用PCR结合甲基化特异PCR方法检测遗传性脆性X综合征

目的 建立一种快速、灵敏、廉价的实验室检测脆性X综合征(FXS)的方法.方法 检测130例样本的(CGG)n重复数.首先,PCR扩增脆性X智力低下基因Ⅰ(FMRⅠ)的(CGG)n重复序列;其次,进行甲基化特异性PCR (MS-PCR),对男性不能有效扩增和女性样本的基因组DNA进行亚硫酸氢钠脱氨基修饰,以修饰后的DNA为模板,用两套不同的引物对(甲基化特异性引物对和非甲基化特异性引物对)扩增FMRⅠ基因的(CGG)n重复序列区,PCR产物测序.结果 所有样本的(CGG)n重复数都在13～47之间;测序结果表明FMRⅠ基因中的非甲基化的C碱基转变为T碱基,甲基化的C碱基保持不变.结论 PCR结合MS-PCR方法可以同时检测FMRⅠ基因的 (CGG)n重复数和CpG岛的甲基化情况,为临床检测FXS提供了依据.     

非同位素PCR方法检测脆性X 综合征FMR-1基因CGG重复序列数目

目的 建立一种可靠、简便的非同位素PCR方法检测脆性X综合征的突变基因。方法 采用生物素标记的CGG寡核苷酸探针，检测通过PCR扩增的FMR-1基因中CGG三核苷酸重复序列数目。从而判断所检样品中FMR—1基因是否正常。结果 该法可检测出正常人及携带者的CGG重复拷贝数。结论 该方法可以简便、安全、可靠地检测FMR—1基因中(CGG)n重复拷贝数，从而确定为正常人或携带者，可作为临床上筛查脆性X综合征的首选方法。     

男性脆性X综合征快速筛查

脆性X综合征（FraX）是较为常见的遗传性智力低下疾病，发病率仅次于唐氏综合征的遗传性智力低下综合征，在xq28存在两个脆性位点FRAXA，FRAXE，以FRAXA为最常见，该病是由于脆性X智力低下基因FMR。功能丧失所致，约占全部儿童的0．05％，呈X隐性遗传，占X连锁智能低下的40％，分子遗传学研究表明，脆性X综合征最主要的致病突变FMR，基因5＇-非翻译区中不稳定的三核苷酸重复序列（CGG）。扩增。（CGG）。拷贝数在60～200时，携带者虽表型正常，但在传代中易发生进一步扩展，称为前突变，不导致疾病，但通过女性传递时拷贝数会大幅度增加，转变成全突变。     

慢性进行性舞蹈病一家系五例临床及基因分析

目的:调查舞蹈病的家系资料并对其成员外周血白细胞DNA进行基因分析.方法:对该病进行家系调查,应用巢式PCR及琼脂糖凝胶电泳技术检测患者、HD症前高风险成员、正常人(CAG)n片段的长度,进行基因分析.结果:该家系符合常染色体显性遗传病特征,患者DNA电泳显示两条扩增区带,正常人显示一条扩增区带.该家系中2例高风险成员未见基因异常,DNA电泳显示一条扩增区带.结论:舞蹈病为常染色体性遗传病,患者相关基因分端(CAG)n重复序列存在异常扩增.     

先天性智力低下儿童脆性X综合征的基因分析

目的探讨先天性智力低下与脆性X综合征智力低下基因1(Fragile X mental retardation gene 1,FMR-1)关系.方法应用复式PCR一次性扩增FMR-1基因的(CGG)n的重复区,检测CGG重复序列的大小,分析FMR-1基因状态:正常、前突变、全突变,对脆性X综合征可疑患儿进行快速筛查.结果在253例不明原因的先天性智力低下患儿中,检出脆性X综合征携带者(FMR-1基因前突变者)14例(2男12女),脆性X综合征患者(FMR-1基因全突变者)9例,阳性率达9.09%.结论脆性X综合征是引起儿童先天性智力低下的重要病因之一,应对不明原因的智力低下儿童进行大面积筛查,尤其是对有智力低下家族史的孕妇进行产前筛查,防止患儿出生.     

