Silencing of CD21 expression in synovial lymphocytes is independent of methylation of the CD21 promoter CpG island

The complement receptor II (CD21) recognises the complement component C3d of immune complexes. Expression of the CD21 gene is tightly regulated during B lymphocyte differentiation. Only mature B lymphocytes express CD21 but not pro-, pre-, or plasma B lymphocytes. Previously we found that pro-, pre-, and intermediate B lymphocytes contain a methylated CpG island and do not express CD21. CD21-expressing mature B lymphocytes, plasma B lymphocytes, and nonlymphoid cells carried a demethylated CD21 CpG island. Furthermore, we found that synovial lymphocytes from patients with rheumatic disease show reduced expression of CD21. This observation tempted us to analyse the methylation status of the CD21 CpG island in peripheral blood mononuclear cells and synovial fluid mononuclear cells derived from these patients. While methylation is involved in silencing CD21 in early types of B lymphocytes, we found the CD21-CpG island to be demethylated in peripheral blood mononuclear cells and synovial fluid mononuclear cells of patient DNA.     

Methylation profiling in acute myeloid leukemia

Aberrant methylation of multiple CpG islands has been described in acute myeloid leukemia (AML), but it is not known whether these are independent events or whether they reflect specific methylation defects in a subset of cases. To study this issue, the methylation status of 14 promoter-associated CpG islands was analyzed in 36 cases of AML previously characterized for estrogen-receptor methylation (ERM). Cases with methylation density of 10% or greater were considered positive. Seventeen cases (47%) were ERM(+) while 19 cases were ERM(-). Hypermethylation of any of the following, p15, p16, CACNA1G, MINT1, MINT2, MDR1, THBS1, and PTC1 (2 promoters), was relatively infrequent (6% to 31% of patients). For each of these CpG islands, the methylation density was positively correlated with ERM density (rank order correlation coefficients, 0.32-0.59; 2-tailed P < or = .058 for each gene). Hypermethylation of MYOD1, PITX2, GPR37, and SDC4 was frequently found in AML (47% to 64% of patients). For each of these genes as well, methylation density was positively correlated with ERM density (correlation coefficients 0.43 to 0.69, P < or = .0087 for each gene). MLH1 was unmethylated in all cases. Hypermethylation of p15, MDR1, and SDC4 correlated with reduced levels of expression. There was an inverse correlation between age and the number of genes methylated (P = .0030). It was concluded that CpG-island methylation in AML results from methylation defects in subsets of cases. These results have potential implications for the classification and prognosis of AML and for the identification of patients who may benefit from treatment with methylation inhibitors.     

Hypermethylation of E-cadherin in leukemia

E-cadherin gene is often termed a "metastasis suppressor" gene because the E-cadherin protein can suppress tumor cell invasion and metastasis. Inactivation of the E-cadherin gene occurs in undifferentiated solid tumors by both genetic and epigenetic mechanisms; however, the role of E-cadherin in hematologic malignancies is only now being recognized. E-cadherin expression is essential for erythroblast and normoblast maturation, yet expression is reduced or absent in leukemic blast cells. This study examined the messenger RNA (mRNA) and protein expression of the E-cadherin gene in bone marrow and blood samples from normal donors and patients with leukemia. We found that all normal donor samples expressed E-cadherin mRNA, whereas both samples of acute myelogenous leukemia and chronic lymphocytic leukemia had a significant reduction or absence of expression. However, normal blast counterparts expressed only a low level of E-cadherin surface protein. Sodium bisulphite genomic sequencing was used to fully characterize the methylation patterns of the CpG island associated with the E-cadherin gene promoter in those samples with matched DNA. All of the normal control samples were essentially unmethylated; however, 14 of 18 (78%) of the leukemia samples had abnormal hypermethylation of the E-cadherin CpG island. In fact both alleles of the E-cadherin gene were often hypermethylated. We conclude the E-cadherin gene is a common target for hypermethylation in hematologic malignancies.     

Allelic silencing at the tumor-suppressor locus 13q14.3 suggests an epigenetic tumor-suppressor mechanism

Genomic material from chromosome band 13q14.3 distal to the retinoblastoma locus is recurrently lost in a variety of human neoplasms, indicating an as-yet-unidentified tumor-suppressor mechanism. No pathogenic mutations have been found in the minimally deleted region until now. However, in B cell chronic lymphocytic leukemia tumors with loss of one copy of the critical region, respective candidate tumor-suppressor genes are down-regulated by a factor >2, which would be expected by a normal gene-dosage effect. This finding points to an epigenetic pathomechanism. We find that the two copies of the critical region replicate asynchronously, suggesting differential chromatin packaging of the two copies of 13q14.3. Although we also detect monoallelic silencing of genes localized in the critical region, monoallelic expression originates from either the maternal or paternal copy, excluding an imprinting mechanism. DNA methylation analyses revealed one CpG island of the region to be methylated. DNA demethylation of this CpG island and global histone hyperacetylation induced biallelic expression, whereas replication timing was not affected. We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. Accordingly, deletion of the single active copy of 13q14.3 results in significant down-regulation of the candidate genes and loss of function, providing a model for the interaction of genetic lesions and epigenetic silencing at 13q14.3 in B cell chronic lymphocytic leukemia.     

p16基因启动子区甲基化在人嗜铬细胞瘤和副神经节瘤中的变化

目的 检测嗜铬细胞瘤(PHEO)和副神经节瘤(PGL)中p16基因突变和启动子区DNA甲基化改变,分析其与患者临床特征之间的关系.方法收集34例(PHEO 20例、PGL14例)组织标本及患者临床资料,通过甲基化特异性PCR(MSP)测定p16基因启动子区甲基化状态,DNA测序检测基因序列以及RT-PCR方法测定其mRNA表达.结果 (1)34例肿瘤组织中未发现p16基因纯合缺失及点突变;(2)35.3%(12/34)的患者存在p16基因高甲基化,p16基因甲基化阳性标本中,PHEO和PGL分别占25%和75%,两者差异有统计学意义(P=0.005);p16基因甲基化在恶性、单发肿瘤、发病年龄早的亚组中有增高趋势(P＞0.05);(3)甲基化与非甲基化肿瘤组织之间p16 mRNA表达无统计学差异;不同特点的肿瘤中其mRNA表达亦无统计学差异,但恶性肿瘤p16 mRNA表达与良性肿瘤相比有下降的趋势(0.83±0.65对1.12±0.81,P=0.278).结论人PHEO和PGL中,p16基因纯合缺失和突变并不常见,但p16基因启动子区甲基化是其失活的主要形式.     

Hepatitis B viral DNA is methylated in liver tissues

The mechanisms that regulate hepatitis B virus (HBV) replication within the liver are poorly understood. Given that methylation of CpG islands regulates gene expression in human tissues, we sought to identify CpG islands in HBV-DNA and to determine if they are methylated in human tissues. In silico analysis demonstrated three CpG islands in HBV genotype A sequences, two of which were of particular interest because of their proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2). Human sera with intact virions that were largely unmethylated were used to transfect HepG2 cells and HBV-DNA became partially methylated at both islands 1 and 2 by day 6 following exposure of HepG2 to virus. Examination of three additional human sera and 10 liver tissues showed no methylation in sera but tissues showed methylation of island 1 in six of 10 cases and of island 2 in five of 10 cases. The cell line Hep3B, with integrated HBV, showed complete methylation of island 1 but no methylation of island 2. In conclusion, HBV-DNA can be methylated in human tissues and methylation may play an important role in regulation of HBV gene expression.