Aberrant DNA methylation of PTPRG as one possible mechanism of its under‐expression in CML patients in the State of Qatar

Abstract Background Several studies showed that aberrant DNA methylation is involved in leukemia and cancer pathogenesis. Protein tyrosine phosphatase receptor gamma (PTPRG) expression is a natural inhibitory mechanism that is downregulated in chronic myeloid leukemia (CML) disease. The mechanism behind its downregulation has not been fully elucidated yet. Aim This study aimed to investigate the CpG methylation status at the PTPRG locus in CML patients. Methods Peripheral blood samples from CML patients at time of diagnosis [no tyrosine kinase inhibitors (TKIs)] (n = 13), failure to (TKIs) treatment (n = 13) and healthy controls (n = 6) were collected. DNA was extracted and treated with bisulfite treatment, followed by PCR, sequencing of 25 CpG sites in the promoter region and 26 CpG sites in intron‐1 region of PTPRG. The bisulfite sequencing technique was employed as a high‐resolution method. Results CML groups (new diagnosed and failed treatment) showed significantly higher methylation levels in the promoter and intron‐1 regions of PTPRG compared to the healthy group. There were also significant differences in methylation levels of CpG sites in the promoter and intron‐1 regions amongst the groups. Conclusion Aberrant methylation of PTPRG is potentially one of the possible mechanisms of PTPRG downregulation detected in CML.

Epigenetic silencing is a phenomenon whereby gene transcription is suppressed through DNA methylation (a process that may regulate gene function) resulting in decreased protein expression. Several studies have suggested that hypermethylation might play a role in disease progression in CML. Hypermethylation of several genes was associated with the progression of CML, its pathogenesis and response to treatment (Heller et al., 2016;Jelinek et al., 2011;Machova Polakova, 2013;Toyota et al., 2001;Wang et al., 2009Wang et al., , 2010. Protein tyrosine phosphatase (PTP) superfamily of enzymes represents a natural regulatory mechanism of the tyrosine kinase family. They have the ability to remove phosphate groups from phosphorylated tyrosine residues leading to an equilibrium status in normal populations. Based on their cellular localization, PTPs are classified as receptor-like and nonreceptor. Even though receptor-type protein tyrosine phosphatases (PTPRs) share similar basic structure, distinct PTPRs have specific targets and, may thus, play alternative roles in cell regulation (Du & Grandis, 2015;Jiang, den Hertog, & Hunter, 2000;Tonks, 2006). Of these, protein tyrosine phosphatase receptor gamma (PTPRG) was described as a tumor suppressor gene in several tumors and its expression level was found to be significantly downregulated in CML patients and correlates with its promoter methylation in both patients and cell lines (Della Peruta, 2010;Tomasello et al., 2020). Moreover, PTPRG expression is restored in patients who respond optimally to TKIs treatment, and its expression remains low in patients who fail treatment (Vezzalini et al., 2017). More recently, we identified a single healthy controls (n = 6) were collected. DNA was extracted and treated with bisulfite treatment, followed by PCR, sequencing of 25 CpG sites in the promoter region and 26 CpG sites in intron-1 region of PTPRG. The bisulfite sequencing technique was employed as a high-resolution method. Results: CML groups (new diagnosed and failed treatment) showed significantly higher methylation levels in the promoter and intron-1 regions of PTPRG compared to the healthy group. There were also significant differences in methylation levels of | 3 of 11 ISMAIL et AL.
Nucleotide Polymorphism (SNPs) (rs62620047) in PTPRG (Y92H) in patients who failed Imatinib Mesylate (IM) treatment . While the molecular and flow cytometry characteristics of PTPRG were studied (Vezzalini et al., 2017), the contributing epigenetic mechanisms that influence the PTPRG activity in CML patients remains unclear and warrants further investigation.
The aim of this study is to investigate the methylation patterns of PTPRG gene in a cohort of CML patients in Qatar where resistance to IM treatment has been reported to be significantly higher than other parts of the world (Al-Dewik, Jewell, Yassin, El-Ayoubi, & Morsi, 2014).

