Molecular aspects of Huntington's and triplet repeat conditions Flashcards
What are triplet repeats
repetitive sequences in genome (they are common in genome)
e.g. CAGCAGCAGCAG
What does a longer repeat mean in neuromuscular and neurodegenerative disorders
Longer repeats=more severe disease phenotype
What type of disease is Huntington’s disease
What gene is it to do with
Autosomal dominant
Expansion of the CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene
What is a normal CAG allele repeat size
<27
Myotonic Dystrophy type 1 symptoms
Myotonia (difficulty relaxing clenched hands or a hand shake)
Muscular dystrophy
Cataracts
Testicular atrophy
frontal balding
Cardiac conduction defects
Cause of Myotinic dystrophy type 1
expansion of a CTG trinucleotide repeat in the 3’UTR of the DMPK gene
What happens to CTG repeats and what does this lead to
transcribed into mRNA, but not translated into protein
-Large mRNA molecules accumulate in the nucleus. These acquire a new toxic function by trapping essential cellular RNA-binding proteins
Normal range of repeat size in Myotonic dystrophy type 1
5-35
difference between mechanism of disease of Huntington’s and Myotonic dystrophy type 1
Huntingtons: CAG expansion in exon 1 of HTT gene (coding region)
Myotonic dystrophy:
CTG expansion in 3’UTR of DMPK (non-coding regions)
Why can triplet repeats expand
Due to errors in DNA replication and repair (this accounts for normal variation in repeat length)
When a triplet repeat reaches beyond a certain size, what happens
It becomes more unstable and more likely to expand
Can full expansion mutations be inherited from unaffected parents
Yes- by unaffected parents with an intermediate or permutation allele
Does it make a difference if there is transmission of repeat instability from mother vs father
Yes
What is repeat instability
Repeats can expand in subsequent generations
What is ‘anticipation’
An intermediate repeat or a small expansion can increase in size in subsequent generations
Results in a more severe phenotype in child than in the parent
Who is transmission of MDtype1 more likely to be from
Mother
Who is more likely to have children of larger expansions of CTG triplet
Female carriers of unstable repeat sizes
How does congenital DM1 occur
Almost exclusively via maternal transmission
Where does expansion of CTGs take place for DM1
In the oocyte
Who is large expansions of CAG repeat in Huntington’s Disease more likely passed down from
Father
Who is likely to have children with larger expansions in CAG repeats in Huntington;s
Male carriers of unstable repeat sizes
Who is more likely to have Greater risk of Juvenile Onset HD
Those with fathers who are carriers of unstable repeat sizes
Where does expansion of CAGs in Huntington’s take place
Spermatogenesis
When may a single allele be detected when using PCR to interpret HD1+HD3 results
- patients may be homozygous for this repeat size (i.e. inherited an allele with 17 repeats from each parent)
- Patient may have an expansion that is not detectable by this method
When would you use HD2 and HD5
When you have a single gene detected when using PCR to interpret HD1 and HD3 results
What does using HD2 and HD5 tell you and not tell you.
How would you use them
How to use them:
-They amplify DnA across the CAG AND the CCG repeat (cCG is one adjacent)
-HD2 primer is fluorescently labelled. PCR fragment sizes can be detected by capillary electrophoresis
What they can and can’t tell you:
Can’t be used to size the repeat but useful to confirm alleles have been detected
If there is only one allele detected when using HD1+3 and HD2+5 tests, what does that tell you
Patient may be homozygous for this repeat size (i.e. inherited an allele with 17 repeats from each parent)
Patient may have an expansion that is not detectable by this method
When is triplet prime PCR used
When is It particularly important
Used in HD for individuals homozygous for normal alleles on both HD1+HD3 and HD2+HD5 PCRs
Particularly important for possible cases of Juvenile HD
What are the primers used in Triplet Primed PCR
HD_TPF
HD_TPR
HD Tail
What does the HD_TPF primer do
Binds to specific sequence upstream of the CAG repeat
What does the HD_TPR primer do
Binds to the repeat sequence (CAG)
Has a ‘tail’ with unique sequence
What does the HD Tail primer do and its role
Binds to the HD_TPR tail
-Amplifies the different sized products generated
Why can TP-PCR produce fragments of different sizes
Because the HD_TPR primer can bind anywhere on the repeats
Why is HD_TPR at a limiting concentration
Prevents gradual shortening of product size
What are products of TP-PCR separated by
Capillary electrophoresis
how can you tell there’s an expansion present in a TP-PCR graph
Normal results show a clear end to the triplet peaks
Positive (expansion) results show peaks continue until they tail off
