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. 2023 Feb;191(2):357-369.
doi: 10.1002/ajmg.a.63027. Epub 2022 Nov 8.

Tissue mosaicism, FMR1 expression and intellectual functioning in males with fragile X syndrome

Emma K Baker  1   2   3 , Marta Arpone  1   2   4 , Minh Bui  5 , Claudine M Kraan  1   2 , Ling Ling  1 , David Francis  6 , Mathew F Hunter  7   8 , Carolyn Rogers  9 , Michael J Field  9 , Lorena Santa María  10 , Víctor Faundes  10 , Bianca Curotto  10 , Paulina Morales  10 , Cesar Trigo  10 , Isabel Salas  10 , Angelica M Alliende  10 , David J Amor  2   11 , David E Godler  1   2
Affiliations

Tissue mosaicism, FMR1 expression and intellectual functioning in males with fragile X syndrome

Emma K Baker et al. Am J Med Genet A. 2023 Feb.

Abstract

Fragile X syndrome (FXS) is caused by hypermethylation of the FMR1 promoter due to the full mutation expansion (full mutation [FM]: CGG ≥ 200 repeats) and silencing of FMR1. Assessment of mosaicism for active-unmethylated alleles has prognostic utility. This study examined relationships between FMR1 methylation in different tissues with FMR1 messenger ribonucleic acid (mRNA) and intellectual functioning in 87 males with FXS (1.89-43.17 years of age). Methylation sensitive Southern blot (mSB) and Methylation Specific-Quantitative Melt Aanalysis (MS-QMA) were used to examine FMR1 methylation. FMR1 mRNA levels in blood showed strong relationships with FMR1 methylation assessed using MS-QMA in blood (n = 68; R2 = 0.597; p = 1.4 × 10-10 ) and buccal epithelial cells (BEC) (n = 62; R2 = 0.24; p = 0.003), with these measures also showing relationships with intellectual functioning scores (p < 0.01). However, these relationships were not as strong for mSB, with ~40% of males with only FM alleles that were 100% methylated and non-mosaic by mSB, showing methylation mosaicism by MS-QMA. This was confirmed through presence of detectable levels of FMR1 mRNA in blood. In summary, FMR1 methylation levels in blood and BEC examined by MS-QMA were significantly associated with FMR1 mRNA levels and intellectual functioning in males with FXS. These relationships were not as strong for mSB, which underestimated prevalence of mosaicism.

Keywords: DNA methylation; FMR1; epigenetics; fragile X syndrome; intellectual disability; mosaicism.

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Conflict of interest statement

Dr. David E Godler is as an inventor on patents related to the technologies described in this publication and is an Executive Director of Epigenetic, Diagnostic & Genetic screening (E.D.G). Innovations & Consulting that receives funds from this intellectual property. He has also acted as a paid consultant for Bellberry, Ltd. and Actinogen Medical, Pty, Ltd. No other authors have conflict or competing interests to declare.

