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Flashcards in Fragile X Syndrome Deck (82):
1

Give an overview of Fragile X syndrome.

- Fragile X syndrome is one of the most common forms of mental retardation with a prevalence of 1 in 4,000 to 1 in 6,000 males where it causes moderate to severe intellectual and social impairment.
- Fragile X is the most common known cause of autism.
- A fragile site called FRAXA is expressed on the X chromosome at Xq27.3, typically in 2-40% of blood cells in affected males.
- Females with a full mutation have a variable phenotype, ranging from apparently normal to moderate mental and social impairment with or without fragile site expression. Variable phenotype in females is possibly due to differences in the proportion of active and inactive normal and mutated X chromosmes in the relevant tissues.
- FRAXA is caused by mutations in the Fragile X mental retardation 1 gene (FMR1). The term fragile X has been used to define the syndrome at the FRAXA locus. Further investigations have described additional fragile sites in the Xq region including FRAXE which is associated with a form of X-linked mental retardation involving the FMR2 gene. This may be misclassified as fragile X mutations by cytogenetic investigations.
- The pathogenic mutation in the majority of fragile X cases is a large expansion of the CGG repeat tract in the first untranslated exon of FMR1. Full mutations result in hypermethylation of the DNA in and around the CGG tract and this causes inactivation of the FMR1 gene.
- The FMR1 gene encodes the Fragile X Mental Retardation Protein (FMRP) which is thought to be involved in regulating protein synthesis.
- Mutations involving expanded, unstable DNA repeat sequences are often referred to as 'dynamic mutations' - other examples of such mutations are Huntington disease and Myotonic dystrophy.
- There are several reports of FMR1 deletion and point mutations within RNA binding domain. These account for less than 1% of fragile X cases.

2

What fragile site causes fragile X syndrome? What other fragile sites are known to exist?

- A fragile site called FRAXA is expressed on the X chromosome at Xq27.3, typically in 2-40% of blood cells in affected males.
- FRAXA is caused by mutations in the Fragile X mental retardation 1 gene (FMR1).
- The term fragile X has been used to define the syndrome at the FRAXA locus.
- Further investigations have described additional fragile sites in the Xq region including FRAXE which is associated with a form of X-linked mental retardation involving the FMR2 gene. This may be misclassified as fragile X mutations by cytogenetic investigations.

3

Mutations in what gene causes fragile X syndrome?

FRAXA is caused by mutations in the Fragile X mental retardation 1 gene (FMR1).

4

What is the locus of FRAXA?

A fragile site called FRAXA is expressed on the X chromosome at Xq27.3, typically in 2-40% of blood cells in affected males.

5

What is the incidence of fragile X syndrome and what are it's most prominent features?

- Fragile X syndrome is one of the most common forms of mental retardation with a prevalence of 1 in 4,000 to 1 in 6,000 males where it causes moderate to severe intellectual and social impairment.
- Fragile X is the most common known cause of autism.

6

What gene does the FRAXE site involve?

Further investigations have described additional fragile sites in the Xq region including FRAXE which is associated with a form of X-linked mental retardation involving the FMR2 gene. This may be misclassified as fragile X mutations by cytogenetic investigations.

7

What is the pathogenic mutation involved in the majority of fragile X cases? How does this mutation result in disease?

- The pathogenic mutation in the majority of fragile X cases is a large expansion of the CGG repeat tract in the first untranslated exon of FMR1. Full mutations result in hypermethylation of the DNA in and around the CGG tract and this causes inactivation of the FMR1 gene.

- The FMR1 gene encodes the Fragile X Mental Retardation Protein (FMRP) which is thought to be involved in regulating protein synthesis.

8

What protein does the FMR1 gene encode? What is thought to be the function of this protein?

The FMR1 gene encodes the Fragile X Mental Retardation Protein (FMRP) which is thought to be involved in regulating protein synthesis.

9

What are 'dynamic mutations'?

Mutations involving expanded, unstable DNA repeat sequences are often referred to as 'dynamic mutations' - other examples of such mutations are Huntington disease and Myotonic dystrophy.

10

Roughly what % of fragile X cases are caused by FMR1 deletion and point mutations within the RNA binding domain?

There are several reports of FMR1 deletion and point mutations within RNA binding domain. These account for less than 1% of fragile X cases.

11

When were trinucleotide expansions first described?

In the early 1990's

12

What are trinucleotide repeats?

