- 學位論文
利用毛細管電泳法分析SMN基因以診斷脊髓肌肉萎縮症
Analysis of SMN Gene by Capillary Electrophoresis for Diagnosis of Spinal Muscular Atrophy
摘要
脊髓肌肉萎縮症(spinal muscular atrophy, SMA)為發生頻率相當高的遺傳性疾病，而決定此疾病之遺傳因子位於第五對染色體(5q13)上的運動神經原存活基因(survival motor neuron; SMN)，此SMN基因分成2型：telomeric SMN (SMN1)和centromeric SMN (SMN2)，兩者於序列上相當類似，cDNA僅有位於exon 7(c.840C＞T)和exon 8(G＞A)上之2個核苷酸不同。根據研究指出，大約94%的SMA病人缺乏SMN1基因，表示此疾病的發生主要與SMN1的存在與否有關，但目前另有研究指出SMN2含量多寡會影響疾病的嚴重程度，因此同時定性及定量SMN1與SMN2為目前臨床診斷SMA迫切需求的方法，本研究建立了第一篇以毛細管電泳法(capillary electrophoresis, CE)定量分析exon 7上之SMN1/SMN2基因的方法，並且另開發一簡單且方便的通用多重聚合酶連鎖反應，只需共用一條螢光通用引子，即可將所有基因進行螢光標示。當在與DHPLC分析結果比較後，發現數個檢體以DHPLC方法檢測時，僅測得SMN2基因，但本CE方法卻測得SMN1與SMN2基因，然而再藉由定序確認後，發現其結果與CE所得相符，證實本CE方法之準確性優於DHPLC。 然而缺乏SMN1之病人，除了指整段SMN1基因喪失外，另有基因轉移之可能，目前大部分的臨床診斷技術只針對SMN1基因缺失，忽略了基因轉移，若能同時定量分析exon 7和8上之SMN1/SMN2基因，將可診斷病因為SMN1基因缺失或基因轉移，因此本研究依此目標建立同時分析exon 7和8上之SMN1/SMN2基因的毛細管電泳法。除了94%的SMA病人缺乏SMN1外，另有6%的病人為基因上之其他外顯子具有突變所致，相關外顯子包括exons 1、2a、2b、3、4、5、6、7和8，共9個外顯子，因此若要更準確地診斷SMA，方法除了可定量SMN1/SMN2兩基因外，還必須可同時偵測其他外顯子是否具有突變，本研究依此目標建立一可同時分析SMN基因上之所有外顯子片段的法。 本研究循序所開發之四種方法皆可被臨床應用於診斷SMA疾病，再與臨床目前使用的DHPLC檢驗方法比較時，我們的方法擁有更高準確性，而本研究除了可用於定量SMN1/SMN2基因外，另可偵測基因轉移與突變，對SMA疾病可提供全面性的資訊。
並列摘要
Spinal muscular atrophy (SMA) is the high frequently inherited disease. The SMA-determining gene group, survival motor neuron gene (SMN), has been found located in the region of chromosome 5q13. There were two highly homologous genes identified, including telomeric SMN (SMN1) and centromeric SMN (SMN2). These 2 genes possessed only two differences in the cDNAs, substitution of single nucleotides in exon 7 (c 840 C＞T) and 8 (G＞A). Absence of the SMN1 gene has been reported in approximately 94% of clinically typical SMA-affected patients. This indicates that SMA is only controlled by the SMN1 gene, but the SMN2 copy number has been related to the disease severity and life length. Therefore, quantification of SMN1/SMN2 is a powerful tool for diagnosis of SMA or related carriers. In this study, we presented the first capillary electrophoresis (CE) method for the separation and quantification of SMN1 and SMN2 genes of exon 7. Additionally, an ingenious and convenient method, universal multiplex PCR, was established with only one universal fluorescence-labeled primer needed in the PCR reaction for all the genes. When our results were compared with those obtained by using DHPLC, some samples were analyzed as having only SMN2 gene by DHPLC, but having both SMN1 and SMN2 by our CE method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. That demonstrated our CE method possessed a better accuracy than DHPLC. Nevertheless, the absence of SMN1 indicates the deletion of whole gene or genetic conversion. Recent methods were mostly developed with focusing on the analysis of whole gene deletion, but gene conversion was ignored. Simultaneous quantification of SMN1/SMN2 in exon 7 and 8 is a good strategy, not only to calculate the copy number, but also to judge the gene conversion. Therefore, we established a CE method which could be applied to simultaneous analysis of SMN1/SMN2 in exon 7 and 8. Additionally, approximate 6% of affected SMA individuals whose SMN1 was still present, but they were found with small deletions or point mutations in other exons, including exons 1, 2a, 2b, 3, 4, 5, 6, 7 and 8. An accurate method for diagnosis of SMA should combine both the quantification of SMN1/SMN2 in exon 7 and detection of mutations in other exons. Here, we have established a CE-universal multiplex PCR method for simultaneous analysis of 9 gene fragments containing all sequences of exons 1, 2a, 2b, 3, 4, 5, 6, 7 and 8 as well as some intron sequences at the splicing site. The method could not only be used for quantification of the copy numbers of SMN1/SMN2 genes in exon 7/8, but also for determination of intragenic mutations in other exons. These CE methods could apply to diagnose SMA in clinical. When compared with DHPLC which was the most frequently used for diagnosis of SMA in clinical, our method possessed a higher accuracy than that. Gene conversion and intragenic mutations in other exons were also detected by our methods that supplied more accurate diagnosis of SMA in clinical. These methods were very suitable for diagnosis of SMA in clinical.