- 學位論文
生殖細胞染色體基因轉位之偵測與分析
Detection and analysis of germ-line chromosomal rearrangements
摘要
人類的基因體序列被定序出來後，狗、小鼠、雞等物種的序列也隨之被定序完成。過去實驗室的生物資訊團隊，以人類基因體序列為參考序列，進行兩兩物種之序列比對。將完全一樣且大於50 bp的序列稱為Conserved Sequence Tag (CST)。再利用一些篩選的條件從CST中挑出比較大的片段稱為Synteny Identity Markers (SIMs)。接著，實驗室的生物資訊團隊利用Human-Dog (HD)、Human-Mouse (HM)和Human-Chicken (HC)三種SIMs，取其共同保留的片段稱為Evolutionary Frozen Spots (FS)。 本篇的主要分為兩部份： 第一部份是從上述的保留區段 (SIMs, FS)中挑出3個HD 的SIMs和7個FS。探討這些區段於其他物種間的保留程度。實驗共選用哺乳類、鳥類、爬蟲類、兩棲類、魚類等13個物種的基因體DNA (genomic DNA) 做材料。結果發現這些區段在不同物種間皆具有高度保留性，甚至在一些低等的脊椎動物中也被保留下來。 這些保留區段所屬的基因大部分都比較大且多半和發育有關。其中部份基因曾經被發現在體細胞中有基因轉位 (translocation)的現象，且常造成癌症。 第二部份，欲探討這些發生在體細胞的基因轉位是否也會發生於生殖細胞中。選用的研究對象為常造成嬰兒及孩童型癌症的EWSR1和MLL。當EWSR1發生基因轉位時常造成孩童型的骨癌，而MLL主要造成嬰兒及孩童型的血癌。 利用Inverse PCR (i-PCR)偵測在精蟲細胞 (sperm cells)中EWSR1及MLL是否發生基因轉位。實驗結果共發現14個基因轉位現象，包含5個EWSR1的基因轉位和9個MLL的基因轉位。EWSR1的5個基因轉位對象，分佈於5個不同的染色體上，其中2個座落於基因區 (genic region)，3個座落於非基因區(intergenic region)。而MLL的9個基因轉位對象，分佈於7個不同的染色體（第一條染色體和第七條染色體各有2個），其中4個位於基因區；5個位於非基因區。 分析其產生融合蛋白 (fusion protein)的可能性，發現其中有兩個基因轉位後的mRNA預測序列為in frame狀態。所以假使這樣的基因轉位發生於體細胞，則有表現融合蛋白的可能性。而其他mRNA預測序列為out of frame狀態的基因，則有可能造成表現出的蛋白為斷裂蛋白 (disrupted protein)。 進一步探討其造成基因轉位的機制，由於斷裂處兩邊的序列相似度不高，故初步排除同源性重組 (Homologous recombination) 的可能。分析週邊Alu的位置，未發現斷裂處同時座落於兩條染色體的Alu上。故推測這些基因轉位可能也不是由 Alu-mediated recombination造成的。另外，於14個基因轉位的斷裂處皆有發現1-12個相同的核甘酸同時存於兩條發生基因轉位的染色體上，此現象可能為非同源性末端接合 (Non-Homologous End Joining，簡稱NHEJ) 中常見的microhomology usage現象，故推測這些基因轉位有可能為NHEJ造成的。 從以上結果可知，以i-PCR技術偵測精蟲細胞中ESWR1和MLL兩個基因的確有發現基因轉位的現象。故發生於體細胞中的基因轉位也會發生於生殖細胞。 未來期望能偵測在不同人的精蟲細胞中基因轉位發生的頻率。並期望以此篇的技術與結果做為基礎，針對更多染色體上的不同位點，探討是否也於精蟲細胞中有發生基因轉位。最後，期望能連結精蟲細胞的基因轉位和嬰兒型和孩童型癌症的關連性，也希望將此篇結果應用於臨床的診斷。
並列摘要
When comparing the human reference genome with the genomes of dog, mouse and chicken, we identified sequences longer than 50 basepairs (bp), termed conserved sequence tags (CSTs), that are absolutely conserved. From the CST datasets we further defined the synteny identity markers (SIMs) and constructed the synteny maps between two vertebrate genomes. The extremely conserved DNA fragments in the vertebrated genomes examined are represented as frozen spots (FSs) when the overlapped human-dog (HD), human-mouse (HM) and human-chicken (HC) SIMs are longer than 50 bp. To address whether these elements are conserved in other species, we selected 3 HD SIMs and 7 FS, and examined their presence in other mammals, reptiles, amphibians and fish by the polymerase chain reaction (PCR) assay using the conserved human sequences as primers. Results from multispecies analysis indicate that these absolutely conserved genic and intergenic elements are well conserved in lower vertebrates, additional to other mammalian species. Each conversed vertebrate gene may portrait its own evolutionary history of purifying selction. Bioinformatics analysis revealed that the majority of FS-containing genes are large in size and have a role in embryonic development with transcriptional activities. Approximately 10% of the FS-containing genes have been implicated in cancer-associated chromosomal translocations. We therefore initiated the study of possible germline translocations in sperm. Human EWSR1 and MLL were used as the anchoring genes to interrogate germline chromosomal translocations by the inverse PCR method. Altogether in the male germ cells, we detected five translocation events of the EWSR1 gene juxtaposed with 5 different chromosomes and nine of the MLL gene with seven different chromosomes. Potentially, these germline translocations led to the disruption of six translocation partner genes (TPGs); two with EWSR1 and four with MLL. Close inspection of the juxtaposed genes suggests two potential chimeric fusion products, if they are expressed in somatic cells. Germline chromosomal translocation is unlikely to be mediated by either allelic or non-allelic homologous recombination, as we compared sequences flanking both sides of the translocation breakpoints. In the current study we identified short stretches of homologous nucleotides, 1-12 bp long, at all the breakpoints, which are present in both of the unarranged chromosomes. This observation suggests that microhomology usage of the non-homologous end joining (NHEJ) process may be mechanistically involved in the germline chromosomal translocation. This germline event may occur preferentially in the less conserved region of the genome, except for exonic translocations.