臺灣博碩士論文加值系統

摘要
嚼食後的檳榔渣存留有咀嚼者的口腔上皮脫落細胞，因此乃刑案現場之重要跡證。口腔上皮細胞為刑事檢驗及親子鑑定常用之檢體，醫用之口腔切片組織也被用在空難後之人別身分辨識與確認。但眾所週知的是嚼食檳榔會造成口腔癌及相關的口腔疾病，且腫瘤組織的基因組DNA序列常出現突變或不穩定的現象，究竟刑事所用的DNA標誌系統在口腔癌組織的穩定性如何？會產生哪些變異型態？而在未罹患口腔癌之嚼食檳榔者的口腔細胞是否有可能產生前期性的口腔傷害，而致使這些刑事鑑定所用的多種基因座的基因穩定性受到挑戰。此一穩定性的調查與確認，直接關係著人別鑑識結果是否誤判，因此就刑事應用而言，是非常有必要，也是本研究的主要目的之一。另一方面，從嚼食後的檳榔渣無法萃取出咀嚼者的DNA，因此本研究的另一個目的為發展出一套有效的萃取方法，以解決實務警察機關所面臨的困難。
受測者分為三群，口腔癌患者100人，嚼食檳榔者100人，無嚼食檳榔習慣者100人(作為控制組)，合計300人。蒐集其口腔細胞與血液細胞，萃取DNA，以商業化之鑑驗盒配合DNA 自動定序儀與其分析軟體來比對同一人的口腔與血液細胞內的基因標誌，最後再以統計(ANOVA, x2, Scheffe test)分析計算、比較所有出現變異之基因座。所分析的基因座均為現今刑事實驗室常用的基因標誌，主要有兩種商業化之鑑驗盒，其一為AmpliType HLA-DQA1/PM multiplex system，包含HLA-DQA1及PM(LDLR，GYPA，HBGG，D7S8，GC)合計6個基因。另 一為AmpFlSTRTM multiplex system, 包含9個STR基因座(D3S1358、vWA、FGA、TPOX、TH01、CSF1PO、D5S818、D13S317、D7S812)與一個性別基因。而在檳榔渣之人類DNA的萃取方法研發方面，則先比較分析三種傳統方法(salt/chloroform, 5% chelex-100 resin, QIAamp)萃取存放八週之模擬檢體所得到的結果，並根據其分別的萃取原理找出失敗的可能原因，再以化學藥劑(PVP與CTAB)結合抑制檳榔渣中的多酚類與多醣類物質之策略來研發萃取方法。
在口腔癌檢體方面，共發現兩種DNA變異型態，其一為major allelic imbalance；另一為allelic alterations (又分為expansion, contraction, un-classified三種亞型)。整體不穩定率為33%，兩種變異型態之嚴重度均與病理期成正比例。長度多型之標誌總共發生44個major allelic imbalance與25個allelic alterations，序列多型之標誌僅發現一個基因不吻合。而在嚼食檳榔者之檢體方面，長度多型之標誌發現兩個基因座出現major allelic imbalance，但仍能清楚辨識為異質型組合；序列多型標誌則全部吻合。因此，對於口腔癌檢體而言，序列多型標誌比長度多型標誌穩定；而對於嚼食檳榔者之檢體而言，兩種系統均可使用。統計分析結果則顯示：口腔癌檢體之九個STR基因座的allelic imbalance平均值與其對應的控制組有顯著差異 (p value 均小於0.05 )，但基因座彼此之間則無發生率之顯著差異(p value = 0.19)；口腔癌患者中之嚼食檳榔因素方面，嚼食者在兩種變異型態均比未嚼食者出現顯著差異之高發生率，但癌組織所發生的變異型態種類則不受嚼食檳榔因素所影響(p value 分別為0.15與0.57)。另一方面，所研發的”PVP/CTAB萃取法” 對於陳舊之檳榔模擬檢體與存放四年之50個刑案檳榔檢體，其成功率分別為100%與92%，因此本法對於咀嚼後檳榔渣人類DNA之萃取確實有效。

