Methylation status analysis of cell cycle regulatory genes (p16INK4A, p15INK4B, p21Waf1/Cip1, p27Kip1 and p73) in natural killer cell disorders

Abstract

Natural killer (NK) cell disorders are rare diseases. Genetic abnormalities of the several tumor suppressor genes, including p15INK4B, p16INK4A/p14ARF, p53, p73, and Rb genes have been reported. Deletions and point mutations of these genes are frequently detected in these diseases. It has been reported that tumor suppressor genes are inactivated by DNA methylation of the promoter region and/or first exon of the genes in a variety of human cancers. In this study we analyze the methylation status of the genes associated with cell cycle regulation, including p16INK4A, p15INK4B, p21/Waf1/Cip1, p27/Kip1, p73, and p14ARF, by methylation specific (MS) PCR and/or bisulfite sequencing. We examined 29 cases of NK cell disorders (five aggressive NK cell leukemia/lymphoma, three blastic NK cell lymphoma/leukemia, five nasal NK cell lymphoma, three myeloid/NK cell precursor acute leukemia, 13 chronic NK lymphocytosis). We found methylation of the first exon of the p16INK4A gene in two cases (one aggressive, one blastic), and methylation of the p14ARF gene in one aggressive NK cell leukemia. Bisulfite sequencing revealed that methylation of the p15 and p27 genes was rare in these disorders. MS-PCR suggested that the p73 and p21 genes were methylated in seven cases, respectively (p73: one blastic, one nasal, five chronic; p21: one myeloid/NK, one aggressive, one nasal, and four chronic); bisulfite sequencing confirmed that methylated alleles of these genes were dominant in the samples except three cases (one myeloid/NK, one aggressive, and one chronic) in which methylated alleles of the p21 genes were less than 34% of all alleles. These results suggested that inactivation of the cell cycle regulatory genes by DNA methylation could be associated with tumorigenesis in NK cell disorders, not only aggressive subtypes but also chronic subtype.

Leukemia and Lymphoma of Natural Killer Cells

“…Homozygous deletion of p15 and p16/p14 were identified in approximately 30% of the cases studied 170,171 , and mutation of the FAS gene 172,173 and methylation of p73 174 , SHP1 175 , hMLH1 176 , p16 176 , and RARb 176 have been identified in more than half of the cases. Mutation of b-catenin [177][178][179] and methylation of p21 and p15 were observed less frequently 177,180 , but mutation of N/K/H-RAS and N/c-MYC genes were found in only a small minority of cases 171,[177][178][179] . Ethnicity also affects genetic alterations in NK-cell lymphoma including differences between Japanese and Chinese patients for p73 174,180 , p53 178,179,181 , and c-Kit 182 alterations.…”

The role of p73 in hematological malignancies

“…71 However, in a more recent study, this frequency was only 25% in patients with aggressive and chronic types of NK-cell proliferations. 84 Aberrant methylation of P73 gene has also been described in one out of three examined primary central nervous system lymphomas. 72 Most recently, tumor cells in cutaneous T-NHL were also found to show widespread promoter hypermethylation associated with P73 inactivation, among other tumor suppressor genes, such as BCL7a or PTPRG.…”

Natural killer-cell malignancies: diagnosis and treatment

“…Epigenetic inactivation by promoter hypermethylation of multiple genes has been observed. 53,71 Partial deletion of the Fas gene at the death and transmembrane domains was found in some cases, which disrupted the Fas-ligand/Fas signaling pathway, thereby protecting the lymphoma cells from apoptosis. 72 The relative roles of these genetic aberrations in NK-cell lymphomagenesis remain to be further defined.…”