Alzheimer病患者早老素-1基因第5外显子突变特征分析

目的　探讨早老素-1 基因突变在散发性Alzheim er 病(sporadic Alzheim er's disease, SAD)患者发病机理中的作用。方法　应用聚合酶链反应-单链构象多态性(polym erase chain reaction-singlestrand conform ation polym orphsim ,PCR-SSCP)及DNA 直接测序技术检测68 例SAD 患者和65 名正常老年人的早老素-1 基因第5 外显子。结果　发现68 例SAD患者中有4 例患者的SSCP发生泳动异常,DNA 序列分析发现:这4 例SAD 患者的130 号密码子发生了CTG→ATG 错义突变(388 位点发生C→A突变),使氨基酸由亮氨酸变为蛋氨酸(Leu 130 Met);157 号密码子发生了GTG→CTG 错义突变(469 位点发生G→C突变),使氨基酸由缬氨酸变为亮氨酸(Val157 Leu);有11 例患者的SSCP表现为一条单链电泳迁移率明显增快,DNA 序列分析发现:这11 例SAD患者的130 号密码子发生了CTG→ATG 错义突变(388 位点发生C→A 突变),使氨基酸由亮氨酸变为蛋氨酸(Leu 130 Met);154 号密码子发生了TGC→TGT 同义突变(462 位点发生C→T)突变。结论　我们发现在SAD患者中存在早老素-1 基因第5 外显子突变,该突变点可能为中国人SAD 患者早老素基因突变点之一。     

中国人阿耳茨海默病早老素-1基因测序一例

阿耳茨海默病 (Alzheimer's disease,AD)可分为早发性、晚发性家族性 AD(familial Alzheimer's disease,FAD)和散发性 AD(sporadic Alzheimer's disease,SAD)。国外研究发现 ,70 %～ 80 %的早发性 FAD与 Presenilin- 1(PS- 1)基因突变有关[1 ] 。我们在一个 FAD家系患者中发现了外显子 46 5 19nt T→A杂合性点突变 ,报告如下。1　材料与方法1.1　检测对象　先证者 ,女 ,47岁 ,1993年因进行性智能障碍来我院就医 ,诊断为 FAD[2 ]。 2例 SAD患者系本院住院患者 ,均符合美国精神疾病诊断统计手册第 3版修订版 (DSM- - R)和史玉…     

PAX6基因5′端非编码区剪接突变能够引起先天性无虹膜症

目的对一先天性无虹膜症家系进行了致病基因PAX6的突变分析。方法PCR反应扩增PAX6基因的所有外显子,PCR产物进行SSCP（单链构象多态性）分析,通过患者与正常人带型的差异来确定突变发生的外显子,对有差异SSCP带型的PCR产物进行直接测序找到突变位点。PCR产物进一步亚克隆到pGEM-T载体,测序验证突变位点。结果发现基因突变为PAX6基因第2内含子和第3外显子之间的剪接识别位点＂AG＂中的碱基A的丢失（IVS2-2delA）。结沦PAX6基因5′端非编码区剪接突变能够引起先天性无虹膜症。     

聚合酶链反应-单链构象多态性分析在家族性高胆固醇血症患者家系调查中的应用

目的　探讨聚合酶链反应 -单链构象多态性分析 (polymerase chain reaction- single strainconformation polymorphism,PCR- SSCP)在家族性高胆固醇血症 (familial hypercholesterolemiac,FH)患者家系分析中的应用价值。方法　对于经 PCR- SSCP筛查、DNA序列分析证实的 4例 FH患者 (1例纯合子 FH外显子 7发生点突变 ,1例杂合子点突变位于外显子 14,2例杂合子点突变位于外显子 4的 3′部分 ) ,用 PCR- SSCP分析各家系成员共 2 3例 ,并对基因型和表型进行比较。结果　各家系成员均从基因水平明确了诊断 ,2 3例个体中 ,除先证者外 ,还发现 1例纯合子 ,8例杂合子。结论　 PCR- SSCP可对 FH先证者家系进行分析 ,并对其家系成员早期诊断 ,以便提供咨询和指导。     

