肝豆状核变性基因12号外显子突变特征的研究

目的 研究中国人肝豆状核变性（ＷＤ）基因１２号外显子的突变特征,为建立直接基因诊断的方法提供理论依据。方法 应用聚合酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）技术,对４４例无亲缘关系的确诊患者和６０名正常对照组进行ＷＤ基因第１２号外显子的突变检测,并用ＤＮＡ测序证实其突变性质和位置。结果 ８例患者在１２号外显子检测到２种错义突变,占１８％（８／４４）,其中６例为Ｔｈｒ９３５Ｍｅｔ突变,２例为Ｇｌｙ     

Wilson病ATP7B基因突变研究

目的：研究肝豆状核变性（WD）ATP7B基因常见突变种类和形式,以期建立WD的ATP7B基因突变热点的检测平台。方法：提取30名WD患者外周血基因组DNA,聚合酶链反应（PCR）扩增ATP7B基因第5、8、12号外显子,并进行DNA直接测序。结果：30例WD患者共检测到6种ATP7B基因突变,均为点突变,8号外显子检测到5种突变,突变频率40.00%;12号外显子检测到1种突变,突变频率23.33%;5号外显子未检测到突变。结论：ATP7B基因第8和12号外显子可能是WD基因突变热区,以Arg778Leu和Arg952Lys为高频突变位点,WD患者的基因突变检测应首选第8和12号外显子。     

Wilson病基因突变类型与临床表型的关系

目的 探讨Wilson病（WD）基因突变类型与临床表型的关系。方法 应用聚合酶链反应-单链构象多态分析（PCR-SSCP）和DNA测序方法,对84例WD患者进行WD基因全长21个外显子的突变检测。结合统计学方法,着重分析并探讨中国人WD基因突变热点Arg778Leu基因型与临床表型的关系。结果 18例患者检出Arg778Leu纯合突变。29例患者在一条染色体上检出Arg778Leu杂合错义突变。其中,在另一条染色体上,3例检出移码突变;15例突变未明;余11例检出其他错义突变。就理论而言。与错义突变相比。移码突变可导致更严重的功能缺陷及表型。不能与错义突变混合进行统计学分析。另15例患者因有一条染色体突变未明亦被排除在外。因此,相对Arg778Leu纯合错义突变进行统计学分析时,只能选择11例发生复合错义突变的患者做为Arg778Leu杂合突变病例组。经统计学分析。携带Arg778Leu纯合突变的患者和携带Arg778Leu杂合突变的患者,其起病年龄,血清铜蓝蛋白水平及临床类型均差异有显著意义,P值小于0.05或0.001。结论 Arg778Leu突变不是一种温和的突变。它可导致患者症状出现较早并较严重。     

Identification of a mutation hotspot in exon 8 of W ilson disease gene by cycle sequencing

Objective To screen for mutation hotspot of Wilson disease (WD) gene in Chinese population.Methods Cycle sequencing was used to detect mutation in exon 8 of WD gene in 30 patients with Wilson disease. Results The same missense mutation, Arg779Leu, was identified in 14 WD patients, four of whom were homozygous and the other heterozygous for this mutation.The frequency of this mutation in Chinese patients was 30%.Conclusion The codon 779 (CCG→CTG) of exon 8 of WD gene was one of mutation hotspots in Chinese.     

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     

CCC2基因缺陷型酵母对中国人Wilson病突变热点致病性的研究

【目的】应用CCC2基因缺陷型酵母研究中国人Wilson病（Wilson disease，WD）突变热点Arg778Leu的致病性，在蛋白水平上探讨中国人Wilson病的发病机制。【方法】应用RT-PCR和TOPOTA克隆技术分段克隆ATPTB的全长cDNA，USE定点诱变技术制备突变体，应用酵母功能互补分析的方法研究Arg778Leu的致病性。【结果】本研究应用USE定点诱变技术成功制备了ATPTB的Arg778Leu突变体，在CCC2缺陷型酵母内表达人的野生型ATPTB可以弥补酵母内CCC2蛋白的运铜功能，使CCC2缺陷型酵母在铜、铁及亮氨酸缺陷型培养基上生长良好；对突变体的研究表明Arg778ku只能部分代偿CCC2蛋白的运铜功能。【结论】本试验证明CCC2基因缺陷型酵母是研究人ATPTB的一种良好的细胞模型，证明了中国人WD的常见突变Arg778Leu是一种致病性的突变。     