Trinucleotide repeats ar blocks of 3 bases repeated over and over again. Repeats below a certain length are stable in meiosis, however, above a certain threshold level the repeats are unstable and expansions and contractions may occur. there is a bias towards expansion with the likelihood of expansion dependant upon the length of the repeat. These unstable repeats are virtually never transmitted unchanged from parent to child and the degree of expansion can depend on the sex of the transmitting parent.

13

How many exons does the FMR1 gene contain? How big is it?

The FMR1 gene contains 17 exons and contains 38kb of sequence.

14

Describe the FMR1 gene.

- The FMR1 gene contains 17 exons and contains 38kb of sequence.
- The CGG repeat is highly polymorphic in the normal population - 5-58 CGG repeats and is usually interspersed by one or more single AGG interruptions.
- Most of the repeat length variation occurs at thr 3' end of the CGG tract sequence.
- These AGG interruptions are thought to infer DNA stability. The loss of AGG interruptions leads to alleles with longer 'pure' CGG repeat tracts which are prone to expansion.

15

What is thought to be the purpose of the AGG interruptions usually found withing the CGG repeat tract of the FMR1 gene? What is the consequence of the loss of AGG interruptions?

- The CGG repeat is highly polymorphic in the normal population - 5-58 CGG repeats and is usually interspersed by one or more single AGG interruptions.
- These AGG interruptions are thought to infer DNA stability. The loss of AGG interruptions leads to alleles with longer 'pure' CGG repeat tracts which are prone to expansion.

16

What size of CGG repeats is usually descrived as a full fragile X mutation? What is the consequence of a full fragile X mutation?

200 Repeats, Methylated - Clinical features in males and approximately 50% of females.

17

Where is FMR protein normally expressed? What is the function of FMR protein thought to be?

FMRP is normally expressed in many tissues but is most abundant in neurons and appears to play a role in the functional and structural maturation of the synapses.

18

What is the full mutation size range for the fragile X allele? What will the effect of this allele be?

200 repeats and methylated
- All males will have LD and around 50% of females.

19

Describe intermediate FMR1 gene alleles.

Intermediate: 46-55 repeats- possible expansion in future generations.

Intermediate/Premutation: 56-58, one report of a patient with 56 CGGs expanding to a
full mutation in offspring.

Intermediate alleles have a CGG repeat in the size range of 50-58. They tend to be stably transmitted. Any change of repeat tends to be small. There is one report of an expansion from maternal 56 repeats to a full mutation in son. The number of CGG repeats in a tract determines its stability, pure repeats of more than 35 CGGs are more likely to become unstable. Intermediate alleles are the most challenging to report, diagnose, council etc.

20

Describe premutation FMR1 gene alleles.

Intermediate/Premutation: 56-58, one report of a patient with 56 CGGs expanding to a
full mutation in offspring.

Premutation: 59-approx. 200 repeats, high chance of expansion in
offspring of females.

Premutration alleles have CGG repeats in the range of 59-200. They are unmethylated with normal transcript and protein levels, but are extremely unstable during transmission to the next generation. Expansion from premutation to full mutation occurs only by maternal transmission and this is dependant on the length of the maternal premutation. Premutation alleles may be associated with subtle symptoms. There is an increased risk of fragile X associated tremor/ataxia syndrome (FXTAS) and fragile X associate primary ovarian failure (FXPOI). Women with premutation alleles are also at risk of having an affected child due to the potential for the allele to expand. The prevalence of premutation alleles in the general population is about 1 in 800 for males and 1 in 250 for females.

21

What symptoms may manifest in carriers of fragile X premutation alleles?

Premutation alleles may be associated with subtle symptoms. There is an increased risk of fragile X associated tremor/ataxia syndrome (FXTAS) and fragile X associate primary ovarian failure (FXPOI). Women with premutation alleles are also at risk of having an affected child due to the potential for the allele to expand.

22

What is the prevalence of fragile X premutation alleles in the general population?

The prevalence of premutation alleles in the general population is about 1 in 800 for males and 1 in 250 for females.

23

What boundary for the fragile X premutation range do the ACMG use?

Recommendations from the American College of Medical Genetics (2005) use the lower boundary of the premutation range of 55 CGG repeats, irrespective of sequence composition. UK laboratories follow the recommendations made by CMGS where the lower end boundary is 59 repeats.

24

Why is there a low pick up rate for fragile X?

Because most labs test a very broad range of patients.

25

How are most fragile X referrals reported?

Most referrals can be reported following sizing PCR. However, some require further testing by Southern blotting/Asuragen AmplideX FMR1 PCR.

26

What percentage of full FMR1 mutation patients also show some mosaicism?

Approximately 15-20% of full FMR1 mutation patients also show some mosaicism.

