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…


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