P15INK4b gene methylation and myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are clonal disorders, which frequently undergo leukemic transformation. It was recently shown that the promoter of the p15INK4b but not the p16INK4a gene is frequently and selectively hypermethylated in MDS. The p15INK4b gene is a cyclin dependent kinase inhibitor gene, which is actively transcribed after TGFbeta exposure. Methylation of the p15INK4b gene is significantly correlated with blastic bone marrow involvement, and sequential analyses have shown that methylation increases with disease evolution toward AML. These data strongly suggest that p15INK4b gene methylation is a mechanism allowing leukemic cells to escape to inhibitory signals from the bone marrow environment, however the exact role of p15INK4b gene methylation in disruption of the signal mediated by TGFbeta remains to be investigated.     

Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes.

We previously reported that the hypermethylation of the p15INK4B gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15INK4B gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15INK4B gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15INK4B methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15INK4B-methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15INK4B methylation were detected in both patients. Furthermore, X-chromosome inactivation (XCI) pattern of each colony was simultaneously determined by MSP-based human androgen receptor gene analysis (HUMARA-MSP), and all p15INK4B-methylated colonies showed the same XCI pattern, which was dominant among the colonies, while p15INK4B-unmethylated colonies showed both patterns of XCI, in each of the two patients. We then examined the methylation status of the p15INK4B gene of granulocyte (PB-PMN) fractions from 10 patients with available peripheral blood cells. In all four patients with p15INK4B-methylated BM-MNCs, their PB-PMNs showed p15INK4B methylation. These results suggest that p15INK4B methylation in hematopoietic cells in MDS patients is restricted to the MDS clone but not necessarily to blast cells.     

低剂量亚砷酸及维甲酸方案对儿童骨髓增生异常综合征p15INK4B基因的影响

目的 探讨低剂量亚砷酸及维甲酸双诱导方案对骨髓增生异常综合征(MDS)患儿p15INK44B基因表达的影响及患儿p15INK4B基因甲基化水平的变化.方法 11例MDS患儿接受低剂量亚砷酸及维甲酸双诱导方案治疗,应用实时荧光定量聚合酶链反应(PCR)方法检测治疗前后p15INK4B基因的表达,应用亚硫酸氢钠修饰及实时荧光定量PCR方法检测p15INK4B基因的甲基化水平.结果 11例MDS患儿经过双诱导方案治疗后,9例获益.治疗后10例患儿p15INK4B基因表达水平显著升高,治疗前后p15INK4B基因甲基化水平分别为(1.68±0.58)％、(1.44±0.65)％,二者差异无统计学意义(t=-0.885,P＞0.05).结论 低剂量亚砷酸及维甲酸双诱导方案治疗儿童MDS疗效较好,该方案的作用机制可能与p15INK4B基因表达升高有关,但p15INK4B基因表达升高与去甲基化无相关性.     

异硫氰酸苯己酯诱导Molt-4细胞p15基因去甲基化的实验研究

 目的　研究异硫氰酸苯己酯（PHI）对急性T淋巴细胞性白血病Molt-4细胞p15基因的去甲基化作用及转录激活作用。方法　采用甲基特异性聚合酶链反应（MSP）检测PHI作用前后Molt－4细胞株p15基因甲基化状态的变化情况;RT-PCR检测p15基因的mRNA的表达变化;Western blotting检测p15蛋白的表达变化。结果　PHI作用于Molt-4细胞5 d后,p15基因的异常甲基化现象被逆转,基因的甲基化程度减弱;基因转录激活,p15 mRNA、p15蛋白表达呈浓度依赖性增加。各组p15 mRNA条带灰度值与β-actin比值为：空白对照组（0.17±0.12）,PHI 10 μmol／L组（0.29±0.14）,PHI 20 μmol／L组（0.55±0.07）,PHI 40 μmol／L组（0.93±0.13）,各加药组与空白对照组相比,差异均有统计学意义（P＜0.05）。结论　PHI有DNA去甲基化的作用,能诱导沉默的p15基因重新表达。     

Mutations in the p53 gene in myelodysplastic syndromes.

We have examined p53 alleles in 151 DNAs from patients with myelodysplastic syndrome using single-strand conformation polymorphism analysis of polymerase chain reaction products. We focused our study on the four highly conserved regions of the p53 gene and detected five patients with aberrantly migrating fragments. We confirmed the putative mutation in each case by direct sequencing analysis. Of these five patients, three had chromosome 17 monosomy associated with p53 mutation, one patient showed one mutated p53 allele and one wild-type allele, and the last patient demonstrated only the mutant allele, suggesting a homozygous state. Unlike many other types of human cancers, point mutations in the p53 tumor-suppressor gene appear to be a rare event in myelodysplastic syndromes.     

Roles of p15Ink4b and p16Ink4a in myeloid differentiation and RUNX1-ETO-associated acute myeloid leukemia

Inactivation of p15(Ink4b) expression by promoter hypermethylation occurs in up to 80% of acute myeloid leukemia (AML) cases and is particularly common in the FAB-M2 subtype of AML, which is characterized by the presence of the RUNX1-ETO translocation in 40% of cases. To establish whether the loss of p15(Ink4b) contributes to AML progression in association with RUNX1-ETO, we have expressed the RUNX1-ETO fusion protein from a retroviral vector in hematopoietic progenitor cells isolated from wild-type, p15(Ink4b) or p16(Ink4a) knockout bone marrow. Analysis of lethally irradiated recipient mice reconstituted with RUNX1-ETO-expressing cells showed that neither p15(Ink4b) or p16(Ink4a) loss significantly accelerated disease progression over the time period of one year post-transplantation. Loss of p15(Ink4b) alone resulted in increased myeloid progenitor cell frequencies in bone marrow by 10-month post-transplant and a 19-fold increase in the frequency of Lin(-)c-Kit(+)Sca-1(+) (LKS) cells that was not associated with expansion of long-term reconstituting HSC. These results strongly suggest that p15(Ink4b) loss must be accompanied by additional oncogenic changes for RUNX1-ETO-associated AML to develop.     