27

What are the 2 types of mosaicism seen in some cases of fragile X syndrome?

2 types of mosaicism:
1). Repeat size mosaicism - both full mutations and premutations are present.
2). Methylation mosaicism - full mutations which are variably methylated - these individuals will be affected but tend to be higher functioning.

A caveat is usually put into premutation reports stating that there is a small possibility that the patient is a tissue mosaic, having a premutation in his/her leucocyte DNA, but having a full mutation in other tissues. The possibility of tissue mosaicism could be investigated by analysing DNA from other tissues.

28

Describe FXTAS.

- Fragile X-associated Tremor and Ataxia Syndrome (FXTAS) is a late onset progressive cerebellar ataxia and intention tremor (gait ataxia, parkinsonism and autonomic dysfunction).
- It has been reported in both males and females with premutation alleles (lower risk in females).
- 1/3 of males over 50 with a fragile X premutation will develop this phenotype.
- Approximately 5% of men presenting with apparent sporadic ataxia may have FXTAS.
- FXTAS is not found in males with full mutation alleles.
- An RNA gain of function mutation has been suggested for FXTAS based on the observation of elevated levels of CGG-containing FMR1 mRNA along with either no detectable change in FMRP, or slightly reduced FMRP levels in peripheral blood leucocytes and brain regions of premutation carriers. The mechanism underlying the elevated FMR1 mRNA levels is unknown, although it may involve a toxic gain of function of mRNA.
- Nuclear inclusions are found in the brains of maleswith FXTAS.
- FXTAS has been reported in females with premutations although its' penetrance and expression appear to be much lower than in males.

29

Approximately how many males over 50 with a fragile X phenotype will develop the FXTAS phenotype?

Approximately 1/3 of males over 50 with a fragile X premutation will develop the FXTAS phenotype.

30

Describe the hypothesised cause of FXTAS.

An RNA gain of function mutation has been suggested for FXTAS based on the observation of elevated levels of CGG-containing FMR1 mRNA along with either no detectable change in FMRP, or slightly reduced FMRP levels in peripheral blood leucocytes and brain regions of premutation carriers. The mechanism underlying the elevated FMR1 mRNA levels is unknown, although it may involve a toxic gain of function of mRNA.

31

Describe FXPOI.

- FXPOI stands for Fragile X-associated Primary Ovarian Pailure.
- The mean age of menopause in Western populations is around 51 years. Early menopause is classified as cessation of mensies before 40yrs.
- 24% of premutation carriers manifest POF. Other causes of POF include Turner syndrome, chromosome rearrangements, survival of childhood cancer that was treated with chemotherapy.
- Women with the premutation have higher levels of FSH and low AMH levels.
- It is considered appropriate to test for premutations upon recieving an appropriate request. The main benefit of testing individuals is to the relatived of the proband found to have FXPOI who would be at risk of FXPOI/POF themselves, and at risk of having offspring with fragile X syndrome.
- 6.5% of women with POF have an FMR1 premutation.
- 13% of women with a family history of POF have a premutation (compared to 3% of women without a family history of POF).

32

What is the benefit of fragile X premutation testing?

The main benefit of premutation testing is to the relatived of the proband found to have a premutation who would be at risk of FXPOI/POF themselves, and at risk of having offspring with fragile X syndrome.

33

Describe PND for fragile X. Why may prenatal diagnosis be required for parents with fragile X mutation alleles? How is fragile X PND usually carried out?

- Females with a premutation allele are at risk of transmitting a full expansion mutation to their offspring.
- CGG-PCR and Southern blotting are typically used to test amniocytes or chorionic villus samples (CVS) from at risk fetuses.
- The amount of DNA from amnios and CVS is typically low.
- The result of PND is required urgently (10 days).
- Possibility of MCC - test fetal and maternal DNA with markers to check for contamination.
- Methylation pattern in fetal DNA may not be representative. 20% of CVS DNA may be partially- or unmethylated.
- Reccommend that female carriers of an allele of 56 CGG repeats or greater should be offered PND.
- Fluorescent CGG PCR is carried out to see if the normal allele has been inherited from the mother. Due to the risk of potential flase negative MCC testing is also performed.
- Asuragen AmplideC PCR is utilised with gene specific and repeated primed PCRs.
- Linkage analysis can be used whereby PCR can be carried out with markers flanking the FMR1 gene. This can be used to determine whether the fetus has inherited the high risk allele. The markers used are highly informative and show negligible recombination with the CGG repeat.
- An EcoR1/Eag Southern Blot can also be set up if there is sufficient DNA available. Blots should inform about the size of the expansion and the methylation status.
- In all tests it is essential to use normal and expansion controls to determine fetal allele size as accurately as possible.
- BGPs advise against issuing an interim report.
- The risk of expansion to a full mutation in the offspring is stated in reports.

