DEHP诱导的隐睾鼠胰岛素样因子3基因CpG岛甲基化状态研究

目的 研究胰岛素样因子3(insulin-like factor 3,INSL3)基因在邻苯二甲酸二已酯(DEHP)致小鼠隐睾中DNA甲基化的状态.方法 运用"MethPrimer"软件对INSL3基因进行分析.预测CpG岛,通过甲基化特异性PCR(methylation-specific PCR,MS-PCR)检测INSL3基因CpG岛的甲基化状况,并应用RT-PCR检测INSL3基因在正常组与实验组睾丸组织中的表达情况.结果实验组INSL3基凶第一外显子区存在CpG岛甲基化;实验组INSL3基因mRNA表达显著下降.结论 INSL3基因第一外显子区CpG岛的异常甲基化导致INSL3在隐睾鼠睾丸中表达降低,可能与隐睾的发生密切相关.     

Upstream CpG island methylation of the PAX3 gene in human rhabdomyosarcomas

BACKGROUND: Adult tumors can be characterized by hypermethylation of CpG islands associated with 5'-upstream and coding regions of specific genes. This hypermethylation can also be part of the aging process. In contrast, much less is known about gene hypermethylation in childhood cancers, where methylation changes are not part of the aging process but likely represent developmental dysregulation. PAX3 is an important gene in muscle development and muscle-producing neoplasms such as rhabdomyosarcomas. PROCEDURES: We examined the methylation status of a PAX3 5'-CpG island in rhabdomyosarcoma subtypes and in normal fetal skeletal muscle. PAX3 methylation was analyzed in 15 embryonal rhabdomyosarcomas, 12 alveolar rhabdomyosarcomas, and in six normal skeletal muscle samples, using semi-quantitative PCR analysis of DNA digested with methyl-sensitive restriction enzymes. RESULTS: The CpG island in the upstream region of the human PAX3 gene was hypermethylated in the majority of ERMS examined (13 of 15 tumors, mean of 52% methylation), whereas most ARMS (9 of 12 tumors) and all normal muscle samples showed relative hypomethylation (both 18% mean methylation). Various CpG sites differ in contribution to overall PAX3 CpG island methylation, with methylation at a HaeII site being inversely correlated with PAX3 expression. CONCLUSIONS: PAX3 CpG island methylation appears to distinguish embryonal subtype of rhabdomyosarcoma from alveolar, and methylation at certain sites within this CpG island is inversely correlated with PAX3 expression. In addition to exemplifying developmental dysregulation, methylation of PAX3 has potential in the development of an epigenetic profile for the diagnosis of rhabdomyosarcoma.     

儿童血浆促酰化蛋白的测定及意义

目的：介绍一种简易、快速、特异性高的人血浆促酰化蛋白(acylationstimulatingprotein,ASP)的检测技术,并阐明ASP测定意义。方法：161例研究对象,其中健康儿童98例,单纯性肥胖症患儿63例,年龄2～6岁,ELISA方法测定血浆ASP的浓度,波长490nm。结果：该方法最佳线性范围:ASP浓度0.5～10ng/mL,平均批内和批间变异系数(CV)分别为8.17%和9.72%。正常学龄前儿童血浆ASP浓度为69.14±25.58nM;单纯性肥胖症儿童血浆ASP浓度为95.64±36.24nM,明显高于健康儿童(P<0.001);ASP与BMI呈正相关。结论：该检测方法能简易、快速对血浆ASP进行准确定量,在ASP基础和临床相关疾病的研究中有重要的意义。     

巢式PCR法检测肝母细胞瘤中p15和p16基因启动子异常甲基化

目的：DNA甲基化被认为是反映细胞内DNA转录状态的重要遗传学标记。该研究旨在对小儿肝母细胞瘤中p15和p16基因5′启动子区CpG岛异常甲基化的相关性进行评估,并分析其与小儿肝母细胞瘤发生发展的关系。方法：采用甲基化特异性PCR法,对30例小儿肝母细胞瘤、癌旁和远癌正常组织进行巢式PCR扩增,经凝胶电泳和测序方法检测目的片段。结果：30例小儿肝母细胞瘤组织中p15和p16基因5′CpG岛有30%(9/30)和67%(20/30)、癌旁组织中有20%(6/30)和57%(17/30)、远癌正常组织有13%(4/30)和33%(10/30)异常甲基化。在肝母细胞瘤中发现1例p15E2纯合缺失,缺失率为3%(1/30),E1和部分I1区域未见缺失;p16E2和部分I2区域中3例杂合缺失,缺失率为10%(3/30),E1和部分I1区域未见缺失。结论：p15和p16基因5′启动子区CpG岛的异常甲基化可能在肝母细胞瘤发生发展中扮演了重要角色,其主要机制可能是启动子区CpG岛甲基化抑制了基因的转录。     

