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A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene
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Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions.

Elaine M Cleary1, Suvankar Pal2, Tara Azam3

  • 1South East Scotland Genetics Service, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom; Euan Macdonald Centre for MND Research, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom.

Molecular and Cellular Probes
|June 12, 2016
PubMed
Summary
This summary is machine-generated.

Detecting C9orf72 hexanucleotide repeat expansions in ALS patients is challenging due to their repetitive nature. Optimized PCR methods, including repeat-primed assays, improve detection accuracy for genetic diagnostics.

Keywords:
Amyotrophic lateral sclerosisC9orf72Genetic testingRepeat-primed PCR

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Area of Science:

  • Genetics
  • Molecular Biology
  • Neuroscience

Background:

  • C9orf72 hexanucleotide repeat expansions are a common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia.
  • The GC-rich and repetitive nature of these expansions poses significant challenges for standard PCR-based detection methods.
  • Existing PCR assays often fail to accurately detect expansions, especially in the presence of genomic variations, hindering precise diagnosis and understanding of pathogenic ranges.

Purpose of the Study:

  • To develop and optimize robust PCR-based assays for detecting C9orf72 hexanucleotide repeat expansions.
  • To overcome the limitations of current methods in identifying expansions, particularly in genetically variable regions.
  • To establish a reliable diagnostic strategy for identifying pathogenic repeat expansions in ALS patients.

Main Methods:

  • Optimized PCR conditions using betaine as a co-solvent and specific thermal cycling parameters (slow ramping, high denaturation temperature).
  • Developed a flanking PCR assay to detect the presence or absence of expansions.
  • Developed repeat-primed PCR assays targeting both the 3' and 5' ends of the C9orf72 repeat expansion.
  • Combined flanking and repeat-primed assays for enhanced detection sensitivity and specificity.

Main Results:

  • The optimized PCR conditions and combined assay strategy successfully detected C9orf72 repeat expansions greater than approximately 100 repeats.
  • The assays demonstrated robustness even with genomic variability at the 3' end of the repeat region.
  • Repeat expansions were identified in 47 out of 442 Scottish ALS patients using the developed assays.

Conclusions:

  • The developed PCR assays, combining flanking and repeat-primed approaches, offer a robust method for detecting C9orf72 hexanucleotide repeat expansions.
  • These optimized assays can accurately identify expansions exceeding ~100 repeats, even with challenging genomic variations.
  • The combined assay strategy is recommended for clinical diagnostic settings to improve the detection of C9orf72-associated ALS.