- 學位論文
蘭嶼達悟族原住民之遺傳疾病研究-高胱胺酸尿症之分子酵素研究
Genetic Disease Studies of Orchid Tao Aborigines-Molecular enzymatic studies of homocystinuria
摘要
高胱胺酸尿症 ( Homocystinuria,HCU ) 是一種胱硫醚β合成脢 ( Cystathionine beta-synthase,CBS ) 缺陷的遺傳疾病。HCU患者在臨床診斷中通常可以很明顯的發現在血漿中同半胱胺 ( Homocysteine ) 與甲硫胺酸 ( Methionine ) 皆異常的偏高。而HCU患者較典型的臨床症狀包括骨骼異常 ( Skeletal abnormalities )、水晶體異位 ( Ectopia lentis ) 與心血管疾病 ( Thrombotic vascular complications ) 等症狀。在先前許多的研究指出CBS之缺陷最普遍的原因是因為錯異的突變，而這些突變往往導致蛋白質的折疊錯誤而造成蛋白質失去生化功能或是影響了酵素活性。在先前的研究中發現蘭嶼達悟族原住民高胱胺酸尿症患者帶有 CBS D47E 這個位置的突變，而本次研究要去探討的是 CBS D47E 這個位置的突變對於 CBS 的蛋白質結構、代謝機制與酵素活性有何影響。在本次研究中發現蘭嶼達悟族原住民高胱胺酸尿症患者帶有 CBS D47E 突變的狀況下，我們推測其 CBS 蛋白質仍然有表現，但是沒有功能，而可能的機制是因為 CBS D47E 突變使蛋白質摺疊錯異導致 CBS 蛋白質結構改變進而影響酵素活性。
關鍵字
高胱胺酸尿症並列摘要
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.