Prenatal diagnosis of fragile X syndrome complicated by full mutation retraction

Abstract

Fragile X syndrome is the most common single gene cause of intellectual disability in boys. It is usually caused by expansion of a CGG repeat sequence in the 5 0 untranslated region, which is associated with methylation and silencing of the FMR1 promoter and adjacent regions. A repeat size of less than 45 is considered normal, 45-54 repeats are termed "gray zone" (GZ) or intermediate alleles, and 55-200 are considered premutation (PM). These allele types are unmethylated. In contrast, alleles with more than 200 repeats, termed full mutations (FM), are methylated [Maddalena et al., 2001]. PM and FM alleles are unstable and usually expand when maternally transmitted. Figures regarding the risk of expansion based on the size of the maternal allele are available and widely used in counseling couples requesting prenatal diagnosis in pregnancies at risk of the condition [Jin and Warren, 2000;Nolin et al., 2003]. Conventionally, prenatal testing involves fetal sex determination by FISH, followed by sizing of the FMR1 allele either through Southern blot or long-range PCR. More recently, methylation testing of Fragile X Related Epigenetic Elements (FREE) located on the 5 0 and 3 0 ends of the FMR1 promoter, has started to be used particularly in the postnatal setting to accurately measure the fraction of methylated alleles [Godler et al., 2010;Godler et al., 2012]. Methylation testing for allele sizes between 150 and 250 CGG repeats is particularly important, because the technical error typically associated with Southern blot CGG sizing in this range is AE30-50 CGG repeats [Zhou et al., 2004]. High levels of methylation of the FREE biomarkers are negatively correlated with verbal cognitive abilities and FMR1 protein expression (FMRP), the latter essential for normal neurodevelopment [Godler et al., 2010;Godler et al., 2012].We present a case where prenatal diagnosis for fragile X syndrome in a male fetus was complicated by transmission of a smaller CGG repeat expansion, that is, a FM retraction. The proband in this family is a FM female carrier with 29 and 390-907 CGG repeats. She has completed secondary education, is in full-time employment, and does not have learning difficulties. She has two male cousins with fragile X syndrome who were severely affected, and a female cousin with FM with mild learning difficulties. She requested prenatal diagnosis and underwent chorionic villus sampling (CVS) at 12 weeks gestation. Male sex of the fetus was determined using X and Y-specific FISH probes. Southern blot testing of uncultured chorionic villi DNA (trophoblast and mesenchymal cells) showed a fragile X allele in the PM range (155 repeats; Fig. 1A). Subsequent Southern blot on cultured CVS DNA (mesenchymal cells) showed only a FM smear (320-730 repeats; Fig. 1B). Given the discordant results, amniocentesis was performed and Southern blot testing of amniotic fluid (AF) DNA showed a mosaic PM (180 repeats)/FM (257-923 repeats) result (Fig. 1C). Testing of a cheek brush sample from the mother confirmed 29 and 390-907 CGG repeats ...

Fragile X syndrome: a review of clinical and molecular diagnoses

“…In a study by Nolin et al, by screening 1040 FMR1 pedigrees, repeat contraction had been observed in 2.3% of maternal and 5.7% of paternal transmissions [64]; previous work of the same research group had already shown a similar rate of contraction for maternal PM alleles (3,1%) [65]. Retraction occurs post zygotically because of an excision of a variable number of trinucleotides, resulting in a mosaic normal size/GZ/PM/FM [68, 69]. Generally, mosaics have a higher percentage of the larger allele [69].…”

“…The prevalence of mosaicism in FXS males largely varies among studies, in the range of 12–41% [6, 67]. Both repeat-size mosaicism (e.g., FM/PM, FM/GZ) and methylation mosaicism have been described; in the latter, FM has varying degrees of methylation from tissue to tissue [6, 68–70]. Somatic FM/deletion mosaicism has also been reported because deletion can occur mitotically during embryonic cell divisions, usually before the 11th week, with the result of two distinct subpopulations of cells carrying the deletion and FM alleles [71].…”

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

“…Further testing identified that the GZ allele had arisen as a result of a postzygotic retraction event in some of the cells, which was confirmed at birth in multiple tissues. There has also been a prenatal report of mosaicism in a male fetus that demonstrated instability/retraction from FM to PM range and placental tissue mosaicism [ 20 ]. Another study summarised 26 cases reported in the literature regarding likely retraction and repeat size mosaicism, suggesting that this phenomenon is not uncommon [ 16 ].…”

Prenatal diagnosis of fragile X syndrome: Small meiotic recombination events at the FMR1 locus

“…On the other hand, several reports on contractions mention a size mosaicism in favor of a postzygotic event. Thus, observation of a single normal allele on CVS DNA does not protect against the possibility of a size mosaicism, occasionally including a FM allele, in some fetal tissues. This eventuality undoubtedly warrants checking the CGG repeat profile on a source of fetal DNA such as amniocytes or cord blood cells.…”