Molecular Aspects of Myotonic Dystrophy Part 3 Flashcards
What are currently the main theories for how the expanded CTG repeat leads to the multi-systemic phenotype in DM?
1) . Haploinsufficiency
- The presence of the expansion somehow prevents transcription or translation of the gene and thus gene expression
- Reduction in protein levels leads to the phenotype
2) . Change to chromatin structure
- The presence of the repeat at the genomic level alters the expression of surrounding genes possibly due to a change in the local chromatin structure - this then leads to the DM phenotype
3) . RNA toxicity / gain of function
- Rather than just coding for protein the RNA obtains a new abnormal function within the cell that gives rise to some form of toxicity at the cellular level.
Is DMPK transcription affected in DM?
No, transcription of DMPK is not affected by the expansion. mRNA containing the expansion is not exported from the nucleus therefore preventing translation.
What evidence is there to support/oppose the theory that the DM phenotype is the result of haploinsufficiency?
- Transcription of DMPK is not affected by the expansion.
- mRNA containing the expansion is not exported from the nucleus therefore preventing translation.
- Level of DMPK protein is reduced in cell lines from DM1 patients.
However….
- Mice with only 1 copy of DMPK show some muscle weakness and cardiac defects, but do not replicate the multi-systemic phenotype of DM.
- No DMPK point mutation found in DM1 patients which would be expected if the phenotype was specifically linked to haploinsufficiency.
- The differing functions of DMPK and CNBP but similar phenotypes of DM1 and DM2 also suggest that the mechanism is not as straight forward as haploinsufficiency.
What evidence is there to support/oppose the theory that the DM phenotype is the result of changes in chromatin structure?
- The main evidence for a local area effect around the CTG repeat expansion in DMPK is the reduced level of transcription of the neighbouring gene SIX5.
- Secondary structures formed by the CTG repeat lead to a more condensed chromatin structure in the region followed by an increase in local CpG methylation and therefore the repression of local transcription.
- The 3’ end of DMPK contains the regulatory region of the SIX5 gene.
- Reduced SIX5 transcription is seen in DM1 cell lines.
However…..
- Mouse models heterozygous for SIX5 develop cataracts, but no other symptoms of DM. If the cataracts in SIX5 heterozygous mice are due to SIX5 deficiency we wouldn’t expect DM2 patients to also develop cataracts.
- Doesn’t explain the entire phenotype or the similarity to DM2.
What evidence is there to support/oppose the theory that the DM phenotype is the result of RNA toxicity?
- The majority of the DM phenotype is now thought to be due to a novel, toxic function of the expanded CUG/CCUG mRNA.This would explain the similarity between the phenotypic features of DM1 and DM2. Similarity between DM1 and DM2 phenotypes due to common mutation type rather than the function of the mutated gene.
- Transgenic mice expressing expanded CUG tracts have a phenotype including muscle weakness, myotonia, aberrant glucose metabolism and some signs of CNS tauopathy.
- Expanded mRNA is not exported to the cytoplasm and forms aggregates/foci within the nucleus. Abnormal interaction with RNA foci and RNA binding proteins leads to the DM phenotype.
- The musclebind-like (MNBL) family of RNA binding proteins have been shown to be sequestered by the CUG containing RNA foci. Results in depletion of MNBL proteins in the cell. 3 family members MNLB1, 2 and 3 that are involved in the regulation of alternative splicing. Mouse models with MNBL1 and MNBL2 knockouts show a range of DM phenotypes. MNBL1 knockout = myotonia, cataracts, lethargy, cardiac conduction. MNBL2 knockout = myotonic discharges, sleep disturbance, cognitive impairment
- Not all of DM phenotype can be explained by the loss of MNBL. The level of CUGBP and ETR3-like factor (CELF) family of proteins has been found to be up-regulated in DM1 cells. These proteins normally bind to single stranded CUG repeats, but do not bind to the expanded RNA foci. CELF1 in the presence of the expanded CUG repeats is phosphorylated increasing stability. CELF proteins also regulate alternative splicing. Overexpression in mouse models gives a phenotype with myopathy and cardiomyopathy. Overexpression not as clear in DM2 cells.
What is the majority of the DM phenotype now thought to be caused by?
The majority of the DM phenotype is now thought to be due to a novel, toxic function of the expanded CUG/CCUG mRNA.
What RNA binding proteins have been shown to be sequestered by the CUG containing RNA foci in muscular dystrophy?
- The musclebind-like (MNBL) family of RNA binding proteins have been shown to be sequestered by the CUG containing RNA foci. Results in depletion of MNBL proteins in the cell. 3 family members MNLB1, 2 and 3 that are involved in the regulation of alternative splicing. Mouse models with MNBL1 and MNBL2 knockouts show a range of DM phenotypes. MNBL1 knockout = myotonia, cataracts, lethargy, cardiac conduction. MNBL2 knockout = myotonic discharges, sleep disturbance, cognitive impairment
- Not all of DM phenotype can be explained by the loss of MNBL. The level of CUGBP and ETR3-like factor (CELF) family of proteins has been found to be up-regulated in DM1 cells. These proteins normally bind to single stranded CUG repeats, but do not bind to the expanded RNA foci. CELF1 in the presence of the expanded CUG repeats is phosphorylated increasing stability. CELF proteins also regulate alternative splicing. Overexpression in mouse models gives a phenotype with myopathy and cardiomyopathy. Overexpression not as clear in DM2 cells.