白介素-1遗传多态性和Alzheimer病

淀粉样前体蛋白基因、早老素 - 1基因、早老素 - 2基因的突变可导致家族性 Alzheimer病 (FAD)的发生 ,散发性Alzheimer病 (SAD)与许多遗传易感性基因相关 ,其中最常见的是载脂蛋白 E基因的 ε4等位基因。近来的研究表明 ,AD与白介素 - 1(IL- 1)基因多态性相关。IL- 1水平在 AD脑内增高 ,IL- 1的过表达与 β-淀粉样斑块形成有关 ,并与其他危险因素如高龄、头外伤等共同作用 ,促进 SAD的发生。研究表明 ,IL- 1在 AD的发生、发展过程中具有重要作用     

冠心病新致病基因MEF2A第一、第八外显子突变的检测

目的研究冠心病新致病基因MEF2A在中国人群突变情况。方法利用聚合酶链反应-单链构象多态性(polym erase chain reaction-single strand conform ation polymorph ism,PCR-SSCP)和DNA测序技术对156例冠心病(CAD)患者第1外显子及第8外显子进行基因突变检测。结果MEF2A基因第1外显子区域6例患者SSCP泳动异常,第8外显子区域7例患者SSCP泳动异常,但DNA直接测序未发现MEF2A基因第1、8外显子区域基因突变。结论冠心病患者在MEF2A基因第1、8外显子未发现新的突变,PCR-SSCP结果与DNA测序结果并非平行关系,SSCP同样存在假阳性。     

新疆维吾尔族阿尔茨海默病患者脑啡肽酶基因与载脂蛋白E基因多态性的关联分析

目的 探讨脑啡肽酶(neprilysin,NEP)基因rs3736187位点突变及其与载脂蛋白E(apolipoprotein E,ApoE)基因相互作用在新疆维吾尔族人群散发性阿尔茨海默病(sporadic Alzheimer disease,SAD)发病机制中的作用.方法 应用聚合酶链反应-限制性片段长度多态性方法 检测了111例维吾尔族SAD患者和117名维吾尔族正常老年人NEP基因和ApoE基因多态性分布特征.结果 (1)NEP基因T等位基因频率在AD组高于对照组(x2=5.005,P＜0.05),携带T等位基因个体出现AD的危险性高于携带C等位基因的个体.(2)ApoE基因ε4等位基因频率AD组高于对照组(x2=4.218,P＜0.05),携带ε4等位基因个体出现AD的危险性高于未携带ε4等位基因的个体.(3)NEP基因的T等位基因与SAD发病相关且不受ApoE基因型影响.结论 NEP基因和ApoE基因的基因多态性与新疆维吾尔族SAD发病有关联.NEP基因可能是新疆维吾尔族SAD发病独立的易感基因.     

家族性帕金森病α-共核蛋白基因第3、4外显子的初步研究

目的 分析家族性帕金森病与α-共核蛋白基因的相关性，在其第3、4外显子中寻找相关突变或多态。方法 收集中国帕金森病家系，采用单链构象多态性(single strand conformational polymorphism，SSCP)与异源双链(heteroduplex analysis，HA)分析相结合的方法，筛查α-共核蛋白第3、4外显子中是否存在致病性突变。结果 SSCP和HA分析第3、4外显子未见单双链泳动异常。基因测序发现第4内含子5′端的第23位和67位分别插入1个c和t。结论 (1)α-共核蛋白基因的第3、4外显子不是中国家族性帕金森病的突变热点；(2)发现α-共核蛋白基因第4内含子在不同人群中有两个多态位点(23ins c和67ins t)。     