| Patient recruitment, characteristics, and sample collection
Informed consent was obtained from all participants. The study was approved by both Ministry of Public Health and institutional review board of Hamad Medical Corporation (Project No. 11118/11). This study adhered to the World Medical Association's Declaration of Helsinki (1964Helsinki ( -2008 and its amendments for Ethical Human Research including confidentiality, privacy, and data management. A total of 26 adult CML patients and 6 matched healthy controls that were confirmed to have normal complete blood count (CBC) and negative for BCR-ABL1 translocation were included in this study.
The peripheral blood samples were collected in EDTA tubes for newly diagnosed patients before starting TKIs and at time of failure for patients who relapsed or failed treatment according to European Leukemia Net (ELN) guidelines 2013. The CML patients' response to TKIs treatment was assessed based on the hematologic, cytogenetic, and molecular response results according to (ELN 2013) (Baccarani et al., 2013;Steegmann et al., 2016). Resistance to treatment was defined as showing lack of one of the following; hematological response and/or Ph + >95% by 3 months, BCR-ABL1 >10% and/or Ph + >35% by 6 months, BCR-ABL1 >1% and/ or Ph + >0 by 12 months after the start of treatment.

| DNA isolation and bisulfite conversion
Total DNA was isolated from total leucocytes with Maxwell ® 16 DNA Purification Kits as per manufacture guidelines (Khokhar, Mitui, Leos, Rogers, & Park, 2011). Purity of extracted DNA was assessed by a Nano Drop spectrophotometer 2000 (Thermofisher Scientific), a ratio of 1.8-2.4 was considered acceptable and for optimal results, an absolute quantity of 200-500 ng of DNA was used.
The DNA samples were then treated with sodium bisulfite according to the manufacturer's instructions (EpiTect Bisulfite Kit, QIAGEN). The bisulfite treatment catalyzes the deamination of all the unmethylated cytosine (uC), nucleotides to uracil (U), or thymidine (T) nucleotides and leaves the methylated cytosine (mC) unchanged. For optimal results, the amount of starting DNA in the bisulfite modification process was kept at 200-500 ng.

PCR (BSP), and Gradient Polymerase Chain reaction
The University of California, Santa Cruz (UCSC) Genome Browser (UCSC, 2013) was utilized to identify the possible "CpG sites" flanking the cytosine phosphate guanine (CpG) region followed by "Bio Edit Sequence Aliqment Editor" tool to identify the forward and reverse primers. Finally, BiSearch is a primer-design and search tool utilized to ensure amplification of specific PCR products (Table S1) (BiSearch, 2019). Gradient PCR was performed to find the specific annealing temperature for the selected gene. Specific products 321bp and 218bp were detected at 60°C for promoter and intron-1 regions of PTPRG, respectively. Bisulfite treatment was performed followed by Sanger sequencing (Table S2).

| Bisulfite sequencing
The PCR products were sequenced with an ABI PRISM BigDye terminator sequencing kit v1.1 (Life Technologies) and directly analyzed by an automated ABI 3130 Genetic Analyzer (Life Technologies).

| Methylation analysis
The analysis of the methylation status of CpG sites in the region amplified by PCR was performed using the ESME (Epigenetic Sequencing Methylation) Analysis Software (Lewin, Schmitt, Adorjan, Hildmann, & Piepenbrock, 2004). The software uses a genomic sequence as a reference and four-dye trace sequencing data as tests. The ratio of C to T at CG sites was determined after correction for incomplete conversion to determine extent of methylation. Furthermore, the percentage of methylation was calculated as the peak height of C versus the peak height of C plus the peak height of T for each CpG site as shown in the computer-generated sequencing chromatogram extracted from the Chromas program (Version 2.32, Technelysium). The ABI sequencing data files (*.ab1) along with reference fasta-files (*.fa) were run through the ESME software. A single C at the corresponding CpG site was considered as 100% methylation, a single T as no methylation and overlapping C and T as partial methylation (0%-100%) (Jiang et al., 2010).
The methylation levels (0%-100%) were converted to (0-1) for plotting purposes (Mallona, Diez-Villanueva, & Peinado, 2014). The genomic co-ordinates were identified for the CpG sites (Table S3). The methylation data obtained was represented diagrammatically as follows: a horizontal series represented a single sample, circles referred to CpG sites and shape filled indicated the methylation of the site.

| Statistical analysis
Descriptive statistics in the form of median range and frequency, and percentages were calculated. For continuous outcomes and categorical independent variables, T test for independent samples was used to test the mean differences for two groups and one-way ANOVA with Bonferroni post hoc analysis was employed to test the mean differences for three groups using SPSS 24. All p values presented are two-tailed, and p values < .05 are considered statistically significant.

| Participants' characteristics
Out of the 26 CML patients, 13 were newly diagnosed (ND) with CML and 13 failed treatment (F) (

| Hypermethylation of the promoter region of PTPRG
T test was performed to study the methylation pattern of promoter of PTPRG in both cases and controls. The results revealed a significant difference in promoter methylation levels between CML (ND and F groups) and the healthy group (t (30) = 5.7, p < .001) (CML: mean = 6.77, SD: 2.87; Healthy: mean = 0.00, SD: 0.00). Additionally, we tested the differences between the three groups (ND, F and H). One-way ANOVA and Bonferroni post hoc test results indicated that the ND and F groups had significantly higher methylation compared with the H group (p < .001). There was no significant difference between ND and F groups ( Table 2).

