非致死性骨骼發育不全與致死性骨骼發育不全是常見的新生兒遺傳性疾病。其中，非致死性骨骼發育不全的發生率為1/4,000~5,000，致死性骨骼發育不全的發生率為0.95/10,000。FGFR2、FGFR3、COL1A1、COL1A2以及COL2A1基因若發生序列變異，則為已知造成非致死性以及致死性骨骼發育不全的原因。針對骨骼發育不全，目前使用的傳統基因檢測，為一次鑑定不同的基因或是外顯子的序列變異，因此，傳統的檢驗方法在臨床應用上是十分耗費金錢以及時間。隨著精準醫療逐漸被重視，偵測嚴重的遺傳性疾病，是需要以一種快速及價格合理的方法完成檢測。幸運的是，次世代定序在人類基因體計畫完成後成功發展，並且結合生資分析後，能夠達到以非侵入性的方法進行基因檢測，能夠在產前時期就偵測出遺傳性且具危害性的序列變異。在本篇研究中，我們目標是建立一個針對分布於5個常見影響骨發育不全基因的87個變異位點所設計的目標基因定序平台。首先，我們分析14個家庭的原始定序資料。在生資分析的結果中，能夠確認原始資料能夠符合我們所設定的標準，有80%的讀數是在目標區域，也有85%的位點的讀數會到達至少20%的平均深度的讀數。另外，在這14個家庭中，我們能夠在游離DNA中偵測到大約是4%的變異比例，並且推估在游離DNA中，胎兒的游離比例DNA為8%左右。此外，我們也能夠以1ng的游離DNA來執行文庫的製備，並且得到精確的變異位點判斷結果。另一方面，我們使用陽性檢體混合正常檢體模擬游離DNA狀態，以確認此平台的序列變異比率檢測最低限制。從數據中顯示，此平台能夠在給予總讀數為80萬的情況下，正確偵測出模擬2.5%游離DNA的位點變異，並且達到變異熱點的讀數能夠得到深度為50以上。此平台的敏感度及特異度皆為100%，此平台變異位點的敏感度及特異度分別為100%以及99.91%。最後，本研究的最終目的就是能夠將這個平台廣泛應用於臨床檢測，已達到產前預防及幫助治療策略的選擇。

Non-lethal and lethal skeletal dysplasia are common inherited neonatal disorders which the disease frequencies are approximately 1/4,000~5,000 and 0.95/10,000, respectively. Sequence mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 are known to cause non-lethal or lethal skeletal dysplasia. Traditional genetic screening of skeletal dysplasia was costly and time-consuming due to multiple-genes and -exons for mutation identification. With precision medicine initiative, a fast and yet cost-effective approach is needed to avoid the inheritance of severe disease. Fortunately, the advance of next-generation sequencing (NGS) technology, together with the bioinformatics analysis have been applied to noninvasive prenatal testing (NIPT) to detect the inherited and deleterious mutations in the fetal at the prenatal stage. In this study, we have established an amplicon-based target sequencing panel that covers 87 hotspots in 5 genes. At first, we tested the performance on 14 families. Results from bioinformatics analysis showed that the panel meets the criteria nicely. On average, an on-target rate of more than 80 % and a uniformity of more than 85 % were obtained. The performance data from 14 families showed that we can accurately call the paternal variant at ~8% of fetal DNA in the plasma and as little as 1 ng cell free DNA for library preparation was sufficient to gain reliable results. In addition, the test limit was assayed by spiking-in test and the results showed that while the total reads are about 0.8M, we can call the FGFR3 (c.1138G>A) variant at ~2.5 % of positive sample fraction in the pooling sample. Furthermore, a hotspots depth more than 4000x and alternative allele reads more than 50 were obtained. The sensitivity and the specificity of the panel are both 100% and of the variants calling are 99.91% and 100%, respectively. The ultimate goal of current research is to apply the skeletal dysplasia target-screening panel in the prenatal diagnosis to provide the best prevention and treatment strategies.

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