Leber遗传性视神经病变家系的线粒体基因突变分析

为探讨Leber遗传性视神经病变（Leber′s hereditary optic neuropathy,LHON）家系线粒体DNA（mtDNA）常见致病原发突变的频谱,用聚合酶链反应（polymerase chain reaction,PCR）和单链构象多态性（single-stranded conformational polymorphism,SSCP）以及DNA测序的方法,对13个家系22位临床诊断为LHON的患者及其母系亲属21人的线粒体DNA进行检测,同时检测71例正常人作为对照。临床拟诊为LHON的13个家系中,11个家系存在mtDNA位点11778 G→A突变,另2个家系存在14484位点T→C突变。说明中国LHON病人存在线粒体DNA 11778或14484位点突变,其中14484位点突变在国内尚未见报道。 Abstract:The purpose of the study is to investigate the frequency of common pathogenic primary mitochondrial DNA mutations in pedigrees of Leber′s hereditary optic neuropathy (LHON).Mutations were determined by polymerase chain reaction (PCR),single-stranded conformational polymorphism (SSCP) and DNA sequencing.Twenty-two patients with suspicion of LHON and twenty-one their maternal relatives underwent molecular genetic evaluation.Seventy-one normal individuals underwent molecular genetic evaluation as control at the same time.Members from 13 families with suspicion of LHON,11 families had nucleotide position nt11778 G→A mutations.Another 2 families had nt14484 T→C mutations.It is concluded that the point mutations at nucleotides 11778 and 14484 are primary LHON mutations,but the point mutation of nt14484 is rare in Chinese.     

胞质异质性——人类肿瘤组织线粒体基因突变的普遍现象

为了探讨不同肿瘤组织中线粒体基因体细胞性突变的胞质异质性和同质性状态,利用32对重叠引物对149例肿瘤组织和匹配的正常组织的全线粒体基因进行PCR扩增,并同时进行时相温度梯度凝胶电泳扫描突变筛选,基因测序确定突变类型与异质状况。结果表明,不同肿瘤组织中线粒体基因体细胞性突变的异质率不同,口腔癌（65%）和食道癌（64%）具有较高的异质率,其次为乳腺癌（45.9%）。4种转换形式的发生频率Hm→Hm > Hm→Ht > Ht→Hm > Ht→Ht。碱基转换的主要转换形式为Hm→Hm,碱基颠换则以Hm→Ht。认为胞质异质性是人类肿瘤组织线粒体基因突变的普遍现象。Abstract: To explore the status of heteroplasmy and homoplasmy of Mitochondrial DNA somatic mutations in different tumors. DNA from 149 tumors and corresponding normal tissues were extracted and entire mitochondrial genome was amplified using 32 pairs of overlapping primers. The somatic mutations were screened by temporal temperature gradient gel electrophoresis and their heteroplasmic statute were identified by sequencing. The results showed that the incidence rate of heteroplasmy of mitochondrial DNA somatic mutations varies in different tumors. There is a high rate of heteroplasmic mutation in oral cancer (65%) and esophageal cancer (64%), followed by breast cancer (45%). The frequency of four transfer types is Hm (homoplasmy)→Hm (heteroplasmy) > Hm→Ht > Ht→Hm > Ht→Ht. The main transfer forms of transition and transversion mutations are Hm→Hm and Hm→Ht respectively. Heteroplasmy is a common phenomenon in mitochondrial DNA somatic mutations of human tumors.     

Polymorphism analyses of hepatitis B virus X gene in hepatocellular carcinoma patients from southern China

Chronic hepatitis B virus(HBV)infection is one of the major causes of hepatocellularcarcinoma(HCC),and the HBV X(HBx)gene plays a critical role in the molecular pathogenesis ofHBV-related HCC.We have investigated whether there are particular HBx gene mutations associated withHCC in patients from southern China.The HBx gene was examined in 51 paraffin-embedded tumor tissuesamples from patients with HCC and 25 serum samples from the HBV carrier by nested polymerase chainreaction(PCR),single-stranded conformational polymorphism and heteroduplex analysis.The HBx geneswith potentially important mutations from tumor tissue samples were cloned,sequenced and aligned withthe published HBx gene sequence.HBV genotypes in tumor tissue samples were analyzed by nested PCR.Analyses of HBx gene polymorphism showed that 31.3% of HBx gene fragments in tumor tissue sampleshad a special pattern.A common deletion at nt 382-400 of the HBx gene accompanied by 29 point mutationswas detected in four randomly selected tumor tissue samples with this pattern which caused a frame-shiftin the HBx open reading frame with a new stop codon at nt 1818,resulting in an HBx polypeptide chaintruncated at the C end in these cases.Among the four randomly selected samples,three were HBV genotypeB,and one was not detected by our present assay.In another tumor tissue sample,amplification of thefull-length HBx gene yielded a shorter fragment.Sequencing of this fragment revealed a 264 bp deletionbetween nt 1577 and 1840 of the HBV gene.These results suggest that HBx gene mutation occurs frequentlyin HCC samples,and the deletion at nt 382-400 of the HBx gene might play a role in carcinogenesis of HCCin southern China.     

