先天性智力低下儿童脆性X综合征的分子流行病学调查

目的根据脆性X综合征的两个分子生物学标志,建立在先天性智力低下患儿中快速分析脆性X综合征智力低下基因1(Fragile X mental retardation gene 1,FMR1)突变的方法,对先天性智力低下儿童进行脆性X综合征的大面积筛查和诊断,调查智力低下儿童中脆性X综合征的发病率。方法应用复式PCR方法检测FMR1基因的(CGG)n重复区CGG重复序列的大小,判断FMR1基因状态(正常、前突变、全突变),快速筛查脆性X综合征可疑患儿。用甲基化特异性PCR方法,检测FMR1基因启动子区CpG岛的异常甲基化情况,进一步诊断脆性X综合征患者,并用Southern印迹技术进行验证。结果1248例先天性智力低下患儿中,筛查出149名不明原因的智低儿童进行了FMR1基因分析,检出脆性X综合征携带者(FMR1基因前突变者)32例(10男22女),脆性X综合征患者(FMR1基因全突变者)12例(9男3女),脆性X综合征检出率为8.05%。结论复式PCR法灵敏、快速、简便,适应于临床和基层开展脆性X综合征筛查。脆性X综合征在先天性智力低下儿童中的发病率高,应对先天性智力低下儿童进行脆性X综合征FMR1基因的分析。     

用聚合酶链反应技术对脆性X综合征进行筛查与诊断

目的 建立脆性X综合征大面积快速筛查与诊断的方法。方法 用7-deza-dGTP的PCR法扩增脆性位点智力低下1基因（fragile X mental retardation gene 1,FMR1)的（CGG）的重复区，检测CGG重复数的大小，对脆性X综合征进行诊断。结果 在一家系14个成员中，我们筛查出2例脆性X综合征携带者，5例患者；并用甲基化特异性X综合征男性携带者及患者进行快速鉴定；用甲基化特异性PCR法可以对脆性X综合征患者进行快速诊断。两种方法也适用于脆性X综合征的产前筛查与诊断。     

甲基化特异性三重PCR检测FMR1基因突变的研究

目的探讨甲基化特异性三重PCR检测FMR1基因不同突变类型的价值。方法用甲基化特异性三重PCR方法检测了99例病人的FMR1基因,并用半巢式PCR和Southern印迹杂交方法进行比较。结果用甲基化特异性三重PCR检测出70例男性正常基因型、27例女性正常基因型,1例男性全突变基因型,1例女性前突变基因型,与半巢式PCR和Southern印迹杂交方法的检测结果相符。结论甲基化特异性三重PCR能准确检测FMR1突变的不同类型,适用于对脆性X综合征的临床筛查和诊断。     

不同民族人群FMR1基因CGG重复序列检测与分析

目的 检测和分析脆性X综合征致病基因FMR1 CGG重复序列在汉族和壮族人群中的多态性分布。方法 采用PCR扩增技术和聚丙烯酰胺凝胶电泳技术对1060例汉族人（男280人，女780人）和283例壮族人（男85人，女198人）FMR1基因CGG重复序列进行分析，并用Southern blot技术对结果进行了验证。结果 汉族人群中共检测到33种等位基因，其中CGG重复序列范围为6～43，壮族人群共检测到27种等位基因，CGG重复序列范围为6～57，两类人群中最大频率等位基因分别为28和29。结论 应用PCR扩增技术进行了脆性X综合征大面积筛查，中国汉族和壮族人群中FMR1基因CGG重复序列变异分布略有差异。     

应用PCR快速筛查脆性X综合征患儿

目的应用PCR快速筛查脆性X综合征患儿。方法采用PCR和聚丙烯酰胺凝胶电泳技术,对24例不明原因智力低下患儿的脆性X基因（CGG）n重复序列进行检测。结果在24例不明原因智力低下患儿中,筛查出1例脆性X综合征患者。结沦采用PCR技术扩增脆性X基因的（CGG）n重复序列,可对脆性X综合征患者进行快速筛查。     

脆性X综合征的筛查及基因诊断

目的：建立一种简便快速初步筛查脆性X综合征智力缺陷基因FMR－1突变的方法。方法：采用套式PCR技术对新生儿及婴幼儿的足跟血X染色体上基因FMR－1CGG重复序列进行扩增,通过以其拷贝数的鉴定筛查其突变型。结果：共筛查5200全新生儿和婴幼儿,查出1例男婴患者,其母亲是携带者。结论：套式PCR能简便快速地初筛出人群中携带者和可疑患者,对脆性X综合征的早期诊断和产前诊断有应用价值。     

