Fragile X

Question 1

Frequency of frax?

Answer 1

1 in 4,000 to 1 in 6000

Question 2

Where is the fragile site located?

Answer 2

Xq27.3

Question 3

What does the FMR1 gene do?

Answer 3

Encodes for FMRP which is involved in regulation of protein synthesis in many issues- especially functional and structural maturation of synapses

Question 4

What type of mutation causes fragile x?

Answer 4

Dynamic mutations- expansion of unstable DNA repeat sequences
CGG trinucleotide repeat in 5’UTR

Question 5

What % of fragile x is caused by point mutations or deletions?

Answer 5

1%

Question 6

Can unstable repeats be transmitted unchanged?

Answer 6

Virtually never

Question 7

Size of fmr1?

Answer 7

17 exon, 38kb

Question 8

What is the normal size f the 5’UTR?

Answer 8

5-58 CGG repeats interspersed by one or more single AGG interruptions every 9-10 repeats

Question 9

What happens when there is a full mutations?

Answer 9

Increased methylation of FMR1 - no fmrp

Question 10

List ranges….

Answer 10

Normal = 6-49
Intermediate = 50-58
Permutation= 59-200
Full= >200

Question 11

Risks to premutation carriers?

Answer 11

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

Question 12

Frequency of premutation carriers?

Answer 12

1:800 (males) and 1:250 Females

Question 13

Risk to intermediate alleles?

Answer 13

Mostly stable but expansion from intermediate to full in one generation has been reported

Question 14

What percentage of FMR1 mutations are mosaic?

Answer 14

15-20%

Question 15

What are the two types of FMR1 mosaicism?

Answer 15

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

Question 16

What caveat must you say in PM reports?

Answer 16

Possibility of patient being tissue mosaic with a pm in leucocyte DNA but full mutation in others
- can analyse other tissue samples

Question 17

What is FXTAS?

Answer 17

Late onset progressive cerebellum ataxia and intention tremor (gait ataxia, Parkinsonism and autoimmune dysfunction)

Question 18

Pathophysiology of FXTAS

Answer 18

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

Question 19

What percentage of males over 50 with PM

Answer 19

1/3

Question 20

What percentage of women with PM will develop early menopause? (<40 years)

Answer 20

24%

Question 21

Pathophysiology of POF?

Answer 21

Women have higher levels of FSH than healthy individuals and low AMH

Question 22

What % of women with POF have pM?

Answer 22

6.5%

Question 23

Risk of using CvS for PND?

Answer 23

Methylation status is usually not established in CVS- 20% of cvs DNA is unmethylated - determine on size alone

Question 24

Who is PNd offered to?

Answer 24

Female carriers of an allele of 56 CGG repeats or greater

Question 25

Steps in PND?

Answer 25

1) CGG-pcr to determine if normal allele was inherited | 2) McC test

Question 26

Possible MCC tests?

Answer 26

1) Asuragen amplidex pcr 2) Linkage analysis with markers flanking FMR1 3) eco R1/ eag1 southern blot

Question 27

True or false: Risk to women with newly identified premutation alleles with no FH is higher than women with FH?

Answer 27

False - risk significantly reduced | Ref Nolan et al 2003 and 2011

Question 28

Why cytogenetics not great for FRAXA diagnosis?

Answer 28

1) Positive result may not be absolute if only a portion of cells examined showed fragile site 2) analysis complicated by nearby FRAXE

Question 29

What is the limits of pcr across the CGG?

Answer 29

Can pcr up to 50-60 repeats but struggles to pcr beyond 70 repeats Pcr preference for smaller repeats

Question 30

What samples are followed up with asuragen?

Answer 30

Male with no allele or female with 1 allele

Question 31

Limitations of fluorescent pcr?

Answer 31

- 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

Question 32

Describe southern boot for fmr1

Answer 32

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

Question 33

Limitations/ abnormal southern blot

Answer 33

``` Restriction site polymorphisms Mosaicism Skewed x inactivation X chromosome aneuploidy Incomplete digestion ```

Question 34

Describe asuragen amplidex FMR1 pcr kit?

Answer 34

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

Question 35

What size repeat can asuragen detect up to? And can it detect AGG repeats ?

Answer 35

> 1000 CGG repeats | Comprehensive genotyping of AGG

Question 36

Describe TP-PCR

Answer 36

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

Question 37

How much DNA do you use for pcr?

Answer 37

5-40ng

Question 38

NEQAS and EMQN for fmr1

Answer 38

Testing for genotyping, correct interpretation and reporting Lab registers for relevant disease Annual distribution- 3 per disease