一个氨基糖苷类抗生素致聋家系线粒体DNA突变研究

应用PCR、PCR－SSCP和DNA序列分析等分子生物学技术,对一个有明确氨基糖苷类抗生素应用史的母系遗传耳聋家系共8人(包括聋人和听力正常者) 的线粒体DNA进行研究,结果显示,家系中有4份样品存在线粒体DNA 12S rRNA 1 555位点A→G的突变。提示线粒体DNA点突变是导致该家系致聋的主要因素之一。 Abstract:Blood samples were obtained from a pedigree with aminoglycoside antibiotic induced deafness.DNA was extracted from the isolated leukocytes.The mitochondrial DNA fragments were detected by PCR－SSCP and DNA sequencing.It was found that four individuals from the pedigree carried 1 555 A→G mutation.From our results,mitochondrial DNA mutation may be one of major factors in aminoglycoside antibiotic induced deafness.     

脑胶质瘤中P53基因突变及其与染色体17p杂合性丢失的比较

为进一步阐明P53基因突变和染色体17p杂合性丢失(LOH)的关系及两者与脑胶质瘤发生发展的相关性。应用PCR－SSCP、DNA序列测定及RFLP分析方法对55例脑胶质瘤中的P53基因突变及染色体17p的杂合性丢失进行了检测。发现P53基因在高级别星型细胞肿瘤(III－IV级)、低级别星型细胞肿瘤(I－II级)和非星型细胞肿瘤中的突变频率分别为：53％(9/17)、7％(1/15)和9％(2/23)。而55例肿瘤组织对应的淋巴细胞中未见P53突变。22％的胶质瘤丢失1个17p等位基因,这部分肿瘤中P53基因的突变频率为50％(6/12),而在43例持有2个17p等位基因的肿瘤中,P53基因的突变频率降为14％,两组相比差异显著(P<0?025)。结果提示P53基因突变是星型细胞肿瘤演变过程中的一个常见遗传事件,可能标志着肿瘤的恶性进展;散发性脑胶质瘤中的P53突变是体细胞型的突变;另外,丢失1个17p等位基因的肿瘤中有50％缺少P53基因突变,提示染色体17p上可能存在另一个参与了部分肿瘤恶性演变的肿瘤抑制基因。 Abstract：To further illustrate the roles of P53 gene and loss of heterozygosity (LOH) on chromosome 17p in the development of malignant gliomas,mutations in the P53 gene were analyzed in 55 gliomas of various malignant grades and histological types by the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique and were confirmed by sequencing.Loss of heterozygosity (LOH) for chromosome 17p was also assessed using restriction fragment length polymorphism (RFLP) analysis in same tumors.The mutations did not follow a random distribution among various different subtypes,but occurred in 9 of 17 high-grade astrocytomas (53％),in 1 of 15 low-grade astrocytomas (7％) and in 2of 23 (9％) non-astrocytic tumors.Most of the mutations were missense ones and 42％ (5/12) occurred at the sites of CpG dinucleotide.P53 mutations were not detected in any of the 55 leukocyte DNA samples from patients with brain tumors.These studies demonstrated that P53 inactivation is a common genetic event in astrocytoma progression that may signal the transition from benign to malignant tumor stages.P53 gene mutations in sporadic human brain tumors are somatic in origin (i.e.,nonprenatally determined).The majority of glomas (43/55) analyzed here retained both 17p alleles.The frequency of P53 mutations was 14％ in this group of tumors and increased to 50％ (6/12) in tumors with one 17p allele (P<0.025).Allelic loss for chromosome 17p occurred in 6 of 12 gliomas independently of mutations in the P53 gene.Absence of P53 mutations in 50％ of the tumors with one allele suggests that a tumor suppressor gene other than P53 may be located on chromosome 17p and involved in progression to malignancy of some gliomas.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

