Sequence analysis and genotypes of glutamate rich protein of Plasmodium falciparum isolates from different malaria endemic areas in China

To sequence the gene encoding glutamate rich protein (GLURP) and identify the genotypes of geographically different Plasmodium falciparum (P. f ) isolates from China. Methods The gene of R2 repeat region of GLURP was amplified by nested polymerase chain reaction and cloned into T-vector. The nucleotide sequence of GLURP gene was determined by automatic sequencer (Dideoxy termination method) and analyzed by DNA Star software. Results At least 7different GLURPgenotypesranging from 600 bp to 1 500 bp were found in Yunnan and Hainan provinces. R2 region of GLURP gene consisted of several repeat units. Each repeat unit was composed of 19-20 residues which were shown to be highly conserved. GLURP gene was also size polymorphic due to differences in the number of repeat units, whereas the repeat sequence was conserved. Sequence analysis showed that DNA sequences and deduced amino acid sequences were highly homologous among the geographically dispersed isolates or various isolates from the same geographical region. No obvious differences were found in the GLURP gene sequences among geographically different isolates. Conclusion GLURP gene is highly structure conserved and size polymorphic, and so is useful in searching for malaria vaccine candidate antigen and developing a genotyping method for malaria research.     

A missense mutation S228P in the CRYBB1 gene causes autosomal dominant congenital cataract

Background Congenital cataract is a highly heterogeneous disorder at both the genetic and phenotypic levels. This study was conducted to identify disease locus for autosomal dominant congenital cataracts in a four generation Chinese family. Methods Family history and clinical data were recorded. All the members were genotyped with microsatellite markers which are close to the known genetic loci for autosomal congenital cataracts. Two-point Lod scores were obtained using the MLINK of the LINKAGE program package （vet 5.1）. Candidate genes were amplified by polymerase chain reaction （PCR） and direct cycle sequencing. Results The maximum Lod score of Zmax=2.11 was obtained with three microsatellite markers D22S258, D22S315, and D22S1163 at recombination fraction θ= 0. Haplotype analysis showed that the disease gene was localized to a 18.5 Mbp region on chromosome 22 flanked by markers D22S1174 and D22S270, spanning the β-crystallin gene cluster. A c.752T→C mutation in exon 6 of CRYBB1 gene, which resulted in a heterozygous S228P mutation in predicted protein, was found to cosegregate with cataract in the family. Conclusions This study identified a novel mutation in CRYBB1 gene in a Chinese family with autosomal dominant congenital cataract. These results provide strong evidence that CRYBB1 is a pathogenic gene for congenital cataract.     

Thermal injuries induce gene expression of endogenous c-fos, c-myc and bFGF in burned tissues

Objective To investigate the expression sequence and distribution characteristics of the protooncogenes cfos,c-myc and endogenous basic fibroblast growth factor(bFGF) genes in burned tissues,and to explore the possible effects of changes in these genes‘ functions on wound healing.Methods Partial-thickness burns of 30% TBSA were esablished on backs of Wistar rats.In situ hybridization and histological methods were used to detect expression of c-fos,c-myc and bFGF genes in normal and burned tissue at 3h,6h,1d,3d,7d and 14 d postburn. Results Although expression of c-fos and c-myc genes and bFGF gene could be found in normal skin,the expression of all three were markedly induced by burn wounds and the expression models in sequence and distribution were quite different.Expression of c-fos gene increased and peaked at 6h.Signals were mainly localized in both nuclei of dermal fibroblasts and monocytes.The expression of bFGF gene increased at 6h and peaked at 1 d postburn,and was distributed in the cytoplasm of fibroblasts.C-myc gene peaked 3 d postburn and was also distributed in the cytoplasm of fibroblasts.Conclusions These results indicated that thermal injury could induce the expression of c-fos.c-myc and bFGF at gene level,showing phasic control and regional distribution.The phasic expression of these genes suggests that there is an interaction between protooncogenes and bFGF,which may play an important role in wound healing.The different expressions of c-fos and c-myc play an inducing role in regulating bFGF,and in turn affect wound healing.     

