Single-tube methylation-specific duplex-PCR assay for rapid and accurate diagnosis of Fragile X Mental Retardation 1-related disorders.

Abstract:

AIM:Molecular diagnosis of fragile X syndrome demands assessment of fragile X mental retardation 1 (FMR1) CGG repeat size and methylation status, while predicting disease transmission risk requires determination of AGG interruption pattern. There is currently no single assay that provides all three categories of information. We describe a single-tube methylation-specific triplet-primed PCR assay for concurrently assessing methylation state, repeat size and structure of CGG repeat(s). METHODS:Differentially labeled primers specific for methylated and unmethylated FMR1 alleles were used to amplify bisulfite-modified DNA, followed by capillary electrophoresis. Twenty-four reference DNAs and 107 patient samples were analyzed to evaluate assay performance. RESULTS:Repeat size, AGG interruption pattern and methylation state were correctly identified in all tested samples. The assay also detected skewed X-inactivation when present in females, and somatic mosaicism in fragile X males. CONCLUSION:When used in a molecular diagnostic setting, this novel assay could significantly minimize the need to reflex patient samples for Southern analysis.

journal_name

Expert Rev Mol Diagn

authors

Rajan-Babu IS,Teo CR,Lian M,Lee CG,Law HY,Chong SS

doi

10.1586/14737159.2015.1001749

subject

Has Abstract

pub_date

2015-03-01 00:00:00

pages

431-41

issue

3

eissn

1473-7159

issn

1744-8352

journal_volume

15

pub_type

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