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.     

A novel polymorphism in the FMR1 gene: implications for clinical testing of fragile X syndrome

Fragile X syndrome is the most common cause of inherited mental retardation among males. In most cases, the molecular basis of fragile X syndrome is the expansion and subsequent methylation of a CGG trinucleotide repeat in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. Laboratory diagnosis usually relies on a combination of Southern blot and polymerase chain reaction analyses. In this case report we describe an unusual Southern blot result in a patient who presented with developmental delay and had a normal CGG repeat number by polymerase chain reaction analysis. Further investigation revealed a novel G3310C transversion in the FMR1 gene resulting in a new recognition site for the BssHII restriction enzyme. This novel restriction site could potentially mimic a partial deletion of the FMR1 gene on Southern blot analysis and thus represents a possible pitfall in the diagnosis of fragile X syndrome.     

Development of a novel, accurate, automated, rapid, high-throughput technique suitable for population-based carrier screening for Fragile X syndrome.

PURPOSE: To develop a high-throughput, automated, accurate method suitable for population-based carrier detection of fragile X syndrome. METHODS: We developed a new method called capillary Southern analysis that allows automated high-throughput screening for expanded fragile X mental retardation 1 (FMR1) alleles. Initially samples are analyzed by a multiplex polymerase chain reaction that contains an internal control to establish gender. All females heterozygous for two normal alleles are reported as normal without further analysis. All females homozygous at the FMR1 locus (24% of all analysis) are then analyzed by capillary Southern analysis. Theoretically this method can detect expansion as high as 2000 CGG repeats, although in our series the largest nonmosaic FMR1 present was 950 CGG repeats. After assay development, we performed capillary Southern analysis on 995 female and 557 male samples submitted for fragile X syndrome testing by polymerase chain reaction and Southern blot. RESULTS: The polymerase chain reaction/capillary Southern analysis technique identified 100% of six female premutation carriers, seven full mutation carrier females, one premutation male, and five affected males. There was only one discrepancy between analysis by polymerase chain reaction/Southern blot and analysis by polymerase chain reaction/capillary Southern analysis. A single female sample appeared to be heterozygous for a premutation allele by polymerase chain reaction/capillary Southern analysis but was negative by Southern blot. It is possible this patient is a mosaic for the premutation allele, but because samples were deidentified, we were unable to determine whether this was a true false positive. CONCLUSION: We have developed an automated, high-throughput technique capable of detecting carriers of fragile X syndrome with 100% sensitivity and at least 99.5% specificity. This should allow population-based carrier detection for the most commonly inherited form of mental retardation.     

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。     

Trinucleotide CGG repeat in the FMR1 gene in Chinese mentally retarded patients.

The fragile X syndrome of mental retardation is related to the number of trinucleotide CGG repeats at the 5'-untranslated region of the FMR1 gene located on the X-chromosome. We have studied X-chromosomes from 649 unaffected Chinese subjects and 324 patients with mild mental retardation. All study subjects were unrelated. The CGG repeat number was analysed by electrophoresis of a polymerase chain reaction followed by gel transfer and hybridisation with a 32P-labeled (CCG)5 probe. The DNA samples having detectable CGG expansion were further analysed by Southern blot analysis with probe StB12.3 after restriction digestion by EcoR I and Eag I. For the unaffected Chinese subjects, a different distribution pattern of CGG allele size from Caucasians was observed. It was a bimodal pattern and the CGG repeat number ranged from 19 to 54. The most common CGG repeat allele was 29 compared with 30 in Caucasians. The second mode appeared at 36 repeats. There was mild statistical difference in the repeat patterns between the mentally retarded patients and unaffected subjects, although the essential features were similar. Among the mentally retarded patients, one male had an unmethylated full mutation and one female had a full mutation. The fragile X prevalence was 0.6%, which is lower than two previous studies in Chinese mentally retarded patients utilising cytogenetic analysis. Our results indicate that a large-scale screening program would be worthwhile to determine the prevalence of the fragile X syndrome in the Chinese population.     

Fragile X syndrome

Fragile X syndrome, the most common genetic disorder associated with mental retardation is caused by an expansion of the unstable CGG repeat within the FMR1 gene. Although overgrowth is not the main hallmark of this condition, the fragile X syndrome is usually included in the differential diagnosis of children with mental retardation and excess growth. This review highlights the most recent advances in the field of fragile X research.     