Genetic environment of β-lactamase genes of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates from patients with lower respiratory tract infection in China

Background Extended-spectrum β-lactamase （ESBL）-producing Klebsiella pneumoniae （K.pneumoniae） is one of the most popular pathogens that cause refractory respiratory tract infection.The genetic environment,including insertion sequences and the types of promoter,plays a key role in exploration of the mechanism of prevalence and dismission of the ESBL-producing K.pneumoniae isolates.The aim of the investigation was to target analysis the genetic environment and promoter sequences of blaCTX-M,blaSHV and blaTEM,the most popular β-lactamase genes harbored by ESBL-producing K.pneumoniae isolates.Methods From February 2010 to July 2011,158 of 416 K.pneumoniae isolates producing ESBL from patients with lower respiratory tract infection were collected from seven tertiary hospitals from Beijing,Anhui,Fujian,Liaoning,Hebei and Inner Mongolia Autonomous Region in China.The genetic environment including promoters of 10 types of blaCTX-M,18 types of blaSHVand 2 types of blaTEM were analyzed by amplification and direct sequencing with various sets of PCR primers.Results ISEcp1 was located upstream of the 5＇ end of the blaCTX-M gene in 130 （97.0％） out of 134 K.pneumoniae isolates harboring blaCTX-M and provided a conserved promoter to blaCTX-M.A non-coding sequence preceded by kdpC and recF was identified in all of the blaSHV genes except blaSHV-12 and blaSHV-2a.IS26 was also found upstream of 1 blaCTX-M-15,10 blaSHV-1 strains,4 blaTEM-1 and all of the blaSHV-2,blaSHV-2a,blaSHV-5 and blaSHV-12.Eighty-seven of 91 strains harboring blaTEM-1 carried a copy of Tn2 and IS26-blaTEM-1 fragments were also detected in 4 strains.With respect to K.pneumoniae,the genetic environment of blaCTX-M-38,blaSHV-142 and blaTEM-135 were firstly elaborated,and four kinds of novel genetic environment of blaCTX-M-3,blaCTX-M-15 and blaTEM-1 have been detected as well.Conclusions Perfective implementation of the genetic environment information of β-lactamase gene needs to be further explored and supplemented.ISEcp1 and IS26 elements ar     

基因芯片－同时研究数以千计的生物技术

The introduction of microarray technology represented a paradigm shift in the way that biological science is performed. This shift involved a switch from conducting purely hypothesis driven research to hypothesis generating research. It is no longer necessary for a biologist to attempt to determine which gene or class of genes they would like to study. Instead, researchers can now look at an entire collection of genes, in deed for several organisms, all the potential genes, to determine which are implicated in a particular process. Once a key set of genes are identified, then the biologist can return to more classical means to undertake a hypothesis driven approach. Coincident with this change in focus is a change in terminology. No longer do we simply study gene expression, with microarrays, we now study genomics : gene expression on a global scale. Originally, microarrays were used to study processes such as cell cycle regulation, or to profile diseases such as cancer; areas of research that studied gene expression as a matter of course. Now however, as the technique grows in popularity, and the cost has been reduced through economies of scale, new areas of research are beginning to employ microarrays. Microarrays are now being used in agricultural research, toxicology, and food safety. In addition, although, arrays were originally designed to assay gene expression, they have now been adapted to study genetic mutation, protein expression, and protein function. The past couple of years have seen the introduction of arrays of protein, cells, tissues and even small molecules. Each of these tools will allow microarrays to find footholds into ever more areas of research.This review will attempt to give a general overview of microarray technology and then look at the application of microarrays to two aspects of disease, genetic based disease and infectious disease. Issues of experimental design and data analysis will also be addressed.     

FBN1 mutation in Chinese patients with Marfan syndrome and its gene diagnosis using haplotype linkage analysis

Objectives To analyze the FBN1 mutations in Chinese patients with Marfan syndrome (MFS) and to make a genetic diagnosis based on haplotype linkage analysis for MFS.Methods Nine MFS families (17 patients) were analyzed with single strand conformation polymorphism (SSCP) and sequencing. Four primers were designed for the flanking sequences of FBN1 gene and used for haplotype-segregation analysis of MFS (B).Results SSCP band alteration was detected in the PCR products for exon 25 in MFS(A) Ⅱ : 1.Direct sequencing revealed a small 13 bp deletion; the deleted sequence is gccTcTgcaccca at bases 3243 -3456 of the cDNA in exon 25. This mutation was novel. MFS(B) families were analyzed using the haplotype linkage technique. The data suggested that MFS(B) families were linked to the FBN1 gene. The proband‘ s daughter was an asymptomatic patient.Conclusion The combination of mutation detection and chromosome haplotype analysis can provide better evidence for a genetic diagnosis of MFS.     

