Prenatal Diagnosis of Fragile X Syndrome in a Twin Pregnancy Complicated by a Complete Retraction

Abstract

Fragile X syndrome (FXS) is usually associated with a CGG repeat expansion >200 repeats within the FMR1 gene, known as a full mutation (FM). FM alleles produce abnormal methylation of the FMR1 promoter with reduction or silencing of FMR1 gene expression. Furthermore, premutation (PM: 55⁻199 CGGs) and full mutation alleles usually expand in size when maternally transmitted to progeny. This study describes a PM allele carried by the mother decreasing to a normal sized allele in a male from a dichorionic diamniotic (DCDA) twin pregnancy, with the female twin inheriting FM (200⁻790 CGGs), PM (130 CGGs) and normal-sized (39 CGGs) alleles. Further evidence of instability of the maternal PM allele was shown by a male proband (older brother) mosaic for PM (CGG 78 and 150 CGGs) and FM (200⁻813 CGGs), and a high level of FMR1 promoter methylation, between 50 and 70%, in multiple tissues. The fully-retracted, normal-sized allele was identified by PCR CGG sizing in the male twin, with no evidence of a FM allele identified using Southern blot analysis in multiple tissues collected postnatally and prenatally. Consistent with this, prenatal PCR sizing (35 CGGs) showed inconsistent inheritance of the maternal normal allele (30 CGGs), with single-nucleotide polymorphism (SNP) linkage analysis confirming that the abnormal FMR1 chromosome had been inherited from the mother's PM chromosome. Importantly, the male twin showed no significant hypermethylation of the FMR1 promoter in all pre and postnatal tissues tested, as well as normal levels of FMR1 mRNA in blood. In summary, this report demonstrates the first postnatal follow up of a prenatal case in which FMR1 mRNA levels were approaching normal, with normal levels of FMR1 promoter methylation and normal CGG size in multiple pre and postnatally collected tissues.

Keywords: FMR1 gene; expansion; fragile-X syndrome; methylation; mosaicism; retraction.

Figure 1

Pedigree and sizing of the CGG repeat using Southern blot and PCR. (A) Pedigree of the studied family with squares and circles symbolising males and females, respectively. CGG size is indicated by numbers below each participant’s ID (determined on postnatal tissues); (B) Southern blot analysis of the PstI restriction sites (not methylation sensitive) using the FMR1 specific and PBS control probes. (C) Capillary electrophoresis sizing using standard PCR, as previously described [8]. NS: normal CGG size (<44 repeats); PM: premutation size (55–199 repeats); FM: full mutation (≥200 repeats). The x and y axes in panel C represent fragment sizes of bp and fluorescence units of capillary electrophoresis profiles from CGG sizing by standard PCR. uCVS: uncultured chorionic villus sampling; Amnio: amniocyte DNA; Bl: blood DNA; cCVS: cultured chorionic villus sampling DNA; uCD: umbilical cord DNA; CD.BL: cord blood DNA.

Figure 2

Chromosome X SNPduo comparison. A SNPduo comparative analysis of Illumina core exome single-nucleotide polymorphism (SNP) microarray genotyping data was performed using samples from Twin 1 (male) and the male proband. The output analysis showed that the two male siblings share identical alleles (identified by State 2) for chromosome regions Xpter (p11.4) and Xp11.23 (qter), including the FMR1 gene. This demonstrates the inheritance of the same X chromosome, with the exception of a small region of recombination (identified by State 0) for chromosome region Xp11.4 (p11.23).

Figure 3

High resolution melt derivative plots from Methylation Sensitive Quantitative Melt Analysis (MS-QMA) analysis of Fragile X-Related Epigenetic Element 2 (FREE2) methylation in the male twin (III-2). Derivative plots in pre and postnatal tissues (A–F) represent cluster results based on similarities of melt curve placement and shape as determined using the Applied Biosystems^® High resolution Melt Software (Thermo Fisher Scientific, Global). Positive and negative controls (A) and (D) have been included for comparison with the results for the male twin’s samples (B, C, E and F), providing details about the presence or absence of the abnormally methylated (M) and unmethylated (UM) FMR1 alleles. The numbers above each panel represent technical replicates. Note: for each plot X-axis represents melting temperature, while y-axis represents fluorescence released, with each fluorescence unit (FU) equal to 1000 units generated by the Applied Biosystems ^® High resolution Melt Software (Thermo Fisher Scientific, Global).