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Flashcards in Rett Syndrome Deck (45):
1

What gene is affected in Rett syndrome?

- MECP2
- Mutations identified in 80-95% females with classic Rett syndrome
- In 99.5% of cases the mutations are single occurrences
- Mostly females affected, parents can be germline mosaics and the degree of presentation in a female can be affected by the level of skewed X inactivation
- Exon 1 discovered in 2004 - total of 4 exons

2

Who is mostly affected by Rett syndrome?

- Mostly females affected, parents can be germline mosaics and the degree of presentation in a female can be affected by the level of skewed X inactivation

3

How many exons does MECP2 contain?

- Exon 1 discovered in 2004 - total of 4 exons

4

How many mutations have been found in MECP2?

about 390 (RettBASE)

5

Describe the mutation spectrum of MECP2

- Approximately 390 reported pathogenic mutations found in MECP2.
- Most cases are sporadic with approximately 95% of cases being caused by a de novo mutation on the paternal copy of MECP2.
- Screening of Rett patients has shown that 90-95% will have a pathogenic mutation - 8 common mutations that account for 70% of these and a further 9% are a result of small deletions in a c-terminal hot spot.
- Mutations in exons 3-4 account for about 80% of classic Rett, 50% for the preserved speech variant of atypical Rett syndrome and lower percentages for other atypical variants
- Large deletions have been found in classical and atypical Rett and frequently involve exons 3 and 4. This is probably as a result of unequal recombination as there are two highly repetitive regions, one in exon 4 and the other in intron 2. Recombination between these regions could lead to the deletion of exons 3 and 4.
- It has been suggested that de novo mutations occur in male germ cells resulting in affected daughters
- CpG dinucleotides are susceptible to mutations - in one study seven of the eight identified MECP2 variants involved a C to T transition at a CpG site.
- Large deletions are more commonly found in individuals with a classic Rett syndrome phenotype (30%) than an atypical phenotype (7%).
- Pathogenic mutations in exon 1 are rare. There have been no mutations identified in exon 2.

6

What % of Rett syndrome cases are caused by a de novo mutation on the paternal copy of MECP2?

Most cases are sporadic with approximately 95% of cases being caused by a de novo mutation on the paternal copy of MECP2

7

What % of classic Rett syndrome cases do mutations in MECP2 exons 3-4 account for?

Mutations in exons 3-4 account for about 80% of classic Rett, 50% for the preserved speech variant of atypical Rett syndrome and lower percentages for other atypical variants

8

What is thought to cause the large deletions in MECP2 exons 3 and 4 that are often observed in Rett syndrome patients?

Large deletions have been found in classical and atypical Rett and frequently involve exons 3 and 4. This is probably as a result of unequal recombination as there are two highly repetitive regions, one in exon 4 and the other in intron 2. Recombination between these regions could lead to the deletion of exons 3 and 4.

9

What exons are MECP2 mutations usually seen in?

- Mutations in exons 3-4 account for about 80% of classic Rett, 50% for the preserved speech variant of atypical Rett syndrome and lower percentages for other atypical variants.
- Pathogenic mutations in exon 1 are rare. There have been no mutations identified in exon 2.

10

Are MECP2 de novo mutations more likely to come from the maternal or paternal allele?

- It has been suggested that de novo mutations occur in male germ cells resulting in affected daughters.
- In a study of 19 Rett cases 70% of mutations were from the paternal allele. This may explain the high number of females seen with the condition and the lower number of males as the fathers will not pass their X chromosome onto their son.

11

What may explain the high number of females seen with the condition and the lower number of males?

- It has been suggested that de novo mutations occur in male germ cells resulting in affected daughters.
- In a study of 19 Rett cases 70% of mutations were from the paternal allele. This may explain the high number of females seen with the condition and the lower number of males as the fathers will not pass their X chromosome onto their son.

12

Where have missense mutations in MECP2 been identified?

Missense mutations have been found predominantly in the Methyl Binding Doman (MBD). This region binds symmetrically methylated CpGs.

13

Where have nonsense mutations in MECP2 been identified?

Nonsense mutations - more scattered between the Methyl Binding Domain (MBD) and the Transcription Repression Domain (TRD). TRD recruits co-suppressor complexes that mediates the repression of transcription.

14

Where have deletions in MECP2 been identified?

Deletions tend to be found in what is referred to as the 'deletion prone region' and almost all cause a loss of the terminal region of the protein. This region affects codons 366-388 and these small deletions make up about 7% of all MECP2 mutations.

15

What mutations are you most likely to find in the Methyl Binding Domain of MECP2?

Missense mutations have been found predominantly in the Methyl Binding Doman (MBD).