| Methylation patterns in the 25 CpG sites of promoter region of PTPRG
One-way ANOVA was performed to compare methylation status in each CpG site in the promoter region between the three groups (ND, F, and H). There were significant differences in 2 out of 25 CpG sites (13 and 143) among the groups (F (2, 29) = 7.0; p = .003 and F (2, 29) = 4.35; p = .022 respectively). Bonferroni post hoc test results indicated that methylation in CpG 13 for the ND and the F groups was significantly higher compared to the H group (p = .002 and p = .035 respectively). Furthermore, methylation in CpG 143 for the F group was significantly higher compared to the H group (p = .045). (Figure 1). No significant differences were found in the rest of the CpG sites.

| Hypermethylation of intron-1 region of PTPRG
T test was performed to study the methylation pattern of intron-1 of PTPRG in both cases and controls. The results

Region
Groups One-way ANOVA showed significant differences in methylation between the three groups (ND, F and H) for intron-1 region [F (2, 29) = 53.590, p = .001]. Bonferroni post hoc test results indicated that the methylation status for the ND and the F groups was significantly higher than the H group (p < .001). There was no significant difference between the ND and the F groups (Table 2).

Intron-1 region of PTPRG
One-way ANOVA was also performed to compare methylation levels in each CpG sites in the intron-1 region between the three groups; ND, F, and H. The results indicated that there were significant differences in 23 out of 26 CpG sites (Figure 2 and Table 3). Bonferroni post hoc test revealed that the methylation levels were significantly higher amongst the ND and the F groups compared to the H group in most of the intron-1 CpG sites except CpG 70, CpG 94, CpG 155 and CpG 161 (in the ND group) and CpG 173 (in the F group). In addition, the F group had significantly higher methylation levels in the CpG sites 94 (p = .003) and 155 (p = .01) compared to the ND group.

| DISCUSSION
This is the first prospective study to evaluate epigenetic mechanisms of PTPRG regulation amongst CML patient's population where the rate of IM resistances is higher than other reported parts of the world (Al-Dewik, Al-Dewik, Morsi et al., 2016;Patel et al., 2017). It addresses the important role of PTPRG as a regulatory element in BCR-ABL1-mediated oncogenesis. Our study provides an evidence of the involvement of the epigenetic modification of PTPRG in the pathogenesis of CML. PTPRG was found to be significantly hyper methylated compared to the control (Figures 1 and 2).
Protein tyrosine phosphatase receptor gamma is known to induce a reduction of protein BCR-ABL-specific tyrosine phosphorylation of its direct downstream targets/substrates such as CRKL and of STAT5 (Della Peruta et al., 2010). In the current study, we expanded the methylation coverage of PTPRG via studying two regions of its promoter; 321bp andintron-1 218 bp using advanced molecular technique such as Sanger sequencing. In the same context, Della Peruta et al., documented earlier that upregulated PTPRG expression is associated with a reduction in methylation levels in 166 bp of PTPRG using Methylation-specific PCR technique (Della Peruta et al., 2010). More recently, we demonstrated that in PTPRG-negative CML cell lines, the methylating enzyme DNA (cytosine-5)-methyl transferees 1 (DNMT1) is overexpressed, bind to PTPRG promoter and is responsible for its hypermethylation, while its inhibition or downregulation correlates with PTPRG re-expression (Tomasello et al., 2020). Our findings revealed that the methylation occurs more frequently in the intron-1 region compared to the promoter region in CML patients besides showing a significant increase of the methylated percentage at the CpG sites in both promoter and intron-1 regions compared to healthy individuals (Table 3). Interestingly, our findings indicated and confirmed that the hyper methylated pattern of PTPRG gene in CML patients acts as an early promoter for CML formation and to be dependent on BCR-ABL1 titers. It may well contribute as a BCR-ABL1 independent resistance molecular event.
We analyzed 51 CpG sites in PTPRG in CML and healthy control groups for methylation. Overall, the frequency of methylated CpG sites was significantly higher in CML cases compared to healthy controls, suggesting the potential involvement of CpG methylation sites in CML (Figures 1 and 2). Interestingly, two CpG sites in the intron-1 region were found to be significantly hyper-methylated amongst failed groups compared with newly diagnosed. In the newly diagnosed group, the frequency of CpG site methylation was significantly different from the healthy group, suggesting that CpG site methylation have a central role in the molecular events leading to CML. These findings support the assumption that the CML disease is not only driven by the BCR-ABL1 translocation (Lecca & Sorio, 2016). Moreover, we also observed a significantly higher methylated CpG sites in the failed group compared to the healthy group, indicating that CpG site methylation may be important for disease progression (Table 3).
Several studies have documented the effect of DNA methylation pattern of regulatory genes on various cellular activities such as cell proliferation and survival, as well as cell-signaling molecules in CML (Behzad et al., 2018). Jelinek et al., 2011 studied the Methylation levels of 10 genes in CML patients and found that the frequency of methylated genes ranged from 11%to 86% as follows: ABL1 (86%), CDH13 (79%), NPM2 (74%), PGRA (66%), TFAP2E (63%), DPYS (54%), PGRB (52%),OSCP1 (30%), PDLIM4 (21%), and CDKN2B (11%), suggesting an aberrant methylation of DNA associated with the progression of the disease (Jelinek et al., 2011). Another study using a whole methylome approach in 36 CML patients, found that 31 genes were uniquely hyper methylated in CML and 42 genes that became hyper methylated with the progression of CML. Remarkably, the same group showed that utilizing DNA methylation inhibitor such as azacytidine in blastic crisis CML patients resistant to Imatinib Mesylate (IM) could reverse the aberrant hypermethylation associated with progression of CML to blast crisis and supports the use of this drug as an epigenetic therapy (Byun, 2007). In another study with CML cell line K562 and its IM resistant variant (K562-R) the methylation levels were found to be significantly higher and that the gene expression levels were significantly lower for MLH1, RPRM, FEM1B, and THAP2 in K562-R cells when compared to parental K562 cells. Further, treatment of the K562-R cells with epigenetic drugs, such as 5-azacytidine (AzaC) reduced resistance to Imatinib Methylate (REN, 2012). In another study, SOX30 methylation has been correlated with disease progression in patients with chronic myeloid leukemia (Zhang et al., 2019). Protein tyrosine phosphatase receptor gamma expression has been shown to be downregulated by RAS activation, while its upregulation has been observed in hypo-methylation condition in in childhood acute lymphoblastic leukemia (ALL) (Xiao et al., 2014). Finally, PTPRG methylation has also been reported in solid cancer (Cheung et al., 2008;Wang & Dai, 2007). Eddy et al., suggested that PTPRG inton1 methylation could be a biomarker for early detection of colorectal cancer (van Roon et al., 2011).