强直性肌营养不良症DMPK基因CTG重复序列与Alu±1kb单倍型研究

强直性肌营养不良(myotonic dystrophy,DM)是由于DMPK基因3′非翻译区CTG重复序列异常扩展所致的、主要累及神经肌肉系统的常染色体显性遗传病。在该基因的第8内含子中还存在一个Alu重复序列的1kb插入/缺失多态性,即Alu±1kb多态性。为了帮助阐明汉族人群中DM突变的起源,并为解释DM在不同群体中发病率的差异提供更多依据,本文从300例已知CTG拷贝数的正常汉族群体中随机挑选60例,首先通过PCR扩增确定其Alu±1kb多态性,然后对Alu±1kb和CTG双杂合的标本,采用长PCR方法先行扩增含Alu±1kb和CTG重复序列的DNA片段,再分别对含Alu(＋)和Alu(－)的DNA片段中的CTG拷贝数进行常规PCR分析,以确定二位点的单倍型。结果表明60例正常人中二位点间呈连锁不平衡。其单倍型为:（CTG）5均与Alu(＋)连锁;多数(CTG)11～14与Alu(－)连锁;在两个(CTG)≥19的等位基因中一个与Alu(＋)连锁,另一个与Alu(－)连锁。各民族相关资料的比较提示,汉族人群中(CTG)11～14与非洲黑人的起源可能不同;(CTG)19～30/Alu－1kb在汉族人群中的频率远比欧洲人群的高;(CTG)19～30/Alu－1kb与(CTG)19～30/Alu＋1kb在汉族人群中是以一定比例共存的;(CTG)19～30在不同民族间的起源不尽相同;如果从(CTG)5到(CTG)19～30的假设成立的话,则很可能是一个较为复杂的过程。 Abstract：Myotonic dystrophy(DM),an autosomal dominant disease of the neuromascular system,is caused by expansion of the CTG repeats in the 3＇untranslated region of the DMPK gene.In the eighth intron of this gene,there is another polymorphism－the insertion/deletion of 1kb Alu repeat(Alu±1kb).In order to help elucidate the origin of DM mutation in Chinese Han patients,and explain the difference of incidence of DM in different populations,60 normal Han individuals were randomly chosen from 300 Chinese,whose CTG copy number had been previously ascertained.The polymorphism of Alu±1kb of the 60 cases were firstly analyzed by in vitro amplification;then the 22 cases in which both sites were heterozygous were analyzed as following:the fragments containing both Alu±1kb and CTG repeat sequence were first amplified by long PCR method;and then the CTG copy numbers were analyzed in the Alu(＋)and Alu(－)alleles.In the 60 cases studied,a remarkable linkage disequilibrium between CTG triplet repeats and Alu±1kb were observed.All the(CTG)5 alleles were linked with Alu(＋),and most of the(CTG）11－14 alleles were linked with Alu(－);one of the two alleles of(CTG）≥19 was linked with Alu(＋),the other was linked with Alu(－).This suggests that the origin of(CTG)11－14 in Chinese Han may be different from that of African Blacks;the frequency of (CTG)19－30 /Alu－1kb in Chinese Han might be much higher than that in Europeans Caucasians.The(CTG)19－30/Alu－1kb and(CTG)19－30/Alu＋1kb coexist in Chinese Han in some proportion;the origin of (CTG)19－30 in different population may be different;if the hypothesis of(CTG)5 to(CTG)19－30 is true,then the progress should have been a relatively complicated process.     

