脊髓性肌肉萎縮症之運動神經元存活基因套數檢測：變性高效能液體層析法與多重連接依賴式探針擴增法之分析比較

Copy Number Analysis of Survival Motor Neuron Genes SMN1 and SMN2 in Spinal Muscular Atrophy: Comparison between Denaturing High Performance Liquid Chromatography (DHPLC) and Multiplex Ligation-dependent Probe Amplification (MLPA)

李建宏(Chien-Hong Lee) ； 呂淑娟(Shu-Chuan Lu) ； 王美嘉(Mei-Chia Wang) ； 朱大成(Da-Chang Chu) ； 張璧月(Pi-Yueh Chang) ； 甯孝真(Hsiao-Chen Ning) ； 吳竹蘭(Tsu-Lan Wu) ； 孫建峰(Chien-Feng Sun)

《Journal of Biomedical & Laboratory Sciences》 21卷4期 (2009/12) Pp. 119-126

https://doi.org/10.30046/JBLS.200912.0002

摘要

脊髓性肌肉萎縮症(Spinal Muscular Atrophy, SMA)是一種體染色體隱性遺傳的的疾病，國人帶因率約爲2%~4%。如果夫妻能知道自己都是帶因者，且在懷孕時進行產前診斷，就比較有機會可以選擇適當的醫療處置。SMA的檢測主要是偵測運動神經元存活基因(Survival Motor Neuron Genes, SMN1 and SMN 2)的基因套數，正常人至少有2個SMN1基因，一般的帶因者只有一個SMN1基因，而SMA的患者沒有SMN1基因。SMN2基因套數多寡在正常人與帶因者均不造成影響，但卻與SMA患者的嚴重程度有關，通常SMA越嚴重者，SMN2基因套數越少。目前市面上用來偵測SMA帶因情形的方法有數種，其中包括多重連接依賴式探針擴增法(Multiplex Ligation-dependent Probe Amplification, MLPA)與利用變性高效能液相層析法(Denaturing High Performance Liquid Chromatography, DHPLC)進行分析。但是，根據之前不同實驗室的篩檢報告可知，此兩種方法所得到的帶因率，並不是完全一致。因此，本研究希望能藉由同時使用此二方法分析同一批檢體，以確認其適用性。本實驗共收集60個檢體，包括健康組37個、帶因組14個與SMA患者9個，同時以兩種方法進行分析。結果顯示，96.7%(58/60)的檢體經此二方法分析可得到相同的臨床診斷，但是有兩個檢體的結果判讀卻不一致。其中一例的MLPA判讀爲SMN1:SMN2=2:3的正常型，但是DHPLC分析的結果無法確定爲SMN1:SMN2=2:3的正常型或是SMN1:SMN2=1:2的帶因型。另外一例在MLPA判讀爲SMN1:SMN2=1:1的帶因型，但是在DHPLC的結果卻無法確定爲SMN1:SMN2=2:2的正常型或是SMN1:SMN2=1:1的帶因型。這兩個檢體在MLPA重複分析時都出現一致的結果，並無判讀上的困擾。由以上結果顯示，雖然DHPLC有操作時間較短、操作流程較簡便與試劑成本較低廉等優勢，但是MLPA在判讀SMN1與SMN2的基因套數的可信度與再現性卻明顯優於DHPLC。因此，MLPA似乎是較適合的SMA帶因者篩檢的方法。但若受限於成本問題，實驗室亦可考慮先用DHPLC進行篩檢，再將一些有判讀困擾的檢體，用MLPA做確認，以獲得可靠的篩檢結果。

並列摘要

Objective: Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder. The carrier rate is estimated at between 2% to 4% among the Taiwanese population. If both parents realize their carrier status and include SMA genotyping in the prenatal care, they may have the opportunity to receive proper intervening manifestation. Detection of SMA is to determine the copy numbers of survival motor neuron genes 1 and 2 (SMN 1 and SMN 2). Normal persons have two or more copies of the SMN1 gene, while SMA carriers have only one copy. No SMN1 gene is present in SMA patients. In contrast, the copy number of the SMN2 gene does not affect normal subjects or SMA carriers, but for SMA patients, the severity of SMA depends on the number of the SMN2 genes copy numbers. Several methods are currently available for the detection of SMA carriers, including multiplex ligation-dependent probe amplification (MLPA) and denaturing high performance liquid chromatography (DHPLC) methods. However, according to the data published previously in Taiwan, different carrier rates would be reported if different methods were used. Therefore, the present study was conducted to evaluate the two most commonly used methods in analyzing the same batch of clinical samples. A total of 60 specimens were collected from 37 healthy subjects, 14 carriers, and 9 SMA patients. Identical molecular diagnosis results were obtained by the two methods except in two cases. One case showed SMA-free results (SMN1:SMN2=2:3) with the MLPA method, while ambiguous results of SMA-free results (SMN1:SMN2=2:3) or carrier (SMN1:SMN2=1:2) types were obtained by using the DHPLC method. Similarly, the other patient was diagnosed as a carrier (SMN1:SMN2=1:1) with the MLPA method, but normal (SMN1:SMN2=2:2) or carrier (SMN1:SMN2=1:1) with the DHPLC method. Although the DHPLC method has many advantages, including short turn-around time, ease of operation, and lower cost; the MLPA method is able to produce more definite results in the determination of copy numbers for the SMN1 and SMN2 genes with better reliability and reproducibility. In conclusion, the MLPA method appears to be more suitable than the DHPLC method in the correct diagnosis of SMA carriage. For laboratories that reagent expenditures are a great concern, it is suggested that the DHPLC method may be used primarily to screen for all associated specimens, and the MLPA method may be used subsequently to confirm the results for those ambiguous results generated by the DHPLC method.

並列關鍵字

DHPLC ； SMN1 ； SMN2 ； spinal muscular atrophy ； SMA ； MLPA