Expression of GAS7 in childhood CNS tumors

Expression of growth arrest-specific (Gas) genes is observed during growth arrest in terminally differentiating cells during development of peripheral nerves. Gas7 is expressed predominantly in the brain and is required for neurite formation. Human GAS7 is located on chromosome 17p11.3 close to or within the putative breakpoint of isochromosome 17q (i(17q)) in medulloblastoma, indicating a potential role as a tumor suppressor gene, lost by formation of i(17q). However in the present study, the expression of GAS7 was detected in 20 of 29 childhood medulloblastoma samples regardless of the presence of i(17q). Therefore, GAS7 is not likely to be a tumor suppressor gene in medulloblastoma development.     

Genetic alterations in pediatric medulloblastomas detected by comparative genomic hybridization.

Children with medulloblastomas show diverse clinical courses even when receiving similar treatments. In this study, comparative genomic hybridization, which allows the detection of losses and gains in DNA copy number along the entire genome, was used to investigate the genetic alterations in 6 cases of medulloblastoma with adequate follow-up periods and similar treatments, and in a medulloblastoma cell line. In the cell line, the number of aberrations was the highest of all samples examined. In 6 clinical samples, frequently altered regions (more than 3 cases) observed in all medulloblastomas were gains of 7q and 17q (4 cases each), and of 2p, 2q and 7p (3 cases each). High-grade amplification was observed at the loci 8q, 17q and 21q, each in a single case. The case with the most favorable outcome 9 years after surgery had the smallest number of chromosomal changes among the cases examined. Our results may indicate that further acquisition of genetic alterations detected by comparative genomic hybridization are associated with unfavorable prognosis in patients with medulloblastomas.     

Methylation status of the retinoblastoma gene (RB1) in osteosarcoma: no evidence for hypermethylation

Alterations of the retinoblastoma (RB1) tumor suppressor gene are not only associated with retinoblastoma but also with several other malignancies including osteosarcoma. Besides direct sequence alterations, hypermethylation of a CpG island in the promoter region can cause inactivation of the RB1 gene as it has been shown in retinoblastomas. We examined the methylation status of the RB1 gene in 25 osteosarcoma specimens by using the methylation-sensitive restriction enzymes SacII and SmaI. The restriction fragments were hybridized with clone p123, which is a 1.8-kb genomic subclone that spans the RB1 CpG island including the promoter region and exon 1. Whereas we reconfirmed hypermethylation of the RB1 gene in a sporadic retinoblastoma, no hypermethylation could be detected in the 25 osteosarcoma specimens, suggesting that hypermethylation of the RB1 promoter is not of major importance during osteosarcoma genesis.     

Risk stratification in medulloblastoma: screening for molecular markers

BACKGROUND: The present study evaluates molecular markers for patients at risk of poor or no response to medulloblastoma. The aim of the study is to optimize therapy stratification. PATIENTS AND METHODS: 69 snap-frozen medulloblastoma samples were examined. C-MYC amplification was determined by fluorescence in situ hybridisation (FISH) analysis. Methylation specific PCR revealed the level of promoter methylation status of the tumor suppressor genes CASP8 (Caspase 8), TIMP3, CDH1 (E-Cadherin), CDKN2A (p16) and MGMT. Expression of GAS7 was evaluated by RT-PCR. RESULTS: C-MYC amplification: 4/69 tumors displayed high level amplification; 53/69 tumors displayed low level (< 4 copies) or no amplification; 12/69 samples were not predictive. In patients with c-MYC amplification a tendency towards unfavorable outcome was observed (p = 0.3). Promoter methylation status: CASP8: in 36/40 tumors methylated; TIMP3: 1/38 methylated; MGMT 0/44 methylated; CDKN2A: 1/46 methylated; E-cadherin 3/36 methylated; no association between methylation status and clinical outcome. GAS7: Detection of specific RNA in 20/29 medulloblastoma samples. CONCLUSION: No significant association between amplification of c-MYC and clinical outcome was observed. Promoter methylation of tumor suppressor genes is non-randomly distributed with a high level of methylation of CASP8. Recent studies show that silencing of CASP8 by methylation could be overcome by interferon gamma providing a possible therapeutic mechanism. GAS7 was shown to be a marker of mature neuronal cells with potential antitumorigenic capacity in neuronal tumors. In medulloblastoma 20/29 of the tumors examined express GAS7. Therefore a tumor suppressing function of GAS7 is improbable.     