34

Who should be offered PND for fragile X?

Reccommend that female carriers of an allele of 56 CGG repeats or greater should be offered PND.

35

What technologies are commonly used for fragile X PND?

- CGG-PCR and Southern blotting are typically used to test amniocytes or chorionic villus samples (CVS) from at risk fetuses.
- Fluorescent CGG PCR is carried out to see if the normal allele has been inherited from the mother. Due to the risk of potential flase negative MCC testing is also performed.
- Asuragen AmplideC PCR is utilised with gene specific and repeated primed PCRs.
- Linkage analysis can be used whereby PCR can be carried out with markers flanking the FMR1 gene. This can be used to determine whether the fetus has inherited the high risk allele. The markers used are highly informative and show negligible recombination with the CGG repeat.
- An EcoR1/Eag Southern Blot can also be set up if there is sufficient DNA available. Blots should inform about the size of the expansion and the methylation status.
- In all tests it is essential to use normal and expansion controls to determine fetal allele size as accurately as possible.

36

Why might different risk estimates be appropriate when conselling women with no family history of fragile X syndrome?

It has been previously found that women who carry newly identified premutation alleles with no family history, the full mutation expansion rates are dramatically lower than that for women in families with fragile X. This suggests that different risk estimates be appropriate when conselling women with no family history of fragile X syndrome.

37

Describe molecular testing for fragile X syndrome. What problems make testing for fragile X using cytogenetic methods difficult?

- Cytogenetic methods involving detection of the FRAXA fragile site at Xq27.3 were initially used.
- However, positive cytogenetic results were not always absolute. One reason for this was that only a proportion of cells examined showed fragile site.
- Analyses are also complicated by the presence of a nearby FRAXE fragile site which cannot reliably be distinguished from the FRAXA site.
- Molecular diagnostic methods are now used to detect FRAXA and FRAXE. Most diagnostic labs routinely test for FAXA, but only a few specialist labs test for FRAXE.

38

What kinds of referral indications for FRAXA testing are labs likely to recieve?

1). Diagnostic referrals:
- Patients with mental retardation of unknown etiology, developmental delay, autism.
- Most diagnostic fragile X referrals are from community paediatricians.
- Patients with ataxia (?FXTAS) - from neurologists.
- Patient with POF/early menopause (?FXPOI) - from gynaecologists/fertility centres.

2). Carrier testing referrals:
- Known family history of fragile X
- From clinical genetics

3). Prenatal diagnosis:
- For known female carrier
- From clinical genetics

39

Describe the desirable qualities of a fragile X diagnostic test. What testing technology is generally used for fragile X testing?

- High specificity and sensitivity
- Detection of normal, premutation and full mutation alleles
- Detection of mosaics
- Detection of homozygous females
- Rapid, cost effective, easy to use workflow for lab, robust, reproducible, high throughput

- Generally, CGG-PCR is used as a primary screen followed by Southern blotting or Asuragen AmplideX FMR1 PCR.

40

Describe the use of CGG-PCR.

- PCR across the FMR1 CGG site is initially used for sizing the CGG repeat in all referred fragile X diagnostic and carrier tests.
- DNA is extracted from EDTA blood samples.
- PCR product is labelled and run on the Beckman CEQ8000 or ABI3700).
- It is essential to include size markers to accurately determine the allele size of the test cases.
- PCR can easily detect up to 50-60 repeats but it can sometimes struggle to PCR beyond 70 repeats. Some other PCR techniques can amplify fragments up to 120 CGG repeats.
- Any method used in a diagnostic setting must be validated on a range of controls of knwon sizes.

- From PCR across the CGG repeat we can report males with one allele and females with two alleles (normal CGG repeat range).
- If the situation is still not clear we then progress to second line testing for all other cases, or those with known family history of fragile X syndrome.

- This method of testing will not be able to detect large expansions.
- If there is no PCR product this could be due to poor DNA quality, a large expansion or there could be a SNP in the primer binding site which could cause preferential amplification of the normal allele.
- This PCR is not quantitative and homozygous females cannot be distinguished from those that have one allele in the normal size range and one allele that is too big to amplify.
- This PCR cannot detect rare point mutations or deletions and these account for up to 1% of all fragile X cases. In order to detect these sequencing would have to be performed.
- There is the posibility of a false negative result in the case of mosaics. It would not indicate the presence of a normal allele or premutation allele alongside an expansion. For this reason when an indicidual has known family history CGG-PCR should be accompanied by a second line test due to the mosaicism risk.

41

What fragile X referral cases can be reported directly from the CGG-PCR?

- From PCR across the CGG repeat we can report males with one allele and females with two alleles (normal CGG repeat range). We then progress to second line testing for all other cases, or those with known family history of fragile X syndrome.

42

What are the limitations of fragile X testing via PCR across the CGG repeat?

- This method of testing will not be able to detect large expansions.
- If there is no PCR product this could be due to poor DNA quality, a large expansion or there could be a SNP in the primer binding site which could cause preferential amplification of the normal allele.
- This PCR is not quantitative and homozygous females cannot be distinguished from those that have one allele in the normal size range and one allele that is too big to amplify.
- This PCR cannot detect rare point mutations or deletions and these account for up to 1% of all fragile X cases. In order to detect these sequencing would have to be performed.
- There is the posibility of a false negative result in the case of mosaics. It would not indicate the presence of a normal allele or premutation allele alongside an expansion. For this reason when an indicidual has known family history CGG-PCR should be accompanied by a second line test due to the mosaicism risk.

43

What are some possible reasond for PCR failure in the CGG-PCR fragile X test? What are the possible solutions to a failed PCR?

- Pipetting error
- Degradation of the PCR mastermix
- Presence of a larger sized allele not detected by
- Typically the PCR is repeated, possibly with a different dilution of DNA. If the same result is obtained then second stage testing will be initiated.

44

What would you do if your results for a CGG-PCR showed a female with a 2nd allele below 5,000?

- Repeat the PCR or proceed to 2nd stage testing.
- CGG-PCR often preferentially amplifies the smaller allele making it difficult to confidentally call the second larger allele without using a repeat or a 2nd line testing method.

45

In what cases of fragile X testing should 2nd line testing be used?

2nd stage testing required:
1). When PCR fails to amplify the expected number of FMR1 alleles - 1 in males, 2 in females.
2). If the patient has a family history of FRAXA.

46

What are the common 2nd line testing methods used for fragile X testing?

1). Southern blot analysis - can detect changes in size and methylation status of FMR1 expansion alleles. This method can use radioactively labelled probes or those that have been fluorescently tagged. Southern blotting has several disadvantages, including the fact that several types of blot may be required. EcoR1 and Eag1 used for large expansions and PST for small expansions. % of gel critical to allow optimal resolution of bands.
2). Asuragen AmplideX FMR1 PCR kit - replacement for Southern blotting - can detect changes in the size of FMR1 alleles.
3). Other techniques have been utilised by diagnostic labs such as a methylation specific PCR.

47

What probes are EcoR1 and Eag1 digests probed with?

OX1.9 - this can be used for determining the type of expansion in terms of methylation status of the alleles. The distinction between a premutation and a full mutation is more likely due to methylation status rather than the exact size of the repeat. Methylated or inactive alleles are cut only by the EcoR1 enzyme to give a 5.2kb fragment. Unmethylated or active alleles are cut by both enzymes to give a smaller 2.8kb fragment.

48

In the Southern blot 2nd line fragile X test what enzyme cuts methylated alleles?

Methylated or inactive alleles are cut only by the EcoR1 enzyme to give a 5.2kb fragment. Unmethylated or active alleles are cut by both enzymes to give a smaller 2.8kb fragment.

49

In the Southern blot 2nd line fragile X test what enzyme cuts unmethylated alleles?

Unmethylated or active alleles are cut by both the EcoR1 and Eag1 enzymes to give a smaller 2.8kb fragment.

50

In the Southern blot 2nd line fragile X test what size expansions can an Ecor1/Eag1 blot detect? What are some of the limitations of using this double digest?

CGG expansions of 80 - >1000 repeats. Using the double digest can distinguish between a premutation and a full mutation but it may be difficult to accurately determine those alleles close to the intermediate size range or small pre-mutations. For this it may be necessary to perform a second blot using a PST1 digest.

51

If on a fragile X double digest Southern blot (using Ecor1/Eag1) you are struggling to accurately determine those alleles close to the intermediate size range or small pre-mutations what technique may you want to use?

Using the double digest can distinguish between a premutation and a full mutation but it may be difficult to accurately determine those alleles close to the intermediate size range or small pre-mutations. For this it may be necessary to perform a second blot using a PST1 digest.

52

Look at fragile X Southern Blots in lecture.

See lectures.

53

What does the Pst1 fragile X Southern blot digest detec?

