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.
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.
Authors: Grace X Y Lim; Minli Yeo; Yvonne Y Koh; Tri Indah Winarni; Indhu-Shree Rajan-Babu; Samuel S Chong; Sultana M H Faradz; Ming Guan Journal: PLoS One Date: 2017-03-09 Impact factor: 3.240
Authors: Indhu-Shree Rajan-Babu; Mulias Lian; Felicia S H Cheah; Min Chen; Arnold S C Tan; Ethiraj B Prasath; Seong Feei Loh; Samuel S Chong Journal: Expert Rev Mol Med Date: 2017-07-19 Impact factor: 5.600