TET2 enzymatic ally converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. Genetic mutations of TET2 gene were associated with leukemia, whereas TET1 down regulation has been shown to promote malignancy in breast cancer. To expand on this concept, we studied methylation status in TET2 gene in Breast Cancer (BC) samples. TET2 Messene or nonsense mutations were detected in 53% (16/30) of patients. In contrast, only 1/30 patient had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A in the sites most frequently mutated in Breast Cancer. Using bisulfate pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutants and wild-type BC cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We found that two non-CpG island promoters, AIM2 and SP140, were hyper ethylated in patients with mutant TET2. These were the only two gene promoters (out of 14,475 genes) previously found to be hyper ethylated in TET2 mutant cases. However, total 5-methylcytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases (median = 14.0% and 9.8%, respectively) (p = 0.016). Thus, TET2 mutations affect global methylation in BC but most of the changes are likely to be outside gene promoters.
Keywords: TET2; DNA Methylation; Breast Cancer; Mutation; Promoter
PCR: Polymerase Chain Reaction;
BC: Breast Cancer;
TET: Ten Eleven Translocation
TET2 [ten-eleven translocation (TET) oncogene family member 2] is a tumor suppressor gene on chromosome 4q24 . TET2 mutations were first described in myeloproliferative neoplasms (MPN) [1-7]. As reported for TET1 , TET2 also converts 5-methyl-cytosine to 5-hydroxymethylcytosine  in embryonic stem cells, and thus mutations of TET2 were proposed to contribute to leukemogenesis by altering epigenetic regulation of transcription through DNA methylation. Disrupting hematopoietic differentiation [10,11]. Furthermore, in murine models, TET2 deficiency impairs hematopoietic differentiation with expansion of myeloid precursors [12,13]. The exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. In contrast, Figueroa et al. studied TET2 mutant AMLs and identified a hyper methylation phenotype, including 129 differentially methylated regions . These studies were conducted using microarray-based screening methods for DNA methylation analysis, which might have false positive and negative findings. To examine this issue in more detail, we used bisulfite pyrosequencing, which is one of the most reliable ways to analyze DNA methylation for in- dividual genes, and probed the DNA methylation status of 21 promoters of interest as well as global DNA methylation levels in a cohort of 30 BC patients. Our data suggest that effects of TET2 mutations on DNA methylation are primarily outside gene promoters.
Patients and Methods
We analyzed peripheral blood samples prior to treatment from 30 patients with BC referred to The Loqman Hakim Hospital Cancer Center in Tehran. All patients gave informed consent for the collection of residual tissues as per institutional guidelines.
For TET2 gene analysis, polymerase chain reaction (PCR) and direct sequencing of exon 3– 11 was performed starting from 20 ng of genomic DNA, as previously described . PCR amplicons were sequenced by Beckman Coulter Genomics (Danvers, MA). All TET2 mutations were scored on both strands. Sequence traces were analyzed with SeqMan Pro (DNASTAR, Inc., WI) and reviewed visually. Previously annotated single nucleotide polymorphisms in the HapMap database (www. hapmap.org) were discarded. SIFT software  was used to determine the probability that a particular amino acid substitution is tolerated.
Quantitative DNA methylation analyses by bisulfite pyrosequencing
We used bisulfite pyrosequencing to quantitatively assess DNA methylation  for 10 promoter CpG islands frequently abnormal in MDS [ER (ESR1), NOR1 (OSCP1), p15 (CDKN2B), PM2, ECAD (CDH1), CDH13, OLIG2, PGRB, PGRA and RIL (PDLIM4)],  and 11 promoter regions of genes reported by others to be differentially methylated by TET2 mutations (hypomethylated:C9orf16, PSMD6, LRRC32, TMEM34, FSD1NL; hypermethylated: AIM2 and SP140 from Ko et al. and ACOX3, SLC39A14, ZNF662 and DNM3 from Figueroa et al.). Long interspersed nuclear element-1 (LINE-1) was also analyzed to measure global repeat element methylation. We tested samples for which a sufficient amount of DNA was available for bisulfite treatment. The number of patients with successful results (mostly >90% success rate) varied slightly for each gene.