Figures

FIGURE 1
FIGURE 1
Intergroup comparisons of FMR1 mRNA levels in blood of males with fragile X syndrome (FXS) and typically developing controls (CGG < 44). Normalized non‐transformed (a) and log‐transformed (b) FMR1 mRNA levels in blood of 70 males with FXS and typically developing controls (CGG < 40). For all log‐transformed plots FMR1 mRNA values of 0 were removed. p Values for the intergroup comparisons were derived using the Wilcoxon rank‐sum test. Methylation Specific‐Quantitative Melt Analysis (MS‐QMA) methylation ratio versus log transformed (n = 45) (c) and non‐transformed (n = 68) (c) normalized FMR1 mRNA levels in blood of males with FXS. The solid line represents the line of best fit for a pairwise linear regression (c,d). Solid dots colored in blue represent full mutation (FM)‐only and red mosaic for PM and FM alleles. The difference in numbers between relationships for log‐transformed versus non‐log transformed mRNA values is due to exclusion of all zero mRNA values for log‐transformed mRNA relationships; vertical lines represent methylation thresholds established using pairwise linear regression (Table 1).
FIGURE 2
FIGURE 2
Relationships of FMR1 promoter methylation between different tissues assessed using Methylation Specific‐Quantitative Melt Analysis (MS‐QMA) and mSB. (a–c) relationships between FMR1 promoter methylation in blood, buccal epithelial cells (BEC) and saliva analyzed using MS‐QMA. (d–f) relationships between FMR1 promoter methylation analyzed using methylation sensitive Southern blot (mSB) in blood and MS‐QMA in blood, BEC, and saliva. MS‐QMA FMR1 methylation % = methylation ratio multiplied by 100. Blue dots = FM‐only and red dots = mosaic for PM and FM alleles; blue ovals highlight ceiling effect observed on mSB.
FIGURE 3
FIGURE 3
Relationships between FMR1 methylation assessed using Methylation Specific–Quantitative Melt Analysis (MS‐QMA) and methylation sensitive Southern blot (mSB) and mRNA levels in blood with standard intellectual functioning scores in the combined cohort of males with FXS. (a–c) relationships between FMR1 promoter methylation assessed using MS‐QMA and standard Verbal IQ (VIQ), Performance IQ (PIQ), and standard Full Scale IQ (FSIQ) scores, respectively. (d–f) Relationships between FMR1 promoter methylation assessed using mSB and standard VIQ, PIQ, and FSIQ scores, respectively. (g–i) Relationships between normalized FMR1 mRNA levels and standard VIQ, PIQ and FSIQ scores, respectively. Open circles represent PM/FM allelic group; black circles represent males with only FM alleles 100% methylated by mSB; blue circles represent males with only FM alleles less than 100% methylated by mSB; red circles represent males with only FM alleles where mSB was not performed due to insufficient DNA being available; gray circles represent all males with only FM alleles.
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References

    1. Aliaga, S. M. , Slater, H. R. , Francis, D. , Du Sart, D. , Li, X. , Amor, D. J. , Alliende, A. M. , Santa Maria, L. , Faundes, V. , Morales, P. , Trigo, C. , Salas, I. , Curotto, B. , & Godler, D. E. (2016). Identification of males with cryptic fragile X alleles by methylation‐specific quantitative melt analysis. Clinical Chemistry, 62(2), 343–352. - PubMed
    1. Alliende, M. A. , Urzua, B. , Valiente, A. , Cortes, F. , Curotto, B. , & Rojas, C. (1998). Direct molecular analysis of FMR‐1 gene mutation in patients with fragile Xq syndrome and their families. Revista Medica de Chile, 126(12), 1435–1446. - PubMed
    1. Arpone, M. , Baker, E. K. , Bretherton, L. , Bui, M. , Li, X. , Whitaker, S. , Dissanayake, C. , Cohen, J. , Hickerton, C. , Rogers, C. , Field, M. , Elliott, J. , Aliaga, S. M. , Ling, L. , Francis, D. , Hearps, S. J. C. , Hunter, M. F. , Amor, D. J. , & Godler, D. E. (2018). Intragenic DNA methylation in buccal epithelial cells and intellectual functioning in a paediatric cohort of males with fragile X. Scientific Reports, 8(1), 3644. - PMC - PubMed
    1. Baker, E. K. , Arpone, M. , Aliaga, S. M. , Bretherton, L. , Kraan, C. M. , Bui, M. , Slater, H. R. , Ling, L. , Francis, D. , Hunter, M. F. , Elliott, J. , Rogers, C. , Field, M. , Cohen, J. , Cornish, K. , Santa Maria, L. , Faundes, V. , Curotto, B. , Morales, P. , … Godler, D. E. (2019). Incomplete silencing of full mutation alleles in males with fragile X syndrome is associated with autistic features. Molecular Autism, 10(1), 21. - PMC - PubMed
    1. Biancalana, V. , Glaeser, D. , McQuaid, S. , & Steinbach, P. (2015). EMQN best practice guidelines for the molecular genetic testing and reporting of fragile X syndrome and other fragile X‐associated disorders. European Journal of Human Genetics, 23(4), 417–425. - PMC - PubMed

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