北京地区传播的SARS冠状病毒N区基因变异分析

SARS冠状病毒的N蛋白具有很强的免疫原性 ,是机体产生特异性体液反应主要针对的靶分子之一。收集并提取了我院收治的北京地区 1 2位确诊SARS患者的病毒RNA样本 ,用反转录巢式聚合酶链式反应分 3个片段扩增出N基因全序列 ,用pGEM T载体克隆后进行DNA序列分析。结果发现 ,在 1 2条病毒N基因序列中 ,与北京地区最早报告的BJ0 1株SARS冠状病毒N基因序列相比 ,有 3个核苷酸替换 ,分布于 2个序列位点 ,分别为 2 841 7G→C(aa1 0 6S→T)和 2 8433C→T(aa1 1 1F→F同义突变 ) ,均为新发现的变异位点。结果表明 ,北京地区传播的SARS冠状病毒N基因存在变异序列 ,但发生频率较低 ,提示N基因的结构稳定对病毒的生存较为重要。     

猪CACNA1S基因部分序列的克隆、测序及SNP检测

CACNA1S是钙离子通道主效亚基α1亚单位的编码基因,该基因突变会导致人（Homo sapiens）低钾性周期性麻痹和恶性高温综合征．目前CACNAIS基因的研究主要集中在人和模式动物上,而在家畜中的研究少见报道．本研究以金华猪（115头）、皮特兰猪（30头）、金华猪与皮特兰猪杂交产生的金皮F2代杂种猪（126头）、大约克猪（23头）为研究对象,根据人CACNA1S基因序列设计引物,对猪基因组DNA进行PCR扩增、克隆测序,并与人相应序列作同源性比较,然后采用PCR-SSCP技术检测序列中可能存在的单核苷酸多态（single nucleotide polymorphism,SNP）位点,并对有合适内切酶存在的突变点应用PCR-RFLP技术进行验证．结果表明：（i）用6对引物对猪基因组DNA进行扩增,共克隆得到猪CACNAIS基因约5211bp的DNA序列,其中外显子区域,猪与人同源性为82．6％,序列已递交GeneBank收录（登录号DQ767693）;（ii）在克隆得到的DNA序列中,共检测到57个单核苷酸突变点,其中外显子区域存在24个;（iii）突变位点的PCR-RFLP验证与PCR-SSCP检测结果一致,经与GenBank公布的猪CACNAIS基因EST小片段（Bx914582,Bx666997）比较,相应长度核酸序列内本实验检测到的11个SNP位点中,有8个位点的碱基突变与2个EST片段间存在的碱基差异相同．     

大鳞副泥鳅和泥鳅GNB2L1基因的克隆、表达及系统进化分析

由G蛋白β2亚基类似物1基因(GNB2L1)编码的蛋白激酶C受体(RACK1)是一个高度保守的锚定蛋白,属于WD40结构域蛋白家族成员,在细胞信号转导等生命过程中发挥着重要作用。本文采用RACE技术和基因克隆技术分别对大鳞副泥鳅(Paramisgurnus dabryanus)和泥鳅(Misgurnus anguillicaudatus)精巢组织的GNB2L1基因c DNA序列进行了克隆。序列分析表明,大鳞副泥鳅GNB2L1基因c DNA序列全长1 115 bp,开放阅读框(ORF)长965 bp,编码317个氨基酸;泥鳅GNB2L1基因c DNA序列的开放阅读框长965 bp,编码317个氨基酸;两种泥鳅GNB2L1基因编码的蛋白与其他鱼类的RACK1蛋白的同源性为94%~97%,且不同进化地位物种的GNB2L1基因均由8个外显子和7个内含子组成。以GNB2L1基因为标记基因,构建的鱼类系统发育树显示,大鳞副泥鳅和泥鳅在进化上的亲缘关系最近。RT-PCR结果显示,GNB2L1基因在大鳞副泥鳅成体各组织中均有表达,且在脑组织的表达量高于其他组织。以上结果表明,GNB2L1基因为一个进化保守基因,可能在大鳞副泥鳅的细胞活动中发挥着重要作用。     

