Methylation Status and Polymorphism of Asparagine Synthetase Gene in Acute Lymphoblastic Leukemia Cells.

Asparagine synthetase (ASY) is an enzyme expressed ubiquitously in mammalian cells. Expression of the gene, however, is repressed in acute lymphoblastic leukemia (ALL), and the cells are unable to synthesize sufficient asparagine. Depletion of asparagine by L-asparaginase leads to cell death in ALL; therefore, asparaginase has been used as treatment of ALL. Here, we show the methylation status of ASY gene in ALL clinical samples, and report a novel insertional polymorphism in the gene. The ASY gene has a CpG island located from −313 to +336 including 49 CpG sites. We analyzed the methylation status in the region by sodium bisulfite conversion of genomic DNA followed by PCR analysis. A total of 18 of 22 (82%) bone marrow samples of ALL at diagnosis were methylated in the CpG island. After chemotherapic induced-remission, all the bone marrow samples were unmethylated including the 18 patients whose ALL samples were methylated. Experiments by others using an ALL cell line showed that methylation of the ASY gene silenced the gene. In addition, we discovered either a 14 bp or 28 bp inserted sequence between +83 and +84 located in the 1st intron of the gene. Methylation status of the gene was independent of the insertion. To calculate the frequency of the insertion, 92 ALL samples were analyzed by PCR and sequencing. 69 samples (75%) did not contain the insertion in either allele, 22 samples (24%) contained the insertion in one allele, and only 1 sample (1%) contained the insertion in both alleles. To examine whether the insertion was specific for leukemic cells, we analyzed 71 non-ALL samples. 48 samples (68%) did not contain the insertion in either allele, 12 samples (27%) contained the insertion in one allele, and 4 samples (6%) contained the insertion in both allele. These results showed that the insertion is a germline polymorphism; we are exploring if this insertion changes expression of the gene.

In summary, we found a novel insertional polymorphism in the ASY gene. Also, our data for the first time suggests that ALL cells cannot express ASY because the gene’s CpG island is methylated, thus explaining why ALL cells are sensitive to L-asparaginase. Notably, 18% of ALL samples were not methylated, and these patients might be expected to be resistant to L-asparaginase therapy suggesting that a simple test could be of therapeutic value.