Fragile X and the mystery of the ghost genotype

1. Fragile X and the mystery of the ghost genotype Judith Pagan Jan 2011

2. Fragile X syndrome • X-linked disorder • CGG repeat expansion in the 5’UTR of the FMR1 gene • Full mutations (>200 repeats) result in hypermethylation and transcriptional silencing • Prenatal diagnosis is offered to premutation and full mutation females

3. Laboratory testing strategy for Fragile X PND • PCR across the FMR1 CGG repeat • PCR microsatellite markers linked to the FMR1 gene • QF-PCR identify the sex, exclude maternal cell • contamination of the fetal tissue • DNA sent to Glasgow for Southern blotting to confirm • the FMR1 PCR results

4. Fragile X prenatal diagnosis • 37 year old premutation carrier (~ 113 repeats) • son was affected with Fragile X syndrome, the presence • of a full mutation had been confirmed molecularly • presented with a twin pregnancy and chorionic villus • biopsies were performed at 12 weeks gestation • planned to terminate any affected fetuses on PCR • result alone (without Southern blot)

5. fetal fronds DNA specimens CVB A twin 1 clean and sort B A clean and sort B twin 2 Chorionic villus biopsy and DNA extraction • A large bulk extraction, B second smaller bulk extraction

6. FMR1 CGG PCR results dad twin 1 twin 2 mum son • twin 1 = affected male, twin 2 = affected female

7. DXS998 FRAXAC dad twin 1 twin 2 mum son Conflicting result: FMR1 linked markers • twin 1 = affected male, twin 2 = affected female

8. DXS998 FRAXAC dad twin 1 twin 2 mum son Conflicting result: FMR1 linked markers twin 1 = affected male, twin 2 = affected female

9. DXS998 FRAXAC dad twin 1 twin 2 mum son Conflicting result: FMR1 linked markers twin 1 = affected male, twin 2 = affected female

10. twin 1 twin 2 twin 1 twin 2 Conflicting result: QF-PCR data • twin 1 and twin 2 were monozygotic males

11. Conflicting result: QF-PCR data • chromosome 13,18, 21 trace for twin 2

12. Conflicting result: QF-PCR data • sex chromosome trace for twin 2

13. Conflicting result: QF-PCR data • sex chromosome trace for twin 2

14. dad twin 1 twin 2 mum son FMR1 CGG PCR results: extractions B • twin 1 and twin 2 are both affected males

15. DXS1187 dad A twin 2 B twin 2 mum QF-PCR results for twin 2: extractions A and B • low level additional paternal • allele in A but not B • low level additional maternal • allele in A but not B • biparental inheritance suggests • that a second genotype is • present in specimen A for twin 2

16. D13S305 dad A twin 2 B twin 2 mum QF-PCR results for twin 2: extractions A and B • the additional paternal allele is • absent from A and B • the additional maternal allele is • also absent from A and B • together this excludes parental • DNA contamination of A

17. A B QF-PCR results for twin 2: extractions A and B D21S11 • low level additional peak • present in A • subtle peak height differences • indicate the presence of a low • level additional genotype • subtle difference seen with • multiple markers including • DXS998 and FRAXAC dad twin 2 twin 2 mum

18. 1 2 3 4 5 6 7 1 premutation ♀ 2Fragile X♂(CVB) 3 twin 1 (CVB A) 4 twin 2 (CVB A) 5 full mutation ♂ 6 normal ♂ 7 normal ♀ 5.2kb 2.8kb Fragile X Southern blot • presence of a full mutation confirmed in twin 1 and 2 • normal female allele (paternal) only in twin 2

19. Karyotype results • both twin 1 and twin 2 were male (46,XY) • mosaicism for the female cell line was not observed in • twin 2

20. Could the presence of the third distinct ghost genotype be the result of a vanishing twin?

21. Twinning and premature ovarian failure (POF) • ~1 in 8 pregnancies begin as multiples yet twins only • account for ~1 in 90 live births • resorption of one or more concepti during early • pregnancy is well documented (vanishing twin) • ultrasound from IVF pregnancies show that embryo • resorption usually occurs by 7 to 8 weeks • POF occurs in ~20% of FMR1 premutation carriers • Fragile X carrier females have a reported 4 fold (10%) • increase in dizygotic twinning in as a result of POF

22. sac A contains a viable fetus • sac B is small and non-viable • Note: At CVB sampling (~ 12 weeks gestation), sac B should have been resorbed and would be invisible by scan however residual cellular material may persist. A B Vanishing twin syndrome: ultrasound scan at 7 weeks gestation

23. twin 1 monozygotic twinning dizygotic twinning CVB sampling twin 2 fetal resorption residual cellular material from resorbed fetus Proposed mechanism: vanishing twin syndrome

24. Conclusions • twins 1 and 2 were monozygotic and male • a 3rd female genotype was present at low level (~ 5%) in • the biopsy for twin 2, resulted from multiple biopsying • parental DNA admixture was ruled out • all fetal material had inherited the high risk maternal X chromosome, predicted to be affected by Fragile X • dizygotic pregnancy (male and female), followed by a monozygotic twinning event but that the female fetus was reabsorbed

25. 1 2 3 4 5 6 7 1 premutation ♀ 2Fragile X♂(CVB) 3 twin 1 (CVB A) 4 twin 2 (CVB A) 5 full mutation ♂ 6 normal ♂ 7 normal ♀ 5.2kb 2.8kb What about the Southern blot? • the 2.8kb band is significantly stronger than the full • mutation bands in twin 2

26. Interesting observation!!! • 4 set of affected monozygotic twin males have passed • through the Edinburgh genetics service in 10 years • dizygotic twinning is more common in premutation • carrier females • however is the full mutation triggering a monozygotic • twinning event?

27. Report

28. SB report