Modification of the triplet repeat primed polymerase chain reaction method for detection of the CTG repeat expansion in myotonic dystrophy type 1: application in preimplantation genetic diagnosis.
OBJECTIVE
To overcome problems associated with the use of triplet repeat primed polymerase chain reaction (TP-PCR) in preimplantation genetic diagnosis (PGD) of myotonic dystrophy type 1 (DM1).


METHODS
Clinical research study.


METHODS
UCL Centre for PGD and Centre for Reproductive and Genetic Health.


METHODS
Seven couples undergoing PGD for DM1.


METHODS
A modified TP-PCR protocol (mTP-PCR) for the reliable detection of both expanded and nonexpanded alleles in DMPK was optimized using single lymphocytes. Four cycles of PGD were performed with TP-PCR for diagnosis and a further 10 cycles with mTP-PCR.


METHODS
Amplification efficiency, allele dropout, diagnosis rate, and delivery rate.


RESULTS
Preliminary testing showed that the TP-PCR amplification efficiency was higher using lymphocytes versus buccal cells. Single lymphocytes gave very high amplification efficiencies for both protocols (99% to 100%). There were no false-positive or false-negative results for 148 single lymphocytes tested with mTP-PCR compared with 9% (5 out of 54) false-positive results with TP-PCR, indicating the improved accuracy of the modified protocol. In embryos, the diagnosis rate was 95.6% with mTP-PCR and 75% with TP-PCR.


CONCLUSIONS
For PGD of DM1, mTP-PCR is recommended. It may also be applied as a rapid screen for DMPK expansions in individuals with symptoms of DM1, relatives of known mutation carriers, or in prenatal diagnosis.
Kakourou
Georgia
G
UCL Centre for PGD, Institute for Women's Health, University College London, London, United Kingdom.
Dhanjal
Seema
S
Mamas
Thalia
T
Serhal
Paul
P
Delhanty
Joy D
JD
SenGupta
Sioban B
SB
D000483:Alleles; D001483:Base Sequence; D005188:False Negative Reactions; D005189:False Positive Reactions; D005260:Female; D005820:Genetic Testing; D006801:Humans; D008214:Lymphocytes; D008297:Male; D008969:Molecular Sequence Data; D009223:Myotonic Dystrophy / Q000145:classification / Q000175:diagnosis* / Q000235:genetics*; D065636:Myotonin-Protein Kinase; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D019836:Preimplantation Diagnosis / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D018911:Trinucleotide Repeats / Q000235:genetics*
C585517:DMPK protein, human; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases