建立以濾紙血片檢測裘馨氏肌肉失養症基因的方法

Abstract

背景 裘馨氏肌肉失養症（Duchenne muscular dystrophy, DMD）是一種X染色體性聯隱性遺傳的進行性肌肉萎縮症，全世界發生率約每5000名男性新生兒中即有1例。多數患者在9至12歲時需依賴輪椅代步，並常因心肺併發症而在20多歲死亡。裘馨氏肌肉失養症和貝克型肌肉萎縮症（Becker muscular dystrophy, BMD）的傳統基因診斷常需要至少兩種以上的分析方法來檢測突變，既耗時又費力。鑑於巨大的肌肉萎縮蛋白基因（dystrophin gene, DMD gene）以及其複雜的突變模式特徵，DMD基因可視為高通量測序的潛在候選者。此外，基於證據顯示早期診斷有助於接受早期皮質類固醇治療而改善病程，以及近期基因治療的發明，DMD亦有機會被納入全國性新生兒篩檢疾病之中。我們希望建立一個實驗方案，利用新生兒篩檢濾紙血片中萃取出理想品質與數量、不需經擴增的去氧核糖核酸（deoxyribonucleic acid, DNA），經由目標區間次世代定序（targeted next generation sequencing）的技術成功進行肌肉萎縮蛋白基因分析。 材料和方法 我們收集由健康成年志願者、DMD確診患者和新生兒篩檢存檔濾紙血片組成的10個樣本。該研究分為兩個階段。第一階段為微調從濾紙血片萃取的DNA實驗步驟，及比較不同的DNA商業萃取試劑套組之純度及產量：Gentra Puregene Blood Kit（QIAGEN）、QIAamp DNA Micro Kit（QIAGEN）和QIAamp DNA Investigator Kit（QIAGEN）。第二階段包括調整標靶次世代定序實驗步驟來分析肌肉萎縮蛋白基因，以及測試各種市售樣本庫製備(library preparation)試劑套組，如KAPA DNA Library Preparation Kits (Kapa Biosystems, Roche)和ThruPLEX DNA-seq Kit (Rubicon Genomics)，以及各種市售標的外顯子組捕獲（target exome capture）套組，如SeqCap EZ Exome Library v2.0（Roche Nimblegen）和xGen Lockdown Probes（IDT），來優化實驗方案。 結果 萃取出DNA的初始濃度在2.11至131 ng/uL波動。使用QIAamp DNA Investigator Ki可獲得最高產量，平均值為26.29 ng/uL（範圍為3.48-131.00 ng/uL）。由A260/A280比率測試的純度平均值為1.92（範圍為0.74-1.99）。樣本庫的濃度在13.3至45.8 ng/uL之間變化。QIAamp DNA Investigator Kit和KAPA DNA Library Preparation Kits的組合可產生最高濃度的DNA樣本庫，平均值為35.14 ng/uL（範圍為17.2-45.8 ng/uL）。ThruPLEX DNA-seq Kit和KAPA DNA Library Preparation Kits的覆蓋深度分別為88.3×（範圍為32.4×-144.2×）和86.81×（範圍為22.0×-244.5×）。KAPA DNA Library Preparation Kits產出較低的PCR重複率（polymerase chain reaction duplication rate），平均值為0.86（範圍為0.7-1.7）。而比較兩組商業標的探針，xGen Lockdown Probes顯示有較高的覆蓋DMD外顯子組的效率，最高可達244.5×，亦可得到較低的平均PCR重複率（2.89，範圍為0.7-18.3）。然而，比對回exon上的讀序百分比（ on-target reads）在這兩種標的外顯子組捕獲技術中表現皆不佳，僅能達到10。 結論 我們成功建立一可靠診斷策略，利用QIAamp DNA Investigator Kit可由濾紙血片萃取出理想品質與數量、不需經擴增的DNA，接續使用KAPA DNA Library Preparation Kits及xGen Lockdown Probes製備樣本庫後，將可在Illumina MiSeq測序儀上有效鑑定DMD基因。

Background Duchenne muscular dystrophy (DMD) is an X-linked disorder with an incidence of approximately 1 in every 5000 male births worldwide. The majority of DMD patients become wheelchair-bound between ages 9 and 12 years and die at their early 20’s due to cardiopulmonary complications. The traditional genetic diagnosis of Duchenne and Becker muscular dystrophies requires two or more analytical methods to detect mutations and is cost-intensive and time-consuming. Dystrophin gene (DMD) is potentially an acceptable candidate for high throughput sequencing in view of its characteristics of the complex mutational spectrum and the largest size of the human genome. Moreover, based on the bright evidence that early corticosteroid treatment improves outcomes and on new genetic therapies that require early diagnosis for effectiveness, DMD has emerged as a promising candidate for the recommended universal screening panel. We demonstrated a protocol that unamplified DNA (deoxyribonucleic acid) extracted from archived DBS could yield adequate quality and quantity input DNA for DMD gene analysis by targeted exome sequencing. Material and method Ten subjects composed of healthy adult volunteers, patients with diagnosed DMD, and archived dried blood spots (DBS) samples were enrolled. This study was conducted into two phases. Phase I was composed of modification of steps of DNA extraction and comparison of the different commercial DNA extraction kits, Gentra Puregene Blood Kit (QIAGEN), QIAamp DNA Micro Kit (QIAGEN) and QIAamp DNA Investigator Kit (QIAGEN). Phase II consisted of adjustment of process of DMD gene analysis by targeted sequencing and testing of various commercially available library preparation kits, KAPA DNA Library Preparation Kits (Kapa Biosystems, Roche) and ThruPLEX DNA-seq Kit (Rubicon Genomics), and exome capture probes, SeqCap EZ Exome Library v2.0 (Roche Nimblegen) and xGen Lockdown Probes (IDT) for optimization of the protocol. Results The concentration of extracted genomic DNA were fluctuated from 2.11 to 131 ng/uL. The highest yields were obtained using the QIAamp DNA Investigator Kit with a mean of 26.29 ng/uL (range, 3.48–131.00 ng/uL). The quality evaluated by A260/A280 ratio was 0.74-1.99. The concentrations of the libraries were variable from 13.3 to 45.8 ng/uL. The highest concentration of DNA library was constructed by combination of the QIAamp DNA Investigator Kit and KAPA DNA Library Preparation Kits with a mean of 35.14 ng/uL (range, 17.2-45.8 ng/uL). The depth of coverage in ThruPLEX DNA-seq Kit and KAPA DNA Library Preparation Kits were 88.3× (range, 32.4×-144.2×) and 86.81× (range, 22.0×-244.5×), respectively. The low PCR (polymerase chain reaction) duplicate rate was yielded by KAPA DNA Library Preparation Kits with a mean of 0.86% (range, 0.7%-1.7%). The xGen Lockdown Probes revealed greater efficiency in covering the DMD exome with a maximum of 244.5× with lower mean duplicate rate (2.89%, range 0.7%-18.3%) than those with SeqCap EZ Exome Library v2.0. However, on target percentage were both poor in these two exome capture technologies and could only achieve 10%. Conclusion We demonstrated the use of QIAamp DNA Investigator Kit for DBS with Illumina MiSeq sequencers for targeted sequencing by KAPA DNA Library Preparation Kits and xGen Lockdown Probes is a reliable diagnostic strategy to identify DMD gene.