Detection of the DMD gene deletion using DNA extracted from dried blood spots

背景： 裘馨氏肌肉萎縮症（Duchene muscular dystrophy, DMD）或貝克氏肌肉萎縮症（Becker muscular dystrophy, BMD）導因於DMD基因的突變，是造成男性肌肉疾病病變的一種X染色體性聯遺傳隱性疾病。DMD的發生率約每3600-6000名活產男嬰中有1個，確診的個案中約60~70%可在此段基因坐落的位置Xp21.2上發現基因缺失（deletion）或擴增（duplication）。近年來藥物治療日益進步，可以延緩發病患者的症狀，然而目前新生兒篩檢方式為檢測creatine kinase(CK)數值，敏感度有待加強，此症平均診斷年齡約5歲，若能以新生兒篩檢方式在疾病早期便確認診斷，將可望更改善預後。 研究目的： 我們希望可以建立血片萃取DNA進行多重連接擴大反應技術（multiple ligation-dependent probe amplification, MLPA）的方法，以偵測DMD基因是否有缺失或擴增，作為高CK值者的第二線檢驗工具。 實驗方法： 我們首先比較全血（whole blood, WB）標準萃取DNA方式與血片（DBS）萃取出的DNA經過濾、沉澱、DNA kit column、tissue extraction kit等處理方式的檢體，進行MLPA分析後，決定何種DNA處理方式適用於本次實驗，接著進行標準值之建立，最後比對臨床受試者以傳統檢測方式與我們方式的異同，以確認本檢驗的正確性。 結果： 我們發現以血片萃取DNA後直接回溶H2O的方式所獲得的DNA，是進行MLPA最好的檢體處理方式。經檢測8名受試者血片DMD基因缺失的結果發現，4名為DMD基因缺失、1名DMD基因擴增、與3名無DMD基因缺失與擴增，皆與全血DNA進行MLPA後的結果相同，準確率達100%。 結論： 我們建立了以濾紙血片萃取DNA 藉由MLPA的方式檢測DMD基因缺失或擴增的方式，這個方式簡單快速，未來將可用於新生兒篩檢時，針對高CK值個案的第二階段的檢測。Background: Duchene muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) are caused by the DMD gene mutation, which is one of the X-linked myopathy that affects mostly males. The prevalence is one in 3600-6000 in male newborns. About 60~70% of these patients can find mutation in the DMD gene, that located at Xp21.2. According to the advance in pharmaceutical development, the progress of patients can be slow down. Examination of the creatine kinase (CK) is what we use in the newborn screening test nowadays; the sensitivity needed to be improved. The average diagnosis ages of DMD is 5 years old. To improve the prognosis, it can achieve by early detection of patients via newborn screening. Purpose: We hope to establish the method to detect DMD gene deletion or duplication by DNA extracted from the dried blood spot (DBS) using multiple ligation-dependent probe amplification (MLPA) as a second tier for those with high CK values. Method: We compared the MLPA result using DNA extracted from traditional method (whole blood) with DNA extracted from DBS using different methods (direct dissolve, filtration, precipitation, DNA kit column, tissue extraction kit). Then, we chose one method from this experiment to establish the standard values. Finally, we tested the clinical samples using traditional methods with our method to check the accuracy of our method. Results: We found that the best way to handle for MLPA was to dissolve DNA in H2O directly. After testing eight clinical samples, we identified four with DMD deletion, one with DMD duplication, and three with normal DMD gene dosage. The results were comparable to traditional results. The accuracy is 100%. Conclusion: We established the MLPA method to detect DMD gene deletion or duplication using DNA from DBS. This method is simple and fast. In the future, it is suitable be the second tier test for high CK in newborn screen