Single-tube methylation-specific duplex-PCR assay for rapid and accurate diagnosis of<i>Fragile X Mental Retardation 1</i>–related disorders

Rajan‐Babu, Indhu‐Shree; Teo, Clara R.L.; Lian, Mulias; Lee, Caroline; Law, Hai Yang; Chong, Samuel S.

doi:10.1586/14737159.2015.1001749

Cited by 12 publications

(14 citation statements)

References 41 publications

(46 reference statements)

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“…Alternatively, polymerase chain reaction (PCR)-based approaches can be relied upon to characterize CGG-repeat size and/or AGG-interruption patterns, and several methods that are capable of detecting the entire spectrum of FMR1 expansions have now been developed [94,95,96,97,98,99]. Therefore, many diagnostic laboratories employ a first-tier, high-throughput PCR-based approach to exclude non-expansion carriers and reflex only samples with an expansion for confirmatory SB analysis, as testing all samples by SB can be both labor- and time-intensive.…”

Section: Diagnostic Tools For Characterizing the Multiple Moleculamentioning

confidence: 99%

“…The Warner et al TP-PCR approach has now been modified for successful amplification and detection of CGG-repeat expansions in the FMR1 gene [94,97,98,99,101,102,127]. We initially developed a methylated allele TP-PCR (mTP-PCR) to detect all methylated expansions; however, although FM males can be readily identified from the mere detection of mTP-PCR amplicon peak “stutters”, females with methylated PM cannot not be differentiated from those with a FM by mTP-PCR alone [101,102].…”

Section: Diagnostic Tools For Characterizing the Multiple Moleculamentioning

confidence: 99%

“…We also developed a methylation-specific TP-PCR (msTP-PCR) assay that determines the methylation status of FMR1 alleles, in addition to CGG-repeat size and AGG-interruption analyses [99]. This single-tube duplex-PCR is performed on bisulfite-treated DNA, and includes two sets of primers for amplifying the methylated and unmethylated FMR1 alleles simultaneously.…”

Section: Diagnostic Tools For Characterizing the Multiple Moleculamentioning

confidence: 99%

“…In general, the improved TP-PCR methodologies [94,98,99] address most of the limitations of flanking PCR-based tests. Owing to its ability to amplify from within the CGG-repeat region, these methods detect all FMs (irrespective of the actual length of the mutant FMR1 allele), resolve zygosity issues in females, determine both number and position of AGG interruptions, and facilitate accurate sizing of NL to PM alleles.…”

Section: Diagnostic Tools For Characterizing the Multiple Moleculamentioning

confidence: 99%

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Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

Rajan‐Babu

Chong

2016

Genes

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Fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability and autism. Molecular diagnostic testing of FXS and related disorders (fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS)) relies on a combination of polymerase chain reaction (PCR) and Southern blot (SB) for the fragile X mental retardation 1 (FMR1) CGG-repeat expansion and methylation analyses. Recent advancements in PCR-based technologies have enabled the characterization of the complete spectrum of CGG-repeat mutation, with or without methylation assessment, and, as a result, have reduced our reliance on the labor- and time-intensive SB, which is the gold standard FXS diagnostic test. The newer and more robust triplet-primed PCR or TP-PCR assays allow the mapping of AGG interruptions and enable the predictive analysis of the risks of unstable CGG expansion during mother-to-child transmission. In this review, we have summarized the correlation between several molecular elements, including CGG-repeat size, methylation, mosaicism and skewed X-chromosome inactivation, and the extent of clinical involvement in patients with FMR1-related disorders, and reviewed key developments in PCR-based methodologies for the molecular diagnosis of FXS, FXTAS and FXPOI, and large-scale (CGG)n expansion screening in newborns, women of reproductive age and high-risk populations.

