Mutations analysis of STK_(11) gene in Chinese families with Peutz-Jeghers syndrome

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease that is characterized by multiple gastrointestinal hamartomatous polyps and melanin spots on lips and buccal mucosa at young age[1,2]. Previous studies have demonstrated that PJS predisposes carriers to cancers of gastrointestinal tract, uterus, ovary, testis, breast and other extragastrointestinal organs[3—5]. The STK11 gene, encoding a serine/threonine kinase at chro-mosome 19p13.3, was identified in 1998 as the main causativ…     

乙肝病毒前C基因变异对宿主T细胞亚群的影响及其临床意义的研究

目的　通过检测乙型肝炎病毒 (HepatitisBVirus,HBV)变异株感染患者外周血T细胞亚群分布的变化 ,探讨HBV基因组前C区 1896位基因变异与宿主机体免疫水平的关系 .方法　用酶联免疫吸附试验 (ELISA)检测 5 5名患者血清乙型肝炎病毒标志物 (HBVM)HBsAg ,抗 HBs,HBeAg ,抗HBe及抗 HBc;采用定性PCR法检测上述患者血清HBVDNA ;通过限制性片段长度多态性分析法 (RFLP)检测发生前C区 1896位基因突变的HBV变异株 ;采用流式细胞术 (flowcytometry)对上述患者外周血的CD4 + T细胞 ,CD8+ T细胞 ,CD3+ T细胞含量进行检测 ,将检测结果同所设健康对照组进行比较 ,并对数据进行统计学分析 .结果　在 5 5名患者中 ,HBsAg(+) ,anti HBs(- ) ,HBeAg(+) ,anti HBe(- ) ,anti HBc(+)者共 2 2例 ,其HBVDNA检测结果均为阳性 ,在这2 2例患者中 ,有 2 0例被检出为单纯HBV野毒株感染 ,另有 2例被检出为变异株和野毒株混合感染 ;HBsAg(+) ,anti HBs(- ) ,HBeAg(- ) ,anti HBe(+) ,anti HBc(+)者共 33例 ,其中HBVDNA阳性者为 18例 .在 18例HBeAg(- )而HBVDNA (+)的患者中 ,有 17例被检出为HBV前C区 1896位变异株感染 .变异株感染患者外周血CD4 + T细胞含量较之健康对照组有所减低 (P <0 .0 1) ,CD8+ T细胞含量较之健康对照组也有所     

Mutations analysis of STK11 ene in Chinese families with Peutz-Jeghers syndrome

Peutz-Jeghers syndrome (PJS) is an autosomal dominantly inherited disease characterized by multiple gastrointestinal hamartomatous polyps and melanin spots on lips and buccal mucosa, with an increased risk for various cancers. ThePJS gene, a potential tumour suppressor gene, encoding a serine/ threonie kinase (STK11), was mapped to chromosome 19p13.3. To investigate the mutations of STK11 gene in Chinese with PJS, we analyzed its coding sequence in fifteen patientsand twenty unaffected members of six families, including three multigenerational families with PJS and three sporadic families with PJS, by PCR, PCR-DHPLC and DNA sequencing techniques. Ten point mutations were found in the six families, including five missense mutations, one acceptor-splice site mutation, a nonsense mutation and three silent mutations. Our data showed that five missense mutations occurrd at codon 123 (CAG to CAT) in exon 2, codon 161 (ATT to AGT) in exon 4,codon 194 (GAC to GAG) in exon 4, codon 245 (CTC to TTC) in exon 5 and codon 354 (TTC to TTG) in exon 8. One kind of nonsense mutation was detected at codon 37(CAG to TAG) in exon 1. Furthermore, we found an intronic mutation at a splice-acceptor site: a one base substitution from AG to AA in intron 4. These mutations were not detected in 20 normal DNA samples. In three sporadic families, only in one patient, we detected a missense mutation in exon 5. In addition, we found three silent mutations, which may cause polymorphisms of STK11 gene in introns 1(+36), 3(-51) and 5(+27). These results indicated that the point mutation in STK11 might be involved in PJS pathogenesis. Mutation frequency is higher in the families suffering PJS in three or more generations than that of the sporadic cases.     

Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia

DNAs from four out of five patients with acute myeloid leukaemia (AML) tested by an in vivo selection assay in nude mice using transfected mouse NIH 3T3 cells were found to contain an activated N-ras oncogene. Using a set of synthetic oligonucleotide probes, we have detected a mutation at codon 13 in all four genes. The same codon is mutated in an additional AML DNA that is positive in the focus-formation assay on 3T3 cells. DNA from the peripheral blood of one patient in remission does not contain a codon 13 mutation.     

