Triplet Repeats and Imprinting (Diagnostics L4)

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…