Measurement of 5-methyl-cytosine levels by mass spectrometry
DNA hydrolysis was performed as previously described by Song et al. with minor modifications [11,31]. Briefly, one microgram of genomic DNA was first denatured by heating at 100°C. Five units of Nuclease P1, were added and the mixture incubated at 45°C for 1 h. A 1/10 volume of 1 M ammonium bicarbonate and 0.002 units of venom phosphodiesterase 1 were added to the mixture and the incubation continued for 2 h at 37°C. Next, 0.5 units of alkaline phosphatase were added, and the mixture incubated for 1 h at 37°C. Before injection into the Zorbax XDB-C18 2.1 mm x 50 mm column (1.8 μm particle size), the reactions were diluted 10-fold to dilute out the salts and the enzymes. Samples were run on an Agilent 1200 Series liquid chromatography machine in tandem with the Agilent 6410 Triple Quad Mass Spectrometer. LC separation was performed at a flow rate of 220 μL/min. Quantification was done using a LC-ESI-MS/MS system in the multiple reaction monitoring (MRM) mode. We measured 5-methyl-cytosine levels in genomic DNA of TET2 mutant and wild-type cases where sufficient amount of samples are available in quantity for mass spectrometry (12 mutant and 7 wild-type cases).
Statistical analyses were performed using PRISM (GraphPad Software, Inc., CA). Differences in clinical characteristics of patients with or without TET2 mutations were assessed using the Fisher’s exact test, the Mann-Whitney or log-rank analysis. We used Kaplan-Meier tests to calculate and generate survival curves and used the log-rank test to determine significance between the group of TET2 mutant and wild-type. We used the Mann Whitney test to compare continuous variables of DNA methylation and 5-methyl-cytosine levels between TET2 mutant and wild-type cases. All p values were two-tailed and the threshold of statistical significance was p less than 0.05 followed by Bonferroni’s correction when multiple analyses were performed.
TET2 mutation status in BC
We analyzed the nature and frequency of somatic mutations affecting the TET2 coding sequence (exons 3–11) in a cohort of 30 patients with BC according to WHO criteria. TET2 missense or nonsense mutations were detected in 16 out of 30 (53%) patients. Ten patients had a single heterozygous mutation, two had a biallelic or homozygous mutations, three had two mutations, and one patient had three distinct mutations. Altogether, 21 mutations were identified, including 7 missense, 7 nonsense and 7 frame shift mutations. Detailed mutation information is shown in Table 1. Mutations were observed in exon 3 (8
events), exon 5 (1 event), exon 6 (3 events), exon 7 (1 event), exon 9 (1 event), exon 10 (2 events) and exon 11 (5 events). Only 5 out of 21 identified mutations have been reported previously. Six out of seven identified missense mutations were predicted to affect protein function by using SIFT software. We identified IDH2 R140Q mutation in 1 out of 30 BC patients (3%). Mutation at the R882 residue in DNMT3A was found in 21 of the 30 BC patients.. We compared the overall and progression free survival in patients with vs. without mutations and observed no significant differences.
Table 1. TET2 mutation status
Comparison of DNA methylation levels between TET2 mutant and wild-type cases.
Next, we performed bisulfite pyrosequencing to compare DNA methylation status between patients with mutant vs. wildtype TET2 genes (Table 2). Bisulfite pyrosequencing is a highly quantitative and reliable method for methylation analysis of individual CpG sites. We compared TET2 mutant to TET2 wild-type cases to distinguish the effects of TET2 on methylation from the effects of BC transformation. First, we studied DNA methylation levels of 10 promoter CpG islands frequently abnormal in MDS  since these genes are assumed to be most likely to show abnormal DNA methylation levels in their promoters when the DNA methylation machinery is altered. Table 2. DNA methylation levels
In this cohort of 30 BC patients, we found that missense or nonsense mutations of TET2 were detected in 16 out of 30 (53%) patients. Mutations were found to be distributed broadly from exon 3 to exon 11. Furthermore, only 5 out of 21 mutations were the same as previously reported, confirming the marked heterogeneity in mutational status. Overall, in addition to the frequency of mutations, the characteristics of the mutations in this study are in good agreement with what has been reported so far. Missense mutations and frame shift mutations are mainly found in exon 3 of TET2, whereas point mutations are found in exons 4 to 11.