Prognostic significance of molecular genetic markers in childhood brain tumors.

Molecular genetic analysis of several common cancers has yielded information concerning their etiology and prognosis. Restriction fragment length polymorphism (RFLP) studies showing consistent loss of specific DNA sequences in tumor tissue have identified genes, known as tumor suppressors, associated with the etiology of neoplasms of the adult colon, breast, lung, and brain. For several of these tumors, identifiable mutations of these genes in association with multiple chromosomal losses have been linked to a poor clinical outcome. Using RFLP and other techniques, we have determined that one or more tumor suppressor genes on chromosome 17p is involved in the etiology of both medulloblastoma and astrocytoma in children. Loss of chromosome 17p sequences is indicative of prognosis negative for these children. Molecular genetic data therefore supplement and in some cases surpass clinical criteria in predicting prognosis for these childhood tumors.     

Control of beta-catenin/Tcf-directed transcription in medulloblastoma

The beta-catenin, glycogen synthase kinase 3beta (GSK-3beta), and adenomatous polyposis coli (APC) gene products interact to form a network that influences the rate of cell proliferation. Medulloblastoma occurs as part of Turcot's syndrome and patients with Turcot's syndrome, who develop medulloblastomas, have been shown to harbor germline APC mutations. While APC mutations have been investigated and not identified in sporadic medulloblastomas, the status of the beta-catenin and GSK-3beta genes has not been evaluated in this tumor. This study shows that 3 of 67 medulloblastomas harbor beta-catenin mutations, each of which converts a GSK-3beta phosphorylation site from serine to cysteine. The beta-catenin mutation seen in the tumors was not present in matched constitutional DNA in the 2 cases where matched normal DNA was available. A loss of heterozygosity (LOH) analysis of 32 medulloblastomas with paired normal DNA samples was performed with 4 microsatellite markers flanking the GSK-3beta locus; LOH with at least one marker was identified in 7 tumors. Sequencing of the remaining GSK-3beta allele in these cases failed to identify any mutations. Taken together, these data suggest that activating mutations in the beta-catenin gene may be involved in the development of a subset of medulloblastomas. The GSK-3beta gene does not appear to be a target for inactivation in this tumor.     

Epigenetic changes in the DAP-kinase CpG island in pediatric lymphoma

BACKGROUND: Hypermethylation of CpG islands in the promoter region of death-associated protein kinase (DAP-kinase) coupled with the loss of gamma-interferon-induced apoptosis have been reported in B-cell malignancies suggesting a role in pathogenesis or prognosis. Along with B-cell malignancies, pediatric lymphomas also include T-cell non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma, and Hodgkin disease, each with unique prognoses. The purpose of this study was to elucidate epigenetic changes in the DAP-kinase promoter region of pediatric cases to determine associations with aberrant hypermethylation. PROCEDURES: Thirty-nine cases of different lymphoid pathology [10 Burkitt lymphoma, 1 B-cell NHL; 7 T-cell lymphoblastic lymphoma; 4 anaplastic large cell lymphoma (LCL); 2 B-cell LCL; 14 nodular sclerosing Hodgkin disease (NSHD); and 1 B-cell acute lymphoblastic leukemia (ALL)] had methylation-specific polymerase chain reaction performed on bisulfite-treated DNA, which distinguishes the methylation status of the promoter region, and DAP-kinase mRNA expression assays performed on available specimens. RESULTS: In normal lymphocytes, the CpG islands in the promoter region were unmethylated, as were the T-cell lymphoblastic lymphoma and anaplastic LCL. In contrast, 100% of the Burkitt lymphoma (10/10) and B-cell ALL (1/1) were hypermethylated. Of the specimens with mRNA available, 7/8 Burkitt lymphoma had no DAP-kinase mRNA expression compared to normal expression in 3/3 and 4/4 T-cell lymphoblastic lymphoma and NSHD, respectively. CONCLUSIONS: In these pediatric lymphoid tumors, hypermethylation of the DAP-kinase promoter region with associated loss of DAP-kinase gene expression was associated with B-cell malignancies and thus may be important in the development and/or provide a prognostic tool in B- cell lymphomas.     