Nonsense mutations - more scattered between the Methyl Binding Domain (MBD) and the Transcription Repression Domain (TRD).

16

Describe the structural domains of MECP2.

There are 3 functional domains and a nuclear localisation signalling domain in MECP2.

1). Methyl Binding Doman (MBD). This region binds symmetrically methylated CpGs.
2). Transcription Repression Domain (TRD). TRD recruits co-suppressor complexes that mediates the repression of transcription.
3). Tryptophan Tryptophan binding domain which is located closer to the C-terminus. This region binds splicing factors.

17

Describe what MECP2 mutations are seen in males.

- It was previously thought that mutations in MECP2 were lethal in males.
- 3 types of mutations are seen in males:

1). Mutations that are found in classic Rett
2). Mutations that are inherited from the mother which are not found in females with Rett. These have a range of MR severity with a good prognosis.
3). Males with deletions of the whole MECP2 gene and neighbouring genes resulting in a severe phenotype.

- If the male is XXY or somatic mosaicism is present then a milder phenotype will be the result as the proportion of neurones carrying the mut will likely affect the phenotype.

- MECP2 in males can present as severe neonatal encephalopathy and affected individuals will usually die within the first few years of life. This led to the identification of a condition known as MECP2 duplication syndrome and this is the most common form of MECP2 mut found in males.

18

What is the most common form of MECP2 mutation found in males?

MECP2 in males can present as severe neonatal encephalopathy and affected individuals will usually die within the first few years of life. This led to the identification of a condition known as MECP2 duplication syndrome and this is the most common form of MECP2 mut found in males.

19

Describe the molecular genetic pathogenesis of Rett syndrome.

- MECP2 encodes a protein present in two isoforms found in different tissues (protein=MeCP2).
- MeCP2_e1 contains exon1 but alternatively splices out exon 2 thus encoding a distinct N-terminus. This isoform was thought to be predominantly located in the brain.
- MeCP2_e2 doesn't contain exon 1 but does contain part of exon 2. This isoform is predominant in the fibroblast and lymphocyte cells.
- The e1 isoform means the start codon is in exon 1 and the e2 isoform has the start codon in exon 2.
- Initially MECP2 was thought to act as a global transcription repressor but this is not always supported as the phenotype is predominantly neuronal and does not globally affect gene regulation.
- It may be that MECP2 is involved in specific functions in neuronal cells of the CNS; it has also been linked to the regulation of transcription to synaptic activity.
- Suggested role for MECP2 in the repression of Brain Derived Neurotrophic Factor (BDNF) transcription as this can have an effect on synapse development and neuronal plasticity.

20

What protein does MECP2 encode?

- MECP2 encodes a protein present in two isoforms found in different tissues (protein=MeCP2).

21

What exons does MeCP2_e1 contain and where is it primarily found?

- MeCP2_e1 contains exon1 but alternatively splices out exon 2 thus encoding a distinct N-terminus.
- This isoform was thought to be predominantly located in the brain.
- The e1 isoform means the start codon is in exon 1.

22

What exons does MeCP2_e2 contain and where is it primarily found?

- MeCP2_e2 doesn't contain exon 1 but does contain part of exon 2.
- This isoform is predominant in the fibroblast and lymphocyte cells.
- The e2 isoform has the start codon in exon 2.

23

Which MeCP2 isoform is more abundant?

- MeCP2_e1 is more abundant as the e2 isoform is less efficiently translated.

24

What is the function of MECP2?

- Initially MECP2 was thought to act as a global transcription repressor but this is not always supported as the phenotype is predominantly neuronal and does not globally affect gene regulation.
- It may be that MECP2 is involved in specific functions in neuronal cells of the CNS; it has also been linked to the regulation of transcription to synaptic activity.
- Suggested role for MECP2 in the repression of Brain Derived Neurotrophic Factor (BDNF) transcription as this can have an effect on synapse development and neuronal plasticity.

25

Describe the genotype/phenotype correlation of Rett syndrome.

- Studies have given conflicting results.
- Mutations occurring 3' to TRD region often result in a milder phenotype. This is probably due to the late truncating protein formed with this mutation.
- Females with missense mutations - significantly milder phenotype than those patients with truncating (nonsense/frameshift) mutations.
- Observed that patients with deletions have a milder phenotype if the mutation is small and towards the 3' end of the gene.
- Missense mutations may result in a less severe phenotype than truncating mutations.
- The lack of truncating mutations in the MBD may be due to selection against those cells. These muts would be very likely to result in a very severe phenotype and one that may not actually resemble a classic Rett syndrome picture.
- The most significant correlation observed is between that of mutation type and location.
- Missense mutations clustered predominantly in the MBD and truncating mutations within the TRD or 3' end of the gene.