Allows detection of small premutations and enables a more accurate size to be given to small expansions. The OX0.55 probe is used to provide an increased resolution. Pst1 blots are now not commonly used because small premutations can be detected by the fluorescent CGG-PCR.

54

What factors may reult in a poor Southern blot? What factors may affect the interpretation of fragile X tests?

Incomplete restriction enzyme digest or poor hybridisation of the probe will give a poor blot which is very difficult to interpret. Other factors that may affect the interpretation of fragile X tests include:
- X chromosome aneuploidy
- deletions
- duplications
- restriction site polymorphisms
- mosaicism
- skewed X inactivation

55

Describe the Asuragen AmplideX FMR1 PCR kit.

- The Asuragen AmplideX PCR can amplify alleles with far greater than 1,000 CGG repeates and actually characterises normal, intermediate, premutation and full mutation alleles.
- The kit incorporates a novel CGG repeat primer PCR reaction can reconcile allele zygosity and accurately detect full mutations - even if they cannot be amplified as full length amplicons.
- Asuragen technology significantly outperforms previous PCR methods and and provides a 5- to 10- fold faster turn around time, a simpler, more error-free protocol and greater sensitivity than Southern blotting.
- These PCR technologies are expected to reduce the need for Southern blotting to only those samples where methylation information is absolutely required (e.g. only in about 1% of all samples if only full mutations are interrogated).
- Comprehensive genotyping has shown that this test can be used to assess AGG interruptions.
- The kit is able to amplify large premutation and full mutation FMR1 CGG repeat expansions. The kit is therefore able to accurately distinguish between large premutation alleles and full mutations.

56

Can the Asuragen AmplideX FMR1 PCR kit distinguish between large premutation alleles and full mutations?

The kit is able to amplify large premutation and full mutation FMR1 CGG repeat expansions. The kit is therefore able to accurately distinguish between large premutation alleles and full mutations.

57

Describe the two different PCRs that the Asuragen AmplideX FMR1 PCR kit supports.

The kit supports 2 different PCRs:
1). A gene-specific PCR which gives a full length product peak for each allele size.
2). A CGG repeat prime PCR which is a TP PCR comined with a gene-specific PCR.

Both kits will amplify full mutation alleles. Either of the PCRs can be performed or both can be performed together in two sets of tubes.

Typically the CGG repeat prime PCR is carried out unless otherwise directed by the disease scientist. Gene-specific PCR may be carried out for prenatal samples.

58

Describe the features of the gene-specific FMR1 PCR which can be carried out using the Asuragen AmplideX FMR1 PCR kit.

- >1,000 CGG repeats detected
- All full mutations alleles detected to date
- More sensitive than Souther blotting
- Whole blood, prenatal, blood spots

59

Describe the features of the CGG repeated primer FMR1 PCR which can be carried out using the Asuragen AmplideX FMR1 PCR kit. What type of PCR is Asuragen CGG repeat prime PCR?

- Asuragen CGG repeat prime PCR is a triplet prime PCR.
- Triplet prime (TP) PCR is useful for analysing repeat regions that are too large to be amplified by a standard PCR.
- It is commonly used as part of the testing strategy for several diseases such as Huntingtons and myotonic dystrophy in addition to fragile X syndrome.

- Instead of just having a forward and reverse primer like a normal PCR would the TP PCRs have 3 primers. - The first primer (P1) flanks the repeat region and thus acts as a forward primer. P2 has 2 parts, the first hybridises across the repeat region at every repeat and the second part is an extra sequence not found in the human genome which acts as a binding region for the third primer. Primers 1 and 2 produce lots of fragments of different sizes which are then amplified up further by primers 1 and 3. The fragments can then be analysed by capilliary electrophoresis.


- >1,000 CGG repeats detected
- All full mutations alleles detected to date
- More sensitive than Souther blotting
- Definitely respolves zygosity
- Provides absolute CGG repeat quantification
- Whole blood, prenatal, blood spots

60

What are the functions of each primer found in a Triplet prime (TP) PCR kit?

- Instead of just having a forward and reverse primer like a normal PCR would the TP PCRs have 3 primers. -

- The first primer (P1) flanks the repeat region and thus acts as a forward primer.

- P2 has 2 parts, the first hybridises across the repeat region at every repeat and the second part is an extra sequence not found in the human genome which acts as a binding region for the third primer.

- Primers 1 and 2 produce lots of fragments of different sizes which are then amplified up further by primers 1 and 3.

- The fragments can then be analysed by capilliary electrophoresis.

- CGG repeat primer PCR also includes gene-specific primers so a size can be determined for each allele.

