Simultaneous screening of the FRAXA and FRAXE loci for rapid detection of FMR1 CGG and/or AFF2 CCG repeat expansions by triplet-primed PCR
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Date
2021
Authors
Liu, T.
Wang, F.S.
Cheah, F.S.H.
Gu, Y.
Shaw, M.
Law, H.-Y.
Tay, S.K.H.
Lee, C.G.
Nelson, D.L.
Gecz, J.
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Journal article
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Journal of Molecular Diagnostics, 2021; 23(8):941-951
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Timing Liu, Furene S. Wang, Felicia S.H. Cheah, Yanghong Gu, Marie Shaw, Hai-Yang Law, Stacey K.H. Tay, Caroline G. Lee, David L. Nelson, Jozef Gecz, and Samuel S. Chong
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Abstract
Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with intellectual disability and other conditions. Unlike molecular diagnosis of FMR1 CGG repeat expansions in FRAXA, current detection of AFF2 CCG repeat expansions in FRAXE relies on low-throughput and otherwise inefficient techniques combining Southern blot analysis and PCR. A novel triplet-primed PCR assay was developed for simultaneous screening for trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, and was validated using archived clinical samples of known FMR1 and AFF2 genotypes. Population samples and FRAXE-affected samples were sequenced for the evaluation of variations in the AFF2 CCG repeat structure. The duplex assay accurately identified expansions at the FMR1 and AFF2 trinucleotide repeat loci. On Sanger sequencing of the AFF2 CCG repeat, the single-nucleotide polymorphism variant rs868914124(C) that effectively adds two CCG repeats at the 50-end, was enriched in the Malay population and with short repeats (<11 CCGs), and was present in all six expanded AFF2 alleles of this study. All expanded AFF2 alleles contained multiple non-CCG interruptions toward the 50-end of the repeat. A sensitive, robust, and rapid assay has been developed for the simultaneous detection of expansion mutations at the FMR1 and AFF2 trinucleotide repeat loci, simplifying screening for FRAXAand FRAXE-associated disorders. (J Mol Diagn 2021, 23: 941e951; https://doi.org/10.1016/j.jmoldx.2021.04.015)
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© 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0). https://doi.org/10.1016/j.jmoldx.2021.04.015jmdjournal.org. The Journal of Molecular Diagnostics, Vol. 23, No. 8, August 2021