Abstract
Chewed betel-quid (BQ) residues are often considered vital biological evidence at crime scenes, since the human DNA extracted from the residues is actually from buccal epithelial cells and can be associated with suspects. However, BQ-chewing is also a risk factor for oral diseases and/or cancers. Although archived medical oral-specimens can be used to identify specific individuals under adverse conditions, STR markers are known to be unstable in various tumor tissues. On the other hand, the forensic analysis of BQ evidence has been hindered by failures in extraction of human DNA for PCR analysis. Therefore, it is a prerequisite for relevant forensic casework to establish a reliable method for extracting DNA from chewed BQ residues.
The stability of forensic marker systems of both sequence and length polymorphism was first investigated and then used to evaluate the forensic appropriateness of the oral samples of both healthy betel quid (BQ) chewers and the archived clinical specimens from oral cancer patients.
The analyses were performed on buccal samples from 100 BQ-chewers and 100 oral cancer patients, and corresponding peripheral blood samples from the same subject. A group of 100 non-BQ-chewers were used as the control. The genotypes of oral and paired peripheral blood samples were compared, using the commercialized typing systems of HLA-DQA1, PM (including LDLR, GYPA, HBGG, D7S8, and GC loci), and AmpFlSTRTM markers (including 9 STR loci and the Amelogenin gene). As for the development of human DNA extracted from chewed BQ-residues, three conventional methods (salt/chloroform, 5 % Chelex-100 resin, and QIAamp) were first tested for extraction of human DNA from 33 mock BQ samples, which had been stored for less than two months, and 50 four-year old forensic BQ samples. PCR amplifications from the HLA-DQA1&PM and the STR loci were then used to test the quality of the extracted DNA.
In the group of 100 oral cancer patients, two types of DNA instability were found. They were major allelic imbalances, and allelic alterations including expansion, contraction and the un-classified types (can not be confirmed as the expansion or the contraction). The overall percentage of the cancerous subjects demonstrating DNA instability was 33% (5 patients possessing both types of DNA instability). Both types of DNA instability showed a tendency of increasing with the severity of the pathological stage of oral cancer. Forty-four occurrences of major allelic imbalance were found in 21 cancer patients. The statistical result revealed that there was no significant difference in the allelic imbalanced occurrence among the nine STR loci. Allelic alterations were found in 17 patients, within which 12 individuals had the expansion, 5 had the contraction, and 3 had the un-classified type. Further, among these 17 patients, 3 were found to acquire multiple allelic alterations at multiple loci. For the DNA marker of sequence polymorphism, one discordant result at D7S8 was found in the 600 DQA1/PM-marker loci. The 100 healthy BQ-chewers had consistent oral swab and paired blood sample genotypes analyzed with both DQA1/PM and STR marker systems, although two loci with major allelic imbalance were detected. However, the two imbalanced alleles were virtually half lost, and could still be recognized as heterozygous alleles. The statistical results of ANOVA, x2, and Scheffe tests indicated that the means of allelic imbalance at the 9 STR loci of the oral cancerous group revealed a significant difference from those in the control group.
The findings herein suggest that when cancerous specimens were tested, the HLA-DQA1/PM system with point polymorphism appears more reliable than the STR system with length polymorphism. Our results also indicate that healthy BQ-chewers’ oral cotton swabs containing buccal epithelial cells are useful for forensic purposes using the HLA-DQA1, PM, and STR marker systems.
In order to solve the problem in PCR analysis of DNA from old BQ samples, we developed a DNA extraction method based on the use of polyvinyl pyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB), which bind to two common classes of PCR inhibitors in plants, polyphenols and polysaccharides, respectively. The result showed that this “PVP/CTAB” method is completely successful for the mock BQ samples, and 92% (46 out of 50) successful for the four-year old forensic BQ samples. To our best knowledge, this is the first report of a reliable method for the extraction of human DNA for PCR from chewed BQ residues. This method should provide a useful means for forensic identification in countries where betel-chewing is common.

DNA Alteration Analysis and Stability Evaluation of Forensic Marker Systems in Buccal Cell Samples
Chinese abstract…………………………………………………………3
English abstract………………………………………………………… 5
Chapter 1 DNA alterations at the STR loci in samples of oral cancer tissue and betel quid chewers’ oral epithelial cells ………. 7
Introduction…………………………………………………………..7
Materials and methods………………………………………………10
Results……………………………………………………………… 15
Discussion………………………………………………………….. 19
Summary…………………………………………………………… 31
References………………………………………………………….. 32
Chapter 2 DNA stability in HLA-DQA1/PM markers and genetic instability in STR markers: implications for forensic application………………………………………………...35
Introduction………………………………………………………….35
Materials and methods…………………………………………….…37
Results and discussion……………………………………………….40
Summary…………………………………………………………… 49
References………………………………………………………….. 50
Chapter 3 Extraction of human DNA for PCR from chewed residues
of betel quid using a novel“PVP/CTAB”method…………53
Introduction………………………………………………………..53
Materials and methods…………………………………………….55
Results and discussion……………………………………………..61
Summary…………………………………………………………..69
References………………………………………………………...70
Appendix
Part A Association analyses of monoamine oxidase (MAO) genes and sexually aggressive behavior ………………………72
Introduction………………………………………………………..72
Materials and methods……………………………………………..75
Results……………………………………………………..………79
Discussion…………………………………………………………..82
Summary……………………………………………………………93
References…………………………………………………………. 94
Part B Case-control study of catechol O-methyltransferase (COMT) polymorphisms in sexually aggressive behavior…………97
Introduction…………………………………………………………97
Materials and methods………………………………………………99
Results and discussion……………………………………………. 101
Summary…………………………………………………………..106
References…………………………………………………………107

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