X连锁迟发性脊柱骨骺发育不良快速基因诊断方法的研究

目的探讨建立X连锁迟发性脊柱骨骺发育不良（SEDT）快速基因诊断的方法。方法发现一个4代68人累及8例患者的SEDT大家系,呈X连锁隐性遗传。在应用PCR和DNA测序方法对SEDL进行基因突变分析后,提取外周血淋巴细胞RNA,应用RT-PCR扩增cDNA直接测序,建立快速基因诊断方法。结果RT-PCR结果显示,家系8例患者均为SEDL基因外显子6插入突变（c.370-371ins A,）,并发现1例无症状患儿携带相同突变（症状发生前）,6例女性携带者为杂合突变,家系其他成员和正常对照中均未见该插入突变。结沦RT-PCR检测扩增SEDL基因cDNA直接测序是一种快速基因诊断的方法。     

X-连锁迟发性脊椎骨骺发育不良遗传学研究进展

X-连锁迟发性脊椎骨骺发育不良(X-linked spondyloepiphyseal dysplasia tarda SEDL,OMIM313400)是一种罕见的遗传性骨软骨发育不良性疾病,遗传方式为X连锁隐性遗传。临床特点为轻中度非匀称性矮小和早发骨关节炎。SEDL的致病基因—SEDL基因定位于:Xp22.2,cDNA全长2836bp,编码含140个氨基酸残基的蛋白质,其功能尚未完全明确。51.2％的SEDL基因突变发生在外显子4和外显子5,有多种类型的突变可导致SEDL的临床表型,其中缺失突变最常见,占56.1％。SEDL基因型与临床表型之间有一定的相关性,但也存在表型异质性。     

X-连锁迟发性脊椎骨骺发育不良遗传学研究进展

X-连锁迟发性脊椎骨骺发育不良(X-linked spondyloepiphyseal dysplasia tarda SEDL，OMIM 313400)是一种罕见的遗传性骨软骨发育不良性疾病，遗传方式为X连锁隐性遗传。临床特点为轻中度非匀称性矮小和早发骨关节炎。SEDL的致病基因—SEDL基因定位于Xp22、2，cDNA全长2836bp，编码含140个氨基酸残基的蛋白质，其功能尚未完全明确。51．2％的SEDL基因突变发生在外显子4和外显子5，有多种类型的突变可导致SEDL的临床表型，其中缺失突变最常见，占56．1％。SEDL基因型与临床表型之间有一定的相关性，但也存在表型异质性。     

X-连锁迟发性脊椎骨骺发育不良SEDL基因新突变

目的　研究X 连锁迟发性脊椎骨骺发育不良 (X linkedspondyloepiphysealdysplasiatarda ,SEDL)的发病机理。方法　应用聚合酶链反应 单链构象多态及变性聚丙烯酰胺测序凝胶电泳技术 ,并结合DNA序列分析方法 ,对 5例SEDL患者及 3 0名正常对照SEDL基因的全部编码外显子及其邻近序列进行突变分析。结果　在 1例SEDL患者中发现了致病突变 ,并经DNA序列分析证实 ,SEDL基因第 5内含子剪接受体处IVS5 2— 1delAG紧接第 6外显子 3 2 2— 3 3 2delTTTTCAATGAA共 13个碱基缺失。结论该突变系国内外尚未见报道的新突变 ,这一突变可引起SEDL。     

X-连锁迟发性脊椎骨骺发育不良SEDL基因剪接受体突变导致潜在剪接位点激活

目的 深入研究X-连锁迟发性脊椎骨骺发育不良（X-linked spondyloepiphyseal dysplasia tarda,SEDL）的发病机理，为最终防治本病提供依据。方法 应用逆转录-PCR及克隆测序方法对1例涉及SEDL基因第5内含子剪接受体缺失的SEDL患者进行mRNA表达研究。结果 该患者存在2个不同片段长度的mRNA表达产物，与GenBank正常序列进行BLAST比较后发现，393bp的表达产物是第6外显子内一个新的潜在剪接位点激活后形成的产物；433bp的表达产物与8号染色体的部分基因组序列完全一致。结论 SEDL基因第5内含子剪接受体位点及其后的第6外显子共13个碱基的缺失突变导致第6外显子内一个新的潜在剪接受体位点激活，使转录后的mRNA丢失了第6外显子内47bp的编码序列，并使紧接其后的2个密码子产生移码，导致翻译的提前终止（D109-S123del；S124fsX126）。另外，该突变可能激活了8号染色体上假基因SEDLP2的转录，从而部分地补偿了SEDL蛋白的功能。     