Psychopathology and personality traits in patients with treated Wilson disease grouped according to gene mutations

Wilson disease (WD) is a recessively inherited copper storage disorder mainly affecting liver and brain. Genotype/phenotype correlations have been report ed but as yet not regarding psychic symptoms. Our aim was to investigate if a correlation might exist between genotype and phenotype concerning psychopathology and/or personality traits in patients with treated WD. Nine homozygous and three compound heterozygous Swedish patients were retrospectively investigated, representing four different mutation settings. Psychopathological symptoms were studied using the Comprehensive Psychopathological Rating Scale (CPRS), personality traits using the Karolinska Scales of Personality (KSP) and mutations were analyzed by manifold sequencing. Psychopathological symptoms: Patients with the Trp779Stop mutation had the lowest scores on the total CPRS, due to less pronounced reported CPRS items, as compared to the other three groups of patients. Compound heterozygotes for the His1069Gln/Arg1319Stop mutation showed the highest total CPRS scores. Personality traits: Patients homozygous for the Trp779Stop and the Thr977Met mutations had high scores on Psychopathy related scales whereas patients with His1069Gln/Arg1319Stop mutations had the lowest scores on these scales. Serum ceruloplasmin levels were undetectable in all patients with the Trp779Stop and Thr977Met mutations. The results show a trend towards a genotype/phenotype correlation regarding psychopathological symptoms and personality traits in treated patients with WD. If replicable, these results might contribute to the elucidation of the possible clinical importance of functionally deleterious gene mutations in WD psychopathology and personality traits.     

家族性与散发性肥厚型心肌病基因突变的对比研究

目的 研究中国家族性及散发性肥厚型心肌病（HCM）患者的致病基因突变位点的异同。方法 对10个家系中36例HCM患者及50例散发的HCM患者进行B肌球蛋白重链（MYH7）、肌钙蛋白T（TNNT2）及肌球结合蛋白C（MYBPC3）扫描,聚合酶链式反应扩增,双脱氧末端终止法测序。结果家族性HCM患者中,3个家系的13例HCM患者发现MYH7错义突变,分别为18号外显子发生G12601A突变（Arg663His）、23号外显子发生G15373A突变（Glu924Lys）、20号外显子发生T13659C突变（Ile736Thr）,散发的50例HCM患者中,有1例发现MYH7的20号外显子上T13659C突变。所有HCM患者均未发现TNNT2基因突变。家族性HCM患者中有2个家系共4例发现MYBPC3基因突变：2例为18号外显子上的Arg502Trp、2例为13号外显子碱基插入突变,即在7425～7426间插入CGGCA,导致Arg346fs移码突变;50例散发性HCM患者中未发现此基因突变。结论 MYH7和MYBPC3可能为我国家族性HCM的主要致病基因之一,而我国散发性HCM患者MYH7和MYBPC3基因突变率低;TNNT2可能不是我国HCM患者的主要致病基因。     

PCR技术对Wilson病基因进行定点突变的研究

【目的】利用聚合酶链反应 (PCR)技术对Wilson病 (WD)基因进行体外定点突变的研究。【方法】采用PCR定点突变技术 ,首先设计两对引物 ,将突变位点设计在引物上 ,通过重叠延伸法两次PCR扩增 ,扩增片段上含有所需要的突变位点 ,最后将扩增片段克隆入pRc/CMV载体中。【结果】DNA测序表明在预期位点已经发生突变 ,WD基因第 778位密码子由精氨酸 (Arg)残基突变为亮氨酸残基 (Leu) ,用PCR定点突变技术成功构建Wilson病基因突变体。【结论】PCR技术诱导定点突变准确、高效。Wilson病基因突变体的构建成功 ,为进一步研究该突变位点导致Wilson病的发病机制和Wilson病蛋白的结构和功能的关系奠定了基础。     

青少年Gitelman综合征2例临床特点及基因分析

目的研究2例Gitelman综合征患者的临床特点及其SLC12A3基因的突变特点,以提高对该病的认识。方法回顾2例临床诊断为Gitelman综合征的青少年男性患者的临床表现、实验室检查结果等,并对SLC12A3基因进行测序,以确定其突变位点。结果2例患者均表现为不同程度的乏力,低血钾、低血镁、低尿钙,及高血浆肾素活性、高醛固酮水平。应用氯化钾缓释片、门冬氨酸钾镁针、钙镁片治疗后,患者乏力有所缓解,但是易反复。基因检测共发现3个SLC12A3基因的突变位点,均为错义突变：Thr163Met、Gly196Val、Arg871His。患者A存在杂合突变（Thr163Met、Arg871His）,患者B存在纯合突变（Gly196Val）。结论SLC12A3基因检测有助于早期明确诊断Gitelman综合征。     

