scholarly journals Simplified Molecular Diagnosis of Fragile X Syndrome by Fluorescent Methylation-Specific PCR and GeneScan Analysis

2006 ◽  
Vol 52 (8) ◽  
pp. 1492-1500 ◽  
Author(s):  
Youyou Zhou ◽  
Josephine MS Lum ◽  
Gare-Hoon Yeo ◽  
Jennifer Kiing ◽  
Stacey KH Tay ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Southern Blot ◽  
Molecular Diagnosis ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Fragile X ◽  
Molecular Diagnostic ◽  
Methylation Specific Pcr ◽  
Specific Pcr ◽  
Cgg Repeats

Abstract Background: Fragile X syndrome (FXS), the most common cause of inherited mental impairment, is most commonly related to hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5′ untranslated region of the FMR1 gene. Southern blot analysis is the most commonly used method for molecular diagnosis of FXS. We describe a simplified strategy based on fluorescent methylation-specific PCR (ms-PCR) and GeneScan™ analysis for molecular diagnosis of fragile X syndrome. Methods: We used sodium bisulfite treatment to selectively modify genomic DNA from fragile X and normal lymphoblastoid cell lines and from patients. We then performed ms-PCR amplification using fluorescently-labeled primers complementary to modified methylated or unmethylated DNA. Amplification products were resolved by capillary electrophoresis. FMR1 mutational status was determined by a combination of fluorescent peak sizes and patterns on the GeneScan electropherogram. Results: DNA samples from male and female persons with known NL, PM, and FM FMR1 CGG repeats were analyzed. Each FMR1 genotype produced a unique GeneScan electropherogram pattern, thus providing a way to identify the various disease states. The number of CGG repeats in all NL and PM alleles were determined accurately. Analysis by both the new assay and Southern blot of a family segregating with FXS showed complete concordance between both methods. Conclusions: This simplified molecular diagnostic test, based on fluorescent methylation-specific PCR, may be a suitable alternative or complement to Southern blot analysis for the diagnosis of FXS.

scholarly journals The fragile X syndrome: 13 years of experience

2011 ◽  
Vol 65 (3-4) ◽  
pp. 67-72
Author(s):  
Zanda Daneberga ◽  
Zita Krūmiņa ◽  
Baiba Lāce ◽  
Daiga Bauze ◽  
Rita Lugovska
Keyword(s):  
Fragile X Syndrome ◽  
Dna Analysis ◽  
Clinical Symptoms ◽  
Cpg Island ◽  
Fragile X ◽  
Psychomotor Development ◽  
Molecular Diagnostic ◽  
Years Of Experience ◽  
Male Population ◽  
Cgg Repeats

The fragile X syndrome: 13 years of experience Fragile X syndrome (FXS; MIM #300624; FRAXA, Xq27.3) is well known and a common cause of X-linked mental retardation. The syndrome is caused by dynamic mutation of FMR1 gene CpG island CGG repeats. Clinically FXS patients demonstrate delayed developmental milestones, particularly speech, attention-deficit/hyperactivity disorder, autistic features, and psychomotor development delay. Dysmorphic face and macroorchidism are important features in the postpubertal age. We present our 13-year experience with FXS patients who were confirmed by molecular diagnostic. Phenotype-genotype evaluation was made for 12 male FXS patients. Genotype-phenotype analysis did not reveal significant correlation between clinical symptoms observed in FXS patients and genotypes obtained from leucocytes DNA analysis. The prevalence of the fragile X syndrome in the Latvian male population was estimated to be 1/6428 (95% CI 5538-7552) or 15.55/100 000 males (95% CI 13.24 - 18.05). The prevalence of the fragile X syndrome among mentally retarded male patients was estimated to be 2.67%. The low number of diagnosed patients with fragile X syndrome demonstrated in our study led to the conclusion that fragile X syndrome is generally clinically unrecognised.

scholarly journals Diagnosis of the fragile X syndrome in males using methylation-specific PCR of the FMRI locus