61

Does TP PCR give a true indication of the size of the repeat?

No. Usually a cut off point is given where the samples that amplify less than this are considered normal and the samples that amplify further are classed as expanded.

62

What alternative methods should be performed in addition to TP PCR in order to give an indication of the actual size of the repeat?

TP PCR does not give a true indication of the size of the repeat. Usually a cut off point is given where the samples that amplify less than this are considered normal and the samples that amplify further are classed as expanded. An alternative method such as sizing PCR or Southern blotting should also be performed to give an indication of the actual size of the repeat.

63

How can the spacing of AGG interuptions can be determined using the Asuragen AmplideX FMR1 kit?

True. The spacing of AGG interuptions can be determined using the Asuragen AmplideX FMR1 kit. The repeat primer PCR element of the Asuragen kit means that there is a repeat for every CGG repeat. The presence of an AGG prevents primer binding resulting in a location specific loss of signal that can be mapped to the allele location. AGG interruptions may be useful in predicting the clinical significance of the AGG expansions. Currently diagnostic labs in the UK do not AGG interruption data when reporting fragile X results. However, this may be introduced in the future to aid genetic counselling, especially if little family history is provided.

64

What quality issues affect fragile X testing / molecular diagnostic testing generally?

- Essential to use sizing controls when carrying out a fragile X test in order to compare the test cases and determine an accurate allele size.
- Best practice guidelines give useful reccomentations for both testing and reportig fragile X cases.
- Labs also take part in EQA schemes to test the accuracy of genotyping, interpretation and reporting.

65

After the electrophoresis stage of the MLPA what readout will you get?

- The products are differentiated by size using a capillary electrophoresis machine.
- After electrophoresis a trace is produced showing multiple peaks with each peak representing a probe.
- Capillary electrophoresis is a sensitive method with the peak height produced from each probe representing the amount of that probe present in the amplified reaction.
- The relative amount of each product reflects the copy number of the target sequences within the sample DNA.
- The peak heights from the undigested reaction are compared to the peak heights of the digested reaction to quantify the methylation. In a normal sample half of the 15q11-q13 probes will have been digested after binding to the unmethylated paternal chromosome so the peak heights in the digested reaction should be approximately half that of the untreated reaction.

66

Describe how you would go about analysis if MS-MLPA results.

- Export size and peak height raw data to a specialised spreadsheet.
- Several companies and research institutions have developed their own MLPA software. MRC Holland provide a modified Excel program called Coffalyser for MLPA analysis which is free to download and is specifically tailored to their kits. The National Genetics Reference Laboratory (NGRL) in Manchester also provide spreadsheets for MLPA analysis.
- These analysis tools contain all the formulae for MLPA data analysis and only require data input.

67

Describe PWAS MS-MLPA analysis.

1). Each PWAS MS-MLPA reaction contains:
- 14 control probes from regions of the genome that should have normal copy number.
- 31 probes from the commonly deleted 15q11-q13 region.
- 3 probes from just outside the deleted region.
- All of these probes are shown in the copy number analysis.

2). Methylation analysis relies on the result from 9 probes:

- 5 of the probe sites are normally maternally methylated and paternally unmethylated and contain an Hha1 site. These are used to quantify the methylation of the region. Should be differentially methylated in normal individuals.

- 4 probes act as methylation controls. 1 is methylated on the maternal and paternal chromosome so should not be digested. 3 are always unmethylated so act as digestion controls. The presence of the digestion control peaks in the sample implies incomplete digestion and that sample should be repeated.

68

Describe PWAS MS-MLPA copy number results.

- The analysis software produces a dosage quotient (DQ) for each probe by comparing the peak heights to the internal and external controls.
- Dosage quotients give an indication of copy number compared to the normal control samples.
- A DQ of 1 means that the patient has the same copy number as the control.
- A DQ of 1.5 represents a 2:3 ratio with the control representing a duplication in the patient DNA.
- A DQ of 0.5 represents a deletion in the patient.
- DQ values are unlikely to fall exactly on these values and so there is a range that will be accepted. A DQ outside of this range likely represents a poor reaction. However, if the reaction is repeated and produces the same DQ again it may represent mosaicism.

69

What does a DQ of 1 mean?

- A DQ of 1 means that the patient has the same copy number as the control.

70

What does a DQ of 1.5 mean?

- A DQ of 1.5 represents a 2:3 ratio with the control representing a duplication in the patient DNA.

71

What does a DQ of 0.5 mean?

- A DQ of 0.5 represents a deletion in the patient.

72

Describe PWAS MS-MLP methylation results.