26

What kind of Rett syndrome phenotype would you expect to see in females with missense mutations?

- Females with missense mutations - significantly milder phenotype than those patients with truncating (nonsense/frameshift) mutations.

27

True or false? Patients with deletions have a milder phenotype if the mutation is small and towards the 3' end of the gene.

True. It has been observed that patients with deletions have a milder phenotype if the mutation is small and towards the 3' end of the gene.

28

Describe the testing strategy used for testing for Rett syndrome.

- Bidirectional sequencing should detect approximately 85-90% of mutations found in classical Rett syndrome and 30-40% in those patients with atypical Rett.
- Analysis using Mutation Surveyor software.
- All samples will be sequenced for all 4 exons and have duplications and deletions tested for using MLPA.
- MLPA results are analysed using the t-test PO15-spreadsheet obtained from Andrew Wallis.

29

Descripe why you need to consider SNPs when setting up a new service. How would you go about doing this?

- There have been reports of false negative results due to preferential amplification of the normal allele due to a PCR primer polymorphism, which could be present in the mutant allele.
- Primers are checked for the presence of new SNPs using the SNP screening program available from NGRL.
- The SNP screening program uses the latest build of the human genome from NCBI and BLAST to locate where the primers bind and a dbSNP database cross references the primer positions with all SNPs contained in their database.
- Need to repeat for all primers each time the database is updated.
- Frequency data is occasionally available for any SNPs identified.

30

Describe the sequencing procedure used in Notts to test for Retts.

Sequencing - the same PCR conditions are used for template generations of exons 2-3 and 4 while exon 1 is subjected to a different mastermix and PCR conditions. The primers for all amplicons are tagged. Sequencing is carried out using ABI Big Dye Terminator kit and sequenced on the ABI 3130. The templates are sequenced using universal primers.

31

Describe how Rett sequencing data is analysed for potential variants in Notts.

- Data is analysed for potential variants using a comparison between test and control data.
- Quality scores assigned to the overall sequence.
- GenBank files are used for analysis. The annotatio ns that are present from the GenBank files are maintained in the mutation surveyor software and the software can use these - for example exonic regions and known variants.
- Regions of interest (ROI) can be defined and checked to ensure this region is covered.
- We can upload known variants that we have identified as probable polymorphisms to the system so that we can confirm it as a known polymorphism immediately.
- Both heterozygous and homozygous mutations can be identified - should be able to detect insertion/deletions but if these are complicated they often have to be called manually.

32

What other Rett syndrome analysis is carried out other than sequencing?

- Also carry out dosage analysis using MLPA in order to identify deletions and duplications rather than missense muts ect.
- Have control probes from autosomes, X chromosome probes, MECP2 probes, probes in genes associated with atypical Rett syndrome and X and Y chromosome probes.

33

What referral types are you likely to get with Rett syndrome.

1). Female/Male diagnostics - these can be accepted from GPs, Paeds, and clinical genetics:
- If a variant is identified in the affected child, parental samples should be requested, regardless of the pathogenicity of the variant.
- Testing should also be offered to other female relatives of the proband, where a mutation has been identified.
- In male relatives where there is a possible Rett phenotype, testing should also be offered.

2). Parental samples - referral should only be accepted from Clinical Genetics:
- Identification of gene variant of known or unknown clinical significance - testing of parental samples is recommended.
- Variants of unknown significance this testing could shed light on the pathogenicity.
- Variants with known clinical significance testing of the parents could help determine the risk to siblings or future offspring of the couple - even if the variant isn't identified there is a risk of germline mosaicism in the parents.

3). Prenatal diagnosis:
- Risk to the fetus will depend if the mutation has been identifed in the parents. If the mother carries a mutation then the risk will be 50%.
- If the familial mutation has not been identified in the parents then there is still a risk that they are germline mosaics.
- There have been studies where no mutation has been identified in the parents but the child born had the same mutation as their affected sibling.
- Therefore counselling will be important as the procedure of sampling is not without risk.
- Any result has to be checked for MCC. This will determine if the result is due to the presence of maternal cells in the fetal sample.
- This is carried out using a panel of microsatellite markers.

34

Describe X-inactivation.

- One of the 2 copies of the X chromosome present in female somatic cells is inactivated.
- Provides a mechanism for dosage compensation.
- Occurs early in embryogenesis.
- Inactivation takes place at the X inactivation centre (XIC) - methylation.
- X inactivation is not always complete.
- Females may present with a mild or sometimes full expression of an X-linked recessive disorder.
- By chance the X chromosome bearing the normal genes has been inactivated in more than half the cells and this is referred to as skewed X inactivation.

35

How can skewed X inactivation be tested for?

