent wheezing, and early transient wheezing. 45 Other studies showed the correlation of fine particle air pollutant exposure with DNA methylation of several CpG sites in nitric oxide synthase genes in children, 46 and a relationship between indoor exposure to NO 2 and severe asthma in children with high ADRB2 methylation levels. 47 We assessed the influence of clinical parameters in patients with mastocytosis on their DNA methylation profile. We did not find a correlation between age, sex, or BMI with any of the analyzed markers of DNA methylation or demethylation in patients with mastocytosis, but we revealed a significant correlation between Alu and BMI in the control group. We also found lower but not significant levels of 5-mC, and simultaneously significantly higher levels of 5-hmC, in patients with allergic symptoms compared to patients without allergies.
Moreover, we analyzed the correlation between the 5-mC methylation marker and 5-hmC demethylation in the study group. As a result, a positive correlation was confirmed in the control (although not significant), but in the group of patients, there was also a positive and significant correlation. Importantly, the group of patients was not homogeneous in this respect; that is, a group of patients with high 5-mC values, and one with low 5-mC values was clearly visible.
Therefore, an additional analysis was performed grouping the patients in terms of the presence of allergy, where a significant relationship was maintained only in the patients without allergy (p = 0.011).
This phenomenon may indicate an imbalance between DNA methylation and demethylation processes in patients with mastocytosis and allergy, and point toward the existence of epigenetic alterations which precede or arise from mastocytosis.
The results of our study have a few limitations including the statistical differences in the age, gender, and BMI of the controls and mastocytosis patients. However, both populations were similar in terms of age and BMI, with a similar range, and clinical conditions with no additional diseases. We plan to replicate the results in a further study in a larger, independent population.
In conclusion, this study demonstrated decreased DNA demethylation in the blood DNA of patients with ISM. The presented data may suggest a role or involvement of DNA methylation in the pathology of mastocytosis but these results should be confirmed in a larger, independent population. Moreover, the observed possible role of allergy as an important epigenetic modifier suggests that the function of mast cells in ISM patients without allergy symptoms may be more impaired due to epigenetic changes, which requires further studies. The possible association between KIT mutations and methylation status that was observed in human mast cell lines requires further investigation in human studies.