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. 2019 Oct 25;9:15315. doi: 10.1038/s41598-019-51618-7

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Relationships between FREE2 methylation analysed using MS-QMA and methylation of two HpaII sites examined by AmplideX mPCR in thirteen cases identified to have abnormal methylation using MS-QMA. (A) Organization of the FMR1 5′ region including the CGG expansion (sequence numbering from GenBank L29074 L38501) in relation to FMR1 and ASFMR1 transcription start sites, Fragile X Related Epigenetic Element 2 (FREE2) targeted by MS-QMA, the FMR1 CpG island and methylation sensitive restriction sites and corresponding fragments using routine fragile X by Southern blot testing, two HpaII sites targeted by AmplideX methylation PCR. Relationships between FREE2 methylation % (encompassing 12 CpG sites) analysed using MS-QMA with methylation percentage of two HpaII methylation sensitive sites targeted by AmplideX mPCR, with (B) methylation % of the smallest allele.