SEDL基因及其突变体真核表达载体的构建与鉴定

目的构建SEDL基因及其突变体与增强型绿色荧光蛋白(EGFP)表达载体的融合表达质粒pEGFP-C3-SEDL并获得表达。方法分别提取X连锁迟发性脊柱骨骺发育不良(SEDT)患者和正常对照外周血淋巴细胞RNA,RT-PCR方法扩增SEDL基因cDNA,双酶切后克隆至pEGFP-C3空载体,构建表达质粒pEGFP-C3-SEDL。双酶切和DNA测序鉴定后,转染COS-7细胞,通过流式细胞仪和荧光显微镜观察重组蛋白表达情况。结果 DNA测序显示重组真核表达载体pEGFP-C3-SEDL构建成功,SEDL基因c.370-371ins A突变位点被成功克隆到突变体重组质粒中。荧光倒置显微镜观察证实重组质粒均能在细胞内进行蛋白表达。结论 SEDL基因及其突变体真核表达载体的成功构建为其进一步研究SEDL基因突变致SEDT的分子机制奠定了基础。     

X-连锁迟发性脊椎骨骺发育不良家系SEDL基因突变研究

目的：确定中国汉族中一个X－连锁迟发性脊椎骨骺发育不良（spondyloepiphyseal dyskplasia tarda,SEDL)大家系SEDL基因突变类型，探讨SEDL发病的分子基础。方法：用聚合酶链反应扩增产物双向直接测序方法检测了患者构成SEDL基因可读框的第3－6外显子及相邻侧翼区的DNA序列，将测序结果与GenBank公布的SEDL基因正常序列对比找出突变。然后在家系其他成员中证实该突变。结果：在2例患者（Ⅳ15、Ⅴ3）SEDL基因第2内含子剪接受体处发现了IVS2－2A→C突变，4个外显子的核苷酸序列未见改变。该突变在4例女性携带者得到证实，她们的基因型表现为野生型与突变型杂合现象。家系中2名未受累男性和15名无关健康个体未检测到这一突变。在该家系还检测出4个无症状的携带者。结论：首次发现SEDL基因IVS2－2A→C突变。该突变引起SEDL基因第2内含子3'端剪接受体改变，使之不能与外显子3正常剪接，可能是SEDL发病的分子基础。检测该突变可进行基因诊断，有重要的临床价值。     

X连锁迟发性脊椎骨骺发育不良家系SEDL基因突变分析

目的 鉴定中国西南地区一个4代迟发性脊椎骨骺发育不良大家系的分子遗传缺陷.方法 采用X染色体荧光标记微卫星标记物进行连锁分析,并通过直接序列分析筛查SEDL基因突变.结果 DXS987与DXS8051之间呈现连锁(最大LOD值:3.82;θ=0),致病基因定位于Xp22.2-Xp23.1;序列分析发现SEDL基因第4外显子发生点突变(c.239A＞G),导致在第80位编码氨基酸由组氨酸置换为精氨酸(H80R).结论 SEDL基因与此中国迟发性脊椎骨骺发育不良大家系表型完全连锁,并发现该基因新的致病突变(H80R).     

X-连锁迟发性脊椎骨骺发育不良家系基因突变研究

目的 研究X-连锁迟发性脊椎骨骺发育不良(X-linked spondyloepiphyseal dysplasia tarda,SEDL)患者的发病机理,并探讨该病的快速基因诊断方法.方法 应用逆转录聚合酶链反应,结合序列分析方法,对一个X-SEDL家系2例患者及育龄女性进行SEDL基因突变分析.结果 cDNA序列分析显示患者为G209A突变,并对突变所在第4外显子进行PCR扩增并测序进一步证实.患者女儿为该突变的携带者.结论 由于SEDL基因较小,直接对患者提取总RNA,逆转录后直接进行PCR扩增、测序,可直接发现基因阅读框内的多种类型的突变,相对于针对每一个外显子单独扩增检测更加直接、快速.     