男性脆性X综合征快速筛查

脆性 X 综合征(FraX)是较为常见的遗传性智力低下疾病,发病率仅次于唐氏综合征的遗传性智力低下综合征,在xq~(28)存在两个脆性位点 FRAXA,FRAXE,以 FRAXA 为最常见,该病是由于脆性 X 智力低下基因 FMR_1功能丧失所致,约占全部儿童的0.05%,呈 X 隐性遗传,占 X 连锁智能低下的40%,分子遗传学研究表明,脆性 X 综合征最主要的致病突变FMR_1基因5′-非翻译区中不稳定的三核苷酸重复序列(cGG)_n 扩增。(CGG)_n拷贝数在60～200时,携带者虽表型正常,但在传代中易发生进一步扩展,称为前突变,不导致疾病,但通过女性传递时拷贝数会大幅度增加,转变成全突变。脆性 X 综合征患者 FMR_1基因中(CGG)_n 扩增的拷贝数大于200～300,较正常人多5～10倍,同时伴有其周围 DNA序列的异常甲基化,抑制 FMR_1基因表达。通常的检测方法     

广西地区正常人群SCA3／MJD基因多态性研究

目的探讨广西地区汉族正常人群SCA3／MJD基因（CAG）n重复拷贝数的正常变异范围。方法应用荧光PCR及毛细管电泳片段长度分析对广西75名正常人SCA3／MJD基因（CAG）n重复拷贝数进行分析。结果广西地区SCA3／MJD基因（CAG）n在正常人群中的变异范围为13～34个拷贝,集中于13个拷贝,其等位基因频率为52．00％,杂合频率为69．33％,共17种等位基因。结论SCA3／MJD基因（CAG）n重复序列在广西正常人群中呈现多态性,SCA3／MJD基因（CAG）n重复拷贝数正常变异范围存在地区差异。     

河南汉族一脊髓小脑性共济失调家系基因突变检测

目的检测和分析河南汉族一脊髓小脑性共济失调(SCA)家系亚型分型。方法采用聚合酶链式反应(PCR)技术和DNA直接测序法分析该家系患病者SCA1、SCA2、SCA3、SCA6、SCA7、SCA12、SCA17共7种常见SCA亚型基因序列,并与家系中其他正常个体及50例健康个体基因序列进行比较分析。结果检测到该家系4例患者及家系中2例健康成员SCA3基因的1个等位基因三核苷酸序列CAG异常重复扩增,异常重复次数在71~81次。其余6种亚型基因检测无异常。结论该家系SCA患病表现为中国人常见的SCA3亚型,家系中2例健康成员可能为症状前患者。     

CTLA-4基因微卫星多态性与子宫内膜异位症的相关性研究

目的:研究CTLA-4基因微卫星多态性与子宫内膜异位症(EM)的相关性.方法:采用聚合酶链反应技术-扩增片段长度多态性技术,分析56例EM患者和35例对照者CTLA-4基因外显子4的3,非翻译区,包含(AT)n重复序列的特异性等位基因.结果:EM患者CTLA-4基因外显子4的3'非翻译区(AT)n重复序列有12种等位基因.与对照组比较,120 bp等位基因频率在EM组中显著增高,差异有统计学意义(P＜0.01,OR=3.344,95%CI 1.498～7.464);CTLA-4(AT)n 120 bp等位基因频率在EM临床各期分布无明显差异(P＞0.05).结论:CTLA-4基因外显子4的3'非翻译区(AT)n重复序列多态性与EM相关;CTLA-4(AT)n重复序列120 bp等位基因可能是EM的易感基因.     

遗传性脊髓小脑型共济失调7型三家系临床特征及分子生物学研究

目的研究中国人遗传性脊髓小脑型共济失调7型（SCA7）的临床和分子生物学特征。方法应用聚合酶链反应、聚丙烯酰胺凝胶电泳、毛细管电泳等技术,检测临床诊断为脊髓小脑型共济失调（SCA）的184个家系245例患者和71例散发SCA患者以及163名正常人的SCA7基因内CAG三核苷酸重复次数,对异常等位基因片段进行DNA测序,并对其中一个大家系进行连锁分析。结果检出3个SCA7家系（15例患者）,阳性率为1．6％,测序证实异常等位基因的CAG重复次数为38-71次,其他SCA患者以及正常人的SCA7等位基因CAG重复次数为6-15次。其中2个家系存在遗传早现现象,特别在父系遗传时更明显。对其中一个家系进行连锁分析结果在微卫星标记D3S1300处获得两点最大LOD值为2．82（θ=0．00）。结论SCA7是少见的SCA亚型。SCA7基因异常重复突变是SCA7的致病原因,38次CAG重复是目前国内报道的SCA7最小的病理性扩增。     