Association of ghrelin polymorphisms with metabolic syndrome in Han Nationality Chinese

Objective To investigate the association of ghrelin gene polymorphisms with metabolic syndrome in Han Nationality Chinese. Methods A total of 240 patients with metabolic syndrome and 427 adults aged above forty years were recruited. Genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism analysis. Results The allelic frequency of the Leu72Met polymorphism was 17.3% in the patient group and 11.9% in the control group （x^2=7.36, P=0.007）. Metabolic syndrome was more prevalent among carriers of the Met72 variant （43.8 vs 33.1%, age- and sex-adjusted odds ratio=1.57, P=0.01）. No Arg51Gln variants were found in our study subjects. Conclusion Rather than being associated with its individual components, Leu72Met polymorphism is associated with metabolic syndrome in the Han Nationality Chinese. Arg51Gln polymorphism is rare in the Han Nationality Chinese.     

一个复合杂合突变导致的遗传性凝血因子XII缺陷症家系分析

目的：对一个复合杂合基因突变导致的遗传性凝血因子XII（FXII）缺陷症家系进行实验室表型和基因突变分析,寻找致病基因并初步探讨其分子发病机制。方法：检测先证者及其家系成员（共3代5人）血浆凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、凝血因子VIII促凝活性（FVIII:C）、凝血因子Ⅸ促凝活性（FIX:C）、凝血因子XI促凝活性（FXI:C）、XII促凝活性（FXII:C）和FXII抗原含量（FXII:Ag）等指标以明确诊断。采用PCR直接测序法分析先证者F12基因所有的外显子及其侧翼序列,对可疑的突变反向测序进行验证,并对家系成员的相应突变位点区域进行检测。采用ClustalX-2.1-win软件和4个在线生物信息工具（Mutation Taster、Poly-Phen-2、PROVEAN和SIFT）分析氨基酸突变位点的保守性和突变对蛋白质功能的可能影响;用Swiss-Pdb Viewer 4.0.1软件对突变位点进行蛋白模型相互作用分析。结果：先证者APTT为59.1 s,明显延长;FXII:C和FXII:Ag分别降低至4%和5%,其父亲、母亲、女儿的FXII:C和FXII:Ag降低至32%～37%。基因分析发现先证者F12基因第13号外显子存在复合杂合基因突变,即c.1561G＞A杂合错义突变（p.Glu502Lys）和c.1637T＞C杂合错义突变（p.Met527Thr）;其父亲为p.Met527Thr携带者,母亲和女儿为p.Glu502Lys携带者。保守性分析结果表明,Glu502和Met527在同源物种间高度保守;4个在线生物信息学软件对该两个突变的预测结果一致,均预示很可能是有害突变,可引起相关疾病。突变蛋白模型分析显示,野生型Glu502与His365之间形成1条氢键,Glu502替换为Lys502导致氢键的消失;野生型Met527与Leu524之间形成1条氢键,Met527替换为Thr527导致Thr527-Leu524之间增加2条氢键。结论：该先证者F12基因第13号外显子上存在p.Glu502Lys和p.Met527Thr复合杂合突变,推测该突变遗传自具有血缘关系且分别存在p.Glu502Lys和p.Met527Thr杂合子的父母,并与该家系FXII水平降低有关。     

Novel mutations of PRSS1 gene in patients with pancreatic cancer among Han population

Background  A high mortality rate of pancreatic cancer becomes a bottleneck for further treatment with long-term efficacy. It is urgent to find a new mean to predict the early onset of pancreatic cancer accurately. The authors hypothesized that genetic variants of cationic trypsinogen (PRSS1) gene could affect trypsin expression/function and result in abnormal activation of protease activated receptor-2 (PAR-2), then lead to pancreatic cancer. The aim of this study was to elaborate some novel mutations of PRSS1 gene in the patients with pancreatic cancer.

Methods  Totally 156 patients with pancreatic cancer and 220 unrelated individuals as controls were enrolled in this study. The mutations of PRSS1 gene were analyzed by direct sequencing. K-ras Mutation Detection Kit was used to find the general k-ras gene disorder in the pancreatic cancer tissue. Then the clinical data were collected and analyzed simultaneously.

