在两个X连锁显性腓骨肌萎缩症家系中发现同一GJB1基因突变Glu208Lys

在两个X连锁显性腓骨肌萎缩症(Charcot-Marie-Tooth disease, CMT) 家系中进行了GJB1基因的突变分析。提取基因组DNA, PCR(polymerase chain reaction)反应扩增GJB1基因编码序列, 进行单链构象多态性(single strand conformational polymorphism, SSCP)分析, 对有差异SSCP带型的PCR产物进行测序, 结果在两家系中发现同一GJB1基因c.622G→A (Glu208Lys)突变。所发现的突变位点在国内尚未报道。     

K-ras codon 12 mutations may be detected in serum of patients suffering from adeno- and large cell lung carcinoma. A preliminary report

We investigated the presence of K-ras mutations in the serum of 40 patients with respectable stages of adeno- and large cell lung carcinomas. Mutations in codon 12 of the K-ras gene were examined by enriched PCR method in DNA extracts from surgical specimens and serum samples. K-ras mutations were detected in 20 (51%) of 39 analyzed tumours, and in 7 (35%) of 20 patients with K-ras gene mutation positive tumours, a mutation was found in the serum DNA. We also found K-ras mutation in two (10.5%) of 19 serum samples obtained from patients whose tumours were not found to harbor mutation and in one serum sample from patient without tumour sample available for investigation. All of the 14 control healthy persons were negative for serum DNA K-ras mutation assay. Although our results are preliminary they show that K-ras mutation may be detected in serum of patients suffering from adeno- and large cell lung carcinomas and confirm the suggestion that at least a part of a free-cell extracellular blood DNA in cancer patients has neoplastic origin and may become a noninvasive target for genetic investigations of lung cancer patients.     

Mismatch repair gene mutations in Chinese HNPCC patients

To explore the characteristics of DNA mismatch repair gene mutations in Chinese patients with hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome, the MLH1 and MSH2 genes from probands of 76 HNPCC families were sequenced. By doing so, two frame-shift mutations, three splice-site mutations and fourteen missense mutations (thirteen missense mutations and one nonsense mutation) were identified in the MLH1 gene. In addition, one splice-site mutation and six missense mutations were detected in the MSH2 gene. None of these mutations were detected in 100 matched healthy controls. The remaining mutation-negative cases were subjected to large fragment deletion analysis using multiplex ligation-dependent probe amplification (MLPA). By doing so, five large fragment deletions were detected in the MSH2 gene. No large fragment deletions were detected in the MLH1 gene. We conclude that the MLH1 and MSH2 genes in Chinese HNPCC families exhibit broad mutation spectra.     

畸精子症病人的睾丸特异性表达基因SPEM1筛查分析

目的:探讨睾丸特异性表达基因SPEM1突变与畸精子症患者之间的关系.方法:收集从2005年4月至2007年3月临床上不明原因的畸精症患者113份外周血标本以及100份正常生育能力男子的外周血标本,抽提其DNA.然后采用PCR技术、变性高效液相色谱技术(DHPLC)以及测序等手段对全部DNA样本进行该基因的突变筛查.结果:在畸精症患者中发现1个新的未见报道的多态性位点;尚未发现有基因突变或微缺失.结论:SPEM1基因突变或缺失不是引起本组畸精子症病人的主要致病基因.该基因在对畸精子症所致不育的诊断价值尚需进一步研究.     

Infrequent but significant p21/WAF1/CIP1 gene alteration in synchronous or metachronous transitional cell carcinoma

p53 inducible cyclin dependent kinase inhibitor, p21/WAF1/CIP1(p21), played a pivotal role for G1 arrest when cells received genotoxic stimuli. p21 could be a putative tumor suppressor gene, since its dysfunction may lead to accumulation of genomic alteration. We investigated the p21 and p53 status using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, in eight patients who had synchronous or metachronous urothelial tumors. Loss of heterozygosity (LOH) of p21 gene was detected in one coincidental tumor in one case. p21 positive cells were detected by immuno-histochemical staining in all tumors in one case, and in one coincidental tumor in two cases. Among p21 positive cells in these three cases, no p53 mutations were detected, whereas no p21 positive cells were detected in other cases with a p53 mutation. These findings suggested that in transitional cell carcinoma (TCC) p21 gene mutation is infrequent like the p53 gene mutation, but that LOH might be important in the inactivation of p21.     

Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold

Three-way junctions (3H) are the simplest and most commonly occurring branched nucleic acids. They consist of three double helical arms (A to C), connected at the junction point, with or without a number of unpaired bases in one or more of the three different strands. Three-way junctions with two unpaired bases in one strand (3HS2) have a high tendency to adopt either of two alternative stacked conformations in which two of the three arms A, B and C are coaxially stacked, i.e. A/B-stacked or A/C-stacked. Empirical stacking rules, which successfully predict for DNA 3HS2 A/B-stacking preference from sequence, have been extended to A/C-stacked conformations. Three novel DNA 3HS2 sequences were designed to test the validity of these extended stacking rules and their conformational behavior was studied by solution NMR. All three show the predicted A/C-stacking preference even in the absence of multivalent cations. The stacking preference for both classes of DNA 3HS2 can thus be predicted from sequence. The high-resolution NMR solution structure for one of the stacked 3HS2 is also reported. It shows a well-defined local and global structure defined by an extensive set of classical NMR restraints and residual dipolar couplings. Analysis of its global conformation and that of other representatives of the 3H family, shows that the relative orientations of the stacked and non-stacked arms, are restricted to narrow regions of conformational space, which can be understood from geometric considerations. Together, these findings open up the possibility of full prediction of 3HS2 conformation (stacking and global fold) directly from sequence.     

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.     

Cellular and molecular effects of bleomycin are modulated by heat shock in Saccharomyces cerevisiae

To study some mechanisms underlying the stress responses in eukaryotic cells, we investigated the effect of heat shock (HS) on the induction of DNA double strand breaks as well as on potentially lethal and mutagenic events induced by the radiomimetic antibiotic bleomycin (BLM) in Saccharomyces cerevisiae. Haploid wild-type yeast cells in the logarithmic phase of growth were exposed to different concentrations of BLM (0-30 microg/ml, 1.5 h) without and with a previous HS (38 degrees C, 1 h). Immediately after treatments, survival as well as mutation frequency were determined, and quantitative analysis of chromosomal DNA by laser densitometry were performed both immediately after treatments and after incubation of cells during different time intervals in liquid nutrient medium free of BLM. Our results indicate that HS induces resistance to potentially lethal and mutagenic effects of BLM. Quantitative analysis of chromosomal DNA performed immediately after treatments showed the same DNA fragmentation, either upon BLM as single agent or preceded by HS. However, HS pretreated cells incubated during 4 h in liquid nutrient medium free of BLM repaired DNA double strand breaks more efficiently as compared to non-pretreated cells. On this basis, we propose that the observed HS-induced resistance to BLM depends on a regulatory network acting after DNA-induced damage, which includes genes involved in DNA repair, HS response and DNA metabolism.     

Nonsense mutations of the von Willebrand factor gene in patients with von Willebrand disease type III and type I.

von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormalities of the von Willebrand factor (vWF). Genomic DNA from 25 patients with vWD type III, the most severe form of the disease, was studied using PCR followed by restriction-enzyme analysis and direct sequencing of the products. Nonsense mutations (CGA----TGA) were detected in exons 28, 32, and 45 by screening of all the 11 CGA arginine codons of the vWF gene. Two patients were found to be homozygous and five heterozygous for the mutation. Both parents and some of the relatives of the homozygous patients carry the mutation. These are the first reported examples of homozygous point mutations associated with the severe form of vWD. In the three heterozygous probands, one of the parents carried the mutation and had vWD type I. Family studies including parents and family members with or without vWD type I indicated that these three heterozygous patients are likely to be compound heterozygous. Twenty-one individuals from these seven families with vWD type I were found to be heterozygous for the mutation.     

Identification of five rare mutations including a novel frameshift mutation causing beta zero-thalassemia in Thai patients with beta zero-thalassemia/hemoglobin E disease.

6 out of 14 uncharacterized beta-thalassemia alleles from 187 Thai beta-thalassemia/HbE patients were identified by direct sequencing of DNA amplified by polymerase chain reaction. A novel mutation occurring from an insertion of adenosine in codon 95, which results in a shift of the reading frame with terminator at the new codon 101, was detected in one patient. In addition, two frameshift mutations not previously reported among the Thai population were also detected in 3 patients: one with a deletion of thymidine in codon 15 and two with an insertion of cytidine in codons 27/28. A frameshift mutation that occurred from a cytidine deletion in codon 41 was also found in one patient in this study. The remaining case was an amber mutation, GAG-TAG, in codon 43 in exon 2 of the beta-globin gene. These mutations bring the number of mutations known to be present in the Thai population to a total of 20, 15 of which were detected in beta-thalassemia/HbE patients.     

