Detection of Cryptic Fragile X Full Mutation Alleles by Southern Blot in a Female and Her Foetal DNA via Chorionic Villus Sampling, Complicated by Mosaicism for 45,X0/46,XX/47,XXX

Abstract

We describe a female with a 72 CGG FMR1 premutation (PM) (CGG 55-199) and family history of fragile X syndrome (FXS), referred for prenatal testing. The proband had a high risk of having an affected pregnancy with a full mutation allele (FM) (CGG > 200), that causes FXS through hypermethylation of the FMR1 promoter. The CGG sizing analysis in this study used AmplideX triplet repeat primed polymerase chain reaction (TP-PCR) and long-range methylation sensitive PCR (mPCR). These methods detected a 73 CGG PM allele in the proband's blood, and a 164 CGG PM allele in her male cultured chorionic villus sample (CVS). In contrast, the Southern blot analysis showed mosaicism for: (i) a PM (71 CGG) and an FM (285-768 CGG) in the proband's blood, and (ii) a PM (165 CGG) and an FM (408-625 CGG) in the male CVS. The FMR1 methylation analysis, using an EpiTYPER system in the proband, showed levels in the range observed for mosaic Turner syndrome. This was confirmed by molecular and cytogenetic karyotyping, identifying 45,X0/46,XX/47,XXX lines. In conclusion, this case highlights the importance of Southern blot in pre- and postnatal testing for presence of an FM, which was not detected using AmplideX TP-PCR or mPCR in the proband and her CVS.

Keywords: 45,X0/46,XX/47,XXX; CGG; FMR1; X syndrome; cultured chorionic villus; fragile; full mutation; mosaicism; sample; southern blot.

Figure 1

(A) Pedigree of the studied family with CGG sizing determined using an AmplideX TP-PCR. Males are represented by squares, females by circles and triangle sex unknown. A black outline indicates examined and half shaded indicates carrier. A black arrow indicates proband. III:1 Miscarriage at 6 of 40 weeks; (B) AmplideX TP-PCR CGG sizing, with no FM alleles detected in either III:3 male CVS or II:2 proband; (C) II:2 and (D) III:3 AmplideX mPCR, with the upper panel in blue representing CGG sizing based on control digestions and the lower panel in green representing relative methylation based on HpaII methylation sensitive restriction enzyme-based digestion followed by long-range PCR. No FM alleles are detected in either III:3 male CVS or II:2 female. Skewed methylation for the 75 CGG PM allele is suggested by 80% methylation. Note: numbers on each panel next to specific peaks indicate CGG sizes.

Figure 2

(A) Pedigree of the studied family with CGG sizing determined using Southern blot and standard CGG sizing PCR. Males are represented by squares, females by circles and triangle sex unknown. The black outline indicates examined, half shaded indicates carrier, and full shaded indicates affected with FXS. The black arrows indicate proband and a diagonal line indicates being deceased. III:1 Miscarriage at 6 of 40 weeks (B) Southern blot Lane 1 (male PM control), Lane 2 (III:3 male PM/FM CVS), Lane 3 (II:2 PM/FM female), Lane 4 (male normal CGG size control).

Figure 3

G banded chromosome analysis of II:2 proband blood and conventional karyotypes identify mosaic cell lines. (A) 45,X0 was found in 38/60 karyotypes analysed; (B) 47,XXX was found in 19/60 karyotypes analysed; (C) 46,XX was found in 3/60 karyotypes analysed. (D) Diagram of post zygotic nondisjunction and generation of the mosaic cell line 45,X[38]/47,XXX[19]1/46,XX[3]3.

Figure 4

Linkage analysis using chromosomal microarray. (A) Single nucleotide polymorphisms (SNP) duo image for chromosome 1 showing identity by state (sharing one allele) for the whole chromosome. Whole genome comparison confirmed child-parent relationship. Note: Chromosome 1 SNPduo Output A_Culture CVS–B_Proband Mother Average Identity by State (IBS):1.84; (B) SNP duo for chromosome X between the foetus and the X0 cell line showing IBS0, that is sharing no alleles for most of the chromosome including the FMR1 gene. Note: Chromosome X SNPduo Output A_Cultured CVS–B_Proband Mother Average IBS:1.1.547.