Results  There were two patients who carried novel mutations which was IVS 3 +157 G>C of PRSS1 gene in peripheral blood specimens and pancreatic cancer tissue. What’s more, it was surprising to find a novel complicated mutation of exon 3 in PRSS1 gene (c.409 A>G and c.416 C>T) in another young patient. The complicated mutation made No.135 and No.137 amino acid transfer from Thr to Ala and Thr to Met respectively. No any mutation was found in the normal controls while no mutations of k-ras gene were detected in the three patients.

Conclusion  Mutations of PRSS1 gene may be an important factor of pancreatic cancer.

     

一种新型纤维蛋白原β链错义突变导致的遗传性低纤维蛋白原血症

目的：对临床发现的1例遗传性低纤维蛋白原血症进行基因及表型分析,探讨其分子发病机制。方法：采集先证者及其家系成员共2代6人的外周血,采用STAGO自动化血凝仪检测凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、凝血酶时间（TT）、D-D二聚体（D-D）和纤维蛋白降解产物（FDPs）;凝血酶凝固法（Clauss法）与免疫比浊法分别测定血浆纤维蛋白原活性（Fg:C）和抗原水平（Fg:Ag）。PCR扩增纤维蛋白原FGA、FGB和FGG基因的所有外显子和侧翼序列,PCR产物纯化后直接测序进行基因分析。分别用Swiss-PDViewer、在线分析系统对突变蛋白的结构、突变位点的保守性进行预测分析。结果：先证者PT和TT均延长,Fg:C（0.82 g/L）和Fg:Ag（1.19 g/L）明显降低;基因分析发现先证者FGB基因存在c.425T＞G杂合突变,导致纤维蛋白原Bβ链上121位亮氨酸突变为精氨酸（Leu121Arg）。其父亲及儿子也存在该突变位点。蛋白模型分析显示Leu121突变为Arg121后,氨基酸极性发生改变,并且新增与Try117、Met118、Trp125之间的氢键;蛋白同源性分析显示：Leu121在脊椎动物间有较高的保守性。结论：纤维蛋白原Bβ链Leu121Arg杂合突变是引起该家系遗传性低纤维蛋白原血症的主要原因。     

扩张型心肌病与δ-肌聚糖基因突变

目的 :探讨中国人扩张型心肌病患者中δ-肌聚糖 (δ- sarcoglycan,SGCD)基因的突变情况。方法 :通过聚合酶链反应 -单链构象多态性分析 ,结合 DNA测序 ,对 60例扩张型心肌病患者和 60例对照 SGCD基因的所有 9个外显子进行分析。结果 :该基因外显子 9m发现 3种类型的单链构象多态性图谱 ;DNA测序证实其中一种为正常野生型 ,另外两种为832 G/A和 848A/G突变杂合子。 832 G/A突变 ( 2 78Ala→ Thr)仅在对照组检出 1例 ,对照组 A等位基因频率 0 .0 0 8;848A/G突变 ( 2 83Gln→ Arg)共检出 5例 ,G等位基因频率在扩张型心肌病组和对照组中差异无显著性 ( P>0 .2 5 ) ,对照组 G等位基因频率 0 .0 33。结论 :发现 SGCD基因 2个新的突变位点 ;未能证实这 2个位点的突变与扩张型心肌病相关     

安徽地区汉族人家族性与散发性肥厚型心肌病MYH7基因18及20号外显子突变分析

目的分析安徽地区汉族家族性及散发性肥厚型心肌病(HCM)患者的致病基因β肌球蛋白重链(MYH7)突变位点的异同。方法对3个家系中8例HCM患者及20例散发的HCM患者进行MYH7基因18及20号外显子扫描,双脱氧末端终止法测序。结果3个家族性HCM(FHCM)家系8例患者中,有2例患者发现MYH7基因20号及18号外显子发生突变,分别为20号外显子发生Arg723Gly突变;18号外显子发生Arg663His突变;散发的20例HCM患者中,有1例发现MYH7的20号外显子上发生Ile736Thr突变,未发现18号外显子发生突变。结论MYH7基因中18号、20号外显子突变可能是安徽地区FHCM的常见突变位点之一;散发性HCM(SHCM)可能与FHCM具有相似的发病机制,但与FHCM相比,在MYH7基因中突变率较低。     

