A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay.
Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5-Mb duplication and a deletion at chromosome 17p11.2-12 encompassing the peripheral myelin protein 22 gene (PMP22), respectively. We developed a rapid and reliable detection system for duplications/deletions of the PMP22 gene based on measurement of gene copy number. The method involves amplification of a test locus with unknown copy number and a reference locus of known copy number by multiplex PCR (MP), followed by denaturing high-performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, 17 patients with HNPP, and 61 unaffected individuals were analyzed. Using the same competitive MP protocol, the measured PMP22 gene dosage revealed concordant results between DHPLC and capillary electrophoresis analysis. The results of the MP/DHPLC or the MP/capillary electrophoresis assay were all confirmed by PCR-restriction fragment length polymorphism analysis. We concluded that the MP/DHPLC assay is an efficient, accurate, and reliable technique for gene dosage determination of the PMP22 gene for CMT1A duplication and HNPP deletion. This technique further extends the application of DHPLC as an alternative method for the measurement of gene amplifications and heterozygous deletions in different genetic diseases.
Lin
Chia-Yun
CY
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan.
Su
Yi-Ning
YN
Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
Lee
Chien-Nan
CN
Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan.
Hung
Chia-Cheng
CC
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan.
Cheng
Wen-Fang
WF
Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan.
Lin
Win-Li
WL
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan.
Chen
Chi-An
CA
Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan. cachen@ha.mc.ntu.edu.tw.
Hsieh
Sung-Tsang
ST
Department of Anatomy and Cell Biology, National Taiwan University College of Medicine, Taipei, Taiwan.|Department of Neurology, National Taiwan University Hospital, Taipei, Taiwan.
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017931:DNA Primers; D019075:Electrophoresis, Capillary; D018628:Gene Dosage; D006801:Humans; D009185:Myelin Proteins / Q000235:genetics*; D015203:Reproducibility of Results
D017931:DNA Primers; D009185:Myelin Proteins; C073687:PMP22 protein, human