骨髓增生异常综合征ASXL1基因突变的研究

目的 研究骨髓增生异常综合征(MDS)患者中表观遗传调节因子ASXL1基因的突变情况.方法 在DNA水平采用聚合酶链反应(PCR)扩增产物片段直接测序分析法检测53例初发MDS患者及20名健康人ASXL1基因第12外显子突变情况,比较ASXL1突变患者与野生型患者临床及实验室特征;反转录聚合酶链反应(RT-PCR)扩增产物直接测序分析法检测ASXL1突变在mRNA水平的表达.结果 在53例MDS患者中,9例(16.9%)ASXL1基因突变.共检测出6种突变类型,包括4种移码突变(2例p.Glu635ArgfsX15、3例p.Gly646TrpfsX12、1例p.Ala640GlyfsX14、1例p.Gly790TrpfsX10)和2种无义突变(1例p.Gln1063X和1例p.Gln695X).所有突变类型均为杂合突变,其中p.Gly790TrpfsX10和p.Gln695X为新发现突变类型.此外还检测到一种单核苷酸多态性(SNP)位点(4例p.Gly652Ser).20名健康人中检测出pGly652Ser SNP5名和p.Leu1173Leu SNP1名.RT-PCR扩增产物片段直接测序可在mRNA水平检测出移码突变(p.Gly646TrpfsX12).ASXL1突变患者初发时外周血白细胞计数、红细胞计数、血小板计数、血红蛋白、网织红细胞、中性粒细胞绝对值、外周血淋巴细胞比例、T细胞亚群、骨髓原始细胞比例、MDS分型和染色体核型分布与ASXL1野生型患者相比,差异均无统计学意义(均P>0.05).结论 ASXL1基因在MDS患者中的突变频率较高,且可在mRNA水平检测到ASXL1基因突变.     

Inactivation of the p15INK4B and p16INK4 Genes in Hematologic Malignancies

The recently discovered p15^INK4B and p16^INK4 genes encoding cell cycle regulating proteins, map to a region on chromosome 9p21 that is commonly deleted in a variety of malignant diseases. The p16^INK4 gene has now been shown to be a tumor suppressor gene. It is frequently inactivated in cancer and is possibly the second most often mutated gene in human malignant disease after p53. The role of the p15^INK4B and p16^INK4 genes in hematologic malignancies has been the subject of intense investigation since their discovery. In this review we address the function and possible role in tumorigenesis of the p15^INK4B and p16^INK4 genes and discuss their significance as prognostic markers in hematologic malignancies.     

骨髓增生异常综合征基因突变与预后模型研究进展

骨髓增生异常综合征(MDS)是一种克隆性疾病,其特征是复杂的基因异常的积累,这些异常决定了疾病的表型、预后以及向急性髓系白血病转化的风险。不同表型的MDS患者临床表现和预后差异很大,总生存期在数月至数年不等。预后评分系统是重要的分层工具,目前的模型对于预测MDS患者预后非常有效,但这些模型的变量主要集中在骨髓和外周血分析,将基因突变添加到现有模型会提高整体预测能力。探寻将临床和基因组数据合并到一个复杂且高度精确模型中的最佳方法仍是一项正在进行中的工作,最终将建立一个全面的基因-临床模型,将基因组数据的使用转化为临床实践。文章结合第60届美国血液学会(ASH)年会相关报道就MDS的基因突变与预后模型研究进展进行综述。     

骨髓增生异常综合征相关基因突变的研究进展

骨髓增生异常综合征（MDS）是一组骨髓造血干细胞恶性克隆性疾病。随着分子生物学技术在科学研究和临床实践中的广泛应用,已有研究表明基因突变是 MDS 发生及进展的重要原因,MDS 临床异质性大小与基因突变的多样性密切相关,基因研究在 MDS 诊断、分型和预后判断中将发挥越来越重要的作用。文章对近年来 MDS 常见基因突变的研究进展进行综述。     

Hypermethylation of the Ink4b locus in murine myeloid leukemia and increased susceptibility to leukemia in p15(Ink4b)-deficient mice

The Ink4b gene (Cdkn2b) encodes p15(Ink4b), a cyclin-dependent kinase inhibitor. It has been implicated in playing a role in the development of acute myeloid leukemia (AML) in man, since it is hypermethylated with high frequency. We provide evidence that the gene is a tumor suppressor for myeloid leukemia in mice. The evidence is twofold: (1) retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation of the 5' CpG island of the Ink4b gene, and this could be correlated with reduced mRNA expression, as demonstrated by TaqMan real-time PCR. p15(Ink4b) mRNA expression in a leukemia cell line, with hypermethylation at the locus, was induced following treatment with 5-aza-2'-deoxycytidine. (2) Targeted deletion of one allele in mice by removal of exon 2 increases their susceptibility to retrovirus-induced myeloid leukemia. Mice deficient in both alleles were not more susceptible to myeloid disease than those deficient in one allele, raising the possibility that there are opposing forces related to the development of myeloid leukemia in Ink4b null mice.