Genetic diversity at the FMR1 locus in Mexican population

BACKGROUND: Fragile X syndrome is the most frequent cause of inherited mental retardation; it is caused by expansion of CGG repeats in the first exon of the FMR1 gene. Number of CGG repeats varies between 6 and 50 triplets in normal individuals and the most common alleles have 29 or 30 repeats. Allelic patterns in the global population are similar; however, some reports show statistical differences among several populations. Distribution of allelic frequencies for FMR1 locus has not been reported in Mexican population. METHODS: Determination of the CGG repeat number was achieved by polymerase chain reaction (PCR) on modified DNA from 129 unrelated Mexican mestizos (46 FRAXA-negative males with mental retardation and 83 healthy individuals). DNA modification by sodium bisulfite achieves conversion of unmethylated cytosine residues to uracil, which allows efficient amplification by single PCR. Methylation status of FMR1 region for each individual was also established. DNA sequencing of a number of amplified samples was realized to validate the procedure. RESULTS: Molecular analysis of the FMR1 gene showed 23 different alleles. Statistical comparison of allelic length between healthy and affected individuals does not show significant differences. Trinucleotide repeat number varied from 16-40, with modal number of 32 (27.58%), second peak at 30 (25.28%), and minor peak at 34 (10.34%). Together, allelic distribution in the Mexican sample differs significantly from those reported for Caucasian, Chinese, African, Indonesian, Brazilian, Chilean, and Mixtec populations. An excess of large alleles (> or =34 repeats) was evident. CONCLUSIONS: Allele distribution in FMR1 gene from Mexican mestizos is different from that of other reported populations around the world. This unusual modal pattern probably is related to the particular ethnic background of the Mexican population. On the other hand, PCR on modified DNA is a valuable and efficient method for determination of CGG repetitive sequences in FMR1 gene.     

A rapid screening and diagnosis on fragile X syndrome by PCR

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脆性X综合征的PCR筛查与诊断

采用多聚酶链反应（ＰＣＲ）技术结合变性序列胶银染方法对１６９例智力低下（简称智低）疑诊病例及６个脆性Ｘ综合征家系中３３名成员的ＦＭＲ１基因（ＣＧＧ）ｎ重复序列进行了分析,结果发现：（１）智低疑诊病例中３例男性未检出ＰＣＲ阳性产物;（２）脆性Ｘ综合征家系内,５例男性先证者亦未检出ＰＣＲ扩增带;（３）对上述ＰＣＲ结果为阴性的８例男性智低患者（此８例均经Ｓｏｕｔｈｅｒｎ印迹杂交证实为全突变患者）,设立ＦＲＡＸＥ位点引物作内对照,结果仅获得ＦＲＡＸＥ位点的正常扩增产物,故而可排除ＦＲＡＸＡ位点ＰＣＲ产物为假阴性的可能。结果表明,ＰＣＲ分析ＦＭＲ１基因（ＣＧＧ）ｎ重复序列可有效检出前突变携带者,若男性智低患者的ＰＣＲ结果为阴性,在设立内对照基本排除假阴性的前提下,对本病也有提示作用。该方法快速、简便、稳定、可靠,适合于脆性Ｘ综合征的大量群体筛查。     

脆性X综合征中不稳定的DNA序列和异常甲基化研究

采用ＰＣＲ结合序列胶分析的方法，对８２条正常中国人Ｘ染色体ＦＲＡＸＡ位点（ＣＧＧ）ｎ重复序列拷贝数的多态性进行了测定，其范围为２１～３１，高峰为２７。并通过Ｓｏｕｔｈｅｒｎ杂交分析了来自６个Ｆｒａ（Ｘ）家系的１５名成员（ＣＧＧ）ｎ的拷贝数与该重复序列上游ＣｐＧ岛的甲基化状态。Ｆｒａ（Ｘ）患者（ＣＧＧ）ｎ大量扩增并伴随ＣｐＧ岛异常甲基化。Ｆｒａ（Ｘ）携带者女性（ＣＧＧ）ｎ扩增较少，有嵌合现象。其子代或产生更大扩增，或保持原来状态，呈动态突变遗传特征。     

Repeat expansion and methylation-sensitive triplet-primed polymerase chain reaction for fragile X mental retardation 1 gene screening in institutionalised intellectually disabled individuals

INTRODUCTIONFragile X syndrome (FXS) is the most prevalent X-linked intellectual disability (ID) and a leading genetic cause of autism, characterised by cognitive and behavioural impairments. The hyperexpansion of a CGG repeat in the fragile X mental retardation 1 (FMR1) gene leads to abnormal hypermethylation, resulting in the lack or absence of its protein. Tools for establishing the diagnosis of FXS have been extensively developed, including assays based on triplet-primed polymerase chain reaction (TP-PCR) for detection and quantification of the CGG trinucleotide repeat expansion, as well as determination of the methylation status of the alleles. This study aimed to utilise a simple, quick and affordable method for high sensitivity and specificity screening and diagnosis of FXS in institutionalised individuals with ID.METHODSA total of 109 institutionalised individuals at the Center for Social Rehabilitation of Intellectual Disability Kartini, Temanggung, Central Java, Indonesia, were screened in a three-step process using FastFrax™ Identification, Sizing and Methylation Status Kits.RESULTSTwo samples that were classified as indeterminate with respect to the 41-repeat control at the identification step were subsequently determined to be non-expanded by both sizing and methylation status analyses. Two samples classified as expanded at the identification step were determined to carry full mutation expansions > 200 repeats that were fully methylated using sizing and methylation status analyses, respectively, yielding a disease prevalence of 1.83%.CONCLUSIONRepeat expansion and methylation-specific TP-PCR is practical, effective and inexpensive for the diagnosis of FXS, especially in high-risk populations of individuals with ID of undetermined aetiology.     