脆性X综合征FMR1基因CGG重复序列与甲基化的检测

目的建立一种快速、可靠的脆性X综合征的群体筛查方法。方法应用热启动PCR和甲基化特异性PCR（MS-PCR）方法对62例智力低下儿童、12例父母外周血液以及5例高危胎儿的脐带血中FMR1基因CGG重复序列与甲基化状态进行检测。结果采用热启动PCR方法检测79例标本,77例标本的CGG重复数在21～40之间,与正常对照组无明显差异;2例标本未扩增出明显条带。采用MS-PCR方法检测出2例FMR1基因甲基化但CGG重复数在正常范围的患者。结论应用热启动PCR结合MS-PCR方法检测FMR1基因CGG重复数和甲基化,能提高诊断效率,可作为筛查脆性X综合征的首选方法。     

The fragile X syndrome.

The fragile X syndrome is characterised by mental retardation, behavioural features, and physical features, such as a long face with large protruding ears and macro-orchidism. In 1991, after identification of the fragile X mental retardation (FMR1) gene, the cytogenetic marker (a fragile site at Xq27.3) became replaced by molecular diagnosis. The fragile X syndrome was one of the first examples of a "novel" class of disorders caused by a trinucleotide repeat expansion. In the normal population, the CGG repeat varies from six to 54 units. Affected subjects have expanded CGG repeats (>200) in the first exon of the FMR1 gene (the full mutation). Phenotypically normal carriers of the fragile X syndrome have a repeat in the 43 to 200 range (the premutation). The cloning of the FMR1 gene led to the characterisation of its protein product FMRP, encouraged further clinical studies, and opened up the possibility of more accurate family studies and fragile X screening programmes.     

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.     

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.     

Fragile X Syndrome

Fragile X Syndrome (FXS) is a genetic disease due to a CGG trinucleotide expansion, named full mutation (greater than 200 CGG repeats), in the fragile X mental retardation 1 gene locus Xq27.3; which leads to an hypermethylated region in the gene promoter therefore silencing it and lowering the expression levels of the fragile X mental retardation 1, a protein involved in synaptic plasticity and maturation. Individuals with FXS present with intellectual disability, autism, hyperactivity, long face, large or prominent ears and macroorchidism at puberty and thereafter. Most of the young children with FXS will present with language delay, sensory hyper arousal and anxiety. Girls are less affected than boys, only 25% have intellectual disability. Given the genomic features of the syndrome, there are patients with a number of triplet repeats between 55 and 200, known as premutation carriers. Most carriers have a normal IQ but some have developmental problems. The diagnosis of FXS has evolved from karyotype with special culture medium, to molecular techniques that are more sensitive and specific including PCR and Southern Blot. During the last decade, the advances in the knowledge of FXS, has led to the development of investigations on pharmaceutical management or targeted treatments for FXS. Minocycline and sertraline have shown efficacy in children.     

Prenatal diagnosis of the fragile X syndrome: loss of mutation owing to a double recombinant or gene conversion event at the FMR1 locus.

The fragile X syndrome, an X linked mental retardation syndrome, is caused by an expanded CGG repeat in the first exon of the FMR1 gene. In patients with an expanded repeat the FMR1 promoter is methylated and, consequently, the gene is silenced and no FMR1 protein (FMRP) is produced, thus leading to the clinical phenotype. Here we describe a prenatal diagnosis performed in a female from a fragile X family carrying a large premutation. In chorionic villus DNA of the male fetus the normal maternal CGG allele and a normal pattern on Southern blot analysis were found in combination with the FRAXAC2 and DXS297 allele of the maternal at risk haplotype. A second chorionic villus sampling was performed giving identical results on DNA analysis and, in addition, expression of FMRP was shown by immunohistochemistry. We concluded that the male fetus was not affected with the fragile X syndrome. Subsequent detailed haplotype analysis showed a complex recombination pattern resembling either gene conversion or a double crossover within a 20 kb genomic region.     

Four sisters compound heterozygotes for the pre- and full mutation in fragile X syndrome and a complete inactivation of X-functional chromosome: implications for genetic counseling

Fragile X syndrome (FXS) is a neurodevelopmental disorder and a leading monogenic form of cognitive impairment and autism. It is the most common form of inherited mental retardation in males and a significant cause of mental retardation in females. It is caused by the instability and subsequent expansion of the CGG repeat in the promoter region of the FMR1 (fragile X mental retardation 1) gene at Xq27.3. We describe a double consanguineous family with four sisters compound heterozygotes for the full and pre-mutation CGG repeat size. The index case shows clinical features of the affected males with profound mental retardation; the other three sisters also suffer from mental retardation, ranging from mild to severe. Molecular analysis reveals very similar ranges for the CGG expansions for both chromosomes in all four sisters. The phenotypic differences observed in the index case and her sisters are the total inactivation of X premutated chromosome and the total absence of FMRP (fragile X mental retardation protein). This family case raises important issues for genetic counseling in families with consanguinity and with cases of idiopathic mental retardation.     

Guidelines for the diagnosis of fragile X syndrome. National Fragile X Foundation.