Mutation analysis of p63 gene in the first Chinese family with ADULT syndrome

Background ADULT syndrome （acro-dermato-ungual-lacrimal-tooth syndrome） is a rare ectodermal dysplasia disorder known as autosomal dominant inheritance. Recent studies have linked p63 gene mutation to the development of this disease. However, the genetic characteristics of ADULT syndrome were still not well understood. Methods Mutation analysis of p63 gene in the first Chinese ADULT syndrome family was performed using direct DNA sequencing. Results The sequence analysis of exon 8 of p63 gene disclosed a heterozygous G〉A substitution at nucleotide 893 （R298Q） in the proband. In addition, a single nucleotide polymorphism （SNP） rs16864880 in the downstream flanking region （DFR） of p63 exon 8 was also identified in this family. The proband and the paternal side including her father exhibited the C/G genotype at this position. The C/G variant frequency in the paternal was significantly higher as compared with the maternal （6/10 vs 0/6, P=0.034）. Conclusions ADULT syndrome may be caused by the p63 gene mutation, and it might have closer genetic association with the paternal side in this family.     

Novel Species Including Mycobacterium fukienense sp. Is Found from Tuberculosis Patients in Fujian Province,China, Using Phylogenetic Analysis of Mycobacterium chelonae/abscessus Complex

Objective To identify the novel species ‘Mycobacterium fukienense＇ sp. nov of Mycobacterium chelonoe/abscessus complex from tuberculosis patients in Fujian Province, China. Methods Five of 27 clinical Mycobucterium isolates （CIs） were previously identified as M. chelonoe/obscessus complex by sequencing the hsp65, rpoB, 165-235 rRNA internal transcribed spacer region （its）, recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobocterium. Clinical Mycobecterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. Results The 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 165-235 rRNA internal tronscribed spacer region （its）, sodA, and recA genes as compared with the M. obscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. Conclusion The novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. cheloneo/abscessus complex.     

Gene mapping of autosomal dominant retinitis pigmentosa in a Chinese family

Background The autosomal dominant form of retinitis pigmentosa （ADRP） can be caused by mutations in 14 genes and further loci remains to be identified. This study was intended to identify mutations in a Chinese pedigree with ADRP.
Methods A large Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite （STR） markers tightly linked to genes known to be responsible for ADRP were selected for linkage analysis. Exons along with adjacent splice junctions of PRPF31 were amplified by polymerase chain reaction （PCR） and screened by direct sequencing.
Results The caused gene of ADRP was mapped to 19q13.4 between markers D19S572 and D19S877, with a maximum LOD score of 3.01 at marker D19S418 （recombination fraction=0）.
Conclusion The affected gene linked to the 19q13.4 in a Chinese family with ADRP, which is different from other mutations at the same loci in other Chinese families.     

Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccinecandidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli). Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3‘ RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi) , Drosophila melanogaster (D.melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24. 921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E coil JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunitwere firstly sequenced, cloned, and expressed in E coil.     

中国人群中Wilson病家系和正常人D13S4区限制性片段长度多态性分析

用Southern blot技术,以13q22-31上的plE8探针对中国人群中4个Wilson病(WD)家系和10例正常人D13S4区见MspⅠ酶切片段长度多态性(RFL)分析。结果表明:A、B两杂交片段出现的频率分别为46.9％和53.1％,这和国外报道的频率0.49/0.51是一致的,同时对WD家系成员A、B两杂交片段出现频率和正常人相比较,经统计学处理,x~2=0.026<3.8,P>0.05。无显著性差异。并对中国人群中WD遗传异质性、连锁不平衡以及WD家系成员的临床前诊断做了探讨,为WD基因的研究和基因水平的诊断提供了科学依据。     

中国人Wilson病ATP7B基因启动子区的研究

目的研究中国人Wilson病（WD）ATP7B基因启动子区的序列和结构,发现存在的多态和突变情况。方法检测60例无亲缘关系的WD患者和20例正常人的基因组DNA序列并进行分析。结果（1）在正常对照和WD患者的启动子区-190位和-78位（转录起始点为＋1）均发现存在单个碱基的不同;（2）60例WD患者中,3例发现存在-504位G→C的突变,其中2例为纯合突变,1例为杂合突变,在正常对照中未发现此改变。（3）60例WD患者中,2例发现存在-782位A→G的突变,其中1例为纯合突变,另1例为杂合突变,在正常对照中未发现此改变。结论WD基因的转录直接受铜或其他重金属的调节。中国人WD基因启动子区序列存在的2个多态位点均位于已知的转录调控元件结构之外,提示核苷酸多态对启动子调控基因的表达不会产生影响。启动子区的突变也是WD的分子发病机制之一。提示WD基因的分子发病机制是多样又复杂。     

