運用DNA突變分析儀及基因掃描技術於多種基因疾病基因定量分析方法之建立

研究已知有許多遺傳疾病是由特定基因點突變所引起，近來發現愈來愈多的基因疾病是由於基因拷貝數的改變所造成。而Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) 疾病發生的原因即由於位在染色體 17p11.2-12 上之1.5-Mb 區域重複或缺失所造成。由於此區域內含有一周邊髓磷蛋白(Peripheral myelin protein-PMP22)基因，此區域若發生重複或缺失就會造成此兩種不同的疾 病產生。 本計畫所提出的方法是利用多重聚合酶連鎖反應(multiplex PCR)策略，同時放大一未知基因拷貝數的待測區域和另一已知基因拷貝數之參考區域，之後藉由DNA 片段突變分析儀(DHPLC) 或毛細管電泳(CE) 檢測出基因拷貝數的微量差異。在此研究中，我們對總共110 個已經診斷的個體，包括已確診的32 位CMT1A 病人，17 位HNPP 病人及61 位正常人進行分析，實驗使用相同多重聚合酶連鎖反應的流程，結果顯示，無論是使用DNA 片段突變分析儀或是毛細管電泳，兩個系統所定量出來的PMP22 基因拷貝數相符合，並且所有樣品均使用聚合酶連鎖反應-DNA 限制酶片段長度多型性(PCR-RFLP)分析確認結果。 在此計畫中，我們證明結合多重聚合酶連鎖反應與DNA 片段突變分析儀的分析方法在檢測PMP22 基因拷貝數發生重複或缺失所造成CMT1A 或HNPP 疾病時，是一種高效率、高準確率、高可信度的技術，並且除了可以定量基因拷貝數，還可以鑑定一般常見已知的突變基因型以及不同族群之新的突變點位，故此技術未來應可成為臨床基因檢測之一項利器。Many genetic diseases are caused by the presence of point mutations in respective genes; an increasing number of diseases are known to be caused by changes in gene copy number. Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5 Mb duplication and deletion at chromosome 17p11.2-12 encompassing the peripheral myelin protein (PMP22). We developed a rapid and reliable detection system for duplications/deletions of PMP22 gene based on measurement of gene copy number. The method involves amplifications of a test locus with unknown copy number and a reference locus with known copy number by multiplex PCR strategy, following by Denaturing high performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, seventeen patients with HNPP, and sixty-one unaffected individuals were analyzed. By using the same multiplex competitive PCR protocol, the measured PMP22 gene dosage revealed the compatible results in DHPLC and capillary electrophoresis analysis. The results of multiplex PCR/DHPLC or multiplex PCR/capillary electrophoresis assay were all confirmed by PCR-RFLP analysis. We demonstrated that multiplex PCR/DHPLC assay is an efficient, accurate, and reliable technique for the gene dosage determination of PMP22 gene for CMT1A duplication and HNPP deletion. It also can detect the common known mutations and the novel mutations in varied population. Hence the technique could be a power tool in clinical genetic screening