Dynamic changes of hepatitis B virus polymerase gene including YMDD motif in lamivudine-treated patients with chronic hepatitis B

The mutation of YMDD motif of hepatitis B virus (HBV) polymerase gene is the most frequent cause in HBV resistant to lamivudine. The aim of the study was to investigate variation features of HBV polymerase gene in chronic hepatitis B (CHB) patients before and after lamivudine treatment. From the serum samples of five CHB patients before and after 12 months of lamivudine treatment, HBV polymerase gene was amplificated and positive DNA fragments were cloned into JM105 competent cell. Twenty positive clones of every sample were checked with mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and YMDD variants were sequenced. Among five patients after 12 months of lamivudine treatment, M552I mutations in two patients with HBV DNA rebounding and D553G mutation in one non-responder were detected except two patients with negative HBV DNA consecutively. In summary, D553G mutation is probably one of the reasons that caused non-responders during lamivudine treatment. The mutations of YMDD motif occurred after lamivudine treatment are caused by the induction of lamivudine.     

Detection of His1069Gln mutation in Wilson disease by bidirectional PCR amplification of specific alleles (BI-PASA) test

Wilson disease (WD) is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase, ATP7B. The majority of known mutations affecting this gene are frequent in different populations, which may help to introduce rapid diagnostic procedures based on direct DNA analysis into routine clinical practise. The His1069Gln mutation in exon 14 is the most frequent one, accounting for 30-60% of all mutations in Caucasian patients. The aim of the present work was to introduce DNA-based direct analysis into routine molecular screening for the above mutation in Slovak WD patients and to assess its frequency in patients as well as in a control population. Twenty seven clinicaly diagnosed patients from twenty five families, twenty relatives of index patients and three hundred and six control DNA samples were tested using two different DNA-based methods: the earlier described amplification created restriction site (ACRS) for Alw21I in combination with nested PCR and the amplification refractory mutation system (ARMS). In 18 of 25 unrelated patients (72%), the mentioned genetic defect was present in at least one copy. In ten of them (40%), the above mutation was detected in homozygous and in eight individuals (32%) in heterozygous state. In seven WD patients (28%), this mutation was not detected. The allele frequency of His1069Gln in Slovak patients with WD was 56%, which was higher as reported in other populations. In a control group of 306 random DNA samples (612 alleles), the His1069Gln mutation was observed in 3 samples (carrier frequency 1%; allele frequency 0.49%). These frequencies correspond to figures observed in different population of European origin. Taken together, we have provided further evidence that the His1069Gln mutation is the prevalent ATP7B mutation in central-european WD patients. Although both methods used in this study worked in our hands reliably, there are in every-day use some drawbacks and limitations inherent to them (PCR reactions in two tubes, possibility of star activity or not complet digestion by restriction endonuclease, etc.). Therefore we developed a simpler, cost effective and rapid DNA diagnostic test based on bidirectional amplification of specific alleles (BI-PASA), which enables detection of homozygotes (wild and mutant) and heterozygotes, respectivelly, in one PCR reaction. The test was highly sensitive and specific, yielding no false-positive or false-negative results. Its reliability and discriminating power was tested on samples of 27 WD patients and 120 random control DNA's, previously genotyped by above mentioned methods. Comparing results of BI-PASA with ACRS and ARMS tests showed 100% concordance.     

Application of Combined Long Amplicon Sequencing (CoLAS) for Genetic Analysis of Neurofibromatosis Type 1: A Pilot Study

Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.     

Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir.

The same point mutation in the human cytomegalovirus UL97 open reading frame was found in three independently isolated ganciclovir-resistant mutants of strain AD169. Point mutations in the DNA polymerase genes of these strains have been previously identified (N.S. Lurain, K.D. Thompson, E.W. Holmes, and G.S. Read, J. Virol. 66:7146-7152, 1992). All three strains are, therefore, double mutants. To determine the contribution of the UL97 mutation to the high ganciclovir resistance of these mutants, the mutation from the ganciclovir-resistant strain D6/3/1 was transferred to the wild-type strain AD169 to produce the recombinant R6HS. The ganciclovir resistance of R6HS is 4-fold lower than that of D6/3/1 but 10-fold higher than that of AD169. R6HS, like AD169, is sensitive to the nucleotide analogs (S)-1-[(3-hydroxy-2-phosphonylmethoxy) propyl]adenine and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine. Ganciclovir phosphorylation in R6HS-infected cells was at the same reduced level as that found in cells infected with the parental mutant D6/3/1. The same G-to-T transversion at nucleotide 1380 in the UL97 coding sequence is present in both R6HS and D6/3/1. This mutation results in the substitution of isoleucine for methionine at amino acid residue 460. In an alignment of the R6HS UL97 amino acid sequence with the amino acid sequences of a wide range of protein kinase family members, methionine 460 lies within a highly conserved region which may function in nucleotide binding and phosphate transfer.     