Slowdown PCR法产前快速筛查脆性X综合征

目的研究产前快速筛查脆性X综合征的基因检测方法。方法收集临床疑似脆性X综合征的男性智力低下患儿病例,采集相应患儿外周血,同时收集需要进行胎儿脆性X综合征产前筛查和诊断的孕妇病例,在孕20w收集孕妇的羊水。通过slowdownPCR方法检测FMR1基因(CGG)n三核苷酸重复顺序基因组,并采用测序方法进行验证。结果共检测疑似病例30例,通过slowdown PCR方法简化了检测步骤,缩短了检测时间,整个检测过程缩短至2h,检测PCR产物片段与测序结果完全吻合。结论采用Slowdown PCR方法替代传统普通的PCR,提高了基因扩增效率,解决了由于基因CG含量过高导致PCR扩增困难和稳定性差的技术难题。     

孕妇早、中期开展 FMR1基因突变筛查的意义及病例分析

目的:对孕早、中期孕妇进行脆性X智力低下1(fragile X mental retardation 1,FMR1)基因筛查,为携带高风险CGG重复数者提供产前诊断。方法:采集2316名12～21 +6周的孕妇的外周血样,提取基因组DNA,用荧光PCR-毛细管电泳法检测FMR1的CGG重复次数。对3例携带前...     

检测脆性X综合征蛋白以评价FMR1的中等等位基因

脆性 X综合征是遗传性智力低下中最常见的 ,在男性中发病率约为 1 /40 0 0 ,在女性中约为 1 /1 0 0 0 ,这个综合征的遗传基础主要是脆性 X综合征基因 5′端非编码区 CGG重复序列所在的不稳定区的扩展 ,Cp G区的过度甲基化 ,以及 CGG区的扩展。这些引起基因转录的抑制 ,导致了 FMR1蛋白的缺乏。脆性 X综合征蛋白 ( FMRP)的缺乏是引起脆性 X综合征表型的直接原因。在人群中 FMR1基因 CGG重复序列分布范围如下 :正常重复序列为 6～ 2 5个 CGG,前突变为5 2～ 2 0 0 CGG,全突变为 2 0 0以上 CGG。但是 ,一些作者赞同中等等位基因的…     

1035名孕前育龄妇女FMR1基因(CGG)n重复序列多态性分析

目的了解深圳地区孕前育龄妇女脆性X智力低下1(Fragile X Mental Retardation,FMR1)基因(CGG)n重复序列多态性分布特征,为本区脆性X综合征(Fragile X Syndrome,FXS)的预警和孕前筛查提供参考依据。方法收集2017年4月～2018年1月来深圳市龙华区人民医院进行孕前检查的育龄妇女1035名,采用Amp1ideX~(TM) FMR1PCR及毛细管电泳技术对全血标本中FMR1基因(CGG)n重复序列及重复数进行检测,并对检测结果进行统计分析。结果 1035名孕前育龄妇女2070条X染色体中共检出23种不同等位基因,CGG重复数目的变异范围为n=17～63,FMRI基因中最常见的CGG重复数为n=28,占49.32%(1021/2070),其次为n=30和n=29,分别占29.56%(612/2070)和11.16%(231/2070),CGG重复数目n=28的检出频率明显高于n=30和n=29,差异有统计学意义(χ~2=2.6145～4.0352,P0.05)。检出FMR1基因突变5例,突变率为0.24%(5/2070),其中前突变2例(n=63的1例,n=58的1例),占0.10%(2/2070);中间型3例(n=47的2例,n=51的1例),占0.14%(3/2070)。结论深圳地区孕前育龄妇女有一定的FMRI基因突变携带率,加强孕前育龄妇女FMR1筛查,对预防或降低遗传性智力低下并伴有自闭症患儿出生率具有重要意义。     

FMR1基因启动子区CpG岛甲基化与智力低下的相关性研究

目的探讨智力低下基因1启动子区CpG岛甲基化与智力低下的相关性。方法应用甲基化PCR方法对79例智力低下儿童及79例正常儿童的外周血中FMRI基因启动子区CpG岛甲基化状态及（CGG）n进行检测。结果2例智力低下儿童的FMRI启动子区CpG岛发生甲基化,正常儿童基因CpG岛无甲基化;两组（CGG）n重复数分别为33—48、27—43,两者之间比较（P〉0．05）无统计学意义。结论FMRl基因启动子区CpG岛甲基化可能引起智力低下。     

