The establishment of diagnosis procedure and the study of CTG trinucleotide expansion pathway of myotonic dystropy

• Main Authors: Her-Maw Lin, 林和茂
• Other Authors: Shuan-Yow Li
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/91898306566538648923

碩士 === 中山醫學院 === 醫學研究所 === 87 === Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase (DMPK) gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats as well as the age of onset. Determination of the CTG repeat length has been primarily relied on Southern blot analysis on restriction enzyme-digested genomic DNA. The development of PCR technology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. Here we describe a procedure, FTAR-PCR system, including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient since high quality DNA sample can be obtained with only few nucleate cells, including lymphoblastoid cells, buccal cells, and amniotic fluid cells, for the detection of CTG repeat length. Therefore, FTAR-PCR system could be very useful in genetic counseling and prenatal diagnosis as well as prevalence study of other trinucleotide repeat diseases. By studying the linkage disequilibrium between Alu polymorphism and various CTG repeat lengths， Imbert et al. proposed that the DM mutations were derived from the pool of alleles with 19-30 CTG repeats, and themselves derived from a very small number of ancient expansions from the major (CTG)5 allele. However, our data indicated that the expansion pathway could be continuous, that is, the mutation process is random, progressive from (CTG)5 to (CTG)50. Through the study of the relationship between CTG repeat lengths and several single base polymorphism present flanking the CTG region, our results showed that the DM alleles in Taiwanese population have a specific haplotype, which is the same as that observed in Caucasian population. Since our preliminary result is consistent with the continuous model of CTG expansion, a more complete haplotype analysis including additional markers using (CTG)18-40 alleles will further elucidate the CTG mutation pathway.