外周淋巴细胞FMR－1mRNA的检测

采用逆转录ＰＣＲ技术检测了人外周淋巴细胞ＦＭＲ－１ｍＲＮＡ表达。对１０名在临床筛查中被认为可能患有脆性Ｘ综合征的男性学生进行了检测，其中２例可疑男性样品中未检测出ＦＭＲ－１ｍＲＮＡ表达。ＤＮＡ印迹杂交和异常甲基化ＰＣＲ检测证实这２例患者同时伴有ＦＭＲ－１基因５＇ＣｐＧ岛异常甲基化和（ＣＧＧ）ｎ扩展。上述方法的建立为诊断脆性Ｘ综合征提供了一种新的分子生物学手段，并可用于ＦＭＲ－１基因表达研究。     

Trinucleotide repeat expansion of spinocerebellar ataxia (SCA1) found in a Chinese family.

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DNA damage and repair in lymphoblastoid cell lines from normal donors and fragile X syndrome patients

BACKGROUND: Because lymphocytes from fragile X patients have been reported as hypersensitive to bleomycin-induced chromatid breaks and because the number of trinucleotide repeats in families with fragile X syndrome has a propensity to expand, we have investigated the possibility that fragile X cells may be hypersensitive to DNA damage and have a lower capacity for DNA repair. METHODS: Lymphocytes from normal and fragile X syndrome donors were immortalized by Epstein-Barr virus transformation. Characteristics of fragile X syndrome including the folate-sensitive fragile site on chromosome Xq27.3, length of CGG repeat expansion, and FMRP expression in Epstein-Barr virus-transformed lymphoblastoid cell lines were analyzed by standard cytogenetic methods, Southern blot, and Western blot, respectively. Analysis of DNA damage and repair induced by hydrogen peroxide, bleomycin, ethyl methanesulfonate, 4-nitroquinoline-N-oxide, etoposide, and mitomycin C was carried out by single-cell gel electrophoresis assay (known as comet assay). RESULTS: Lymphoblastoid cell lines from fragile X donors had a folate-sensitive fragile site on chromosome Xq27.3, no or low FMRP expression, and expansion of the CGG repeat. Results of comet assay showed that fragile X cells were not more sensitive to mutagen-induced DNA strand breaks and did not have lower DNA repair capacity in comparison with normal cells. Furthermore, one fragile X cell line showed hyposensitivity to DNA strand breaks induced by hydrogen peroxide, bleomycin, and ethyl methansulfonate. CONCLUSIONS: The results of this study do not support the notion that CGG trinucleotide expansion in fragile X syndrome is caused by permanent deficiency in DNA repair.     

精神分裂症与多巴胺D4受体基因多态性的关联分析

目的：在中国汉族人群中寻找散发精神分裂症与多巴胺D4受体（DRD4）基因第3外显子48bp片段可变数串联重复（VNTR）多态性的关系。方法：使用病例－对照的关联分析的方法。对符合诊断标准的510名精神分裂症患者／171名正常对照就DRD4基因第三外显子48bp VNTR的多态性进行检测并进行关联分析。结果（1）所检测人群48bp VNTR多态性表现国2－7次重复,4次重复的等位基因在精神分裂症患者及对照组中分别占78．6％及76．9％,2次重复分别占16．2％及19．3％;（2）精神分裂症患者与正常对照之间的基因型频率分布差异具有显著意义,病例组短重复序列的基因型（2／2,2／3）频率（3．3％）显著低于对照组（10．5％）（X^2=14．88,df=2,P=0.00);（3）病例组含4次重复的基因型比例（95．9％）显著高于对照组（88．3％）（X^2＝13．00,df＝1,P=0.000)。结论：中国精神分裂症人群DRD4基因第三外显子48bp VNTR多态性主要集中于4次重复片段,48bp的重复次数可能与精神分裂症相关,短重复序列减少或含4次重复的基因型增多可能增加对精神分裂症的易感性。