一个新的白血病相关基因LRP16全长cDNA的克隆、序列分析及表达特征

 克隆一个与白血病复发相关基因 (LRP1 6)的全长 c DNA序列 ,对其进行染色体定位、组织表达谱分析 ,并对该基因编码蛋白质进行原核表达 .首先用获得的一段 3kb DNA片段在 NCBI提供的 h ESTs数据库中进行电子杂交并对重叠克隆片段组装 ,再设计引物进行 c DNA末端快速扩增(RACE技术 ) .采用 Northern印迹方法进行组织表达分析 .以高通量基因组序列 (HTGS)数据库为基础进行染色体定位 .对构建的克隆菌用 IPTG诱导重组蛋白表达后进行 SDS- PAGE,同时对重组体测序确证 .钓取了该基因全长 c DNA、推导所编码的氨基酸序列 ,并将该基因定位于染色体1 1 q1 2 .2 .原核表达筛选获得了该基因重组子的一个缺失体 .对 LRP1 6基因全长 c DNA的序列分析提示 ,该基因可能编码两种 N端不同的蛋白质 ,且该基因的转录本可能存在一种丰度较低的剪接体 .     

山猪群体钙调蛋白酶抑制蛋白基因遗传变异研究

目的：研究猪钙调蛋白酶抑制蛋白（CAST）基因在山猪群体的遗传变异情况,为山猪肉质研究奠定基础。方法：应用PCR-SSCP技术和测序方法检测山猪及其杂种猪CAST基因的遗传多态性,并与其他品种猪相应序列进行比较。结果：用pCT1引物在山猪及其杂种群体中检测到2个多态位点（A,B）,用pCT2引物检测到3个多态位点（C,D,E）;序列分析表明,C和E位点共同拥有6处变异。结论：通过与其他猪品种比较,发现山猪的CASTMsp基因型分布与梅山猪的基因型分布完全一样,而与国外品种猪的基因型分布差异明显。     

人TIMP-1 cDNA克隆、真核表达载体构建及在COS-7细胞中的表达

根据 Gen Bank中 TIMP- 1基因的碱基序列 ,用 RT- PCR方法从人的正常肾组织中克隆出包含信号肽在内的 TIMP- 1全长 c DNA序列 .采用 T- A克隆的方法将之插入 p CRR2 .1中间载体 ,DNA测序证实该片段序列与文献报告的完全一致 .利用亚克隆的方法将 TIMP- 1 c DNA片段克隆到 pc DNA3载体上 ,构建出 pc DNA3/ TIMP- 1的真核表达载体 ,通过脂质体 DOTAP转染至 COS-7细胞 ,Northern印迹及原位杂交证实在 COS- 7细胞上获得人 TIMP- 1的高效表达 ,细胞增殖实验表明 TIMP- 1的高产表达可促进 COS- 7细胞的增殖 ,证实了所转染人 TIMP- 1的生物活性     

一例急性间歇性血卟啉病病例报道及文献回顾

目的:对患有急性间歇性血卟啉病先证者及其两位直系亲属进行基因突变的分析。方法:采用PCR和一代测序技术分别对患者的HMBS基因的外显子及其旁翼区进行序列分析。结果:检测出先证者HMBS基因11号外显子的旁翼区发生杂合突变c.651+2AG,为剪切突变;从先证者母亲以及女儿的HMBS基因上检测出同样的突变位点。结论:根据先证者的家族史、临床表现及相关代谢检查结果诊断为血卟啉病;基因检测结果提示先证者为急性间歇性血卟啉病;先证者的母亲和女儿存在同样的突变位点,提示先证者母亲及其女儿均患有急性间歇性血卟啉病。     

LALBA基因SNP与内蒙古白绒山羊经济性状的关联

利用PCR-SSCP和DNA测序技术检测452份内蒙古白绒山羊α-乳白蛋白(LALBA)基因单核苷酸多态性(SNP), 并分析SNP与产绒量、绒厚、绒长和体重性状的关联。结果表明, 仅P2引物位点存在SSCP多态, 其外显子3区域存在1个突变位点: M63868:g.1897T>C。内蒙古白绒山羊群体LALBA基因M63868:g.1897位点以TT型为主, T等位基因频率为0.983, 且处于Hardy-Weinberg平衡状态(P>0.05)。方差分析表明, LALBA基因M63868:g.1897位点多态仅与产绒量存在显著相关(P=0.017); 1897位点TC基因型个体产绒量比TT基因型个体多产绒142.68 g, 高26.21%, 且差异显著(P<0.05)。因此, TC基因型可作为山羊产绒性状标记辅助选择的有效DNA标记。     