- The methylation results are always presented in a graphical format showing the the 15q11-q13 probes, the no digestion controls and then the digestion controls.
- Normal methylation pattern ratio is approx 0.5 for 15q11-q13 compared to the no digestion control because half the sample should be methylated (mother) and half should not (father).
- Angelman syndrome shows no methylation of 15q11-q13 probe sites (ratio of 1) as only have the unmethylated paternal copies of this region.
- Prader-Willi syndrome will show all 15q11-q13 region probes methylated with a ratio of 1 (because only have methylated copies of this region from the maternal copy).

- The methylation values are rarely exactly 0, 0.5 or 1.
- To make methylation results easier to interpret the DQ values for the patient and control samples are copied into another spreadsheet.
- The mean DQ for each of the 15q11-q13 probes is calculated from the normal control samples.
- The probe mean is then divided by the DQ for the equivalent 15q11-q13 probe from the patient.
- This gives the ratio for each patient probe in comparison to the normal controls. The normal patients should look like the normal controls and give a ratio of approximately 1.
- The resulting values for each patient 15q11-q13 probe are then averaged to give a single value.

73

What calculations are performed with the DQ values in order to make the methylation results easier to interpret?

- The methylation values are rarely exactly 0, 0.5 or 1.
- To make methylation results easier to interpret the DQ values for the patient and control samples are copied into another spreadsheet.
- The mean DQ for each of the 15q11-q13 probes is calculated from the normal control samples.
- The probe mean is then divided by the DQ for the equivalent 15q11-q13 probe from the patient.
- This gives the ratio for each patient probe in comparison to the normal controls. The normal patients should look like the normal controls and give a ratio of approximately 1.
- The resulting values for each patient 15q11-q13 probe are then averaged to give a single value.

74

What are the disadvantages of MS-MLPA?

- Sensitive to PCR contaminants
- Sensitive to DNA quality
- Cannot be used for detection of UBE3A point mutations
- Can generate false positives due to the sequence changes under the probe sites, any single probe deletions should be confirmed with another method.
- Cost - £10 per reaction but need to account for normal, water and positive controls on each run, only economic with multiple test samples.
- Still cannot differentiate between UPD and imprinting defect without an IC deletion.

75

Describe microsatellite analysis for UPD detection.

- Performed when MS-MLPA shows abnormal methylation with normal copy number.
- Need parental DNA samples.

- Chromosome 15 microsatellite markers are genotyped for the affected patient and both parents and this can identify UPD.
- Markers are predominantly located within 15q11-q13.
- Some markers external to 15q11-q13 can be used to distinguish isodisomy and large deletion (not necessary if normal copy number found by MLPA).
- If the patient is found to have biparental inheritance it implies that there must be an imprinting defect without a detectable IC deletion. All known IC deletions will be detected by the MLPA but there is always a risk that the patient has a rare undetectable IC deletion that could have a recurrence risk of up to 50% (whereas UPD has a low recurrence risk).

- Marker PCR uses fluorescently labelled primers.
- Products sized by capillary electrophoresis.
- Requires at least 2 fully informative markers to report.

76

When would you perform microsatellite analysis for UPD detection?

When MS-MLPA shows abnormal methylation with normal copy number.

77

If microsatellite analysis for UPD detection is carried out and the patient is found to have biparental inheritance what does this imply?

If the patient is found to have biparental inheritance it implies that there must be an imprinting defect without a detectable IC deletion. All known IC deletions will be detected by the MLPA but there is always a risk that the patient has a rare undetectable IC deletion that could have a recurrence risk of up to 50% (whereas UPD has a low recurrence risk).

78

Describe the types of referrals you are likely to get for Prader-Willi and Angelman syndrome.

1). Diagnostic Prader-Willi syndrome:
- Babies with hypotonia
- Children with some/all phenotypic features, particularly overeating/obesity with learning difficulties

2). Diagnostic Angelman syndrome:
- Children with some/all phenotypic features

3). Prenatal diagnosis:
- Parents who carry chromosome 15 translocations
- Microsatellite analysis
- IC deletion identified in previous child
- MS-MLPA possible but may be limited by DNA quality (methylation status at SNRPN exon 1 established early in embyonic development).

79

What size CGG tract is classed as normal in the FMR1 gene?

- Less than 46 repeats.

80

What size CGG tract is classed as Intermediate in the FMR1 gene?

- 46-55 repeats.

81

What size CGG tract is classed as Intermediate/Premutation in the FMR1 gene?

- 56-58 repeats.

82

What size CGG tract is classed as a full mutation in the FMR1 gene?

- More than 200 repeats.