Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Case Reports
. 2019 Aug 11;20(16):3907.
doi: 10.3390/ijms20163907.

Significantly Elevated FMR1 mRNA and Mosaicism for Methylated Premutation and Full Mutation Alleles in Two Brothers with Autism Features Referred for Fragile X Testing

Michael Field  1 , Tracy Dudding-Byth  1   2 , Marta Arpone  3   4 , Emma K Baker  3   4   5 , Solange M Aliaga  3   4 , Carolyn Rogers  1 , Chriselle Hickerton  3 , David Francis  6 , Dean G Phelan  6 , Elizabeth E Palmer  1 , David J Amor  3   4 , Howard Slater  3   4   6 , Lesley Bretherton  3   4   7   8 , Ling Ling  3 , David E Godler  9   10
Affiliations
Case Reports

Significantly Elevated FMR1 mRNA and Mosaicism for Methylated Premutation and Full Mutation Alleles in Two Brothers with Autism Features Referred for Fragile X Testing

Michael Field et al. Int J Mol Sci. .

Abstract

Although fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥200 cytosine-guanine-guanine (CGG) repeats, and a decrease in FMR1 mRNA and its protein (FMRP), incomplete silencing has been associated with more severe autism features in FXS males. This study reports on brothers (B1 and B2), aged 5 and 2 years, with autistic features and language delay, but a higher non-verbal IQ in comparison to typical FXS. CGG sizing using AmplideX PCR only identified premutation (PM: 55-199 CGGs) alleles in blood. Similarly, follow-up in B1 only revealed PM alleles in saliva and skin fibroblasts; whereas, an FM expansion was detected in both saliva and buccal DNA of B2. While Southern blot analysis of blood detected an unmethylated FM, methylation analysis with a more sensitive methodology showed that B1 had partially methylated PM alleles in blood and fibroblasts, which were completely unmethylated in buccal and saliva cells. In contrast, B2 was partially methylated in all tested tissues. Moreover, both brothers had FMR1 mRNA ~5 fold higher values than those of controls, FXS and PM cohorts. In conclusion, the presence of unmethylated FM and/or PM in both brothers may lead to an overexpression of toxic expanded mRNA in some cells, which may contribute to neurodevelopmental problems, including elevated autism features.

Keywords: AmplideX; DNA methylation; MS-QMA; RNA toxicity; autism; fragile X syndrome; mosaicism; pediatrics.