应用变性高效液相色谱技术筛查一个正常血钾性周期性麻痹家系的SCN4A基因突变

目的 应用变性高效液相色谱(DHPLC)技术筛查一发生Met1592Val替换的正常血钾性周期性麻痹(normoKPP)家系的SCN4A基因,证实该突变为疾病相关突变,并总结其在此家系的临床表型特点。方法 总结一normoKPP家系14例患者的临床特点,应用DHPLC技术筛查SCN4A基因全部24个外显子,并对发现异常洗脱峰者进行测序。结果 该家系具有典型的normoKPP临床特征,无肌强直表现及早显、性别偏移现象,病程呈良性过程,发病早晚与症状严重程度及预后无相关。DHPLC筛查发现在外显子1及24存在杂合二倍体,测序及连锁分析证实位于外显子1的突变为良性多态性,外显子24的Met1592Val替换为疾病相关突变。结论 国人存在Met1592Val突变,此突变可导致normoKPP。normoKPP与高钾性周期性麻痹临床表现相似,两者可存在共同突变。     

PCR-SSCP技术检测中国人肝豆状核变性基因第18、14外显子突变

目的 了解中国人肝豆状核变性（WD）患者基因第18、第14外显子的突变情况，为掌握该痛的突变特点并进行基因诊断提供依据。方法 聚合酶链反应-单链构象多态性（PCR—SSCP）检测中国人WD基因第18、14外显子突变，并对异常带型进行直接DNA测序。结果 45例患者和20例正常人的18外显子PCR—SSCP出现两种泳动带型，异常带型测序证实无突变存在。14号外显子PCR—SSCP出现的带型均一致，结论 14外显子和18外显子可能不是中国人WD基因突变的热区。     

ATP7 B基因突变与肝豆状核变性发病关系的探讨

目的 对中国人肝豆状核变性( WD)患者 ATP7B 全基因测序,分析其突变及与WD的关系,并探讨ATP7B基因复合突变在WD发病中的意义. 方法 对临床确诊的WD患者84例及20 例健康者采集口腔黏膜细胞,提取基因组DNA为模板,应用聚合酶链反应对ATP7B 全部外显子5′ 端→3′ 端扩增;运用 DNA 直接测序法检测突变,并分析ATP7B基因突变临床意义及与预后的关系. 结果 在84例患者中,ATP7B基因突变率为82. 14%,还发现一些国内较少报道的ATP7B基因复合突变,可能与WD相关. 结论ATP7B基因突变有较大的异质性,对该基因突变的检测,能发现有相关ATP7B基因突变的WD患者.     

Compound heterozygous mutation in two unrelated cases of Chinese spinal muscular atrophy patients

Background Infantile proximal spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. Approximately 90%–95% cases of SMA result from homozygous deletion of survival motor neuron gene 1 (SMN1) and 5% cases are caused by compound heterozygous mutation (a SMN1 deletion on one allele and a subtle mutation on the other allele).

Methods In this research, two unrelated patients were clinically diagnosed according to the criteria of proximal SMA. Genetic diagnosis was performed to detect the homozygous deletion of exon 7 of SMN1 by PCR-restriction fragment length polymorphism (RFLP) and genomic sequencing. Multiplex ligation-dependent probe amplification (MLPA) analysis was carried out to measure copy numbers of SMN1, SMN2 and neuronal apoptosis inhibitor protein (NAIP) in the patients. Further sequencing of SMN1 allele-specific PCR (AS-PCR) and SMN1 clones were also performed to analyze the point mutation of SMN1 gene. Additionally, the pedigree analysis of these two families was carried out to identify the transmission of the mutation.

Results The inconsistent results using PCR-RFLP and genomic sequencing showed homozygous deletion of exon 7 of SMN1 and heterozygous deletion accompanied with a suspicious mutation in SMN1 gene, respectively. MLPA analysis of these two cases exhibited one SMN1 copy deletion. One identical c.863G>T (p.Arg288Met) mutation was found in two cases by sequencing the SMN1 clones, which confirmed that both cases were SMA compound heterozygotes. One case showed partial conversion to form hybrid SMN (SMN2 I7/SMN1 E8) identified by clones sequencing and another case carrying 3 SMN2 implied complete conversion from SMN1 to SMN2.

Conclusion p.Arg288Met is more a disease-causing mutation than a polymorphism variation, and children with this mutation may have more severe phenotypes.