Fragile X Syndrome

Question 1

Provide some general details on FRagile X

Answer 1

• 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)

Question 2

What are the developmental impacts of fragile X?

Answer 2

• hypotonia and mild motor delay quite common
• variable speech delay
• mean IQ of 41 (full mutation)
• Autism spectrum disorders
• often need supported living

Question 3

What is the behavioural phenotype for a fragile X patient?

Answer 3

• hyperactivity, impulsiveness, poor concentration
• gaze avoidance/shyness
• repetitive behaviours (hand flapping, hand biting)

Question 4

What is the mode of inheritance for Fragile X?

Answer 4

X-linked recessive

Question 5

What is the Sherman Paradox?

Answer 5

• 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

Question 6

There are over 100 fragile sites in the human genome. What are the fragile sites associated with fragile X syndrome?

Answer 6

• Most common = FRAXA (Xq27.3) and FRAXE (Xq28)

Question 7

What gene is present in the FRAXA region?

Answer 7

• 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

Question 8

Describe the CGG repeat found in the normal population

Answer 8

• 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

Question 9

Can intermediate FMR1 expansions (50-58rpts) cause Fragile X?

Answer 9

• 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

Question 10

Provide some details on the premutation allele in Fragile X

Answer 10

• 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

Question 11

Describe why a variable phenotype is observed in females with the full mutation

Answer 11

• 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

Question 12

Provide details of FXTAS

Answer 12

• 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

Question 13

What are the reproductive options available for Fragile X?

Answer 13

• do nothing
• prenatal test
• preimplantation genetic diagnosis (PGD)
• other IVF donor eggs or sperm

Question 14

What referrals are appropriate for Fragile X testing?

Answer 14

• 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

Question 15

Provide information of FRAXE

Answer 15

• 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

Question 16

Give details on mosaicism in fragile X

Answer 16

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

Question 17

Explain prenatal diagnosis for FRAX

Answer 17

• 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

Question 18

What is the general testing workflow for FRAX?

Answer 18

• CGG-PCR used as primary screen

- this is followed by southern blotting or amplidex FMR1 PCR

Question 19

Describe the process of CGG PCR in FRAX

Answer 19

• 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

Question 20

What are the limitations of CGG PCR?

Answer 20

• 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

Question 21

Southern blotting can detect changes in methylation status of FMR1 expansion alleles - how?

Answer 21

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

Question 22

What 7 factors can produce an atypical southern blot result?

Answer 22

• X chromosome aneuploidy
• dels/dups
• restriction site polymorphisms
• mosaicism
• skewed X inactivation
• incomplete digest
• poor hyb

Question 23

Give details on Asuragen amplidex PCR

Answer 23

• 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

Question 24

What PCR method forms the basis of asuragen amplidex?

Answer 24

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

Question 25

Can the asuragen amplidex kit determine spacing of AGG interruptions?

Answer 25

Yes - thought this info may be useful in predicting premutation expansions

Question 26

What is Premature Ovarian Failure?

Answer 26

• 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

Question 27

How does the full mutation of >200 methylated CGG repeats results in Fragile X?

Answer 27

• 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