Direct DNA analysis of the fragile X mutation has become available with the isolation of DNA probes that detect the unstable DNA sequence containing the CGG repeat. We present the various alternatives of combinations of probes and enzymes that can be used for the diagnosis of fragile X syndrome. An overview is given of all the different available probes. A different protocol is presented for postnatal and prenatal diagnosis of fragile X syndrome. This includes Southern blot analysis as well as direct analysis of the CGG repeat by PCR amplification. We discuss the role of constitutional cytogenetic analysis in the diagnosis of mentally retarded subjects and cytogenetic analysis for the diagnosis of fragile X syndrome.     

Understanding the molecular basis of fragile X syndrome

Fragile X syndrome, a common form of inherited mental retardation, is mainly caused by massive expansion of CGG triplet repeats located in the 5'-untranslated region of the fragile X mental retardation-1 ( FMR1 ) gene. In patients with fragile X syndrome, the expanded CGG triplet repeats are hypermethylated and the expression of the FMR1 gene is repressed, which leads to the absence of FMR1 protein (FMRP) and subsequent mental retardation. FMRP is an RNA-binding protein that shuttles between the nucleus and cytoplasm. This protein has been implicated in protein translation as it is found associated with polyribosomes and the rough endoplasmic reticulum. We discuss here the recent progress made towards understanding the molecular mechanism of CGG repeat expansion and physiological function(s) of FMRP. These studies will not only help to illuminate the molecular basis of the general class of human diseases with trinucleotide repeat expansion but also provide an avenue to understand aspects of human cognition and intelligence.     

Frequency of the fragile X syndrome in Chinese mentally retarded populations is similar to that in Caucasians.

Fragile X syndrome is recognized as the most common inherited cause of mental retardation in western countries. The prevalence of the fragile X syndrome in Asian populations is uncertain. We report a multi-institutional collaborative study of molecular screening for the fragile X syndrome from 1,127 Chinese mentally retarded (MR) individuals. We found that 2.8% of the Chinese MR population screened by DNA analysis had the fragile X full mutation. Our screening indicated that the fragile X syndrome prevalence was very close to that of Caucasian subjects. In addition, we found that 62.5% of fragile X chromosomes had a single haplotype for DXS548-FRAXAC1 (21-18 repeats) which was present in only 9.7% of controls. This unique distribution of microsatellite markers flanking the FMR1 CGG repeats suggests that the fragile X syndrome in Chinese populations, as in the Caucasian, may also be derived from founder chromosomes.     

非同位素PCR及Southern印迹杂交分析检测脆性X综合征FMR-1基因突变

目的建立一种非同位素的检测脆性X综合征智力缺陷基因(FMR-1)突变的方法.方法采用PCR技术结合Southern 印迹杂交技术对FMR-1基因进行分析,进而确定其突变类型.结果共对190例样本进行了检测,其中2例为女性前突变携带者,1例为女性全突变患者.结论非同位素PCR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,可作为临床上对脆性X综合征的筛查的首选方法;而非同位素Southern印迹杂交可对结果不确定者进一步进行检测.     

FMRP检测在儿童脆性X综合征中诊断价值的实验评价

目的对脆性X智力低下蛋白（fragile X mental retardation protein,FMRP）免疫细胞化学诊断方法并进行临床流行病学评价;利用ROC曲线确定适合本地筛查脆性X综合征的外周血淋巴细胞FMRP免疫组化法的诊断临界点。方法对临床拟诊为不明原因智力低下的的41例不明原因的智力低下患儿,同时进行免疫细胞化学方法进行外周血淋巴细胞FMRP表达检测和7-deza—dGTP PCR法两种方法检查,以目前较为公认的7-deza—dGTP PCR法为诊断金标准,对外周血淋巴细胞FMRP细胞免疫组化法（SP法）进行实验评价,计算其灵敏度、特异度、阳性似然比、阴性似然比等统计学指标。采用ROC曲线确定适合本地筛查脆性X综合征的外周血淋巴细胞FMRP表达率的诊断临界点。结果41例不明原因智力低下患儿中染色体核型分析均正常,其中染色体脆性位点检查异常的1例,约为2．4％。7-deza—dGTP PCR法诊断FXS患者11例,约占26．2％,其中男9例,女2例;ROC计算得出最佳的外周血淋巴细胞FMRP表达率的诊断临界点为46％,灵敏度为90．9％,特异度为93．5％。结论外周血淋巴细胞FMRP免疫细胞化学检测是一种可靠的FXS诊断和筛查方法,具有快速、简便、廉价、相对无创伤性等优点,可进行大样本筛查及标本邮寄快递检测,适宜在我国基层开展早期FXS患者诊断和筛查。