Triplet Repeats and Imprinting (Diagnostics L4)
- Created by: Former Member
- Created on: 04-11-19 16:50
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Trinucleotide Repeat Disorders
- Repeating residues of 3 bases
- Polymorphic within the population
- Can expand over a defined threshold resulting in disease - unstable
- Monoallelic disorders i.e. one mutation in one gene
- Many different trinucleotide repeat disorders
- Can show anticipation - can expand further depending which parent inherited from e.g. Myotonic Dystrophy has a materal parent of origin effect and Huntington has a paternal effect
- Can show parent of origin effect - which means
- Intermediate and premutations can result in variable phenotypes
- Example: Huntington's, Friedrich's Ataxia, Myotonic Dystrophy, Fragile X
Molecular Techniques to Detect Trinucleotide Repeats
- Size the repeat length
- Exclude the presence of a disease causing expansion
- Using PCR based methods
PCR Test for Tricnucleotide Repeat Disorders
- Simple, rapid and reliable
- Mutation changes the size of the PCR product in a predictable way
- Can not differentiate between individuals homozygous for an allele or with larger expansion i.e. both alleles 10/10 repeat size
- Results interpreted differently if X-linked disorder
- TP-PCR needed to confirm
- SNP under primer binding site may prevent amplification
Triplet Repeat Primed PCR
- General method for investigation trinucleotide repeat disorders where large expansions are too big to PCR amplify
- A simple fluorescent PCR system that can rapidly identify (but not size) large expansions
- Used to exclude the presence of a large expansion
- TP-PCR gives a characteristic ladder on the fluorescent trace enabling the rapid identification of pathogenic repeats of any size
- The repeat specific 3' terminus of P4 binds at multiple sites within the repeat alleles giving rise to a mixture of products
- A 10:1 molar ratio of P3 to P4 ensures that P4 is exhausted in early amplification rounds
- This reduces priming at repeat sites internal to the PCR products produced in earlier rounds which results in gradual shortening of the average PCR product size
- The primer P3 preferentially binds to the end of products from previous amplification rounds owing to the stabilising effect of the 5' tail sequence
- A long extension time is used to allow complete extension of the larger sized products within the PCR product mixture and conserve the representation of the longer products
Huntington's Disease
- CAG (polyglutamine) expansions in coding region
- Autosomal dominant, late onset disorder
- Genotype/ phenotype correlation e.g. larger expansion = earlier onset
- Parent of origin effect on expansion
- risk expansion to larger alleles through paternal line
- Testing strategy: size PCR and TP-PCR to exclude large expansion
- Symptoms: personality disorders, psychosis, trouble with movement and swallowing
- PCR test: Number of repeats
- 6-26 = normal allele range, normal phenotype and stable
- 27-35 = intermediate allele, normal phenotype and possible instability with increased risk of HD in other family members
- 36-39 = incomplete penetrance allele, may or may not develop HD, unstable upon transmission and increased risk of HD in other family members
- 40 and over = Complete penetrance, Diagnosis of HD - affected, unstable upon transmission and increased risk of HD in other family members
Friedrich's Ataxia
- GAA expansion in intron 1
- Autosomal recessive disorder
- Loss of function of FXN gene (frataxin)- expansion…
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