Investigation of leukemia cells from children with common acute lymphoblastic leukemia for genomic sequences of the primate polyomaviruses JC virus, BK virus, and simian virus 40.

BACKGROUND: An infectious etiology for childhood acute lymphoblastic leukemia (ALL) has long been suspected, although the characteristics of the putative childhood ALL-inducing agent(s) remain a mystery. We describe the testing of ALL leukemia cells for the presence of DNA sequences of the polyomavirus family: JC virus, BK virus, and simian virus 40 (SV40). PROCEDURE: Cryopreserved leukemia cells from 25 children between 2 and 5 years of age at the time of diagnosis and classified as having "common" ALL (B-precursor ALL expressing the CD19 and CD10 surface antigens) were tested for the presence of polyomavirus sequences using standard PCR methods. RESULTS: Human beta-globin gene sequences were detected in 22 of 25 leukemia specimens. However, polyomavirus sequences were not detected in any of the 22 specimens with amplifiable DNA. CONCLUSIONS: The inability to detect JC virus, BK virus, and SV40 virus DNA sequences in any of the 22 specimens with amplifiable DNA suggests that that these members of the polyomavirus family are unlikely to be causally associated with most childhood ALL. Published 1999 Wiley-Liss, Inc.     

人巨细胞病毒UL142基因在临床低传代分离株中的多态性

目的研究人巨细胞病毒(HCMV)UL142基因在低传代分离株中的多态性,探讨其多态性与HCMV先天性感染不同致病性之间的关系。方法27株经荧光定量PCR方法检测HCMVDNA阳性的低传代分离株进行UL142全序列PCR扩增,阳性13株PCR产物进行UL142基因测序及结果分析。结果27株分离株UL142PCR扩增,13株阳性,阳性率48.2%,以Toledo株为参考株序列比较表明,13株UL142可读框长度均与Toledo株相同,为921bp,编码307个氨基酸的蛋白。DNA序列变异均为核苷酸替换,不同临床分离株UL142基因与Toledo株进行同源性比较,结果在核苷酸水平为94.4%~95.9%,氨基酸水平为89.9%~99.6%。二级结构预测分为三种构象。大多数HCMVUL142蛋白重要功能基团位点在所有分离株中均高度保守,仅四个位点在一些分离株中存在缺失或新增。系统进化树分析除Toledo株外,13株核苷酸及氨基酸序列可分为3个基因组。结论13株临床低传代分离株HCMVUL142基因DNA及其编码产物的氨基酸序列比较保守,但仍存在一定多态性。未发现不同临床分离株UL142基因多态性与HCMV先天性感染不同疾病表现的关系。     

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。     

人羊水细胞中SLC25A13基因转录产物的克隆和序列分析

目的：克隆分析citrin蛋白编码基因SLC25A13在人羊水细胞中的mRNA编码区全长,并分析其转录子的序列特征,为从mRNA水平开展 Citrin 缺陷导致的新生儿肝内胆汁淤积症（NICCD）产前诊断提供实验依据。方法：选取1例接受 citrin 缺陷病产前诊断并已证实胎儿为851del4突变携带者的羊水标本;另1例取自无 citrin 缺陷病史患者的羊水细胞标本作为正常对照。抽提体外培养的羊水细胞总RNA,逆转录合成cDNA,通过巢式PCR扩增SLC25A13 cDNA编码区全长。PCR产物克隆后测序分析。结果：从2例羊水细胞标本中成功克隆到SLC25A13 cDNA编码区全长,并发现SLCA型转录子（正常型转录子mRNA）;在正常对照样本中发现 SLCB型转录子（外显子9和10之间CAG插入）;在851del4突变携带者标本中发现SLCC 型转录子（外显子5~11缺失）,未发现含851del4突变等位基因转录产物。结论：SLC25A13 cDNA编码区全长可以从羊水细胞中扩增获得,而外显子5~11缺失型转录子是SLC25A13基因一种新的转录子。在正常对照和包含851del4突变的杂合子胎儿SLC25A13基因转录产物中正常mRNA占据优势,提示胎儿无罹患NICCD风险。这些转录特征可以为NICCD产前诊断提供实验依据。     

Characterization and localization of the human genes for ribosomal ribonucleic acid.

The genetic sequence complementary to ribosomal RNA was separated from human DNA and its characteristics studied. Both 18S and 28S sequences were located on the same isolated strand. DNA-rRNA hybrids have a melting temperature of 80 degrees or 4 degrees less than that of native ribosomal DNA. Ribosomal genes behave as a satellite when complexed with Ag+ in the Cs2SO4 gradient. With in situ hybridization, these rRNA genes can be shown to chromosomally translocate. The study of a highly fluorescent chromosome indicates that the ribosomal DNA does not contribute to the fluorescent nature of the secondary constrictions. This study examines the molecular organization of human ribosomal genes. The chromosomal inheritance of these genes is explored by in situ hybridization.     

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.     

