Molecular Cloning and Functional Study of Acid Alpha-Glucosidase L291P Mutation in Pompe Disease

• Main Authors: Ming-Cheng Weng, 翁銘晟
• Other Authors: Woon Peng Yeong
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/13321723644625134226

碩士 === 慈濟大學 === 分子生物暨人類遺傳學系碩士班 === 101 === Pompe disease (also known as glycogen storage disease type II; GSD-II) is a rare autosomal recessive disease which had an incidence of approximately 1 in 40,000 according statistics reported in Pompe Disease Newborn Screening Program by National Taiwan University Hospital from 2005 to 2008. The disease is caused by loss-of-function mutations in acid alpha-glucosidase gene (GAA) which is located on chromosome 17q25.2-q25.3. GAA is a lysosomal enzyme which degrade glycogen to glucose. Glycogen is mainly stored in liver and muscle cell. Patients with Pompe disease lacked complete GAA enzyme activity which will lead to accumulation of glycogen in lysosome and eventually will lead to cell death. We received blood samples from an infant patient and his parents fromTzu Chi General Hospital, Hualien. We designed 15 primer sets in GAA intronic region to amplify and sequence this gene. Our sequencing results indicated that the father exhibited seven genetic variants and one point mutation (c.1798C>T, p.R600C heterozygote) whereas the mother displayed five genetic variants and one point mutation (c.872T>C, p.L291P heterozygote). The patient had inherited all seven genetic variants and two point mutations in heterozygous form as the result of compound heterozygosity. Study from the 3D structure of GAA protein using Molecular Operating Environment (MOE) computer simulation indicates that p.R600C mutation located within the pocket of the GAA active site, whereas p.L291P located very close to the substrate binding domain. The R600C mutation had been reported which leads to loss of 99% of GAA enzyme activity. The L291P mutation had also been reported but with unknown consequence. Expression study in CHO-K1 cell shown that p.R600C and p.L291P retain 0.3% and 1.6% of GAA enzyme activities respectively. Western blots analysis reveals that p.L291P mutation affects GAA both in the protein level and enzyme activity level. In conclusion, p.R600C and p.L291P mutations contributed to the infantile Pompe disease observe in the patient.