Detection of A Recombinant Hotspot Associated with Charcot-Marie-Tooth Disease Type 1A Duplication in A Large Family by A Improved PCR Method

• Main Authors: Zi-Hwa Zeng, 曾麗華
• Other Authors: Chung-Yee Yuo
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/17934611661000333992

碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 92 === Charcot-Marie-Tooth disease type 1A (CMT1A) also known as hereditary motor and sensory neuropathy (HMSN1A), is common hereditary peripheral neuropathy, almost represented 70 to 90 percent of CMT1 disease. The inheritance of CMT1A is autosomal dominant. It is mainly leading to a tandem DNA duplication of a 1.5-Mb region on chromosome 17p11.2 -12. This region contains the peripheral myelin protein 22 gene (PMP22) which is flanked by homologous proximal and distal CMT1A repeat sequences (CMT1A-REPs). The majority of duplications arise during meiotic recombination following unequal crossing-over between the proximal and distal CMT1A-REPs. The hotspot of crossover breakpoints is most frequently located within a 3.2-kb region between the restriction enzymes EcoRI and SacI. Up to now, several molecular diagnostic methods are currently used for detection of CMT1A duplication, including southern blot, pulsed-field gel electrophoresis (PFGE), fluorescence in situ hybridization (FISH), and real-time quantitative polymerase chain reaction. Above methods still have some limitations for clinical diagnosis. These methods require radioisotope or complicated procedures, and they are time consuming and labor intensive. The aim of this study was to develop a set of quicker, simpler, and accurate molecular method for diagnosis of CMT1A duplication in the recombinant hotspot area. We modified the PCR assay（Chang et al., Clin Chem 44: 270, 1998） by using only a pair of primers and EcoRI to detect the recombination in the whole 3.2-kb hot spot region. Then we applied this assay in a large Chinese family. The results showed that 22 from 43 family members had CMT1A duplication. Duplication was detected in 13 family members who never realize they had the symptom. Besides, the phenotype, such as severity and NCV values, is variable between different generations, even within siblings who had the CMT1A duplication. In summary, the modified PCR method is relatively simple, economic, quick, and highly specific. It can be wildly used for diagnosis of the CMT1A duplication in the recombinant hotspot area, especially for screening whole family members to find the patients who never realize the symptom in order to reduce the disease progression.