- This can be tested for using a PCR of the polymorphic CAG repeat in the first exon of the Androgen Receptor gene (AR).
- DNA is cut by methylation sensitive restriction enzymes HpaII and CfoI. There are also 2 control digestion enzymes, one to help identify the alleles and one that shows digestion is complete.
- X inactivation is random but this assay can determine the degree of skewing - ratios of 75:25
- In some labs the analysis is not always quantitative which will not aid in determining the phenotype.

36

Describe diagnostic Rett syndrome referrals.

1). Female/Male diagnostics - these can be accepted from GPs, Paeds, and clinical genetics:
- If a variant is identified in the affected child, parental samples should be requested, regardless of the pathogenicity of the variant.
- Testing should also be offered to other female relatives of the proband, where a mutation has been identified.
- In male relatives where there is a possible Rett phenotype, testing should also be offered.

37

Describe Rett syndrome referrals involving parental samples.

2). Parental samples - referral should only be accepted from Clinical Genetics:
- Identification of gene variant of known or unknown clinical significance - testing of parental samples is recommended.
- Variants of unknown significance this testing could shed light on the pathogenicity.
- Variants with known clinical significance testing of the parents could help determine the risk to siblings or future offspring of the couple - even if the variant isn't identified there is a risk of germline mosaicism in the parents.

38

Describe prenatal testing for Rett syndrome.

3). Prenatal diagnosis:
- Risk to the fetus will depend if the mutation has been identifed in the parents. If the mother carries a mutation then the risk will be 50%.
- If the familial mutation has not been identified in the parents then there is still a risk that they are germline mosaics.
- There have been studies where no mutation has been identified in the parents but the child born had the same mutation as their affected sibling.
- Therefore counselling will be important as the procedure of sampling is not without risk.
- Any result has to be checked for MCC. This will determine if the result is due to the presence of maternal cells in the fetal sample.
- This is carried out using a panel of microsatellite markers.

39

What alternative testing is available for patients who don't seem to have a detectable Rett syndrome mutation?

- There is alternative testing available as many children with Rett syndrome don't often have a mutation identified. This may be due to the fact that there is a mutation in a different gene, the child may not actually have Rett syndrome, or it may just not be identifiable with the screening methods used.

40

What other genes are associated with Rett syndrome type phenotypes other than MECP2?

CDKL5, ARX and NTNG1, FOXG1, SLC9A6.

- Angelman syndrome - like Rett syndrome presents early in childhood with global developmental delays, speech and communication problems, stereotypical hand movements and autistic behaviour.
- Netrin G1 Gene (NTNG1) - A rare cause of atypical Rett is in the Netrin G1 gene (NTNG1). This gene is involved in the development of the CNS. It is located on Chr 1 and spans 340kb, with 10 exons - 9 of which are coding.
- FOXG1 - Located on 14q13 and has been associated with a congenital form of Rett syndrome that presents within the 1st months of life.
- SLC9A6 - found in cases of XLMR, microcephaly, epilepsy, and ataxia and often present with a phenotype mimicking Angelman syndrome.

41

Describe the presentation of patients with mutations in CDKL5. How would you screen this gene?

- This group of patients is characterised by infantile spasms and jerks.
- Mutations have been identified in patients diagnosed with atypical Rett in the gene CDKL5 located on Xp22.
- All atypical Rett syndrome patients with a CDKL5 mutation have had early onset of seizures before aged 6 months.
- Mutations in this gene result in varying phenotypes.
- Mutations are rare in male patients - possibility not many males have been tested due to lethality of CDKL5 mutations.
- Mutations in CDKL5 associated with severe early onset seizures and patients presenting with this form of atypical Rett should have screening of the CDKL5 gene considered.
- There is an MLPA kit available that tests for dels and dups in a number of these alternative Rett genes such as CDKL5, ARX and NTNG1 gene.

42

If a Rett patient presented with infantile spasms and jerks and early onset seizures before 6 months what gene would you recommend screening in addition to MECP2?

Mutations in CDKL5 associated with severe early onset seizures and patients presenting with this form of atypical Rett should have screening of the CDKL5 gene considered.

43

What is the Netrin G1 Gene (NTNG1) associated with?

Netrin G1 Gene (NTNG1) - A rare cause of atypical Rett is in the Netrin G1 gene (NTNG1). This gene is involved in the development of the CNS. It is located on Chr 1 and spans 340kb, with 10 exons - 9 of which are coding.

44

Where is the FOXG1 gene located? What is it associated with?

FOXG1 - Located on 14q13 and has been associated with a congenital form of Rett syndrome that presents within the 1st months of life.

45

What is another possible diagnosis for individual who have been diagnosed with Angelman syndrome.

Rett syndrome. The 2 conditions have a phenotypic overlap.