Can the proposed RNA toxicity mechanism explain the phenotype seen in myotonic dystrophy?
- The mis-regulation of splicing due to RNA gain of function can explain the multi-systemic phenotype of DM.
- The overall disruption to splice regulation factors has a wide effect on mRNAs which may explain the wide range of phenotypes seen in DM patients.
- The increase in CELF proteins and the reduction in MNBL proteins results in conditions similar to those seen in embryogenesis where alternative splicing is used to regulate the expression of a large number of genes. For example the embryonic form of CLCN1 retains exon 7a thus retaining a premature stop codon.
- Larger repeats lead to increased disruption and therefore a more severe phenotype.
- Nonsense mediated decay will reduce mRNA levels and any protein made will be truncated and non-functional.
Give some examples of genes in which splicing is known to be altered in DM cells.
1) . CLCN1 - myotonia
2) . MAPT (Tau) - insomnia, lethargy
3) . BIN1 - muscle weakness
4) . INSR - Insulin resistance
5) . TNNT2 - cardiac conduction defects
How can the similarities between the phenotypes of DM1 and DM2 be explained?
The similarities between DM1 and DM2 are relatively easy to explain given the common pathogenic mechanism and similar mutation type.
Why does only DM1 display a congenital form?
It is still not clear why only DM1 has a congenital phenotype (even though the longest repeat tracts are seen in DM2 patients).
The difference may be due to the expression patterns and levels of the two genes. A greater level of expression will lead to a greater accumulation of toxic RNA and therefore a more severe phenotype.
At the DMPK locus it is known that an antisense transcript originates in the SIX5 regulatory region and runs towards the 3’ end of DMPK gene and therefor the CTG repeat. Normally an insulator region between SIX5 and DMPK prevents this region from running into DMPK.
It has been suggested that due to changes in the local chromosome structure due to the presence of large repeat expansions CTCF proteins cannot bind to the insulator region and the function of the insulator is therefore reduced.
Without a functional insulator the antisense transcript will include the expanded CTG tract.
The antisense transcript is expressed during embryogenesis and so the levels of CTG containing RNA may be expressed from an earlier age giving rise to the congenital form of DM1.
Without the expression of CNBP during embryogenesis the DM2 repeats only build up later in life and therefore only adult phenotypes are seen.
Explain why DM1 is thought to display a congenital form.
1) . A transcript originating in the SIX5 regulatory region is known to run antisense to the DMPK gene during embryogenesis.
2) . Transcription is usually prevented from reaching the CTG repeat by an insulator element that is bound by CTCF proteins.
3) . The expanded allele is known to increase CpG methylation in the region and therefore prevent CTCF binding.
4) . Without a functional insulator the CTG repeat will be included in the transcript leading to increased expression during embryogenesis.
5) . This may contribute to earlier disease phenotype in DM1.
Describe diagnostic referrals for myotonic dystrophy.
The most common referrals for DM are for diagnostic cases. The age of patients varies and the symptoms may cover one or several of those seen in the DM phenotype - most commonly muscle weakness or delayed development. DM1 is also one of the standard tests for neonates with hypotonia.
All DM1 cases are tested using a combination of sizing PCR and bidirectional TP-PCR. If the TP-PCR fails a negative report can still be issued if the patient has 2 alleles in the normal size range.
Reporting time is 10 working days for routine diagnostic testing and 3 working days if there is a pregnancy involved. Neonates are reported in 5 working days.
Describe presymptomatic referrals for myotonic dystrophy.
After an expansion has been identified in a family it is common to offer presymptomatic testing to the relatives of that patient.
These referrals will only be accepted from the clinical genetics service to ensure correct counselling is provided to patients.
Testing will always use both sizing PCR and TP-PCR.
To make sure we are getting the correct results a positive family control will always be run if the original testing was done before the use of TP-PCR or was performed by another lab. This ensures that TP-PCR will detect the familial expansion giving greater confidence in the results.
TAT 10 working days, 3 if a pregnancy is involved.
Describe prenatal diagnosis for myotonic dystrophy.
Prenatal diagnosis is offered to all patients of child bearing age who are found to have an expansion.
Prenatal samples are initially tested by sizing PCR and TP-PCR.
Important to exclude MCC although due to the sensitivity of TP-PCR to MCC the result will be backed up with microsatellite markers or Southern blotting if the mother carries the expansion.
Microsatellite markers are easy and quick to use and can confirm whether the foetus carries the high risk haplotype. However, use of linked markers requires at least one other family member whose expansion status is known. Usually a previously affected child or grandparent of the foetus.
3 working day reporting time.
