Fragile X Flashcards
Frequency of frax?
1 in 4,000 to 1 in 6000
Where is the fragile site located?
Xq27.3
What does the FMR1 gene do?
Encodes for FMRP which is involved in regulation of protein synthesis in many issues- especially functional and structural maturation of synapses
What type of mutation causes fragile x?
Dynamic mutations- expansion of unstable DNA repeat sequences
CGG trinucleotide repeat in 5’UTR
What % of fragile x is caused by point mutations or deletions?
1%
Can unstable repeats be transmitted unchanged?
Virtually never
Size of fmr1?
17 exon, 38kb
What is the normal size f the 5’UTR?
5-58 CGG repeats interspersed by one or more single AGG interruptions every 9-10 repeats
What happens when there is a full mutations?
Increased methylation of FMR1 - no fmrp
List ranges….
Normal = 6-49 Intermediate = 50-58 Permutation= 59-200 Full= >200
Risks to premutation carriers?
Not affected but high risk of expansion to full mutation for children of female carriers and FXPOI/POF and FXTAS for men/ women
No AGG
Frequency of premutation carriers?
1:800 (males) and 1:250 Females
Risk to intermediate alleles?
Mostly stable but expansion from intermediate to full in one generation has been reported
What percentage of FMR1 mutations are mosaic?
15-20%
What are the two types of FMR1 mosaicism?
1) repeat size mosaicism where both full and pre are present
2) methylation mosaicism - full mutations which are variably methylated - individuals are affected but higher functioning
What caveat must you say in PM reports?
Possibility of patient being tissue mosaic with a pm in leucocyte DNA but full mutation in others
- can analyse other tissue samples
What is FXTAS?
Late onset progressive cerebellum ataxia and intention tremor (gait ataxia, Parkinsonism and autoimmune dysfunction)
Pathophysiology of FXTAS
Males with PM have elevated mRNA levels in blood and brain
Toxic gain of function of mRNA?
Nuclear inclusions found in brains of males with FXTAS
What percentage of males over 50 with PM
1/3
What percentage of women with PM will develop early menopause? (<40 years)
24%
Pathophysiology of POF?
Women have higher levels of FSH than healthy individuals and low AMH
What % of women with POF have pM?
6.5%
Risk of using CvS for PND?
Methylation status is usually not established in CVS- 20% of cvs DNA is unmethylated - determine on size alone
Who is PNd offered to?
Female carriers of an allele of 56 CGG repeats or greater
Steps in PND?
1) CGG-pcr to determine if normal allele was inherited
2) McC test
Possible MCC tests?
1) Asuragen amplidex pcr
2) Linkage analysis with markers flanking FMR1
3) eco R1/ eag1 southern blot
True or false: Risk to women with newly identified premutation alleles with no FH is higher than women with FH?
False - risk significantly reduced
Ref Nolan et al 2003 and 2011
Why cytogenetics not great for FRAXA diagnosis?
1) Positive result may not be absolute if only a portion of cells examined showed fragile site
2) analysis complicated by nearby FRAXE
What is the limits of pcr across the CGG?
Can pcr up to 50-60 repeats but struggles to pcr beyond 70 repeats
Pcr preference for smaller repeats
What samples are followed up with asuragen?
Male with no allele or female with 1 allele
Limitations of fluorescent pcr?
- SNP in primer or low quality DNA
- not quantitative
- cannot detect rare point mutations and deletions
- false negatives for mosaics (<1%) eg normal allele and expansion / pm and expansion
Describe southern boot for fmr1
1) fluoresently tagged or radioactively label FMR1 with OX1.9 probe
2) digest with ecor1 and eag1(methylation sensitive)
3) if unmethylated- cut with both and get 2.8kb product. If methylated only cut with ecor1= 5.2 Kbps
4) run on 1% agarose gel
Use PST for small expansions
Limitations/ abnormal southern blot
Restriction site polymorphisms Mosaicism Skewed x inactivation X chromosome aneuploidy Incomplete digestion
Describe asuragen amplidex FMR1 pcr kit?
Uses a gene specific pcr which shows a full length product peak for each allele size and a CGG repeat primer pcr ( a to- pcr combined with a gene specific pcr
Pcr scan be performed in serperate tubes
What size repeat can asuragen detect up to? And can it detect AGG repeats ?
> 1000 CGG repeats
Comprehensive genotyping of AGG
Describe TP-PCR
P1 = upstream of repeat
P2= binds CGG and contains sequence not found in the human genome that P3 can bind to
P1 and P2 procure lots of fragments that are then amplified by p1&3 and analysed by capillary electrophoresis
How much DNA do you use for pcr?
5-40ng
NEQAS and EMQN for fmr1
Testing for genotyping, correct interpretation and reporting
Lab registers for relevant disease
Annual distribution- 3 per disease