PubMed Disclaimer

Conflict of interest statement

David E. Godler is named as an inventor on patent applications (PCT/AU2010/000169 and PCT/AU2014/00004) related to the technology described in this article. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A) Family pedigree, with expansion sizes from AmplideX testing. None of the individuals had manifested symptoms consistent with fragile X syndrome or fragile X-associated disorders, such as Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) or Fragile X-associated Primary Ovarian Insufficiency (FXPOI). The brothers’ have two half-sisters, one of which was identified as carrying a premutation (PM) allele and the other as carrying a normal fragile X mental retardation 1 (FMR1) allele. B1′s cousin (C1) was identified at 1 year and 11 months of age as carrying an FMR1 PM allele of 66 CGG repeats. (B) Methylation-sensitive Southern blot analysis of the NruI restriction site within the FMR1 CpG island in blood. The DNA samples from the two brothers in question are located in lanes 5 and 6. B2 has a 223 CGG unmethylated allele, while B1 has a 190 CGG repeat unmethylated allele. A typical female (CGG < 44) is in lane 1, while typical males (CGG < 44) are in lanes 3 and 4; and a full mutation (FM) female with a 100% methylated 580 CGG allele is in lane 2. The numbers superimposed on the blot indicate CGG sizing. The lower limit of detection for the Southern blot analysis of FM alleles on a normal allele background is 20%. (C) Photos of B1 and B2 at the ages of 5 and 2 years, respectively; written informed consent was obtained from the mother of the brothers to include these images in the publication (D) Photos of B1 and B2 at the ages of 10 and 7 years, respectively; written informed consent was obtained from the mother of the brothers to include these images in the publication. Note: Comparator DNA used of methylation-sensitive Southern blot analysis in (B) were also sized using standard CGG sizing PCR.
Figure 2
Figure 2
(A) Organization of the fragile X mental retardation 1 (FMR1) 5′ region, including the cytosine-guanine-guanine (CGG) expansion and primer binding site locations for AmplideX CGG sizing PCR, FastFraX High-Resolution Melt Analysis, and Methylation-Specific Quantitative Melt Analysis (MS-QMA), in relation to Fragile X-Related Epigenetic Element 2 (FREE2); the FMR1 CpG island and methylation sensitive restriction site NruI analysed using routine fragile X Southern blot testing; and two HpaII sites targeted by AmplideX methylation PCR. Note: The FMR1 translation start site is indicated in red overlapping with FastFraX 3′ melting curve analysis (MCA) primer. (B) AmplideX triplet-repeat primed long range PCR results for B1 and B2, for blood, buccal, saliva, and fibroblast DNA. Note: a reference sample co-run with the samples in question is included as the AmplideX sizing control in (B).
Figure 3
Figure 3
Derivative curve high-resolution melt profiles and mean methylation output ratios of CpG sites located within the Fragile X-Related Epigenetic Element 2 (FREE2) region assessed using Methylation-Specific Quantitative Melt Analysis (MS-QMA) between different tissues for (A) B1 and (B) B2. (C) DNA samples from lymphoblasts of a fragile X syndrome (FXS) male with the fragile X mental retardation 1 (FMR1) promoter 100% methylated spiked with DNA from a typically developing control (cytosine-guanine-guanine (CGG) < 44) with the FMR1 promoter 0% methylated. These samples were mixed at different ratios for the MS-QMA methylation reference curve, with the expected % of methylation indicated on the plot for each derivative curve profile.
Figure 4
Figure 4
Comparison of fragile X mental retardation 1 (FMR1) mRNA levels in blood in the probands, control, premutation (PM), full mutation (FM) only, and PM/FM mosaic reference cohorts. Note: Control reference ranges included in blood of females (age 22 to 54 years; cytosine-guanine-guanine (CGG) < 44) and males (age 7.7–8.1 years) typically developing (TD) controls; males with PM alleles (3.4 to 23.6 years); and males with FM only alleles and PM/FM mosaic alleles (1.89 to 43.17 years). Control children had no family history of developmental delay and their mothers had CGG size < 44.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Vissers L.E., Gilissen C., Veltman J.A. Genetic studies in intellectual disability and related disorders. Nat. Rev. Genet. 2016;17:9–18. doi: 10.1038/nrg3999. - DOI - PubMed
    1. Clifford S., Dissanayake C., Bui Q.M., Huggins R., Taylor A.K., Loesch D.Z. Autism spectrum phenotype in males and females with fragile X full mutation and premutation. J. Autism Dev. Disord. 2007;37:738–747. doi: 10.1007/s10803-006-0205-z. - DOI - PubMed
    1. Hall S.S., Lightbody A.A., Reiss A.L. Compulsive, self-injurious, and autistic behavior in children and adolescents with fragile X syndrome. Am. J. Ment. Retard. 2008;113:44–53. doi: 10.1352/0895-8017(2008)113[44:CSAABI]2.0.CO;2. - DOI - PubMed
    1. Mazzocco M.M., Kates W.R., Baumgardner T.L., Freund L.S., Reiss A.L. Autistic behaviors among girls with fragile X syndrome. J. Autism Dev. Disord. 1997;27:415–435. doi: 10.1023/A:1025857422026. - DOI - PubMed
    1. Merenstein S.A., Sobesky W.E., Taylor A.K., Riddle J.E., Tran H.X., Hagerman R.J. Molecular-clinical correlations in males with an expanded FMR1 mutation. Am. J. Med. Genet. 1996;64:388–394. doi: 10.1002/(SICI)1096-8628(19960809)64:2<388::AID-AJMG31>3.0.CO;2-9. - DOI - PubMed

Publication types

Substances