臺灣博碩士論文加值系統

背景
SDHD 及 SDHAF2 相關之遺傳性副神經節瘤有特殊的親源遺傳模式。當突變基因由父親遺傳給子代時才會形成腫瘤。這種特殊的遺傳模式可能與第 11 p15.5-15.4 在腫瘤中母源基因的缺失有關，過去已經有人使用熒光原位雜合(Fluorescent In Situ Hybridization)、異質性缺失分析(Loss of Heterozygosity Analysis)、微衛星標記(Microsatellite Marker)、單核苷酸多態性陣列(Single Nucleotide Polymorphism Array)等實驗方式證實。
目的
由於先前的研究方式在 11p15.5-15.4 染色體區域解析度較低。我們使用新的實驗方式，單核苷酸變異探針(Single Nucleotides Variation Probe)，此方式偵測在11p15.5-15.4 染色體區域是否有基因缺失及判定缺失的來源有較高的解析度。
方法
我們收集了 13 個 SDHD 相關之遺傳性副神經節瘤腫瘤和部分 SDHD 患者父母血液、23 個非 SDHD 相關之副神經節瘤腫瘤及 1 個 SDHAF2 相關腎上腺髓質增生(Adrenal Medullary Hyperplasia)檢體。使用單核苷酸變異探針合併次世代定序(Next Generation Sequencing)原理，對 12 個位於 11p15.5-15.4 的母源表現基因進行 1-8 個不同點位單核苷酸變異的序列分析。將患者父母血液及患者腫瘤的單核苷酸變異數據進行比對和判定，我們可以證明腫瘤檢體上是否有母源基因缺失。
結果
在 13 個 SDHD 相關之遺傳性副神經節瘤樣本中，有 12 個樣本的異質性不平衡率(Heterozygosity Imbalance Ratio) 超過百分之 75，只有一個腫瘤沒有異質性不平衡。在收集到父母血液或母親血液的 SDHD 家族中，有四位患者可判定百分之 80 以上腫瘤的基因缺失來自母親，且無證據顯示父親基因缺失。在 SDHAF2相關腎上腺髓質增生(Adrenal Medullary Hyperplasia)檢體中只有 H19 有異質性不平衡。在非 SDHD 相關副神經節瘤腫瘤檢體中，突變基因和假性缺氧 (Pseudohypoxia)有關的檢體，異質性不平衡率也較高。
結論
使用單核苷酸變異探針方式可以證實 SDHD 相關之遺傳性副神經節樣本在 11p15.5-15.4 區域有母源基因的缺失。H19 為可能造成父源遺傳模式的抑癌基 因。11p15.5-15.4 區域有母源基因的缺失與假性缺氧的致癌模式有正相關。

Background
Hereditary paraganglioma-related germline SDHD (succinate dehydrogenase D) and SDHAF2 (Succinate Dehydrogenase Complex Assembly Factor 2) exhibit a striking parent-of-origin effect. The mutant gene must be passed down from the father in order for tumor to occur. This particular pattern of inheritance may be related to the deletion of maternal fragments of 11p15.5-15.4 in tumors, which has been confirmed in the past studies by methods of fluorescent in situ hybridization (FISH), loss of heterozygosity analysis (LOH analysis), microsatellite marker and single nucleotide polymorphism array (SNP array).
Aim
The resolution of previous studies was low. For higher resolution of gene on chromosome 11p15.5-15.4, we designed a novel approach - single nucleotides variation probe.
Methods
We obtained blood samples from some SDHD patients' parents, specimens of 13 paraganglioma associated to SDHD through inheritance, 23 paraganglioma tumors unrelated to SDHD, and one adrenal medullary hyperplasia linked to SDHAF2.On 12 maternally expressed genes located at 11p15.5-15.4, the sequencing analysis of 1-8 single-nucleotide variations was carried out using the single nucleotide variation probe and next generation sequencing (NGS) method. We can establish whether there is a maternal gene deletion in the tumor sample by comparing and analyzing the single nucleotide variation data of the patient's tumor and the blood from the patient's parents.
Results
Only one tumor showed no heterozygosity imbalance among of the 13 hereditary paraganglioma samples related to SDHD, while 12 samples had a heterozygosity imbalance ratio (H/I Ratio) more than 75%. There were four patients who could determine that more than 80% of the gene deletions in the tumor came from the mother and there was no evidence of the father's gene deletion in the SDHD family whose parents' blood or mother's blood was obtained. In SDHAF2 related adrenal medullary hyperplasia (AMH), only H19 had loss of heterozygosity. The heterozygosity imbalance ratio (H/I Ratio) was higher in paraganglioma with mutant gene related to pseudohypoxia in Non-SDHD group.
Conclusions
Using a single nucleotide variant probe (SNP probe) strategy, it was determined that the SDHD related hereditary paraganglioma had maternal gene loss in the 11p15.5–15.4. The tumor suppressor gene H19 might result in paternal inheritance. Positive correlations were found between maternal gene deletions in the 11p15.5– 15.4 and pseudohypoxic oncogenic patterns.

口試委員會審定書 1
中文摘要 2
Abstract 4
目錄 7
第一章、Introduction 10
第二章、Methods 13
2.1 Study Design 13
Maternal expressed gene on 11p15.5-15.4 14
Select SNVs 17
Patient and samples 18
2.2 SDHD Carrier of Family 19
2.3 Single nucleotide variation probe analysis 21
2.4 Data Analysis 22
第三章、Results 25
3.1 Loss of Heterozygosity on Chromosome 11p15.5-15.4 25
SDHD group 25
Non-SDHD group 26
3.2 Maternal loss on chromosome 11p15.5-15.4 29
SDHD proband with parent blood DNA 29
SDHD Proband with mother blood DNA 32
3.3 Located the maternal expressed tumor suppress genes 34
SDHAF2 related adrenal medullary hyperplasia 34
Paraganglioma with chromosome 11 retention 35
Heterozygosity percentage 37
3.4 Loss of heterozygosity in 11p15.5-15.4 related to pseudohypoxia 37
第四章、Discussion 38
4.1 Review of Research Findings 38
H19 - a candidate tumor modifier 39
Single nucleotide variation probe – New methods 42
4.2 Limitations of our study 43
4.3 Implications of the Study 44
4.4 Recommendations for Future Research 45
4.5 Conclusions 46
References 47
Supplement 1. Independent T-test analysis of heterozygosity imbalance ratio of SDHD and Non-SDHD groups 51
Supplement 2. Family 1-3, 47 SNV probes analysis 52
Supplement 3. Family 4-6, 47 SNV probes analysis 53
Supplement 4. SDHD group, 47 SNV probes analysis, heterozygosity percentage 54
Supplement 5-1. Non-SDHD group, 47 SNV probes analysis, heterozygosity percentage (H/I ratio >0.55) 55
Supplement 5-2. Non-SDHD group, 47 SNVs probes analysis, heterozygosity percentage (H/I ratio <0.55) 56
Supplement 6. The H/I ratio of cluster 1A and 1B, Cluster 2 57
Supplement 7. 12 genes SNV coverage (base. pair) and coverage rate 58

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