An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region

Deletions of muscle mitochondrial DNA (mtDNA) have recently been found in patients with mitochondrial myopathy. However, as most of the described cases were sporadic, and individual deletions involved different portions of mtDNA, the mechanism(s) producing the molecular lesions, as well as their mode of transmission, remain unclear. By studying families with mtDNA heteroplasmy, valuable information can be obtained about the role of inheritable factors in the pathogenesis of these disorders. We have studied four members of a family with autosomal dominant mitochondrial myopathy. Multiple deletions, involving the same portion of muscle mtDNA, were identified in all patients. Sequence analysis of the mutant mtDNAs, performed after DNA amplification by the polymerase-chain reaction showed that all the deletions start within a 12-nucleotide stretch at the 5' end of the D-loop region, a site of active communication between the nucleus and the mtDNA. The data indicate that a mutation of a nuclear-coded protein can destroy the integrity of the mitochondrial genome in a specific, heritable way.     

JAK2基因单倍型46/1对中国人群骨髓增殖性肿瘤易感性的影响

从91例骨髓增殖性肿瘤(MPNs)患者及1 028例健康志愿者(均为中国人)外周血中提取基因组DNA,应用等位基因特异性PCR(ASP)技术,检测作为蛋白质酪氨酸激酶JAK2单倍型46/1标签的rs12343867和rs10974944位点的基因型,并结合临床资料,应用SNPstats软件进行统计学分析.实验结果表明:MPNs患者JAK2单倍型46/1出现的几率明显高于健康志愿者(P0.000 1);MPNs患者的JAK2单倍型46/1更易在JAK2V617F突变阳性MPNs患者(n=70)中出现(P0.000 1);具有JAK2单倍型46/1的中国人群患MPNs的风险增加显著,该结果与测试欧美人群的结果一致.     

Loss of Omi mitochondrial protease activity causes the neuromuscular disorder of mnd2 mutant mice

The mouse mutant mnd2 (motor neuron degeneration 2) exhibits muscle wasting, neurodegeneration, involution of the spleen and thymus, and death by 40 days of age. Degeneration of striatal neurons, with astrogliosis and microglia activation, begins at around 3 weeks of age, and other neurons are affected at later stages. Here we have identified the mnd2 mutation as the missense mutation Ser276Cys in the protease domain of the nuclear-encoded mitochondrial serine protease Omi (also known as HtrA2 or Prss25). Protease activity of Omi is greatly reduced in tissues of mnd2 mice but is restored in mice rescued by a bacterial artificial chromosome transgene containing the wild-type Omi gene. Deletion of the PDZ domain partially restores protease activity to the inactive recombinant Omi protein carrying the Ser276Cys mutation, suggesting that the mutation impairs substrate access or binding to the active site pocket. Loss of Omi protease activity increases the susceptibility of mitochondria to induction of the permeability transition, and increases the sensitivity of mouse embryonic fibroblasts to stress-induced cell death. The neurodegeneration and juvenile lethality in mnd2 mice result from this defect in mitochondrial Omi protease.     

Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae

The migration of genetic information from ancestral prokaryotic endosymbionts into eukaryotic nuclei is thought to have had an important role in the evolution of mitochondria and chloroplasts. Here we describe an assay for the detection of movement of DNA between mitochondria and the nucleus in yeast. Because recombinant plasmid DNA replicates after transformation into mitochondria of yeast strains lacking endogenous mitochondrial DNA we were able to propagate the nuclear genetic marker URA3 in mitochondria. As expected, the wild-type URA3 gene in mitochondria failed to complement the uracil auxotrophy (Ura-) caused by a nuclear ura3 mutation. But selection of Ura+ prototrophs from a Ura- strain carrying URA3 on a plasmid in its mitochondria enabled us to detect plasmid movement to the nucleus. Conversely, as the plasmid used also contained the mitochondrial gene COX2 required for respiratory growth, we were able to set up corresponding selections to detect migration of DNA from the nucleus to the mitochondria. Our results show that, in yeast, DNA escapes from mitochondria and appears in the nucleus at a surprisingly high frequency (approximately 2 x 10(-5) per cell per generation). But the rate at which DNA makes the journey in the opposite direction--nucleus to mitochondria--is apparently at least 100,000 times less.     