127例PKU患者PAH基因第12外显子点突变及其频率研究

目的　了解中国人苯丙酮尿症 ( phenylketonuria,PKU)患者的苯丙氨酸羟化酶( phenylalanine hydroxylase,PAH)基因第 12外显子点突变种类和频率。方法　应用单链构象多态性( single strand conformation polymorphism,SSCP)、变性梯度凝胶电泳 ( denaturing gradient gelelectrophoresis,DGGE)、DNA测序分析了 12 7例 PKU患者的 PAH基因第 12外显子点突变种类及频率。结果　 DNA测序分析显示 10例患者存在 R4 13P、S4 11X、R4 0 8W、R4 0 8Q 4种杂合突变 ,其突变频率分别为 2 .76 %、0 .39%、0 .39%、0 .39% ,S4 11X突变为中国人中首次报道。 SSCP分析仅发现 2例 R4 13P杂合突变 ,DGGE分析显示 10例出现 3种类型的异常电泳带型。R4 13P突变在南北方人之间、在经典型 PKU和高苯丙氨酸血症之间的分布差异无显著性。结论　 DGGE对 PAH基因第 12外显子点突变检出率明显高于 SSCP。 DGGE结合 DNA测序是明确 PAH基因第 12外显子点突变种类和频率较好的方法。 R4 13P突变在南北方人中分布无明显差异     

信号转导和转录激活因子6基因多态性与湖北汉族人变应性哮喘的相关性研究

目的　探讨信号转导和转录激活因子 (STAT) 6基因 3′非翻译区G2 96 4A位点多态性和第一外显子GT串联重复序列遗传多态性与湖北汉族人变应性哮喘易感性的关系。方法　用聚合酶链反应 限制性片段长度多态性 (PCR RFLP)技术检测STAT6基因G2 96 4A位点多态性 ;用聚合酶链反应 短串联重复多态性 (PCR STR)技术对STAT6基因第一外显子微卫星进行分型 ,并将PCR产物克隆及测序鉴定 ;采用病例 对照法研究了 1 35例变应性哮喘患者和 1 0 9例对照。结果　 ( 1 )湖北地区汉族人群STAT6基因G2 96 4A位点基因型以GA型最为常见 ;哮喘组与对照组STAT6基因G2 96 4A位点的等位基因频率和基因型频率GG、GA、AA之间差异无统计学意义 (P >0 .0 5)。 ( 2 )STAT6基因第一外显子微卫星多态性共检出GT串联重复次数为 1 3、1 4、1 5、1 6的 4种等位基因 ;第一外显子微卫星的多态性检测出 1 3/1 4基因型在哮喘患者组和正常对照组相比差异具有统计学意义(P =0 .0 0 1 4 )。 ( 3)STAT6基因第一外显子GT二核苷酸串联重复序列多态性中 1 3 GT重复等位基因与 2 96 4A变异体之间存在连锁不平衡 (P =0 .0 0 0 0 2 1 8)。结论　STAT6基因 3′非翻译区G2 96 4A位点多态性与湖北汉族人哮喘易感性无明显相关性 ;第一外显子GT二核苷酸串联重     

Is the presenilin-1 E318G missense mutation a risk factor for Alzheimer's disease?

Nearly all of the presenilin-1 (PSEN-1) mutations are missense mutations leading to Alzheimer's disease (AD). The role of the mutation E318G (a substitution of glutamic acid to glycine) in the PSEN-1 is controversial. It has been found both in AD patients and in non-demented control individuals. Using the polymerase chain reaction and the restriction fragment length polymorphism method, we screened for E318G mutation in a total of 16 familial (FAD) cases, in 64 sporadic neuropathologically confirmed AD cases and in 270 non-demented controls including 35 neuropathologically confirmed individuals. We detected the E318G mutation in four FAD cases, seven sporadic AD cases and 10 control individuals with highly varying onset-ages. Odds ratios for carrying the mutation were 7.6 and 3 in FAD and sporadic AD cases, respectively. Our results suggest that this mutation could be a risk factor in the Finnish FAD and sporadic AD population. It may be in linkage disequilibrium with a pathogenic change somewhere else in the PSEN-1 gene or in close proximity to the PSEN-1 gene.     