人染色体8p11.2亚带ANK1位点附近区域表达序列的分离与克隆

目的分离人染色体８ｐ１１．２亚带ＡＮＫ１位点附近区域基因的表达序列。方法采用ｃＤＮＡ选择法从定位于该区域的人工酵母染色体（ＹＡＣ）中分离基因的表达序列，并从分离的表达序列中选取１个序列用于筛选人胎脑ｃＤＮＡ文库。结果获得７个表达序列，其中５个可与目的ＹＡＣ反杂交。所得的表达序列ＢＨＬＣ１５２与鼠的ＣＡｒＧ盒结合因子基因高度同源，ＢＨＬＣ７４与编码心室肌肌球蛋白的碱性轻链１（ＭＬＣ１）基因高度同源，表明ＢＨＬＣ７４可能编码一种ＭＬＣ１的同工蛋白。分离的其它表达序列与数据库内一些位置和功能尚不清楚的表达序列标签（ｅｘｐｒｅｓｅｄｓｅ－ｑｕｅｎｃｅｔａｇ，ＥＳＴ）仅有部分同源性。用表达序列ＢＨＬＣ１１９筛选人胎脑ｃＤＮＡ文库，获得１个插入片段长１９５１ｂｐ的克隆，序列分析表明它编码１７４个氨基酸，该序列与所有已知的蛋白质序列无同源性。Ｎｏｒｔｈｅｒｎ印迹分析结果显示，在人胎脑总ＲＮＡ中呈现１条约３．５ｋｂ带；组织表达分析表明，此系一广泛表达的基因。结论ＢＨＬＣ７４可能作为与８ｐ相关的心脏病候选基因，其它序列至少可以作为定位在ＡＮＫ１位点附近区域的肿瘤抑制基因的初筛基因。     

The molecular cloning and restriction enzyme mapping of retinitis pigmentosa 3 (RP3) locus

X-linkedretinitispigmentosa(XI-RP)isagroupofseriousgeneticdiseaseswithunknownpathogenesis.AlotofllnkageanalysesforRPfamiliesshowedthattherewereatleasttwogenelociexistingintheXchromosomeshortarm.Thefirstlocus(RPZ)waslocatedinXpll,closetoDXS7;thesecondlocus(RP3)waslocatedinXp21,1cMdistaltoOTCLlj.TherecentresearchdatashowedthatRP3locuswasfinelymappedwithinabout270kbrange,betweenOTCandDXSlll0markersLZj.ThismakestheRP3thefirstchoicetargetofpositionalcloningL'].Inthiswork,wesucceededi…     

Carrier detection and presymptomatic identification of Wilson disease in Chinese by non-isotopic linkage analysis with four short tandem repeat polymorphisms

Ｗｉｌｓｏｎｄｉｓｅａｓｅ（ＷＤ）ｉｓａｎａｕｔｏｓｏｍａｌｒｅｃｅｓｓｉｖｅｄｉｓｏｒｄｅｒｏｆｃｏｐｐｅｒｍｅｔａｂｏｌｉｓｍｗｉｔｈａｗｏｒｌｄｗｉｄｅｆｒｅｑｕｅｎｃｙｏｆｂｅｔｗｅｅｎ１／５０００ａｎｄ１／３００００ｉｎｌｉｖｅｂｉｒｔｈｓ...     

Currently Clinical Views on Genetics of Wilson's Disease

Objective:

The objective of this study was to review the research on clinical genetics of Wilson''s disease (WD).

Data Sources:

We searched documents from PubMed and Wanfang databases both in English and Chinese up to 2014 using the keywords WD in combination with genetic, ATP7B gene, gene mutation, genotype, phenotype.

Study Selection:

Publications about the ATP7B gene and protein function associated with clinical features were selected.

