Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions.
Due to the GC-rich, repetitive nature of C9orf72 hexanucleotide repeat expansions, PCR based detection methods are challenging. Several limitations of PCR have been reported and overcoming these could help to define the pathogenic range. There is also a need to develop improved repeat-primed PCR assays which allow detection even in the presence of genomic variation around the repeat region. We have optimised PCR conditions for the C9orf72 hexanucleotide repeat expansion, using betaine as a co-solvent and specific cycling conditions, including slow ramping and a high denaturation temperature. We have developed a flanking assay, and repeat-primed PCR assays for both 3' and 5' ends of the repeat expansion, which when used together provide a robust strategy for detecting the presence or absence of expansions greater than ∼100 repeats, even in the presence of genomic variability at the 3' end of the repeat. Using our assays, we have detected repeat expansions in 47/442 Scottish ALS patients. Furthermore, we recommend the combined use of these assays in a clinical diagnostic setting.
South 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. Electronic address: eme22@cantab.net.
Elaine M
EM
Cleary
Euan Macdonald Centre for MND Research, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom; Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom.
Suvankar
S
Pal
South East Scotland Genetics Service, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
Tara
T
Azam
South East Scotland Genetics Service, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
David J
DJ
Moore
Euan Macdonald Centre for MND Research, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom.
Robert
R
Swingler
Euan Macdonald Centre for MND Research, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom.
George
G
Gorrie
Euan Macdonald Centre for MND Research, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom; Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom.
Laura
L
Stephenson
Euan Macdonald Centre for MND Research, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom; Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom.
Shuna
S
Colville
Euan Macdonald Centre for MND Research, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom; Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom.
Siddharthan
S
Chandran
South 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.
Mary
M
Porteous
South East Scotland Genetics Service, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
Jon P
JP
Warner
D000483:Alleles; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics; D016477:Artifacts; D001483:Base Sequence; D000073885:C9orf72 Protein; D017931:DNA Primers / Q000378:metabolism; D042622:DNA Repeat Expansion / Q000235:genetics*; D006801:Humans; D009030:Mosaicism; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics*; D012606:Scotland; D012680:Sensitivity and Specificity
D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D017931:DNA Primers; D011506:Proteins
Amyotrophic lateral sclerosis; C9orf72; Genetic testing; Repeat-primed PCR