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Section: Diagnostic Tools For Characterizing the Multiple Moleculamentioning

confidence: 99%

Section: Diagnostic Tools For Characterizing the Multiple Moleculamentioning

confidence: 99%

Section: Diagnostic Tools For Characterizing the Multiple Moleculamentioning

confidence: 99%

Section: Diagnostic Tools For Characterizing the Multiple Moleculamentioning

confidence: 99%

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Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

Rajan‐Babu

Chong

2016

Genes

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“…This method is thus unsuitable for the one-third of PGD couples whose normal alleles are identical in size. 13,23 Although the triplet-primed PCR (TP-PCR) method has been used successfully to detect FMR1 CGG repeat expansions from genomic DNA, [24][25][26][27] and although PGD by TP-PCR has been described for myotonic dystrophy type 1 (ref. 10), PGD by direct TP-PCR has not been reported for FXS.…”

Section: Discussionmentioning

confidence: 99%

Identification of microsatellite markers <1 Mb from the FMR1 CGG repeat and development of a single-tube tetradecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of fragile X syndrome

Chen

Zhao

Lee

et al. 2016

Genetics in Medicine

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Purpose: To develop a single-tube polymerase chain reaction (PCR) panel of highly polymorphic markers for preimplantation genetic diagnosis (PGD) of fragile X syndrome (FXS).Methods: An in silico search was performed to identify all markers within 1 Mb flanking the FMR1 gene. Selected markers were optimized into a single-tube PCR panel and their polymorphism indices were determined from 272 female samples from three populations. The single-tube assay was also validated on 30 single cells to evaluate its applicability to FXS PGD.Results: Thirteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single-tube PCR panel together with AMELX/Y for gender determination. Analysis of 272 female samples confirmed the high polymorphism (PIC > 0.5) of most markers, with expected and observed heterozygosities ranging from 0.31 to 0.87. More than 99% of individuals were heterozygous for at least three markers, with 95.8% of individuals heterozygous for at least two markers on either side of the FMR1 CGG repeat. Conclusion:The tetradecaplex marker assay can be performed directly on single cells or after whole-genome amplification, thus supporting its use in FXS PGD either as a standalone linkage-based assay or as a complement to FMR1 mutation detection.

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Triplet‐Primed PCR Assays for Accurate Screening of FMR1 CGG Repeat Expansion and Genotype Verification

2022

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Fragile X syndrome and other fragile X-associated disorders are caused by the full-mutation (>200 copies) and premutation (55 to 200 copies) expansion, respectively, of the CGG short tandem repeat in the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Clinical diagnostic laboratories use Southern blot analysis and polymerase chain reaction (PCR)-based tests to detect and/or size the FMR1 CGG repeats. The development of sensitive and high-throughput triplet-primed PCR (TP-PCR) assays has diminished the need to subject all samples to Southern blot analysis, which is both labor-and time-intensive. In this article, we describe two direct TP-PCR (dTP-PCR) assays for the detection of FMR1 CGG repeat expansions. We outline a protocol that is based on melting curve analysis of dTP-PCR amplicons for a rapid and cost-effective first-tier screening and identification of individuals with premutation and full-mutation expansions. We also describe a protocol that employs capillary electrophoresis to resolve the dTP-PCR amplicon fragments and to estimate the repeat sizes of normal (5 to 44 copies), intermediate (45 to 54 copies), and premutation alleles, as well as to detect full mutations and determine the structure of the FMR1 alleles.

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Single-tube methylation-specific duplex-PCR assay for rapid and accurate diagnosis ofFragile X Mental Retardation 1–related disorders

Abstract: When used in a molecular diagnostic setting, this novel assay could significantly minimize the need to reflex patient samples for Southern analysis.

Cited by 12 publications

References 41 publications

Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

Identification of microsatellite markers <1 Mb from the FMR1 CGG repeat and development of a single-tube tetradecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of fragile X syndrome

Triplet‐Primed PCR Assays for Accurate Screening of FMR1 CGG Repeat Expansion and Genotype Verification

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