不明原因智力低下儿童脆性X综合征的筛查和基因诊断

目的建立一种快速分析脆性X综合征智力低下基因1(Fragile X mental retardation gene 1,FMR-1)突变的方法,对不明原因智力低下儿童进行脆性X综合征的筛查和诊断。方法应用7-deza-dGTP的PCR法一次性扩增FMR-1基因的(CGG)n的重复区,检测CGGn的重复序列的大小判断FMR-1基因状态(正常、突变前、突变后),对脆性X综合征可疑患儿快速筛查。结果在101例不明原因的先天性智力低下惠儿中,我们发现脆性X综合征患儿13例(男性10例,女性3例)。结论采用7-deza-dGTP扩增GC富集区的PCR法可对高危患儿进行快速筛查,确定携带者和患者。     

优化PCR体系结合毛细管电泳技术检测FMR1基因前突变与全突变

目的 通过优化PCR并结合毛细管电泳,建立高扩增效率、高分辨率的FMR1基因CGG重复序列异常扩增检测体系.方法 选择标准样本和经Southern印迹技术确定(CGG)n的正常、前突变、全突变男性和女性样本15例,进行PCR检测体系的优化.优化的PCR扩增产物经琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳和毛细管电泳等多种方法进行结果比较.结果 经优化的PCR体系可以检测出(CGG)n大于260个拷贝的全突变男性和(CGG)n达到183个拷贝的前突变女性.毛细管电泳能够清晰分辨出相差1个CGG的两个等位基因,结果具有良好的可重复性.结论 该PCR检测体系大幅度提高了普通PCR方法的扩增效率和分辨率,明显降低了对于Southern印迹技术的依赖,可以作为临床筛查FMR1基因突变的首选方法.     

Methylation analysis of CGG sites in the CpG island of the human FMR1 gene.

The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of CGG variation through 642 meioses in Fragile X families

Fragile X syndrome is the commonest familial form of inherited mental retardation. The molecular defect is an expansion of the CGG trinucleotide repeats in the 5' untranslated region of the FMR1 gene that is inherited in an unstable fashion in fragile X families. In an attempt to provide more information about the CGG tract intergenerational variation, we have evaluated 642 transmissions in 175 Fragile X families. PCR and Southern blot (StB12.3) was used to analyse the CGG number. Among premutated alleles, 90.2% showed expansion, two-thirds to a full mutation while the rest remained in the premutation range, 5.5% of alleles did not vary and finally 4.3% of them reduced in size. Premutated females showed an increased risk of expansion to the full mutation depending on the CGG tract. The estimated risk for 80 triplets is more than seven times that of a woman carrying 59 CGG, the risk being 100% for alleles of >100 repeats. Fifty-nine repeats was the smallest allele that expanded to full mutation. Contractions were detected more frequently in males than in females, being statistically significant. This study contributes to the literature by increasing the data available regarding transmissions in Fragile X families and it allows us to perform more precise genetic counselling for women with the CGG repeat in the premutation range.     

Expand Long PCR for fragile X mutation detection

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full-mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin-labeled 5'-(CGG)_5–3' probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.     

Prevalence of the FMR1 mutation in Taiwan assessed by large-scale screening of newborn boys and analysis of DXS548-FRAXAC1 haplotype

If carrier women could be identified in time and take appropriate measures, fragile X syndrome (FXS) can be prevented. Wide screening of women to be or in their early pregnancy was considered a good approach to identify carriers without misdetection. Nevertheless, we argued against the cost-effectiveness of implementing such a screening program in Taiwan, due to the lower carrier rate found in our pilot study. To reliably estimate the prevalence of mutant FMR1 gene in Taiwan, we anonymously screened 10,046 newborn boys using bloodspot polymerase chain reaction (PCR). Among them, the sample from one boy, who was most likely had FXS, failed repeatedly in PCR amplification. The estimated prevalence of premutation (55-200 CGG repeats) and intermediate alleles (45-54 CGG repeats) was 1:1,674 (n = 6) and 1:143 (n = 70), respectively. All these estimates were constantly lower than that reported in Caucasian populations, with variable statistic significance. Furthermore, when comparing analyses of the distribution of alleles at the two most often investigated microsatellite loci, DXS548 and FRAXAC1, between 100 control and 28 unrelated fragile X chromosomes, we found no apparent founder haplotype prevalent among the fragile X patients. Because a few founder haplotypes were reportedly prevalent in two thirds of fragile X alleles in Caucasians and in Chinese from Central China, we thus suggested that lack of founder fragile X chromosomes might result in a relatively low prevalence of mutant FMR1 gene in a population, as observed in Taiwan.