A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome

Patients with a myelodysplastic syndrome (MDS) which has a risk of leukaemic change exhibit a variable clinical course. It has been suggested that the development of leukaemia in patients with MDS may be related to chromosomal abnormalities or genetic alterations: somatic mutation of the N-ras gene is now considered to be a critical step in the genetic basis of human leukaemogenesis. Here we report that DNAs of bone-marrow cells from three out of eight patients with MDS contained an activated N-ras oncogene, as detected by an in vivo selection assay in nude mice with transfected NIH 3T3 cells. Molecular analysis revealed the same single nucleotide substitution at codon 13 in all three transforming N-ras genes. Each of the three patients showed a progression of the disease and a resulting leukaemic change within the following year. Our observation of the mutation at codon 13 in leukaemic cell DNAs from all three cases suggests that activation of the N-ras gene is important in the development of leukaemia in some MDS cases.     

Mitochondrial genome variation and the origin of modern humans

The analysis of mitochondrial DNA (mtDNA) has been a potent tool in our understanding of human evolution, owing to characteristics such as high copy number, apparent lack of recombination, high substitution rate and maternal mode of inheritance. However, almost all studies of human evolution based on mtDNA sequencing have been confined to the control region, which constitutes less than 7% of the mitochondrial genome. These studies are complicated by the extreme variation in substitution rate between sites, and the consequence of parallel mutations causing difficulties in the estimation of genetic distance and making phylogenetic inferences questionable. Most comprehensive studies of the human mitochondrial molecule have been carried out through restriction-fragment length polymorphism analysis, providing data that are ill suited to estimations of mutation rate and therefore the timing of evolutionary events. Here, to improve the information obtained from the mitochondrial molecule for studies of human evolution, we describe the global mtDNA diversity in humans based on analyses of the complete mtDNA sequence of 53 humans of diverse origins. Our mtDNA data, in comparison with those of a parallel study of the Xq13.3 region in the same individuals, provide a concurrent view on human evolution with respect to the age of modern humans.     

Deletion of genes on chromosome 1 in endocrine neoplasia

Recent studies have identified normal cellular DNA sequences which are lost in the development of embryonal and adult tumours. These tumours are thought to arise after a primary mutation in one allele of such a sequence is followed by loss of its normal homologue. In familial cases, the primary mutation is transmitted in the germ line. The secondary mutation may involve a substantial loss of chromosomal material and thus lead to identification of the site of the inherited mutation. We have examined constitutional and tumour genotypes of medullary thyroid carcinomas and phaeochromocytomas which develop in the dominantly inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2) to locate the predisposing gene in this syndrome. We observed deletion of a hypervariable region of DNA on the short arm of chromosome 1 in seven out of fourteen tumours. Analysis of the parental origin of the deleted allele in two families showed that it was derived from the affected parent in one case, which suggests that the deletion does not reflect the site of the inherited mutation in MEN2. The deleted region is distal to the breakpoint commonly detected in neuroblastomas, which share with the tumours of MEN2 embryological origin from neuroectoderm.     

一母系遗传非综合征耳聋家系的线粒体DNA突变分析

在问卷调查及家系随访的基础上,在安徽省淮北市收集到一母系遗传非综合征耳聋家系,利用聚合酶链式反应-限制片段长度多态性分析(PCR-RFLP)和测序技术,检测了该家系成员线粒体DNA(mtDNA)上可导致非综合征耳聋的两个突变热点处(12S rRNA基因上的1 555位点和tRNASer(UCN)基因上的7 445位点)的碱基变化,发现该家系所有母系成员的mtDNA上都有A1555G同质型突变,但7 445位点无异常;进而对该家系两个表型明显不同母系成员(一例具有先天性耳聋表型,另一例听力正常)的mtDNA进行全长测序,结果未在mtDNA上发现除A1555G以外的其他位点突变,只发现了27处多态性序列变化,且两成员的mtDNA无序列差异.说明mtDNA上的A1555G同质型突变是该家系部分母系成员致聋的分子生物学基础之一;推测该家系A1555G突变携带者临床表型的差异可能与mtDNA多态性无关,而更可能是核修饰基因与A1555G突变协同作用的结果.     

Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome

Tumour suppressor genes, whose usual function seems to be controlling normal cell proliferation, have been implicated in many inherited and sporadic forms of malignancies Much evidence supports the concept of tumour formation by loss-of-function mutations in suppressor genes, as predicted by the two-hit model of Knudson and DeMars. The suppressor gene, p53, is affected in such a manner by numerous mutations, which occur in a variety of human tumours. These mutations usually represent the loss of one allele and the substitution of a single base in the other. We have now analysed the p53 gene in a family affected by Li-Fraumeni syndrome, a rare autosomal dominant syndrome characterized by the occurrence of diverse mesenchymal and epithelial neoplasms at multiple sites. In some instances the neoplasms seem to be related to exposure to carcinogens, including ionizing radiation. The Li-Fraumeni family that we studied had noncancerous skin fibroblasts (NSF) with an unusual radiation-resistant phenotype. DNA derived from the NSF cells of four family members, spanning two generations, had the same point mutation in codon 245 (GGC----GAC) of the p53 gene. This mutation leads to substitution of aspartic acid for glycine in one of the regions identified as a frequent target of point mutations in p53. The NSF cell lines with the mutation also retained the normal p53 allele. This inherited p53 mutation may predispose the members of this family to increased susceptibility to cancer.     

人类线粒体转录因子B1和B2与裂殖酵母线粒体转录聚合酶Rpo41相互作用的初步研究

由L02型人肝脏细胞cDNA通过PCR技术获取到线粒体转录因子B1和B2的ORF片段(NM_022366和NM_016020);构建表达质粒pGEX-4t-1/GST-mtTFB1及pMAL-c-2X/MBP-mtTFB2,并转化到大肠杆菌BL21(DE3)中表达,经亲和层析,分子筛和阳离子交换柱得到纯度较高的蛋白.利用体外pull-down实验证实TFB1和TFB2均与来源于裂殖酵母(Schizosaccharomyces pombe)的线粒体RNA聚合酶(Rpo41)存在分子间相互作用.在裂殖酵母中过表达h-TFB1和h-TFB2,利用实时荧光定量PCR检测,2个蛋白在酵母体内均对线粒体基因转录产生影响.     

An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome.

Here we report the identification and characterization of a gene defect causing Waardenburg's syndrome with hearing loss in a large Brazilian family. This demonstrates a mutation causing Waardenburg's syndrome as well as a mutation causing a form of congenital deafness. The mutation was found in the HuP2 gene, a member of the paired domain family of proteins that bind DNA and regulate gene expression. The mutation occurred in 100% of the cases with the disease in this family and was absent in a random sample of 50 unrelated control subjects. Identification of the Waardenburg's syndrome gene and future characterization of its gene product is likely to increase our understanding of the pathogenesis of this disorder and may allow prevention of deafness of this type.     

Frequent mtDNA mutations and its role in gastric carcinogenesis

In order to disclose the relationship between mutations of mitochondrial DNA (mtDNA) and gastric carcinogenesis, we screened the entire mtDNA sequence in 30 cases of human gastric cancer and matched normal tissues by using denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. Our data showed that high frequency (66.7%, 20/30) of mitochondrial genome mutation occurred in gastric cancer. Among these variants, 17 cases (56.7%, 17/30) were identified to be somatic mutation. High level mutant frequency was found in ND4, ND5 coding genes and D-loop control region, which was 36.7%, 26.7% and 30% respectively. Comparing with complexes Ⅲ, Ⅳ and Ⅴof the electron transport chain, we found that variants appeared to be more frequent in the subunit genes of complexⅠ. Most of mutations were base substitutions (85.4%, 41/48). Our results suggested that mutations of subunit genes encoding complexⅠ, especially ND3, ND4 and ND5 genes, might contribute to human gastric carcinogenesis.     

头颈部鳞癌中p53-mdm2基因异常的研究

目的:检测头颈部鳞癌患者p53基因突变、mdm2基因扩增的状况,了解其与头颈部鳞癌患者的性别、鳞癌分级、淋巴结转移等的相关性及两异常基因的相关性。方法:收集50例行手术切除的头颈部鳞癌患者的新鲜肿瘤组织及其相应的癌旁正常组织,提取标本DNA;用PCR-SS-CP-银染法检测p53基因第5~8外显子的突变状况;用dPCR法检测mdm2基因扩增情况;采用SPSS 10.0统计软件包行2检验分析实验结果。结果:在50例头颈部鳞癌患者标本中,检测出17例存在p53基因突变,突变率为34%,所有的癌旁正常组织未发现p53基因突变;在50例鳞癌标本中检测出6例标本存在mdm2基因扩增,扩增率为12%,其中有一例同时存在p53基因突变;p53基因突变、mdm2基因扩增与患者的鳞癌分级、淋巴结转移、性别等相关性分析结果均无统计学意义(P>0.05)。结论:p53基因突变与mdm2基因扩增在头颈部鳞癌中较常见,可能是头颈部鳞癌发生发展的主要分子机制。