Genetic Disease Studies of Orchid Tao Aborigines-Molecular enzymatic studies of homocystinuria

• Main Authors: Lien-Chin Yeh, 葉聯璟
• Other Authors: Chung-Yung Chen
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/4p369v

碩士 === 中原大學 === 生物科技研究所 === 98 === Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine. Human CBS is a heme protein that catalyzes the condensation of serine and homocysteine to form cystathionine in a pyridoxal phosphate-dependent reaction. Missense mutations in the CBS gene are the most common causes of clinical homocystinuria in humans. The D47E mutation was identified in a homocystinuric patient from Orchid Tao Aborigines. To understand how this mutation causes disease in human, we used Epstein-Barr virus (EBV) Transformed Human B Lymphocyte cell lines: heterozygote mutant (+/-), homozygote mutant (-/-) and wild type (+/+). First we performed the D47E gene RFLP using restriction enzymes Alu I and Sfan I to cleave the amplified DNA fragments and run the electrophoresis to distinguish between mutant and normal cell lines. After confirmation of the genotype of these cell lines, we want to know how the D47E mutant affects the CBS protein expression levels. Based on RT-PCR analysis, wild type mRNA levels were higher compared with the mutant cell lines. Next we performed the Western Blot to observe the CBS protein expression levels to confirm the results obtained from RT-PCR. D47E mutant cause protein misfolding determined by Native-PAGE. Finally we want to know if D47E mutant residue is important for structural and functional integrity of CBS enzyme as well as its activity. This biochemical characterization of the D47E mutant could be addressed for further insights into structure-function correlations in CBS.