Genomic imprinting and Wilms' tumor

The selective loss of maternal and reduplication of paternal chromosome 11p15.5 alleles in Wilms' tumors (WTs) points to the existence of a paternally imprinted tumor suppressor gene(s) and/or a maternally imprinted dose-dependent growth-promoting gene(s) in this chromosomal region. Two reciprocally imprinted chromosome 11p15.5 genes, H19, a candidate tumor suppressor gene, and IGF2, a candidate dominant oncogene, have been well-characterized in terms of their imprinting and expression status in WTs. Here we review and extend data indicating that a majority of WTs show a bipaternal epigenotype at these loci, with H19 inactive and IGF2 biallelically active. This can arise either through loss of heterozygosity (LOH) or by a non-LOH pathway involving localized biallelic hypermethylation of H19DNA. Conversion to this bipaternal endpoint has recently been found to affect not only these two genes, but also at least one other imprinted 11p15.5 gene, KIP2. Since 11p15.5 LOH and biallelic H19 hypermethylation can occur both early and late in tumor progression and since early loss is not associated with bilaterality or multifocality of WTs, these types of lesions appear to be permissive rather than rate-limiting in Wilms' tumorigenesis. © 1996 Wiley-Liss, Inc.     

Control of β-catenin/Tcf-directed transcription in medulloblastoma

The β-catenin, glycogen synthase kinase 3β (GSK-3β), and adenomatous polyposis coli (APC) gene products interact to form a network that influences the rate of cell proliferation. Medulloblastoma occurs as part of Turcot's syndrome and patients with Turcot's syndrome, who develop medulloblastomas, have been shown to harbor germline APC mutations. While APC mutations have been investigated and not identified in sporadic medulloblastomas, the status of the β-catenin and GSK-3β genes has not been evaluated in this tumor. This study shows that 3 of 67 medulloblastomas harbor β-catenin mutations, each of which converts a GSK-3β phosphorylation site from serine to cysteine. The β-catenin mutation seen in the tumors was not present in matched constitutional DNA in the 2 cases where matched normal DNA was available. A loss of heterozygosity (LOH) analysis of 32 medulloblastomas with paired normal DNA samples was performed with 4 microsatellite markers flanking the GSK-3β locus; LOH with at least one marker was identified in 7 tumors. Sequencing of the remaining GSK-3β allele in these cases failed to identify any mutations. Taken together, these data suggest that activating mutations in the β-catenin gene may be involved in the development of a subset of medulloblastomas. The GSK-3β gene does not appear to be a target for inactivation in this tumor.     

MYCN AMPLIFICATION IN MEDULLOBLASTOMA

Prominent neuronal differentiation and MYCN amplification occur only in a small percentage of medulloblastomas (primitive neuroectodermal tumor of the cerebellum). In this article, we describe two medulloblastomas that showed a striking degree of neuronal differentiation with islands of mature neurons associated with abundant neuropil. In these differentiated foci, there were no mitoses or atypia, and the histology was reminiscent of a malformative or hamartomatous lesion. Both tumors were assessed for MYCN amplification by the novel technique of differential polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH). In case 1, MYCN amplification was in the 10 - fold range determined by differential PCR, while in case 2 the level of amplification was marked, with 20 - 30 copies compared with diploid controls. FISH analysis also confirmed the presence of MYCN amplification in both tumors. These two cases are of great interest, especially as they show MYCN amplification in medulloblastomas with marked degree of neuronal differentiation. Furthermore, in both cases, there was evidence of a slower and more indolent clinical course, suggesting a more favorable outcome in medulloblastoma with this degree of neuronal differentiation.     