13例肝豆状核变性家系致病基因突变的检测和分析

为了研究Wilson病(wilson disease,WD)基因突变的类型和发生情况,探讨WD疾病的病因、临床特点,通过采用变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)方法结合DNA测序方法对13例无亲缘关系的患者及33住亲属进行A TP7B基因所有外显子及5'端非转录区突变检测.33例DNA标本确认11种基因突变,其中包括9种错义突变(p.R778L、p.R919G、p.T1178A、p.T977M、p.K1010Q、p.C490TERM、p.A874P、p.G943S、p.G943D),1种缺失突变(2790delCAT),1种剪切位点改变(c.1708-1G＞C (intron 4 +1 G＞C)).在13个家系中,发现4例R778L突变,2例2790delCAT,均为杂合型突变,涉及17个等位基因,本组检出率为70.8％(17/24).DHPLC-测序法是WD疾病基因突变筛查较为快捷、全面而且敏感的方法.WD基因突变存在热点区分别为3、8、12号外显子.2790delCAT、p.K1010Q、c.1708-1G＞C (intron 4+1 G＞C)是ATP7B基因新突变类型,p.S406A、p.V456L、p.V 1140A、p.R952K是中国人比较常见的多态类型.     

兔出血症病毒NJ85株衣壳蛋白变异性分析及立体结构预测

用分子克隆技术从兔出血症病毒(RHDV)中国早期流行株NJ85中成功克隆出vp60基因,序列分析表明基因长度为1740nt,编码579aa.利用GenBank数据库,NJ85与WX84、TP二个RHDV中国毒株vp60基因DNA序列的同源性分别为92.7%和97.2%,氨基酸序列的同源性分别为96.1%和98.6%,与其他国家16个毒株的vp60基因DNA序列的同源性在83.7%～97.0%之间,氨基酸序列的同源性在90.5%～99.0%之间,具有高度的同源性. 进一步分析vp60基因的六个分区,A、B、D、F四个区变异率较低,C、E二个区变异率较高.在遗传进化上,历年来的RHDV毒株在氨基酸水平上分析可分为三个支谱系,在核苷酸水平上趋向四个支谱系,谱系没有呈现地域或时间特征.三个中国毒株分布在二个不同的支谱系中.与RHDV同为兔病毒属的欧洲野兔综合征病毒(EBHSV)组成了另一个谱系.运用生物信息学方法分析了NJ85 VP60蛋白的分子量、等电点、疏水性和二级结构,根据同源模型预测分析了三级结构.NJ85 VP60的二级结构以β片层为主,三级结构稳定.病毒衣壳表面有32个杯状凹陷, 由90个二聚体组成,二聚体由VP60单体以A/B5和C/C2两种方式构成,单体在二聚体中的构象有A、B、C三种形式.单体含有S、P两个结构域,两者通过柔性绞链连接,P结构域由VP60的C端部分形成,P分为P1和P2二个亚结构域,P2位于病毒衣壳表面,含有病毒株特异性抗原表位和红细胞结合位点.     

A novel RAB7 mutation in a Chinese family with Charcot–Marie–Tooth type 2B disease

Charcot–Marie–Tooth type 2B (CMT2B) disease is a hereditary motor and sensory neuropathy subtype characterized by prominent loss of sensation, distal muscle weakness and wasting skin ulcers. Recurrent ulcers often require amputation of lower limbs. To date, only four mutations of the RAB7 gene, which encodes the small GTPase, have been associated with CMT2B. A Chinese family with CMT2B was identified. Direct DNA sequencing performed on the affected individuals in this family revealed a novel mutation (p.Asn161Ile) in RAB7. The mutation is located in a potential mutational hotspot region, implicating the importance of this region for RAB7 protein. This is the first report of RAB7 mutation in Asian population.     

Evaluation of ROM1 as a candidate gene in generalised progressive retinal atrophy in dogs

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and other animals. The genetic origin is unknown in most cases. We have screened the coding sequence of the ROM1 gene for disease causing mutations in Tibetan Terriers, Miniature Poodles, Dachshunds and Chesapeake Bay Retrievers by single strand conformation polymorphism analysis (SSCP). Two polymorphisms have been identified by sequencing, one in exon 1 in all examined breeds (position 210: G→A; Gly40Arg and position 252: G→T; Ala53Ser). Another polymorphism was present in exon 2 (position 1150: C→T and position 1195: C→T) segregating in Miniature Poodles. None of these polymorphisms were cosegregating with gPRA rendering a disease causing mutation in the ROM1 gene unlikely.     