| CONCLUSION
Hypermethylation of PTPRG locus might suggest a molecular mechanism independent of BCR-ABL1 function in CML patients. Our data contributes to deepen our understanding of the crucial role of aberrant DNA methylation in CML disease initiation and progression. Potentially, PTPRG methylation could be a biomarker for early detection of CML. However, further studies are needed on the validation of specific aberrant methylation of PTPRG and its prognostic and predictive values for the response to therapy in the CML patients.

ACKNOWLEDGMENTS
This report (publication) was made possible by NPRP grants (28-6-7-17 and 4-157-3-052) from the Qatar National Research Fund (a member of Qatar foundation). The statements made herein are solely the responsibility of the authors. The authors thank their respective institutions for their continued support. The publication of this article was funded by the Qatar National Library, Doha, Qatar. MI started this study as part of a PhD project under the supervision of ND (Local PhD Supervisor), HeM (Director of study) and RC (Other supervisor) at Kingston University London.

CONFLICTS OF INTEREST
The authors declare that there are no conflicts of/or competing interests.

AUTHORS' CONTRIBUTIONS
MI was responsible for performing the experiments; MI, MS, and ND were responsible for analyzing the data. MI and ND designed the experiment. MI, ND, and MS performed statistical analysis. MI, MY, and ND involved in patient's recruitment. MI, ND, AS, MS, MV, GV, MM, MS, HiM, MWQ, HZ, RC, CS, HeM, and ND conceptual work, framework, draft write-up and editing. All authors read and approved the final manuscript.

ETHICAL APPROVAL AND CONSENT APPROVAL
Informed consent was obtained from all participants. The study was approved by both Ministry of Public Health and institutional review board of Hamad Medical Corporation (Project No. 1118/11). This study adhered to the World Medical Association's Declaration of Helsinki (1964Helsinki ( -2008 for Ethical Human Research including confidentiality, privacy, and data management.

CONSENT FOR PUBLICATION
Consent for publication was obtained through ethics approval and consent to participate.

DATA AVAILABILITY STATEMENT
This is a research article and all data generated or analyzed during this study are included in this publication All enquiries should be directed to Dr. Nader Al-Dewik: naldewik@ hamad.qa. T A B L E 3 (Continued)