1999 ◽  
Vol 22 (2) ◽  
pp. 169-172 ◽  
Author(s):  
Sérgio D.J. Pena ◽  
Rosane Sturzeneker
Keyword(s):  
Fragile X Syndrome ◽  
Internal Control ◽  
High Efficiency ◽  
Cpg Island ◽  
Fragile X ◽  
Pcr Amplification ◽  
Sodium Bisulfite ◽  
Chromosome 15 ◽  
Methylation Specific Pcr ◽  
Specific Pcr

We have developed a non-isotopic technique based on methylation-specific PCR (MSP) of the FMR1 promoter for the identification of fragile X full mutations among men. DNA samples were first treated with sodium bisulfite to convert unmethylated, but not methylated, cytosines to uracil, followed by PCR amplification with oligonucleotide primers specific for methylated versus unmethylated DNA. We designed two primer pairs: one produced a 142-bp fragment from the bisulfite-treated methylated CpG island, while the other generated an 84-bp product from the treated non-methylated promoter. In normal males only the 84-bp fragment was seen, while the diagnosis of FRAXA was doubly indicated by the appearance of a 142-bp product together with absence or weak visualization of the 84-bp band. As an indispensable internal control for the efficiency of the sodium bisulfite treatment, we used a primer pair specific for the imprinted maternal methylated version of the CpG island of the SNRPN gene on human chromosome 15. Using the methylation-specific PCR we identified with 100% sensitivity and accuracy eight previously diagnosed FRAXA male patients mixed with 42 normal controls. Because of its simplicity and high efficiency, methylation-specific PCR may become the method of choice for the diagnosis of the fragile X syndrome in mentally retarded males.

scholarly journals A Novel FMR1 PCR Method for the Routine Detection of Low Abundance Expanded Alleles and Full Mutations in Fragile X Syndrome

2010 ◽  
Vol 56 (3) ◽  
pp. 399-408 ◽  
Author(s):  
Stela Filipovic-Sadic ◽  
Sachin Sah ◽  
Liangjing Chen ◽  
Julie Krosting ◽  
Edward Sekinger ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Southern Blotting ◽  
Trinucleotide Repeat ◽  
Fragile X ◽  
Clinical Samples ◽  
Full Mutation ◽  
Cgg Repeat ◽  
Specific Pcr ◽  
Cgg Repeats ◽  
Pcr Technology

Abstract Background: Fragile X syndrome (FXS) is a trinucleotide-repeat disease caused by the expansion of CGG sequences in the 5′ untranslated region of the FMR1 (fragile X mental retardation 1) gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on 2 tests, PCR and Southern blotting; however, performance or throughput limitations of these methods currently constrain routine testing. Methods: We evaluated a novel FMR1 gene–specific PCR technology with DNA templates from 20 cell lines and 146 blinded clinical samples. The CGG repeat number was determined by fragment sizing of PCR amplicons with capillary electrophoresis, and results were compared with those for FMR1 Southern blotting analyses with the same samples. Results: The FMR1 PCR accurately detected full-mutation alleles up to at least 1300 CGG repeats and consisting of >99% GC character. All categories of alleles detected by Southern blotting, including 66 samples with full mutations, were also identified by the FMR1 PCR for each of the 146 clinical samples. Because all full mutation alleles in samples from heterozygous females were detected by the PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940-CGG allele in a background of 99% 23-CGG allele—a roughly 5- fold greater sensitivity than obtained with Southern blotting. Conclusions: The novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify; reproducibly detect low abundance full mutation alleles; and correctly infer homozygosity in female samples, thus greatly reducing the need for sample reflexing to Southern blotting.

scholarly journals Simple molecular diagnostic method for fragile X syndrome in Egyptian patients: pilot study.

2014 ◽  
Vol 61 (2) ◽  
Author(s):  
Nagwa A Meguid ◽  
Manal F Ismail ◽  
Rasha S El-Mahdy ◽  
Maged A Barakat ◽  
Mostafa K El-Awady
Keyword(s):  
Mental Retardation ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Clinical Manifestations ◽  
Recessive Gene ◽  
Molecular Diagnostic ◽  
Major Barrier ◽  
Cgg Repeats ◽  
Egyptian Patients ◽  
Pcr Method

Poor knowledge about Fragile X syndrome (FXS) may be a major barrier to early diagnosis that could improve quality of life and prognosis especially in the developing countries. The aim of this study was to evaluate simple and reproducible method for premutation detection in females of fragile X families for the first time in Egypt. We have developed a rapid modified polymerase chain reaction (PCR)-based screening tool for expanded Fragile X mental retardation 1 (FMR1) alleles. This method utilizes betaine as additive to facilitate FMR 1 gene amplification. We screened fifty three males, thirty two first-degree females; twenty normal healthy controls in addition to six reference samples. Simple PCR method showed 16 males with abnormal CGG repeats, where 10 of their mothers and four sisters had FMR 1 premutation. Consanguineous marriage was present in 66.6% percent of the studied families. Studying the correlation between genotype and clinical manifestations showed premature ovarian failure in 40% and learning disability in 50% of the studied female carriers. FXS has to be ruled out in families with consanguineous parents, before assuming that familial mental retardation is due to autosomal recessive gene defects. Early carrier detection may reduce the number of affected children. In conclusion, more studies are still needed of much larger sample size with known allele sizes in order to guarantee the accuracy of the method used.

scholarly journals An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis

2010 ◽  
Vol 12 (5) ◽  
pp. 589-600 ◽  
Author(s):  
Liangjing Chen ◽  
Andrew Hadd ◽  
Sachin Sah ◽  
Stela Filipovic-Sadic ◽  
Julie Krosting ◽  
...  
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Fragile X ◽  
Full Range ◽  
Cgg Repeat

scholarly journals FMR1 gene mutations in patients with fragile X syndrome and obligate carriers: 30 years of experience in Chile

2016 ◽  
Vol 98 ◽  
Author(s):  
LORENA SANTA MARÍA ◽  
SOLANGE ALIAGA ◽  
VÍCTOR FAUNDES ◽  
PAULINA MORALES ◽  
ÁNGELA PUGIN ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Southern Blot ◽  
Fragile X ◽  
Gene Mutations ◽  
Molecular Technique ◽  
Grey Zone ◽  
Cascade Screening ◽  
Cgg Repeat ◽  
Cgg Repeats ◽  
Age Of Diagnosis

SummaryFragile X syndrome (FXS) is the most common form of inherited intellectual disability (ID) and co-morbid autism. It is caused by an amplification of the CGG repeat (>200), which is known as the full mutation, within the 5′UTR of the FMR1 gene. Expansions between 55–200 CGG repeats are termed premutation and are associated with a greater risk for fragile X-associated tremor/ataxia syndrome and fragile X-associated premature ovarian insufficiency. Intermediate alleles, also called the grey zone, include approximately 45–54 repeats and are considered borderline. Individuals with less than 45 repeats have a normal FMR1 gene. We report the occurrence of CGG expansions of the FMR1 gene in Chile among patients with ID and families with a known history of FXS. Here, we present a retrospective review conducted on 2321 cases (2202 probands and 119 relatives) referred for FXS diagnosis and cascade screening at the Institute of Nutrition and Food Technology (INTA), University of Chile. Samples were analysed using traditional cytogenetic methods and/or PCR. Southern blot was used to confirm the diagnosis. Overall frequency of FMR1 expansions observed among probands was 194 (8·8%), the average age of diagnosis was 8·8 ± 5·4 years. Of 119 family members studied, 72 (60%) were diagnosed with a CGG expansion. Our results indicated that the prevalence of CGG expansions of the FMR1 gene among probands is relatively higher than other populations. The average age of diagnosis is also higher than reference values. PCR and Southern blot represent a reliable molecular technique in the diagnosis of FXS.

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