A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay

• Published in: Journal of human genetics Vol. 51; no. 3; pp. 227 - 235
• Main Authors: Lin, Chia-Yun, Su, Yi-Ning, Lee, Chien-Nan, Hung, Chia-Cheng, Cheng, Wen-Fang, Lin, Win-Li, Chen, Chi-An, Hsieh, Sung-Tsang
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.03.2006
• Subjects: Base Sequence; Capillary electrophoresis; Charcot-Marie-Tooth disease; Chromatography, High Pressure Liquid - methods; Chromosome 17; Chromosome deletion; Copy number; DNA Primers; Electrophoresis, Capillary; Gene deletion; Gene Dosage; High-performance liquid chromatography; Humans; Myelin; Myelin Proteins - genetics; Peripheral myelin protein 22; Peripheral neuropathy; Reproducibility of Results; Restriction fragment length polymorphism
• ISSN: 1434-5161; 1435-232X
• DOI: 10.1007/s10038-005-0350-9

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.