Molecular evidence of presenilin 1 mutation in familial early onset dementia

Early onset familial Alzheimer disease (FAD) has been associated with mutations in three genes, of which presenilin 1 (PSEN1) mutations are the most frequent. We reported previously a variant form of FAD, due to deletion of exon 9 of PSEN1, with spastic paralysis and rigidity. We describe a novel PSEN1 mutation in a family of Japanese origin with six affected individuals of both genders in two generations. The disease is characterized by presenile dementia, which is preceded by spastic paraparesis and apraxia. This mutation, which is predicted to cause a missense substitution of serine for glycine, occurred at codon 266 in exon 8 of PSEN1. The mutation was not found in 200 controls and 200 sporadic AD patients. On this basis alone, it seems this mutation is pathogenic. Our findings provide a new clue to the etiology of the familial early onset dementia.     

A novel PSEN1 gene mutation (L235R) associated with familial early-onset Alzheimer's disease

Mutations in the presenilin 1 (PSEN1) gene are the most frequent cause of familial Alzheimer's disease (AD), with at least 182 different mutations published to date. We report a 48-year-old woman (age at onset 47 years) who presented a progressive alteration of episodic memory, spatial disorientation, apathy, language disturbances and neglect of personal care. Her MMSE score was 20/30. The patient presented an unusually rapid deterioration and at 6 months follow-up her cognitive and functional status had worsened considerably (MMSE score of 11). Cranial MRI showed a bilateral atrophy with temporal and parietal predominance and the quantification of AD CSF biomarkers showed the typical AD signature. Family history evidenced an autosomal dominant pattern of inheritance. Mutational screening was performed by direct sequencing of exons 3-12 of PSEN1. The patient presented the 3/3 APOE genotype. Genetic analysis revealed a nucleotide substitution in exon 7 of PSEN1 gene, producing a missense mutation in codon 235 from leucine amino acid to arginine (L235R). This amino acid is conserved between presenilin-1 and presenilin-2 proteins. The L235R mutation had not been previously reported, although other mutations in the same residue have also been associated with familial early-onset AD, providing support for the importance of this residue for the presenilin-1 function.     

Systematic genetic study of Alzheimer disease in Latin America: mutation frequencies of the amyloid beta precursor protein and presenilin genes in Colombia

Nearly all mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid beta precursor protein (APP) genes lead to early-onset Alzheimer disease (EOAD, onset age at or before 65 years). In order to assess the genetic contribution of these genes in a series of Colombian AD cases, we performed a systematic mutation analysis in 11 autosomal dominant, 23 familial, and 42 sporadic AD patients (34% with age of onset < or = 65 years). No APP missense mutations were identified. In three autosomal dominant cases (27.2%), two different PSEN1 missense mutations were identified. Both PSEN1 mutations are missense mutations that occurred in early-onset autosomal AD cases: an I143T mutation in one case (onset age 30 years) and an E280A mutation in two other cases (onset ages 35 and 42 years). In addition, a novel PSEN1 V94M mutation was present in one early-onset AD case without known family history (onset age 53 years) and absent in 53 controls. The E318G polymorphism was present in five AD cases and absent in controls. In PSEN2, two different silent mutations were detected, including one not reported elsewhere (P129). The majority of the Colombian AD cases, predominantly late-onset, were negative for PSEN and APP mutations.     

中国人遗传性非息肉病性结直肠癌MSH6基因胚系突变的测序研究

目的探讨中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系中MSH6基因胚系突变。方法采用PCR-直接测序的方法检测39个无胚系MSH2及MLH1基因突变、符合不同临床标准的中国人HNPCC家系先证者MSH6基因各外显子胚系突变;对137名正常人胚系基因组DNA进行错义突变相应外显子的测序分析。应用Envision二步法检测有突变的先证者肿瘤组织MSH6蛋白表达。结果在39个HNPCC先证者中共发现6个MSH6基因的胚系突变,分别位于第4、6、9和第10外显子;突变类型为4个错义突变、1个无义突变、1个剪接区的插入突变;对4个错义突变的相应外显子的测序分析显示:137名正常人胚系基因组DNA5例具有第6外显子1163密码子处的c.3488A>T的错义突变,约占3.65%(5/137),为单核苷酸多态性(single nucleotide polymorphism,SNP);其余错义突变在正常人群中均未发现。在6例有MSH6基因胚系突变家系的肿瘤组织中免疫组化染色除1例为SNP的肿瘤组织MSH6蛋白阳性表达外,其余均为阴性表达。经过查询国际HNPCC突变数据库及SNP数据库证实上述突变中5个为国际上尚未报道的病理性突变,1个为新发现的SNP。结论MSH6基因胚系突变在符合不同临床标准的中国人HNPCC中均起一定作用,对无MSH2及MLH1基因胚系突变的先证者行MSH6基因胚系突变的测序分析对确诊HNPCC家系是必要的。     

SIP1基因编码区点突变及多态性在先天性巨结肠症中的检测与分析

目的 检测先天性巨结肠症(Hirschsprung disease,HSCR)患者SIP1(Smad-interacting protein 1)基因编码区点突变及单核苷酸多态性特点,探讨SIP1基因与HSCR的关系.方法 应用聚合酶链反应.单链构像多态性(polymerase chain reaction-single strand conformation polymorphism,PCR-SSCP)和DNA直接测序技术,对50例HSCR、30名正常对照外周血SIP1基因编码区的10个外显子,进行点突变与单核苷酸多态性的检测与分析.结果 HSCR与正常对照突变图谱比较,有1例病例在第7外显子出现杂合性缺失,密码子157位点GTG→GTA置换,引起亮氨酸的同义突变,属于单核苷酸多态性,突变率为2%(1/50).有4例患者在第8外显子出现突变,突变率为8%(4/50).PCR-SSCP银染分析,第2外显子2例出现相同类型泳动变位;第7外显子3例出现相同类型泳动变位;第8外显子7例出现相同类型泳动变位.结论 HSCR有SIP1基因突变,提示SIPI基因与HSCR的发病存在一定程度的关联.     

A mutation screening by DHPLC of PSEN1 and APP genes reveals no significant variation associated with the sporadic late-onset form of Alzheimer's disease

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is usually divided into familial and sporadic forms, according to family history. The familial form has often been reportedly caused by mutations in amyloid precursor protein (APP), presenilin-1 (PSEN1), or presenilin-2 (PSEN2) genes, whereas the genetic component for the sporadic form is less clear. We carried out mutation screening in exons 16 and 17 of APP, and in exons 3, 4, 5, 6, 7, 10 of PSEN1 genes in patients with the sporadic late-onset form of AD (LOAD). The aim of this study was to ascertain whether any variation in these genes, besides that of the well-known apolipoprotein E common polymorphism, could be involved in the onset of the disease. To search for the single nucleotide substitutions, we examined 172 LOAD patients by the denaturing high-performance liquid chromatography (DHPLC) technique. Only one same-sense mutation in exon 4 of PSEN1 gene (N32) was observed in this patient group. We concluded that the variation in the screened exons of the APP and PSEN1 genes, reportedly associated with familial AD, is not present in LOAD.     

肾上腺脑白质营养不良患者ABCD1基因第6外显子突变的初步分析

目的 检测肾上腺脑白质营养不良(adrenoleukodystrophy，ALD)(MIM300100)患者编码ALD蛋白的ABCD1基因[ATP-结合盒(ATP-binding cassette，ABC)超家族中D亚家族1]突变。方法 提取无亲缘关系的14例中国ALD患者及其中2例患者父母基因组DNA。聚合酶链反应(polymerase chainreaction，PCR)扩增ABCD1基因的第6外显子，以琼脂糖凝胶电泳鉴定PCR产物，PCR产物纯化后DNA直接测序。结果 证实3个患者的ABCD1基因第6外显子有突变，其中1例患者为5’末端上游第6个碱基C缺失(1489-6 del C)，推测该突变可能导致剪切错误；1例患者为错义突变1559T→A(L520Q)，这两例患者的母亲均为杂合突变。另1例患者为1548G→A(L516L)的同义突变。结论 首次报告了中国大陆ALD患者ABCD1基因突变．第6外显子无大片段缺失和重组突变。同一血缘族中可有不同的表型存在，提示是否有其它遗传或环境因素参与表型的表达。通过DNA测序方法亦证实其中2例患者之母为ABCD1基因的杂合子。