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系統識別號 U0026-1607201923502500
論文名稱(中文) 藉由次世代定序找出小兒原發性骨質疏鬆可能致病基因
論文名稱(英文) Identifying causative genes for pediatric primary osteoporosis through next generation sequencing
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所碩士在職專班
系所名稱(英) Institute of Clinical Medicine(on the job class)
學年度 107
學期 2
出版年 108
研究生(中文) 陳加祥
研究生(英文) Chia-Hsiang Chen
學號 S97031080
學位類別 碩士
語文別 英文
論文頁數 60頁
口試委員 指導教授-陳芃潔
口試委員-江伯敏
口試委員-陳珮君
中文關鍵字 原發性小兒骨質疏鬆  成骨不全症  次世代定序  Ehlers-Danlos症候群  先天性脊椎骨骨骺發育不全 
英文關鍵字 Osteogenesis imperfecta (OI)  Ehlers-Danlos syndrome (EDS)  Spondyloepiphyseal dysplasia congenita (SEDC)  Next generation sequencing (NGS) 
學科別分類
中文摘要 原發性小兒骨質疏鬆是指兒童在沒有營養不良及骨質流失的狀態下骨質密度不足或是容易骨折。發生原因可能是基因缺陷或不明原因,最常見是成骨不全症(Osteogenesis imperfecta,OI)。而這些疾病的病人有相似的外觀以及臨床表現,可能影響的基因很多,使作確切診斷困難。次世代定序是高通量的技術可以快速分析全基因,而一些成骨不全症的基因也通過此技術被發現.我們希望透過次世代定序找到其他原發性小兒骨質疏鬆相關基因。
在成大醫院追蹤的原發性小兒骨鬆病人納入此研究,採用全外顕子定序,探針選用Nextera Rapid Exome Capture kit 大小37 Mb可涵蓋人類外顯子區域。我們使用電腦程式整合跟原發性小兒骨鬆有關基因,並使用線上軟體PolyPhen 2,PROVEAN, SIFT及MutationTaster來預測單核甘酸多型性(single nucleotide variants,SNVs)對蛋白質功能的影響。
本實驗分析12個臨床診斷原發性小兒骨質疏鬆的病人,包含7個臨床診斷第三、四型成骨不全患者,2個臨床診斷Ehlers-Danlos症候群患者及3個臨床診斷先天性脊椎骨骨骺發育不全(Spondyloepiphyseal dysplasia congenita)患者。在COL1A1,ADAMSTS2及WNT1基因上發現新的突變點。總結來說全外顯子定序對於原發性小兒骨質疏鬆提供一個快速準確的分子診斷。
英文摘要 Pediatric primary osteoporosis is defined as decreased bone mineral density or bony fragility without inadequate dietary intake nutrients or secondary bone loss. Primary osteoporosis occurs due to an intrinsic skeletal defect of genetic or idiopathic origin. Osteogenesis imperfecta (OI) is the most common of the genetic conditions in pediatric primary osteoporosis but several other diseases were also known. OI is both genetically and clinically high heterogenous disease. Differentiate the cause is difficult. The next generation sequencing (NGS) is a kind of high-throughput technology which can analyze the whole genome quickly. Several new genes in OI were discovered via NGS but still other genes in OI are not identified. The aim of this study is to establish an accurate molecular diagnosis of pediatric primary osteoporosis and detect other unknown gene in OI.
Patients with pediatric primary osteoporosis diagnosed or followed at NCKU hospital were recruited as study group. Patients with mutation identified previously or subjects with secondary osteoporosis were excluded in this study. Whole exome sequencing was performed by capturing the fragmented genomic DNA with Nextera Rapid Exome Capture kit which covers ~ 37Mb exonic regions of the human genome. We used the integrated computing programs to call the genetic variants for genes associated with primary osteoporosis. Impact of single nucleotide variants (SNVs) on the functions of proteins was predicted by online software as PolyPhen 2, PROVEAN, SIFT, and MutationTaster.
Total 12 patients diagnosed as primary osteoporosis in 10 family (two siblings) were enrolled in this study, included 7 cases with type III and type IV osteogenesis imperfecta (OI), 2 case with Ehlers-Danlos syndrome (EDS) and 3 cases with Spondyloepiphyseal dysplasia congenita (SEDC). Novel mutations within COL1A1, ADAMTS2 and WNT1 were discovered in this study.
Whole exome sequencing panel provides a fast and accurate method to arrive at a molecular diagnosis in most patients with inherited pediatric primary osteoporosis.
論文目次 English Abstract I-II
中文摘要 III-IV
Acknowledgement V
Content VI
Table Contents VII
List of Figures VIII
Abbreviations IX
1.Background and introduction 1
1-1 Pediatric osteoporosis 2
1-2 Osteogenesis imperfecta (OI) 2
1-3 Ehlers-Danlos syndrome (EDS) 4
1-4 Spondyloepiphyseal dysplasia congenita (SEDC) 4
1-5 Specific aim 5
2.Material and method 6
2-1 Subjects 7
2-2 Variant filtering and exome sequencing 7
2-3 Sanger sequencing 7
3.Result 9
3-1 Patient and clinical diagnosis 10
3-2 Mutation analysis 10
3-2-1 COL1A1 10
3-2-2 WNT1 11
3-2-3 ADAMTS2 11
3-2-4 COL2A1 11
3-2-5 COL5A1 12
4. Discussion 13
5. Tables 18
6. Figures 39
7. Reference 56
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