有和无甘油的聚丙烯酰胺胶在检测突变时的差别

有文献报道在非变性的聚丙烯酰胺中加入甘油可提高SSCP检测的灵敏度。我们的实验结果建议研究者在进行SSCP筛查未知突变时最好采用不加甘油的非变性的聚丙烯酰胺胶,这既省力省钱,又灵敏。在判读SSCP胶时,千万不要看到在双链带位置有一条比正常迁移率慢的带就判定为插入突变。此时要判定突变的性质,最好测序。 Abstract：It was reported that glycerol in the non-denatured SSCP polyacrylamide gel could increase the sensibility of detecting mutation. We detected the mutation of PKD 1 gene in the patients with autosomal dominant polycystic kidney disease.PCR com bined with SSCP(single-strained conformation polymorphism),the non-denatured 10% polyacrylamide gel without glycerol or 10% polyacrylamide gel with 5% glycerol and DNA sequencing method were used.Our results showed that four single strand b ands were found in the non-denatured polyacrylamide gel without glycerol while t wo single strand bands were found in the polyacrylamide gel with glycerol in the same patient.Sequence showed there is a deletion of G in one DNA molecular and a G→A substitution in another DNA molecular in the patient with abnormal shift SSCP bands.Therefore, our experiment suggested that non-denat ured polyacrylamide gel was better than the polyacrylamide gel with glycerol in detection mutation,and it will save labor and money.It also suggeste d that one basedeletion can cause a slow double-strand DNA following the normal double strand band,which was caused by the heterogeneous DNA molecule formed bet ween the normal DNA strand and the one base deletion DNA strand with the protrud ing base.Our results suggest that when judging mutation in SSCP gel,it is not re liable to decide that mutation is inversion according to slow mobility in the ge l,and when the characteristic of mutation need to be judged,it must be sequenced .     

Constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma.

In most patients with isolated unilateral retinoblastoma, tumor development is initiated by somatic inactivation of both alleles of the RB1 gene. However, some of these patients can transmit retinoblastoma predisposition to their offspring. To determine the frequency and nature of constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma, we analyzed DNA from peripheral blood and from tumor tissue. The analysis of tumors from 54 (71%) of 76 informative patients showed loss of constitutional heterozygosity (LOH) at intragenic loci. Three of 13 uninformative patients had constitutional deletions. For 39 randomly selected tumors, SSCP, hetero-duplex analysis, sequencing, and Southern blot analysis were used to identify mutations. Mutations were detected in 21 (91%) of 23 tumors with LOH. In 6 (38%) of 16 tumors without LOH, one mutation was detected, and in 9 (56%) of the tumors without LOH, both mutations were found. Thus, a total of 45 mutations were identified in tumors of 36 patients. Thirty-nine of the mutations-including 34 small mutations, 2 large structural alterations, and hypermethylation in 3 tumors-were not detected in the corresponding peripheral blood DNA. In 6 (17%) of the 36 patients, a mutation was detected in constitutional DNA, and 1 of these mutations is known to be associated with reduced expressivity. The presence of a constitutional mutation was not associated with an early age at treatment. In 1 patient, somatic mosaicism was demonstrated by molecular analysis of DNA and RNA from peripheral blood. In 2 patients without a detectable mutation in peripheral blood, mosaicism was suggested because 1 of the patients showed multifocal tumors and the other later developed bilateral retinoblastoma. In conclusion, our results emphasize that the manifestation and transmissibility of retinoblastoma depend on the nature of the first mutation, its time in development, and the number and types of cells that are affected.     

A nonsense mutation in the Xeroderma pigmentosum complementation group F (XPF) gene is associated with gastric carcinogenesis

XPF/ERCC1 endonuclease is required for DNA lesion repair. To assess effects of a C2169A nonsense mutation in XPF at position 2169 in gastric cancer tissues and cell lines, genomic DNA was extracted from blood samples of 488 cancer patients and 64 gastric tumors. The mutation was mapped using a TaqMan MGB probe. In addition, gastric cancer cell lines were transfected with mutated XPF to explore XPF/ERCC1 interaction, XPF degradation, and DNA repair by a comet assay. The C2169A mutation was not detected in 488 samples of blood genomic DNA, yet was found in 32 of 64 gastric cancer tissue samples (50.0%), resulting in a 194C-terminal amino acid loss in XPF protein and lower expression. Laser micro-dissection confirmed that this point mutation was not present in surrounding normal tissues from the same patients. The truncated form of XPF (tXPF) impaired interaction with ERCC1, was rapidly degraded via ubiquitination, and resulted in reduced DNA repair. In gastric cancers, the mutation was monoallelic, indicating that XPF is a haplo-insufficient DNA repair gene. As the C2169A mutation is closely associated with gastric carcinogenesis in the Chinese population, our findings shine light on it as a therapeutic target for early diagnosis and treatment of gastric cancer.