Alterations of autophagy in the peripheral neuropathy Charcot-Marie-Tooth type 2B

Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7A^WT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7A^T22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7A^V162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.     

Identification of A Novel SBF2 Frameshift Mutation in Charcot–Marie–Tooth Disease Type 4B2 Using Whole-exome Sequencing

Abstract Charcot-Marie-Tooth disease type 4B2 with early-onset glaucoma （CMT4B2, OMIM 604563） is a genetically-heterogeneous childhood-onset neuromuscular disorder. Here, we report the case of a 15-year-old male adolescent with lower extremity weakness, gait abnormalities, foot deformities and early-onset glaucoma. Since clinical diagnosis alone was insufficient for providing pathogenetic evidence to indicate that the condition belonged to a consanguineous family, we applied whole-exome sequencing to samples from the patient, his parents and his younger brother, assuming that the patient＇s condition is transmitted in an autosomal recessive pattern. A frame-shift mutation, c.4571delG （P.Gly1524Glufs＊42）, was revealed in the CMT4B2-related gene SBF2 （also known as MTMR13, MIM 607697）, and this mutation was found to be homozygous in the proband and heterozygous in his parents and younger brother. Together with the results of clinical diagnosis, this case was diagnosed as CMT4B2. Our finding further demonstrates the use of whole-exome sequencing in the diagnosis and treatment of rare diseases.     

The phosphoinositide-3-phosphatase MTMR2 associates with MTMR13, a membrane-associated pseudophosphatase also mutated in type 4B Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by distinctive, focally folded myelin sheaths. CMT4B is caused by recessively inherited mutations in either myotubularin-related 2 (MTMR2) or MTMR13 (also called SET-binding factor 2). MTMR2 encodes a member of the myotubularin family of phosphoinositide-3-phosphatases, which dephosphorylate phosphatidylinositol 3-phosphate (PI(3)P) and bisphosphate PI(3,5)P2. MTMR13 encodes a large, uncharacterized member of the myotubularin family. The MTMR13 phosphatase domain is catalytically inactive because the essential Cys and Arg residues are absent. Given the genetic association of both MTMR2 and MTMR13 with CMT4B, we investigated the biochemical relationship between these two proteins. We found that the endogenous MTMR2 and MTMR13 proteins are associated in human embryonic kidney 293 cells. MTMR2-MTMR13 association is mediated by coiled-coil sequences present in each protein. We also examined the cellular localization of MTMR2 and MTMR13 using fluorescence microscopy and subcellular fractionation. We found that (i) MTMR13 is a predominantly membrane-associated protein; (ii) MTMR2 and MTMR13 cofractionate in both a light membrane fraction and a cytosolic fraction; and (iii) MTMR13 membrane association is mediated by the segment of the protein which contains the pseudophosphatase domain. This work, which describes the first cellular or biochemical investigation of the MTMR13 pseudophosphatase protein, suggests that MTMR13 functions in association with MTMR2. Loss of MTMR13 function in CMT4B2 patients may lead to alterations in MTMR2 function and subsequent alterations in 3-phosphoinositide signaling. Such a mechanism would explain the strikingly similar phenotypes of patients with recessive mutations in either MTMR2 or MTMR13.     

Molecular characterization and expression analysis of Mtmr2, mouse homologue of MTMR2, the Myotubularin-related 2 gene, mutated in CMT4B

Charcot-Marie-Tooth type 4B (CMT4B) is caused by mutations in the myotubularin-related 2 gene, MTMR2, on chromosome 11q22. To date, six loss of function mutations and one missense mutation have been demonstrated in CMT4B patients. It remains to be determined how dysfunction of a ubiquitously expressed phosphatase causes a demyelinating neuropathy. An animal model for CMT4B would provide insights into the pathogenesis of this disorder. We have therefore characterized the mouse homologue of MTMR2 by reconstructing the full-length Mtmr2 cDNA as well as the genomic structure. The 1932 nucleotide open reading frame corresponds to 15 coding exons, spanning a genomic region of approximately 55 kilobases, on mouse chromosome 9 as demonstrated by fluorescence in situ hybridization analysis. A comparison between the mouse and human genes revealed a similar genomic structure, except for the number of alternatively spliced exons in the 5'-untranslated region, two in mouse and three in man. In situ hybridization analysis of mouse embryos showed that Mtmr2 was ubiquitously expressed during organogenesis at E9.5, with some areas of enriched expression. At E14.5, Mtmr2 mRNA was more abundant in the peripheral nervous system, including in dorsal root ganglia and spinal roots.     

