Fragile X Syndrome - Clinical

1. Fragile X SyndromeTest strategy, analysis and interpretation in a familial case involving skewed methylationSarah Burton-JonesBristol Genetics LaboratoryCardiff Training Day 15th October 2008

2. Fragile X Syndrome - Clinical • Accounts for >¼ cases X-linked learning disability • Symptoms (generally milder in females): • Developmental delay, especially speech • Low IQ (20-60, may decrease with age) • Macrocephaly, long narrow face, large ears • Autism; shyness, poor eye contact, social anxiety • Behavioural problems, ADHD • Carriers • ~20% Female carriers experience Premature Ovarian Failure • Males (and females) at risk of FXTAS,~30% males <age 60 • Only female carriers are at risk of having affected offspring • Approx. 800 test requests per year at Bristol lab. • Majority of diagnostic referrals come from Paediatrics

3. Fragile X Laboratory Testing • Detection of expansion in (CGG)n repeat tract in 5’ untranslated region of FMR1 at Xq27.3, FRAXA • Initial screen by FRAXA PCR; primers flank (CGG)n repeat tract, products visualised as peaks on Beckman CEQ analyser, run includes size controls • Default to Southern blot for: PCR failure, female with only one peak, family history of Fragile X • Current CMGS classification guidelines (2005): • Normal <50 (CGG) repeats, modal value = 30 • Intermediate 50-58 repeats • Premutation 59-200 repeats (unmethylated) • Full mutation >200 repeats (methylated) • Recent audit at Bristol showed pick up <2% FM, ~4% PM and <1% IM

4. 29r Female index case Index case referral • 9 year old female • Request form: Accommodative esotropia (form of squint in which one or both eyes turns inward) • Further info obtained: Learning difficulties, language problems, mild social problems • Karyotype and Fragile X test requested • Result of FRAXA PCR/fragment analysis: 1 allele peak, sized at 29 repeats  Southern blot

5. Ox 0.55 kb (Pfxa32) Ox 1.9 kb (StB 12.3) ~ 500kb (CGG)n Centromeric Telomeric FMR1 (38kb) FMR2 ~ 0.8kb ~ 1.0kb ~ 5.1kb ~ 2.8kb (unmethylated) ~ 5.2kb ~ 12kb Routine blot Prenatal blot Sizing blot Eco RI Bst ZI / Eag I (methylation specific) Pst I Probe Hin dIII Bgl II FRAXA Southern blot analysis Not to scale!

6. 29r,102r 5.2kb (Xi) 2.8kb (Xa) EcoRI+BstZI*/Ox1.9 routine Southern blot Normal and fullmutation alleles in patient N, P P/F, - N, P N, - N, N *EagI now used in place of BstZI (isoschizomer)

7. 31r Mother of FM female Father of FM female ? Parental samples (PCR/Fragment Analysis) Both parents Southern blot

8. 29r,102r 5.2kb (Xi) 2.8kb (Xa) N, P N, N N, N N, F Parental samples (Ox1.9 Southern blot analysis) Normal and methylated expansion alleles in mother of proband Premutation with skewed methylation due to Xi, or (small) full mutation?

9. 29r,102r 0.8kb N, P Mother (PstI /Ox0.55 Southern blot for sizing) Expansion in mother sized by marker scale on blot as ~123 repeats Therefore, can we call this a large premutation in the mother, as opposed to small full mutation?

10. Maternal grandmother 23r 82r 32r Maternal grandfather Maternal grandparents (PCR/fragment analysis)

11. 29r,102r 5.2kb (Xi) 2.8kb (Xa) Maternal grandparents (routine Southern blot) Normal and unmethylated premutation alleles in grandmother of proband Some degree of skew visible? N, P N, N I,- N, P

12. Fragile X family pedigree N,- 32r N,P 23r, 82r N,- ?r N,P 31r, ~123r N,F 29r, >200r

13. UKNEQAS scheme 2007 • Question 1: female with similar analysis results to mother of affected girl in this pedigree • Repeat sizes 30 and ~150 (CGG) • ‘Premutation’ expansion fully methylated on Southern blot, or at least showing extreme skew • Report as full mutation, premutation, or not specified? • Best practice guidelines state: • ‘large premutations may be distinguished from full mutations by their unmethylated status on active X chromosomes’ • Led to introduction of new report phrase in Bristol lab: • ‘This result showed an expanded methylated allele associated with apparently complete methylation. However, we are unable to distinguish between a large premutation showing strongly skewed X-inactivation and a small full mutation.’

14. To consider… Is the girl’s mother (31r, 123rM) at risk of POF and/or FXTAS? • Methylation of FMR1 promoter silencestranscription in full mutation cases (synaptic transmission downregulated) • Symptoms in unmethylated-premutation carriers are due to RNA gain of function, overexpression of FMR1 containing expansion, sequestration of CGGBPs • A female full mutation carrier would not normally be counselled for POF risk • Risk of POF does correlate with expansion size1 and those with >100 (CGG) unlikely to have POF. 1 Sullivan et al 2005

15. Conclusions • Report cases of apparent complete skew or ‘methylated premutation’ with care! • An expansion has been detected • Offer prenatal diagnosis • Clinical Genetics team may counsel that there remains a risk of POF and FXTAS Be aware of limitations of PCR and Southern blot testing • DNA obtained from peripheral blood may not reflect methylation status in the brain • Relatively poor sensitivity of blot may misrepresent skew • Unable to distinguish between a truly methylated (full) mutation and a severely skewed premutation • May be necessary to review classification guidelines to clarify definitions of ‘full’ and ‘pre’ and the implications