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. 2019 Apr 5;10(4):279.
doi: 10.3390/genes10040279.

Clinical and Molecular Differences between 4-Year-Old Monozygous Male Twins Mosaic for Normal, Premutation and Fragile X Full Mutation Alleles

Alison Pandelache  1 , Emma K Baker  2   3 , Solange M Aliaga  4   5 , Marta Arpone  6   7   8 , Robin Forbes  9 , Zornitza Stark  10   11 , David Francis  12 , David E Godler  13   14
Affiliations

Clinical and Molecular Differences between 4-Year-Old Monozygous Male Twins Mosaic for Normal, Premutation and Fragile X Full Mutation Alleles

Alison Pandelache et al. Genes (Basel). .

Abstract

: This study describes monozygotic (MZ) male twins with fragile X syndrome (FXS), mosaic for normal size (NS: <44 CGGs), premutation (PM: 55–199 CGG) and full mutation (FM alleles ≥ 200) alleles, with autism. At 4 years of age chromosomal microarray confirmed monozygosity with both twins showing an XY sex complement. Normal size (30 CGG), PM (99 CGG) and FM (388–1632 CGGs) alleles were detected in Twin 1 (T1) by standard polymerase chain reaction (PCR) and Southern blot testing, while only PM (99 CGG) and FM (672–1025) alleles were identified in Twin 2 (T2). At ~5 years, T2 had greater intellectual impairments with a full scale IQ (FSIQ) of 55 and verbal IQ (VIQ) of 59, compared to FSIQ of 62 and VIQ of 78 for T1. This was consistent with the quantitative FMR1 methylation testing, revealing 10% higher methylation at 80% for T2; suggesting that less active unmethylated alleles were present in T2 as compared to T1. AmplideX methylation PCR also identified partial methylation, including an unmethylated NS allele in T2, undetected by standard testing. In conclusion, this report demonstrates significant differences in intellectual functioning between the MZ twins mosaic for NS, PM and FM alleles with partial FMR1 promoter methylation.

Keywords: FMR1 gene; Fragile-X syndrome; expansion; methylation; monozygous twins; mosaicism; retraction.

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Conflict of interest statement

D.E.G. is named as an inventor on patent applications (PCT/AU2010/000169 and PCT/AU2014/000044) related to the technology described in this article. The other authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(A) Pedigree of the studied family, with squares and circles symbolising males and females, respectively. Black and while shapes indicate affected and nonaffected statuses, respectively. A small black circle in a white shape indicates carrier status. CGG size is indicated by numbers below each participant’s ID; (B) Capillary electrophoresis sizing using standard PCR, as previously described [14], with allele sizes indicated by numbers superimposed on the panels.
Figure 2
Figure 2
(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 (the broken lines indicated spliced out regions), Fragile X-Related Epigenetic Element 2 (FREE2), the FMR1 CpG island and two methylation sensitive restriction sites HpaII sites targeted by AmplideX methylation PCR. The FREE2 region is located downstream of the CGG expansion, at the exon 1/intron 1 boundary and includes 12 CpG sites. (B) Southern blot analysis of the DNA sample in question from the two twins (T1 and T2) and their mother (PM II-2) with numbers next to arrows indicating CGG repeat length. Comparator DNA sized using standard CGG PCR [14] from normal allele size males (NM) was included. (C) Mean methylation output ration of CpG sites located within the FREE2 region. Assessed using Matrix-Assisted Laser Desorption/Ionisation-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) and Methylation Sensitive-Quantitative Melt Analysis (MS-QMA) [13]. Note: the error bars for reference ranges represent 1 standard deviation from the mean FREE2 methylation in blood of male controls (CGG < 44), PM males (56–170 CGGs), FM males with typical FXS (213–2000 CGGs) and 4 atypical ‘high functioning’ FM males unmethylated by Southern blot (UFM) (CGG 200–637 CGG) assessed as part of previous studies [13,21,22]. The reference samples were co-run with T1 and T2 DNA extracted from retrospectively retrieved newborn blood spots. AmplideX PCR targeting methylation of two HpaII sites, (D) HEX and (E) FAM channels from capillary electrophoresis of the DNA from the blood of the T2 male case in question. Note: pink background indicates region of >200 CGG repeats where presence of positive FM alleles with methylated HpaII sites and the control digestion (HpaII methylation independent) was detected.
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