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

Zhou, Youyou; Lum, Josephine M.S.; Yeo, Gare-Hoon; Kiing, Jennifer S.H.; Tay, Stacey K.H.; Chong, Samuel S.

doi:10.1373/clinchem.2006.068593

Cited by 39 publications

(37 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PCR was performed using an untagged forward primer and a fluorescence-tagged reverse primer, as previously described. 22 PCR products were analyzed by GeneScan Analysis software (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions. 22 During the period between 2004 and 2009, Southern blot analysis was performed on women with PCR results that demonstrated only a single normal-sized allele or an allele larger than 45 repeats.…”

Section: Cgg Repeat Analysesmentioning

confidence: 99%

Ethnic effect on FMR1 carrier rate and AGG repeat interruptions among Ashkenazi women

Weiss

Orr-Urtreger

Ber

et al. 2014

Genetics in Medicine

View full text Add to dashboard Cite

introductionFragile X syndrome (FXS; OMIM 300624) is the most common form of inherited mental impairment, with a prevalence of approximately 1/4,500 in males. The syndrome is characterized by moderate intellectual disability and behavioral abnormalities, including autistic-like behavior.1,2 The vast majority of FXS cases are caused by an expansion of CGG nucleic-base repeats in the Fragile X mental retardation gene (FMR1). Normal individuals usually have ~30 CGG repeats. An expansion to greater than 200 repeats is associated with abnormal methylation and suppression of FMR1 transcription, causing the absence of the gene product, the FMR protein. 3 The lack of FMR protein, an RNA-binding protein, is responsible for the syndrome. 4 There are four allelic forms of the FMR1 gene with respect to the repeat length. The normal allele form has approximately 5-44 CGG repeats and it is stable on transmission. In these normal alleles, the CGG repeat region is usually interrupted by AGG triplets after every 9 or 10 CGG repeats. These AGG interruptions are believed to maintain repeat integrity by preventing DNA slippage during replication, therefore increasing the repeats' stability.5 The full-mutation allele form has more than 200 repeats, with several hundred to several thousand repeats being typical. In between those two forms fall the intermediate allele form and the premutation allele form. The intermediate allele form falls between the normal and premutation size ranges (45-54 repeats). A proportion of these alleles are unstable and may expand by a few repeats during meiosis. In this range, expansion to the full-mutation allele may occur over two or more generations but not in a single transmission. 6 The premutation allele form is about 55-200 repeats long. It is unstable and commonly expands during transmission. Individuals with the premutations are defined as carriers of the disease. Expansion of an abnormal allele usually occurs when it is transmitted from the mother but not from the father. 7 However, although alleles of 55-59 repeats are defined as premutations, only a very few cases of expansion to a full mutation have actually been reported, emphasizing the fact that such allele sizes are generally stable. 8In general, the risk of expansion of a premutation to a full mutation upon transmission to the offspring is associated with three factors. First, it correlates with the number of CGG repeats.9 Second, the number of AGG interruptions also affects stability on transmission, such that alleles with a high number of CGG repeats that lack AGG interruptions are more prone to expansion. 5,10 A third factor affecting risk of transmission is the genetic background because some haplotypes are associated with an increased instability as compared with others. Purpose: Fragile X syndrome, a common cause of intellectual disability, is usually caused by CGG trinucleotide expansion in the FMR1 gene. CGG repeat size correlates with expansion risk. Premutation alleles (55-200 repeats) may expand to full mutations in female mei...

show abstract

Section: Cgg Repeat Analysesmentioning

confidence: 99%

Ethnic effect on FMR1 carrier rate and AGG repeat interruptions among Ashkenazi women

Weiss

Orr-Urtreger

Ber

et al. 2014

Genetics in Medicine

View full text Add to dashboard Cite

show abstract

“…32 Other approaches for the detection of expansions, methylation status, or expression of FMR1 gene included the use of antibodies against FMR1 protein, 33 interrogation of upstream regions to interpret methylation status of the CGG repeats in the FMR1 gene, 34,35 methylation-specific PCR, 36 or methylation-specific PCR incorporating TP-PCR for identification of methylated CGG alleles in the FMR1 gene. 37,38 Recently, a modified Southern analysis method was used for rapid qualitative population-based screening of FMR1 gene. 25 Two recent enhancements to the classic PCR methods were developed as shown using a combination of betaine and TP-PCR.…”

mentioning

confidence: 99%

Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: Implications for fragile X syndrome carrier and newborn screening

Hantash

Goos

Tsao

et al. 2010

Genetics in Medicine

View full text Add to dashboard Cite

Purpose: Fragile X syndrome is caused by expansion and subsequent methylation of a CGG trinucleotide repeat in the FMR1 5Ј-untranslated region. Southern blot analysis is typically required to determine expansion size for triplet repeat lengths Ͼ200. We describe a triplet-primed polymerase chain reaction-based method using automated capillary electrophoresis detection for qualitative assessment of expanded CGG repeats. Methods: The assay uses triplet-primed polymerase chain reaction in combination with GC-melting reagents and substitution of 7-deaza-2-deoxyGTP for dGTP. Amplicons are resolved by capillary electrophoresis. Results: A distinctive pattern of tapering or "stutter" polymerase chain reaction amplification was evident on capillary electrophoresis in male and female patients harboring all expanded allele lengths examined (up to 2000 CGG repeats) and could be used to differentiate normal, intermediate, premutation, and full mutation alleles. Full mutation alleles exhibited an additional late-migrating amplicon on capillary electrophoresis. Mixing experiments demonstrated sensitivity as low as 1% for detection of the full mutation allele. In a 1275-sample concordance study against our existing polymerase chain reaction platform (with Southern blot analysis for repeat lengths Ն55), the triplet-primed polymerase chain reaction method exhibited 100% concordance for normal, intermediate, expanded, and full mutation alleles. This method also detected the full mutation alleles in DNA isolated from blood spots. Conclusion: This assay provides an accurate assessment of FMR1 repeat status and holds promise for use in carrier and newborn screening. The method distinguishes normal homozygous females from full mutation carrying females. Although the method is not useful for accurate sizing, it supplements the classic polymerase chain reaction method and results in significant reduction in the number of Southern blot analyses required to be performed in the laboratory to accurately assess the FMR1 genotype in all individuals. Genet Med 2010:12(3):162-173.

show abstract

“…Women with two normal-size FMR1 alleles were scored as unaffected heterozygotes, whereas women with one normal-size FMR1 allele were reanalyzed using fluorescent Methylation-specific PCR ( Ms-PCR) and GeneScan analysis (Zhou et al, 2006a), to find out whether they are ''true homozygotes'' or the other allele is expanded and beyond the amplification efficiency of rPCR, due to its high GC content.…”

Section: Subjectsmentioning

confidence: 99%

“…Methylation-specific polymerase chain reaction DNA samples were treated with sodium bisulfite as described by Zhou et al (2006a) and purified using QIAEX II Gel Extraction Kit (Qiagen).…”

Section: Subjectsmentioning

confidence: 99%

“…Each Ms-PCR was performed in a 50 mL reaction volume containing the respective primers at concentrations described (Zhou et al, 2006a) with 0.2 mmol/L dNTPs, 2.5U HotStarTaqÔ DNA polymerase (Qiagen), Q-Solution (Qiagen) at a concentration of either 0.5 · (for nonMet-PCR) or 1.5 · (for Met-PCR and mTP-PCR), 1 · buffer (including 1.5 mM MgCl 2) , and 5 mL of the bisulfitemodified DNA. An enzyme activation step at 95°C for 15 min was followed by 40 cycles of 98°C for 1 min, 60°C for 1 min, 72°C for 2 min, followed by a final extension at 72°C for 10 min.…”

Section: Subjectsmentioning

confidence: 99%

See 1 more Smart Citation

Fragile X CGG Repeat Variation in Tamil Nadu, South India: A Comparison of Radioactive and Methylation-Specific Polymerase Chain Reaction in CGG Repeat Sizing

Nagarathinam

Singh²,

Chong³

et al. 2012

Genetic Testing and Molecular Biomarkers

View full text Add to dashboard Cite

Fragile X syndrome is the most frequent hereditary cause of mental retardation after Down syndrome. Expansion of CGG repeats in the 5¢ UTR of the fragile X mental retardation gene 1 (FMR1) causes gene inactivation in most of the cases. The FMR1 gene is classified into normal 5-44; gray zone 45-54; premutation 55 to < 200; and full mutation ‡ 200 repeats. Precise sizing of FMR1 alleles is important to understand their variation, predisposition, and for genetic counseling. Meta-analysis reveals prevalence of premutation carriers as 1 in 259. No such reports are available in India. About 705 women from Tamil Nadu, South India, were screened for the FMR1 allelic variation by using radioactive polymerase chain reaction-polyacrylamide gel electrophoresis (PAGE) analysis. The women who were homozygous by radioactive polymerase chain reaction (rPCR) were reanalyzed by methylation-specific polymerase chain reaction (Ms-PCR) and GeneScan analysis. The techniques were validated and compared to arrive at a correction factor. Among 122 genotypes, 35 repeat variants ranging in size from 16 to 57 were observed. The most common repeat is 30 followed by 29. One in 353 women carried the premutation. No full mutations were observed. Screening populations with low frequency of premutations may not be applicable. Ms-PCR is more suitable for routine screening and clinical testing compared with rPCR-PAGE analysis.

show abstract

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

Cited by 39 publications

References 10 publications

Ethnic effect on FMR1 carrier rate and AGG repeat interruptions among Ashkenazi women

Ethnic effect on FMR1 carrier rate and AGG repeat interruptions among Ashkenazi women

Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: Implications for fragile X syndrome carrier and newborn screening

Fragile X CGG Repeat Variation in Tamil Nadu, South India: A Comparison of Radioactive and Methylation-Specific Polymerase Chain Reaction in CGG Repeat Sizing

Contact Info

Product

Resources

About