A novel mutation (Cys308Phe) of the LDL receptor gene in families from the South-Eastern part of Poland

The purpose of this investigation was to characterize a new mutation in the LDL-receptor (LDLR) gene in three families with clinically diagnosed familial hypercholesterolemia (FH) from the South-Eastern part of Poland. Mutational screening with exon by exon sequencing analysis was performed in all probands. The novel mutation c986G>T (Cys308Phe) in the exon 7 of LDLR gene was found in three apparently unrelated probands with FH. Analysis of the receptor activity of peripheral blood lymphocytes by binding and uptake of DiL-LDL showed a significant reduction (by 24% versus healthy control) of the fluorescent label in the lymphocytes of patients heterozygous for this mutation. Concentrations of serum LDL-C in probands before treatment were between 9.5 and 10.5 mmol/l. All patients had corneal arcus and tendon xanthoma. Clinically, families were characterized by premature coronary artery disease. This mutation occurred relatively frequently in our group of patients with FH, but this could be explained by a founder effect since we demonstrated their common ancestors.     

The topology,structure and PE interaction of LITAF underpin a Charcot-Marie-Tooth disease type 1C

Background

Mutations in Lipopolysaccharide-induced tumour necrosis factor-α factor (LITAF) cause the autosomal dominant inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1C (CMT1C). LITAF encodes a 17 kDa protein containing an N-terminal proline-rich region followed by an evolutionarily-conserved C-terminal ‘LITAF domain’, which contains all reported CMT1C-associated pathogenic mutations.

Results

Here, we report the first structural characterisation of LITAF using biochemical, cell biological, biophysical and NMR spectroscopic approaches. Our structural model demonstrates that LITAF is a monotopic zinc-binding membrane protein that embeds into intracellular membranes via a predicted hydrophobic, in-plane, helical anchor located within the LITAF domain. We show that specific residues within the LITAF domain interact with phosphoethanolamine (PE) head groups, and that the introduction of the V144M CMT1C-associated pathogenic mutation leads to protein aggregation in the presence of PE.

Conclusions

In addition to the structural characterisation of LITAF, these data lead us to propose that an aberrant LITAF-PE interaction on the surface of intracellular membranes contributes to the molecular pathogenesis that underlies this currently incurable disease.

     

An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy

Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-¹⁴C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations.     

Homozygous Defects in LMNA, Encoding Lamin A/C Nuclear-Envelope Proteins, Cause Autosomal Recessive Axonal Neuropathy in Human (Charcot-Marie-Tooth Disorder Type 2) and Mouse

The Charcot-Marie-Tooth (CMT) disorders comprise a group of clinically and genetically heterogeneous hereditary motor and sensory neuropathies, which are mainly characterized by muscle weakness and wasting, foot deformities, and electrophysiological, as well as histological, changes. A subtype, CMT2, is defined by a slight or absent reduction of nerve-conduction velocities together with the loss of large myelinated fibers and axonal degeneration. CMT2 phenotypes are also characterized by a large genetic heterogeneity, although only two genes---NF-L and KIF1Bbeta---have been identified to date. Homozygosity mapping in inbred Algerian families with autosomal recessive CMT2 (AR-CMT2) provided evidence of linkage to chromosome 1q21.2-q21.3 in two families (Zmax=4.14). All patients shared a common homozygous ancestral haplotype that was suggestive of a founder mutation as the cause of the phenotype. A unique homozygous mutation in LMNA (which encodes lamin A/C, a component of the nuclear envelope) was identified in all affected members and in additional patients with CMT2 from a third, unrelated family. Ultrastructural exploration of sciatic nerves of LMNA null (i.e., -/-) mice was performed and revealed a strong reduction of axon density, axonal enlargement, and the presence of nonmyelinated axons, all of which were highly similar to the phenotypes of human peripheral axonopathies. The finding of site-specific amino acid substitutions in limb-girdle muscular dystrophy type 1B, autosomal dominant Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy type 1A, autosomal dominant partial lipodystrophy, and, now, AR-CMT2 suggests the existence of distinct functional domains in lamin A/C that are essential for the maintenance and integrity of different cell lineages. To our knowledge, this report constitutes the first evidence of the recessive inheritance of a mutation that causes CMT2; additionally, we suggest that mutations in LMNA may also be the cause of the genetically overlapping disorder CMT2B1.