Although these analyses revealed that TET2 does not have mutation “hot spot(s)” as seen for IDH1/2 and DNMT3A in MDS, some locations in TET2 were found to have high frequencies f mutations. We also confirmed that 21 of 30 BC patients had mutation at the R882 residue in DNMT3A. We could not find ignificant differences in overall and progression free survival between TET2 mutant and wild-type cases in this cohort. However, correlation of TET2 mutation and survival is still in question; reported by different studies as superior in MDS  and BC. Larger studies will be needed to confirm the effect of TET2 mutations on survival.
Bisulfite pyrosequencing is one of the most reliable ways to analyze DNA methylation for individual genes, and we find that only two genes, AIM2 and SP140, were hypermethylated in patients with mutant TET2 compared with wild-type TET2. These genes are the only two genes found to be hypermethylated in a previous report that studied 14,475 genes . Recently, AIM2 was reported to have a putative role in reduction of cell proliferation by cell cycle arrest ; therefore, methylation of the promoter might provide a growth advantage to cancer cells.
Table 2. DNA methylation levels
Methylation of SP140 might have an effect on differentiation to specific lineages. Overall, we find rare promoter methylation differences in TET2 mutant cases, but hypermethylation of AIM2 and SP140 may be useful biomarkers of TET2 mutations in BC.
TET2 mutation has been shown to lead to inefficient conversion of 5-methyl-cytosine to 5hydroxymethyl-cytosine. Consistent with this, we found that 5-methyl-cytosine levels of TET2 mutant cases are higher than TET2 wild-type cases. However, this does not seem to translate to increased promoter methylation, with AIM2 and SP140 being notable exceptions. While we did not study the whole genome to be completely confident of this fact, we did investigate the most frequently hypermethylated genes in MDS, and others studied genomewide methylation with similar findings (hypermethylation of only two out of 14,475 genes). We could not confirm hypomethylation in TET2 mutant BC cases. Given the above, our findings suggest that the total methylation level increase in TET2 mutant cases is mostly outside CpG islands and promoters examined so far.
There are several possible explanations for the findings in this study about the impact of TET2 mutations on promoter methylation. First, different TET2 mutations might affect DNA methylation in divergent ways; however, most of the mutations found in this report are predicted to negatively affect protein function. We found no difference in DNA methylation in patients with homozygous, biallelic or frame-shift mutations. To support this, the only two differentially methylated genes in this study, AIM2 and SP140. It is also possible that the effect on promoter DNA methylation of TET2 is not global but very restricted to a few genes such as AIM2 and SP140. However, the TET1 protein has been found to be enriched at most CpG-rich sequences [23,24] and there is no mechanism to explain selectivity. Because the promoters of AIM2 and SP140 are not in CpG islands, the observed effect on DNA methylation could be secondary to other effects of TET2 on gene expression. Indeed, TET1 protein has been found to affect gene expression independent of DNA methylation . Altogether our data suggest that TET2 mutations have effects on global DNA methylation, but we have not been able to detect major effects on promoter methylation (with the limitations previously discussed). It appears likely that TET2 mutations affect DNA methylation in other regions such as gene bodies or intergenic areas. Larger and genome wide studies will be needed to confirm the precise relationship between TET2 mutations and DNA methylation.
In conclusion, we have shown that epigenetic markers that TET2 gene is one of them, are promising biomarkers for breast cancer. The results presented in this thesis do not unambiguously indicate that altered epigenetic regulation is responsible for the unique methylation pattern observed in samples from patients with Breast cancer. Further research is needed to fully understand the biology of Breast cancer. This study highlights the potential for TET2 methylation to be an informative prognostic biomarker for breast cancer survival and sets the scene for a more comprehensive investigation of the molecular basis of this phenomenon.