Fragile X Syndrome Flashcards
Provide some general details on FRagile X
- most common inherited cause of mental retardation
- affected males prevalence = 1 in 5500
- found in 4-8% of boys with an IQ of 35-70
- classic phenotype often subtle in young children (evolves after puberty)
What are the developmental impacts of fragile X?
- hypotonia and mild motor delay quite common
- variable speech delay
- mean IQ of 41 (full mutation)
- Autism spectrum disorders
- often need supported living
What is the behavioural phenotype for a fragile X patient?
- hyperactivity, impulsiveness, poor concentration
- gaze avoidance/shyness
- repetitive behaviours (hand flapping, hand biting)
What is the mode of inheritance for Fragile X?
X-linked recessive
What is the Sherman Paradox?
- Daughter of unaffected male carrier more likely to have affected offspring than mother of the unaffected male carrier
- Risk of expressing mental retardation increasing in later generations
- Hypothesis = premutation exists with no clinical symptoms, with second event required to convert to full mutation. The mutation changes upon transmission by carrier female
There are over 100 fragile sites in the human genome. What are the fragile sites associated with fragile X syndrome?
- Most common = FRAXA (Xq27.3) and FRAXE (Xq28)
What gene is present in the FRAXA region?
- FMR1 gene
- expansion of CGG trinucleotide repeat of 5’ UTR of FMR1 within exon 1 (non-coding) is responsible for >99% of cases
- leads to methylation and inactivation of FMR1 gene
Describe the CGG repeat found in the normal population
- Highly polymorphic in normal population (5-58 repeats)
- Usually interspersed by one or more single AGG interruptions
- These interruptions are thought to confer DNA stability
Can intermediate FMR1 expansions (50-58rpts) cause Fragile X?
- Not to direct offspring as only a premutation can expand to a full mutation in one generation
- However, intermediate alleles can cause effects in subsequent generations as expansion may gradually occur
Provide some details on the premutation allele in Fragile X
- 59-200 (unmethylated) rpts
- Carrier females and non-transmitting males
- Not affected but both sexes at high risk of FXTAS and women also at risk of POF
- High chance of expansion to full mutation for children of female carriers
Describe why a variable phenotype is observed in females with the full mutation
- normal X produces varying amounts of FMR protein (variable X-inactivation)
- levels of FMR protein correlates with degree of cognitive impairment
- prenatal diagnosis to predict phenotype in female fetuses with full mutation is not possible
Provide details of FXTAS
- 1 in 3 males over 50 with fragile X premutation will develop this phenotype
- Penetrance increases with age (80+ yrs = 75%)
- reported in both sexes with premutation (lower risk in females)
- premutation males have elevated mRNA levels in blood and brain leading to toxic gain of function
What are the reproductive options available for Fragile X?
- do nothing
- prenatal test
- preimplantation genetic diagnosis (PGD)
- other IVF donor eggs or sperm
What referrals are appropriate for Fragile X testing?
- males/females with ID, dev delay or autism, especially those with physical/behavioural characteristics of FRAX or family history of FRAX
- males/females >50yrs who have progressive cerebellar ataxia and intention tremor who have family history of FMR1-related disorders
- women with unexplained POF
Provide information of FRAXE
- less common than FRAXA
- caused by triplet repeat expansion (GCC) in FMR2 gene
- tend to have milder phenotype and usually only tested if family history is suggestive of X-linked mental retardation
Give details on mosaicism in fragile X
15-20% of FMR1 mutations are mosaic. 2 types exist:
- Repeat size mosaicism: both full and premutations are present
- Methylation mosaicism: full mutations are variably methylated resulting in affected individuals with higher functioning
Explain prenatal diagnosis for FRAX
- CGG-PCR and southern blotting typically used to check amnio/CVS samples
- MCC testing required
- methylation pattern in fetal DNA may not be representative: 20% of CVS DNA may be partially or unmethylated
- essential to use normal and expansion controls to determine fetal allele size as accurately as possible
What is the general testing workflow for FRAX?
- CGG-PCR used as primary screen
- this is followed by southern blotting or amplidex FMR1 PCR
Describe the process of CGG PCR in FRAX
- Fluorescent PCR across the CGG repeat and capillary electrophoresis
- use controls to allow accurate sizing
- very difficult to amplify using PCR: can PCR up to 50-60 repeats, struggle to PCR beyond 70 repeats
- can report males with one allele and females with two alleles (normal CGG repeat range) - all other cases progress to second line of testing as well as those with family history of FRAX
What are the limitations of CGG PCR?
- cannot detect large expansions, rare point mutations or deletions
- no PCR product could be due to poor DNA quality, large expansion or SNP under primer
- Not quantitative: females homozygous for a normal or an expansion allele
- can get false negatives from mosaics: normal allele + expansion or premutation allele plus expansion
Southern blotting can detect changes in methylation status of FMR1 expansion alleles - how?
By using Eag1 (methylation sensitive restriction enzyme) in combo with EcoR1 and OX1.9 probe
- Methylated/inactive normal alleles cut by EcoR1 only to give 5.2kb fragment
- Unmethylated/active normal alleles cut by both EcoR1 and Eag1 to give smaller 2.8kb fragment
- Full mutation (methylated) will be slightly larger than 5.2kb fragment due to the expansion
What 7 factors can produce an atypical southern blot result?
- X chromosome aneuploidy
- dels/dups
- restriction site polymorphisms
- mosaicism
- skewed X inactivation
- incomplete digest
- poor hyb
Give details on Asuragen amplidex PCR
- can amplify alleles with >1000 CGG rpts
- accurately characterises all allele size categories
- uses novel CGG repeat primed PCR reaction
- faster, simpler and more sensitive than s.blotting
- reduces need for s.blotting to only samples where methylation info absolutely required
What PCR method forms the basis of asuragen amplidex?
Triplet prime PCR (TP-PCR). Uses 3 primers:
- primer1 flanks rpt region and acts as forward primer
- primer2 has two parts: 1st hybridises across rpt region at every rpt and 2nd part is extra sequence not found in human genome
- primer 3 binds to this unique sequence of primer 2
Primers 1 and 2 produce lots of fragments of different sizes which are then amplified up further by primers 1 and 3. Fragments are then analysed by capillary electrophoresis
Can the asuragen amplidex kit determine spacing of AGG interruptions?
Yes - thought this info may be useful in predicting premutation expansions
What is Premature Ovarian Failure?
- cessation of menses before 40yrs
- 21% risk of POF for premutation carriers (background risk = 1%)
- 6.5% of women with POF have FMR1 premutation
- Family history increases risk
How does the full mutation of >200 methylated CGG repeats results in Fragile X?
- Increased methylation of the FMR1 gene means no FMR protein is produced
- FMR protein usually abundant in neurons and appears to play role in functional/structural maturation of synapses
- Loss of FMR protein produces the syndrome: all affected males have LD as do ~50% of females