强直性肌营养不良症DMPK基因CTG重复序列与Alu±1kb单倍型研究

强直性肌营养不良(myotonic dystrophy,DM)是由于DMPK基因3′非翻译区CTG重复序列异常扩展所致的、主要累及神经肌肉系统的常染色体显性遗传病。在该基因的第8内含子中还存在一个Alu重复序列的1kb插入/缺失多态性,即Alu±1kb多态性。为了帮助阐明汉族人群中DM突变的起源,并为解释DM在不同群体中发病率的差异提供更多依据,本文从300例已知CTG拷贝数的正常汉族群体中随机挑选60例,首先通过PCR扩增确定其Alu±1kb多态性,然后对Alu±1kb和CTG双杂合的标本,采用长PCR方法先行扩增含Alu±1kb和CTG重复序列的DNA片段,再分别对含Alu(＋)和Alu(－)的DNA片段中的CTG拷贝数进行常规PCR分析,以确定二位点的单倍型。结果表明60例正常人中二位点间呈连锁不平衡。其单倍型为:（CTG）5均与Alu(＋)连锁;多数(CTG)11～14与Alu(－)连锁;在两个(CTG)≥19的等位基因中一个与Alu(＋)连锁,另一个与Alu(－)连锁。各民族相关资料的比较提示,汉族人群中(CTG)11～14与非洲黑人的起源可能不同;(CTG)19～30/Alu－1kb在汉族人群中的频率远比欧洲人群的高;(CTG)19～30/Alu－1kb与(CTG)19～30/Alu＋1kb在汉族人群中是以一定比例共存的;(CTG)19～30在不同民族间的起源不尽相同;如果从(CTG)5到(CTG)19～30的假设成立的话,则很可能是一个较为复杂的过程。 Abstract：Myotonic dystrophy(DM),an autosomal dominant disease of the neuromascular system,is caused by expansion of the CTG repeats in the 3＇untranslated region of the DMPK gene.In the eighth intron of this gene,there is another polymorphism－the insertion/deletion of 1kb Alu repeat(Alu±1kb).In order to help elucidate the origin of DM mutation in Chinese Han patients,and explain the difference of incidence of DM in different populations,60 normal Han individuals were randomly chosen from 300 Chinese,whose CTG copy number had been previously ascertained.The polymorphism of Alu±1kb of the 60 cases were firstly analyzed by in vitro amplification;then the 22 cases in which both sites were heterozygous were analyzed as following:the fragments containing both Alu±1kb and CTG repeat sequence were first amplified by long PCR method;and then the CTG copy numbers were analyzed in the Alu(＋)and Alu(－)alleles.In the 60 cases studied,a remarkable linkage disequilibrium between CTG triplet repeats and Alu±1kb were observed.All the(CTG)5 alleles were linked with Alu(＋),and most of the(CTG）11－14 alleles were linked with Alu(－);one of the two alleles of(CTG）≥19 was linked with Alu(＋),the other was linked with Alu(－).This suggests that the origin of(CTG)11－14 in Chinese Han may be different from that of African Blacks;the frequency of (CTG)19－30 /Alu－1kb in Chinese Han might be much higher than that in Europeans Caucasians.The(CTG)19－30/Alu－1kb and(CTG)19－30/Alu＋1kb coexist in Chinese Han in some proportion;the origin of (CTG)19－30 in different population may be different;if the hypothesis of(CTG)5 to(CTG)19－30 is true,then the progress should have been a relatively complicated process.     

Molecular characteristics of the ompA gene of serotype B Chlamydia trachomatis in Qinghai Tibetan primary school students

To study the molecular characteristics of Chlamydia trachomatis, the major outer membrane protein gene(omp A) of C. trachomatis from primary school students with trachoma residing in the Qinghai Tibetan area was sequenced and compared with the same serotype in Gen Bank. In Jianshetang Primary School and Galeng Central Primary School in the Galeng Tibetan Township of Qinghai Haidong Sala Autonomous County, scraped samples were collected from the upper tarsal conjunctiva and lower conjunctival sac of both eyes of 45 students with trachoma, stored at 4°C, and transported to Beijing Tongren Hospital by air within 24 h. The samples were screened for C. trachomatis by real-time PCR. The omp A gene from the C. trachomatis-positive samples was amplified by nested PCR. The serotype was confirmed by National Center for Biotechnology Information(NCBI) BLAST search and homology analysis. The entire omp A gene sequence was compared with the corresponding gene sequences of serotype B strains available in Gen Bank. Of the 45 students aged 6–13 years with trachoma, 26 C. trachomatis-positive students were identified by the initial real-time PCR screening(average age,(9.09±1.63) years; sex ratio, 1.0), accounting for 57.78%(26/45). The cycle threshold values for real-time PCR were 16.79–37.77. Half(13/26) of C. trachomatis-positive students had a bacterial copy number of 105. The compliance rate of the omp A gene sequences with the C. trachomatis serotype B strains in Gen Bank was up to 99%. Two novel genetic mutations were found when the omp A gene was compared with those of the 11 serotype B strains in Gen Bank. The two non-synonymous mutations were located at(i) position 271 in the second constant domain, an adenine(A) to guanine(G) substitution(ACT?GCT), changing the amino acid at position 91 from threonine to alanine(Thr?Ala) in all 26 strains; and(ii) position 887 in the fourth variable domain, a cytosine(C) to thymine(T) substitution(GCA?GTA), changing the amino acid at residue 296 from alanine to valine(Ala?Val) in four of the 26 strains. Six mutations were identified relative to ATCC VR-573. The strains could be divided into two gene clusters according to the mutation at nucleotide position 887: CQZ-1(China Qinghai Tibetan-1) and CQZ-2(China Qinghai Tibetan-2). We thus detected two novel serotype B mutant strains of C. trachomatis among study subjects with trachoma.     

Genomic Perspectives on the Emerging SARS-CoV-2 Omicron Variant

A new variant of concern for SARS-CoV-2,Omicron(B.1.1.529),was designated by the World Health Organization on November 26,2021.This study analyzed the viral genome sequencing data of 108 samples collected from patients infected with Omicron.First,we found that the enrichment efficiency of viral nucleic acids was reduced due to mutations in the region where the primers anneal to.Second,the Omicron variant possesses an excessive number of mutations compared to other variants circulating at the sam...     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.