SV40T抗原基因介导尿源性干细胞永生化

目的 建立猿肾病毒40大T抗原(SV40Tag)永生化尿源性干细胞,为组织工程学及再生医学提供稳定的细胞来源.方法 利用脂质体(Lipofectamine2000)介导将含SV40Tag基因质粒pMPH-FRT-SV40T及转座酶质粒(transposase)共转染至临床分离的尿源性干细胞中.潮霉素(Hy-gromycin)筛选后阳性细胞克隆并连续传代,观察细胞形态学以及增殖情况,RT-PCR,免疫荧光等检测转染细胞的生物学特性,细胞裸鼠皮下注射检测其安全性.结果 筛选获得的阳性克隆扩大培养,即永生化尿源性干细胞(imrnorterlized urine derived stem cell,iUSC),增殖能力强,能连续传代培养.RT-PCR证实iUSC表达SV40Tag基因,而用翻转重组酶(Flippers recombination enzyme Flp)处理iUSC可敲除RFT-SV40Tag基因而逆转永生化.应用免疫荧光细胞化学技术检测该细胞株表达干细胞表面标志物CD73,CD44,CD90 (Thy-1),CD29 (Integrin),CD105 (Endoglin),CD166 (ALCAM),BMPR Ⅱ,SSEA4,CD117,CD133;BMP9可诱导其分化为成骨细胞,脂肪细胞,具备干细胞多向分化能力;转染细胞2×106个/点接种裸鼠,连续观察4周无肿瘤形成.结论 脂质体转染技术成功构建SV40Tag永生化尿源性干细胞,该细胞株仍具有干细胞增殖及多向分化潜能,为组织工程学及再生医学的体外研究和发展应用提供了安全稳定的细胞来源.     

Expression of SV40 T antigen gene in the oligodendroglia induced primitive neuroectodermal tumor-like tumors in the mouse brain

Primitive neuroectodermal tumors (PNET) are classified as the embryonal tumors developed in the brain, except for the cerebellum. Although many studies have been reported, the origin and pathogenesis of PNET are still unclear. In this study, we observed the development of undifferentiated tumors indistinguishable from PNET in the transgenic mice which expressed simian virus 40 T antigen (SV40-Tag) selectively in the oligodendroglia under the control of mouse myelin basic protein gene promoter. These PNET-like tumors reproducibly developed in the brain stem of the founder mice and the transgenic progeny derived from one founder mouse. Oligodendroglia-specific expression of SV40-Tag in these transgenic mice was observed by immunohistochemical analysis. Furthermore, expression of the oligodendroglia-specific marker genes was decreased in the tumors as well as in the transgenic brains. These findings suggested that tumors developed in transgenic mice were indistinguishable from PNET, and one of them showed oligodendroglia-like characteristics. Consequently, this transgenic line is a useful animal model to study the pathogenesis of undifferentiated tumor.     

法洛四联症患儿类视黄酸受体α基因启动子区序列分析

目的 对法洛四联症（TOF）患儿类视黄酸受体α（RXRA）基因启动子区序列进行分析,探讨RXRA基因变异与TOF的关联性。方法 采取病例对照研究方法,以2007年4月至2012年12月心导管检查及外科手术证实为TOF患儿为TOF组,以同时期年龄和性别与TOF组匹配的健康儿童为对照组。采集静脉血,提取基因组DNA, PCR扩增RXRA基因转录起始位点（TSS）上游1 417 bp的启动子区序列,扩增产物采用ABI Prism Bigdye系统进行测序。结果 TOF组纳入213例（男135例,女78例）,平均年龄1.8岁;对照组纳入500名(男310名,女190名),平均年龄2.5岁。①RXRA基因TSS上游1 191 bp处检测出1个杂合突变,即-1 191A>AG（TSS定为+1）;检测出3个新发SNP,即-1 287C>CT、-800C>CA及-760C>CT。②利用http://www.cbrc.jp/research/db/TFSEARCH.html网站进行分析,发现在该4个位点及其附近有多个转录因子结合位点。-1 191A>AG导致新的CpG位点产生,-800C>CA导致原有CpG位点消失。这些新产生的CpG位点的甲基化可能会影响转录因子的结合,从而影响RXRA基因转录水平,进一步导致RXRA蛋白水平的变化。结论 TOF患儿RXRA基因启动子区序列变化可能通过影响RXRA表达水平而导致TOF的发生。     

Clinical implications of unstable DNA repeat sequences

In this article we review the clinical and genetic features characteristic of a number of diseases recently explained by a novel genetic mechanism: unstable segments of the genome containing trinucleotide repeat sequences. Disorders identified to date are mostly progressive, and display unusual inheritance patterns such as anticipation. Anticipation is manifested as an earlier age at onset or a more severe phenotype in later generations of a family, and can be correlated to an increased repeat expansion size. Thus in later generations the disease onset can take place in childhood whereas affected individuals in earlier generations had only adult symptoms. Paediatric cases of typically adult disorders have been shown to be caused by exceptionally long repeat sequences. Anticipation has been observed in a number of disorders not yet identified at the molecular level. Such disorders could be caused by repeat expansions, and are presently subject to intense research efforts. If repeat sequence expansions are related to these disorders, the longest expansions should be seen in the childhood cases, making these the optimal cases to study. Various DNA-based methods have been developed for the detection of these mutations, making possible preclinical and prenatal diagnostics as well as detection of novel expansions.