Recent advances in Wilms tumor genetics

The past decade has witnessed substantial growth in our knowledge of the genes and loci that are altered in Wilms tumor. Although Wilms tumor was one of the original paradigms of Knudson's two-hit model of cancer formation, it has become apparent that several genetic events contribute to Wilms tumorigenesis. Recent research has identified targets and regulators of the first Wilms tumor gene, WT1, has uncovered several candidate genes at the second Wilms tumor locus, WT2, and has identified two familial Wilms tumor loci, FWT1 and FWT2. The recent discovery of activating beta-catenin mutations in some Wilms tumors has also implicated the Wnt signaling pathway in this neoplasm. Recurrent abnormalities of other loci, including 16q, 1p, and 7p, have indicated that these sites may harbor Wilms tumor genes. An enhanced understanding of these and other genetic lesions will provide the foundation for novel targeted Wilms tumor therapies.     

The use of gene expression analysis to gain insights into signaling mechanisms of metastatic medulloblastoma

Metastasis is the leading cause of treatment failure in medulloblastoma. Understanding the genetic regulation of metastasis may aid in the development of novel treatments. We therefore performed in silico analysis of the mRNA expression of 83 medulloblastomas compiled from two independent microarray studies by focusing on 135 genes most frequently linked to metastasis in other tumors. We then asked whether expression of these genes correlated with metastasis in the medulloblastoma array data sets. We found the platelet-derived growth factor receptor alpha, early growth response protein 1 and insulin-like growth factor 2 genes as well as several genes associated with MYCC and ERBB2 overexpressed by at least 2-fold in metastatic tumors in both array data sets. We conclude that these genes may interact to promote prometastatic signaling in medulloblastoma.     

Changeability of the fully methylated status of the 15q11.2 region in induced pluripotent stem cells derived from a patient with Prader‐Willi syndrome

Prader‐Will syndrome (PWS) is characterized by hyperphagia, growth hormone deficiency and central hypogonadism caused by the dysfunction of the hypothalamus. Patients with PWS present with methylation abnormalities of the PWS‐imprinting control region in chromosome 15q11.2, subject to parent‐of‐origin‐specific methylation and controlling the parent‐of‐origin‐specific expression of other paternally expressed genes flanking the region. In theory, the reversal of hypermethylation in the hypothalamic cells could be a promising strategy for the treatment of PWS patients, since cardinal symptoms of PWS patients are correlated with dysfunction of the hypothalamus. The genome‐wide methylation status dramatically changes during the reprograming of somatic cells into induced pluripotent stem cells (iPSCs) and during the in vitro culture of iPSCs. Here, we tested the methylation status of the chromosome 15q11.2 region in iPSCs from a PWS patient using pyrosequencing and a more detailed method of genome‐wide DNA methylation profiling to reveal whether iPSCs with a partially unmethylated status for the chromosome 15q11.2 region exhibit global methylation aberrations. As a result, we were able to show that a fully methylated status for chromosome 15q11.2 in a PWS patient could be reversed to a partially unmethylated status in at least some of the PWS‐iPSC lines. Genome‐wide DNA methylation profiling revealed that the partial unmethylation occurred at differentially methylated regions located in chromosome 15q11.2, but not at other differentially methylated regions associated with genome imprinting. The present data potentially opens a door to cell‐based therapy for PWS patients and, possibly, patients with other disorders associated with genomic imprinting.     

Genetic alterations in childhood medulloblastoma analyzed by comparative genomic hybridization

Despite intensive therapy, the survival of children with medulloblastoma remains disappointing. Moreover, children who survive are affected by serious long-term sequelae of treatment that impair their quality of life. In search of chromosomal aberrations indicative of sites involved in oncogenic transformation and in an attempt to find reliable prognostic markers, the authors analyzed 15 medulloblastomas by comparative genomic hybridization. All neoplasms showed chromosomal abnormalities. The most frequent losses were 17p (7/15 tumors), 8p and 11p (6/15), 10p, 1lq, 16q, and 20q (5/15), and 20p (4/15). Gains were recurrently found at 7q (10/15 tumors), 17q and 18q (9/15 tumors), 7p and 13q (7/15), 18p (6/15), and 1q, 4q, 6q. and 9p (5/15 tumors). Four tumors showed loss of 17p together with gain of 17q, suggesting an isochromosome 17q. High-level amplifications were seen at 1p34, 5p15, 13q34, and 18p11 (one tumor each), and at 2p15 in two tumors, one of which was proven to be N-Myc amplification. The overall pattern of alterations found in this study confirms the findings of other studies and adds two novel regions with chromosomal gains, at 13q and 18q. Previous reports on the relation between 17q gain and survival could not be confirmed, whereas amplification of N-myc or L-myc seems to indicate poor clinical outcome.