Results:

Wilson''s disease, also named hepatolenticular degeneration, is an autosomal recessive genetic disorder characterized by abnormal copper metabolism caused by mutations to the copper-transporting gene ATP7B. Decreased biliary copper excretion and reduced incorporation of copper into apoceruloplasmin caused by defunctionalization of ATP7B protein lead to accumulation of copper in many tissues and organs, including liver, brain, and cornea, finally resulting in liver disease and extrapyramidal symptoms. It is the most common genetic neurological disorder in the onset of adolescents, second to muscular dystrophy in China. Early diagnosis and medical therapy are of great significance for improving the prognosis of WD patients. However, diagnosis of this disease is usually difficult because of its complicated phenotypes. In the last 10 years, an increasing number of clinical studies have used molecular genetics techniques. Improved diagnosis and prediction of the progression of this disease at the molecular level will aid in the development of more individualized and effective interventions, which is a key to transition from molecular genetic research to the clinical study.

Conclusions:

Clinical genetics studies are necessary to understand the mechanism underlying WD at the molecular level from the genotype to the phenotype. Clinical genetics research benefits newly emerging medical treatments including stem cell transplantation and gene therapy for WD patients.     

D13S133位点CA重复序列在正常中国人群的多态性分布

目的研究Ｄ１３Ｓ１３３位点ＣＡ重复序列在正常中国人群的多态性分布。方法：应用ＰＣＲ扩增该位点的ＣＡ重复序列，通过变性聚丙烯酰胺凝胶电泳检测扩增产物，测定６０个正常中国人的等位片段。结果：共检测到１５种不同长度的等位片段，多态信息含量（ＰＩＣ值）为０．８８１。结论：该位点在中国人群中具有长度多态性，且其多态性分布与西方人群存在明显差异。     

肝豆状核变性分子生物学研究

【目的】探讨中国人肝豆状核变性(WD)的分子发病机制和基因诊断的方法。【方法】应用基因工程技术对WD进行了10年的分子生物学研究。【结果】①WD的基因定位研究通过RFLP及微卫星多态性分析,应用两位点及多位点连锁软件,建立了中国人WD基因在D     

磁共振小肠造影多序列联合成像在小肠疾病诊断中的应用

目的 探讨小肠MRI造影多序列应用方法及MRI造影对小肠病变的影像诊断.方法 使用Philips Achieva 3.0T MR对临床疑诊小肠病变的34例患者进行MR小肠造影 (MRE) 多序列联合成像.扫描序列包括横断位、冠状位屏气单次激发快速自旋回波T2WI-TSE-BH序列, 冠状位平衡稳态自由进动序列 (BTFE) 、横断位扩散加权成像 (DWI) 序列及横断位、冠状位3D-THRIVE序列增强扫描.以不同等级评估小肠MRI扫描图像质量.判断各序列的应用价值.结果 平扫COR-SSTSE-BH序列压脂图像显示最佳, 评估炎症性肠病的活动性弥散序列应用价值较高, 联合3D-THRIVE序列增强图像能为临床提供定性诊断.结论 MRE多序列联合扫描图像信息量大, 敏感度高, 对诊断小肠疾病有重要价值.     

血清铜及血清游离铜对肝豆状核变性患者及携带者的诊断及治疗监测意义

目的 研究血清铜和血清游离铜在肝豆状核变性(WD)、WD携带者、病毒性肝炎等疾病中的诊断价值.探讨监测血清铜对于WD患者排铜治疗过程中的指导意义.方法 选取2007年7月至2011年1月住院治疗的WD初诊患者80例(脑型60例,肝型20例)、WD携带者30例,病毒性肝炎20例进行血清铜等铜代谢指标检查.WD患者接受二巯基丙磺钠治疗8个疗程,每疗程用改良Young量表进行神经症状评分,进行肝功能、出凝血功能、血清铜、尿铜等检查.患者出院后用锌剂维持治疗.结果进行Logistic回归分析、t检验和方差检验等统计学分析.结果 WD患者、WD携带者、重型病毒性肝炎血清游离铜分别为0.17 mg/L±0.04 mg/L、0.13 mg/L±0.03 mg/L和0.12mg/L,高于正常.血清游离铜与肝功能等级相关,但与神经症状严重程度无关.排铜治疗过程中,神经症状改善不明显或加重者,血清铜升高;神经症状改善者,血清铜降低.长期排铜过程中,血清铜将稳定在0.2 mg/L左右.结论 血清铜和血清游离铜对WD有辅助诊断意义.肝型WD患者肝功能将影响血清铜水平.而脑型WD血清铜并不能直接反映神经症状严重程度.排铜治疗过程中,血清铜升高,提示预后不佳.在排铜治疗过程中,血清铜可作为监测治疗是否有效的指标,可作为排铜治疗目标.