台灣平地人及原住民葡萄糖-6-磷酸去氫鋂的點突變與多形性的分析研究

Abstract

本實驗以前發現有9種G6PD （Glucose-6-Phosphate Dehydrogenase）單鹼基點突變是造成台灣中國人G6PD缺乏症的主因。而?了檢測這些點突變，本實驗室亦發展出PCR-RE （PCR-restriction enzyme） digestion, ASO (allelic specific oligonucleotide) hybridization及PRSM（PCR-mediated restriction site modification）等方法，這些方法雖快速簡單，但只能用於檢測已知位置的單鹼基突變，對於非已知位置的突變，則未有好的偵測方法。本論文的目地主要是應用非放射性PCR-SSCP（nonradioactive PCR-single strain conformation polymorphism）方法，來偵測未知位置的G6PD點突變。我們首先將G6PD缺陷基因用PCR方式分成16段，放大所有表現序列（exons），再用非放射性SSCP依DNA移動速率的差異，區分出正常及含有突變的DNA片段，而後再將此含有突變的DNA片段直接定序即可。我們運用此PCR-SSCP系統找到了台灣第十種G6PD的點突變：exon 9鹼基871 G→A突變，其胺基酸為Val291→Met。 另一方面，由於G6PD所在之Xq28位置?含有多個基因的區域，包括第八凝血因數（coagulation factor VIII）及紅／綠色盲基因（red/green color vision）。我們根據此區域4個DNA多形性位置（polymorphic site）的表現，將其歸納成8種haplotypes，分析台灣原住民4族—阿美、雅美、泰雅、賽夏與平地人及G6PD缺乏症患者，實驗結果顯示台灣原住民及平地人其主要haplotype均相同，且在G6PD的二個多形性位置—鹼基(nt)1311 C→T突變及intron 11 Nla III site的點突變有強烈的連鎖不平衡現象（linkage disequilibrium）；此與Filosas氏所做有關南義大利人的研究結果有很大差異，本實驗顯示在族群的變遷上臺灣族群間彼此關係密切而與地中海區歐洲族群則有明顯差異。另外在G6PD缺乏症患者則發現nt 1376突變與haplotype III有強烈連結關係（closely associated），而nt 871突變則與haplotype XI相連結，由此結果推測台灣nt 871突變產生可能與G6PD Viangchan為同一起源。

We and others have previously reported that there are 9 different types of point mutation found in the G6PD gene of the affected Taiwan chinese. We have used PCR-RE (PCR-restriction enzyme) digestion, ASO (allelic specific oligonucleotide) hybridization and PRSM (PCR-mediated restriction site modification) methods to identify the exact point mutation site in the defected G6PD gene. Although, the above methods are simple and rapid, they couldn't detect the uncharacterized mutation that dosen't belong to the 9 known mutation sites in the affected Chinese subjects. In this thesis, I have modified a newly developed method - nonradioisotopic polymerase chain reaction single-strain conformation polymorphism (PCR-SSCP) - to analyze the unknown mutation in the G6PD gene. First, I have designed 16 sets of oligonucleotide primers to amplify all the exons of G6PD deficient gene. Then the exon that carries the G6PD mutation can be distinguished from the normal exon by its differential mobility. Finally, I have used direct sequencing strategy to identify the mutation site of the G6PD gene. Using this approach, I have detected a novel mutation - nt 871 G->A mutation (Val291->Met) - located at the exon 9 of the G6PD gene. In addition, chromosome band Xq28 where the G6PD gene located contains several genes including the coagulation factor VIII and the red/green color vision. In our lab, we have studied 4 polymorphic sites in this region and combined them into 8 different haplotypes. We have analyzed each haplotype frequency and its linkage among 4 populations of Taiwan aborigine, Taiwan Chinese and G6PD deficiency cases. Our results showed that haplotype (VI+VII) is the dominant haplotype that commonly found in Taiwan aborigines and Taiwan Chinese. In contrast, Filosas et al reported that haplotype (V+IX) is the major dominant type found in Southern Italian. These results support a hypothesis that the blood relation between Taiwan aborigines and Taiwan Chinese is much closer than that between Taiwan chinese and Southern Italian. In addition, we have also found that, in G6PD dificiency cases, the nt 1376 mutation was closely associated with haplotype III, whereas, nt 871 mutation was always linked with haplotype XI. Taken together, our findings may provide some useful information for tracing the origin of various Taiwan aborigines in the future.