運用全外顯子定序探討臺灣族群短縱列重複序列次數分布

Abstract

背景：短縱列重複序列擴張(short tandem repeat expansion)會導致50種以上的遺傳性疾病，其中又以神經退化性疾病佔大多數，然而這些疾病的臨床表現重複性比例高，使得在分子診斷上具有相當的挑戰性。近年，研究人員開發出讀取WES和WGS中的short-read sequencing的軟體，如:ExpansionHunter, exSTRa, STRetch, and TREDPARSE，以偵測序列中重複序列擴張的重複次數，此外，不被侷限只能看單一基因的重複序列擴張，可以同時看到不同基因的重複序列次數分布表現，藉此來判斷個案是否帶有致病性之重複序列擴張次數。 目的：了解臺灣族群之基因重複序列擴張次數分布以及比較ExpansionHunter軟體分析WES數據與傳統GeneScan之檢驗方法。 方法：本研究以病歷回溯之方法分析臺大醫院曾經做過全外顯子定序之700位個案，將其WES資料輸入至ExpansionHunter軟體加以分析輸出之<STR>數據，並與GeneScan做比對。 結果：本研究比對了ATXN1、ATXN2、ATXN3、CACNA1A、TBP、HTT、DMPK、ATN1八個基因之ExpansionHunter輸出值<STR>與GeneScan(PCR)數值，其中ATXN1、ATXN2、ATXN3三組基因之相關係數(r)達0.9以上(顯著水準<0.01)、R2也>0.8(p值<0.01)，而TBP基因組之r =0.313(顯著水準<0.01)及R2=0.053(p值=0.156)為最低；本研究重複序列過度擴張確診者有5位，約佔總人數之0.7%，就EH<STR>與GeneScan(PCR)比較組來看，偵測確診者的靈敏度(sensitivity)達100%。本研究藉由ExpansionHunter軟體共讀取46個重複序列基因、52245個對偶基因(allele)資料，經統計分析後，有半數以上之重複序列基因其分布次數與其他族群有顯著差異(p<0.05)，其中XYLT1基因與其他族群相比之下其重複序列次數偏高(尚在正常值範圍內)。 結論：ExpansionHunter軟體是個很有可看性的預測工具，但若要成為診斷工具可能需要斟酌，就本研究重序列過度擴張確診者其EH<STR>及PCR數據雖都介於全突變型範圍，但數據精準度還有待校正；本研究也運用ExpansionHunter軟體來讀取WES數據以了解臺灣族群重複序列基因次數分布概況，並知曉與其他族群相比下有些重複序列基因次數分布是有所差異的，因此若未來能建立各族群的重複序列次數分布資料庫，是再好不過的了。期待更多研究者能夠開發出更完善的檢驗方法或工具，以達到精準醫療之目的，並提升群體健康福祉。

Background: Short tandem repeat expansion can cause more than 50 genetic diseases, and neurodegenerative diseases are in the majority. However, the phenotype of these diseases are highly similar, so that is a challenge for molecular diagnosis. In recent years, researchers developed many software to read short-read sequencing in WES and WGS, which can detect the number of short tandem repeat expansion in the sequence, such as: ExpansionHunter, exSTRa, STRetch, and TREDPARSE. In addition, it is not limited to detect the short tandem repeat expansion size of a single gene, it can detect the short tandem repeat expansion size of different genes at the same time. Using these softwares to detect whether the case has pathogenic repeat sizes. Purpose: To realize the distribution of the number of short tandem repeat expansion genes in Taiwanese population, and compare the ExpansionHunter software and the traditional GeneScan(PCR) method to analyze short tandem repeat expansion size. Method: It is a retrospective analysis in this study. 700 cases were enrolled those who used to analyze of Whole Exome Sequencing in National Taiwan University Hospital. The WES data were input into ExpansionHunter software, and to analyze the output <STR> data, and then compared with GeneScan(PCR) data. Result: We compared the ExpansionHunter output <STR> data and GeneScan (PCR) data of eight genes including ATXN1, ATXN2, ATXN3, CACNA1A, TBP, HTT, DMPK, ATN1. ATXN1, ATXN2, ATXN3 three genes correlation coefficient (r) is above 0.9 (significant level <0.01) and R2 above 0.9 (p-value<0.01), while the group of TBP gene’s r = 0.313 (significant level <0.01) and R2 =0.053 (p-value=0.156) is the lowest. In this study, 5 patients were diagnosed with short tandem repeats overexpansion, whom the percentage of 700 cases is 0.7%. As far as the comparison group of ExpansionHunter <STR> and GeneScan(PCR), the sensitivity of ExpansionHunter detecting short tandem repeats overexpansion cases is 100%. A total of 46 genes and 52,245 alleles were read by ExpansionHunter software. The distribution of more than half of the short tandem repeats sizes were significantly different from other ethnic groups (p<0.05). Especially, the number of repeat in XYLT1 gene is higher than other ethnic groups (but still in the normal range). Conclusion: ExpansionHunter software is a predictive tool with promising, but it may be considered that if it used to be a diagnostic tool. In our study, although the ExpansionHunter can detected those who had oversize with short tandem repeats, but their <STR>data compared with GeneScan(PCR) data were not accurate, it would need to be corrected. In this study, ExpansionHunter software was used to read WES data to realize the distribution of short tandem repeats sizes in the Taiwanese population, and to know that the distribution of the short tandem repeats sizes in some genes are different from other populations. Therefore, it must be better if the short tandem repeats sizes distribution of various populations can be established in the future . Looking forward to more and more researchers can develop better methods or tools to detect the number of short tandem repeats, in order to achieve precision medicine, and improve the health of the population.