X-连锁迟发性脊椎骨骺发育不良家系SEDL基因突变及基因诊断

目的 确定一个X-连锁迟发性脊椎骨骺发育不良(X-linked spondyloepiphyseal dysplasia tarda,SEDL)家系的基因突变类型;建立一种快速基因诊断方法.方法 采用体格检查、影像学检查及家系分析进行临床诊断.针对SEDL基因的第3～6外显子及其侧翼序列设计4对引物,建立基于PCR的变性高效液相色谱技术(denaturing high performance liquid chromatography,DHPLC)快速基因分型方法.常规酚-氯仿法从该家系3代18名成员的外周血中提取基因组DNA,经PCR/DHPLC分析,筛查出SEDL基因突变所在的片段,对该片段进行序列分析以确定突变位点及类型.结果 DHPLC分析发现该家系的SEDL基因突变位点在第4外显子片段,序列分析证实为c.218C》T突变,导致氨基酸序列S73L改变.在该家系的18名成员中,3例男性患者,5例女性肯定携带者和2例未婚女性携带者均带有该突变,其余表型正常的8名成员中未检测到这一突变.各成员的DHPLC峰型所代表的基因型与表型结果相吻合.结论 首次报告中国人SEDL基因c.218C》T突变,丰富了中国人SEDL基因的突变谱.采用的技术快速、可靠,能对SEDL进行快速基因分型和产前诊断.     

Identification of a novel protein interacting with RPGR

A novel protein, called RPGRIP, has been identified as interacting with the RPGR protein, which is mutated in a severe form of human retinal degeneration, X-linked retinitis pigmentosa (RP3 type). The bovine RPGRIP was identified initially by screening for RPGR-interacting proteins with a bovine retina cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by co-immunoprecipitation of in vitro translated protein and using RPGR mutants. The human RPGRIP gene was isolated and shown to be expressed in retina and testis. Human RPGRIP spans a genomic interval of 34 kb, and consists of 15 exons, some of which are alternatively spliced. It was mapped using monochromosomal and radiation hybrid cell lines to chromosomal region 14q11. The function of RPGRIP is unknown; it shows no homology to proteins of known function, although it is predicted to form two coiled-coil domains at the N-terminus. RPGRIP is a strong candidate gene for causing human retinal degeneration.     

Mutational spectrum of the CHAC gene in patients with chorea-acanthocytosis

Chorea-acanthocytosis (ChAc) is an autosomal recessive neurological disorder whose characteristic features include hyperkinetic movements and abnormal red blood cell morphology. Mutations in the CHAC gene on 9q21 were recently found to cause chorea-acanthocytosis. CHAC encodes a large, novel protein with a yeast homologue implicated in protein sorting. In this study, all 73 exons plus flanking intronic sequence in CHAC were screened for mutations by denaturing high-performance liquid chromatography in 43 probands with ChAc. We identified 57 different mutations, 54 of which have not previously been reported, in 39 probands. The novel mutations comprise 15 nonsense, 22 insertion/deletion, 15 splice-site and two missense mutations and are distributed throughout the CHAC gene. Three mutations were found in multiple families within this or our previous study. The preponderance of mutations that are predicted to cause absence of gene product is consistent with the recessive inheritance of this disease. The high proportion of splice-site mutations found is probably a reflection of the large number of exons that comprise the CHAC gene. The CHAC protein product, chorein, appears to have a certain tolerance to amino-acid substitutions since only two out of nine substitutions described here appear to be pathogenic.     

乙型肝炎病毒S基因密码子最佳化及在毕赤酵母中的高效表达

目的 制备适合酵母表达的乙型肝炎病毒S基因,在毕赤酵母中高效表达重组乙型肝炎病毒S蛋白.方法 选用酵母偏爱的同义密码子替换野生型S基因的酵母稀有密码子,采取基因搭桥法及递归式聚合酶链反应,制备合成基因,捕入酵母表达载体pPICZB.携带有合成S基因的重组质粒转化毕赤酵母菌株KM71 H,经甲醇诱导表达乙型肝炎病毒S蛋白.结果 酶切电泳及DNA测序证实合成的S基因正确克隆到表达载体中;聚丙烯酰胺凝胶电泳及免疫印迹显示优化后的乙型肝炎病毒S基因在毕赤酵母中的重组蛋白表达量远高于野生型基因.结论 密码子优化的S基因能明显提高重组S蛋白在毕赤酵母表达系统中的表达量.     

Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis.

A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).     

Cloning,characterization, and chromosome mapping of the human GlcAT-S gene

We report on the structure, map location, and tissue expression of the human GlcAT-Sgene. The gene covers approximately 85 Kb on chromosome 6 (6q13) between the D6S455 and D6S1673 markers. GlcAT-S is composed of four exons and encodes a 324-amino-acid protein, which shows 89% homology with the rat glcat-s protein and is involved in the biosynthesis of the HNK-1 carbohydrate epitope on glycoproteins. Although GlcAT-Swas considered an interesting candidate gene for the RP25 locus, the absence of any pathogenic mutations in probands of RP25-linked families ruled out that candidacy.