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研究生:劉郁芬
研究生(外文):Yu-Fen Liu
論文名稱:原發性股骨頭缺血性壞死之分子遺傳學研究
論文名稱(外文):MOLECULAR GENETICS STUDY OF IDIOPATHIC AVASCULAR NECROSIS OF THE FEMORAL HEAD
指導教授:蔡世峰蔡世峰引用關係
指導教授(外文):Shih-Feng Tsai
學位類別:博士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:136
中文關鍵詞:股骨頭缺血性壞死基因型檢定基因連鎖分析
外文關鍵詞:avascular necrosis of the femoral headgenotypinglinkage analysis
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本論文乃講述利用「基因連鎖分析法」以尋找、定位及分離「原發性股骨頭缺血性壞死」之致病基因。
首先,我們利用「基因排除法」以測試凝血因子protein C,protein S, 及抗溶血因子plasminogen activator inhibitor (PAI)等三個候選基因是否為「原發性股骨頭缺血性壞死」之致病基因。雙點(two-point)及多點(multipoint)基因連鎖分析之結果顯示, 位在這些候選基因座區域的微衛星標記(marker)之LOD 值皆小於-2,因此我們可以排除這三個參與血栓形成及溶解之基因與此疾病之關連性。
進一步,我們利用「全基因體掃描法」(genome-wide scan)將「原發性股骨頭缺血性壞死」之致病基因在染色體上的位置加以定位。第一階段,我們針對第一個家族進行四百個微衛星標記的基因型檢定(genotyping)。雙點及多點基因連鎖分析之結果顯示,位於標記D12S345及D12S326間之33 cM的區域其LOD值皆大於3,而標記D12S85之LOD值更高達3.45 (q=0)。第二階段,我們利用更高密度的微衛星標記,並合併第一及第二個家族進行分析,以期將33 cM之候選區域加以縮窄定位。結果顯示,標記D12S368在雙點基因連鎖分析中具有最高的LOD 值 (5.22 at q=0.10)。而在假設「致病基因之外顯性與年齡相關」之模式下所進行之多點基因連鎖分析顯示,位於標記D12S1663及D12S85之區域其最大的LOD值高達6.43。因此我們推論,位於染色體12q13,在標記D12S1663及D12S1632間之15-cM的區域,乃「原發性股骨頭缺血性壞死」之致病基因所在的位置。
接著,我們利用「候選基因推論法」及基因定序之策略搜尋染色體12q13之區域,以期尋獲致病基因。基因定序之結果顯示,在三個罹患「原發性股骨頭缺血性壞死」之家族中,所有的患者皆帶有一個位於第二型膠原蛋白(COL2A1)重複序列(Gly-X-Y)結構上,由甘胺酸(glycine)轉換為絲胺酸(serine)之胺基酸變異。在第一個家族中,COL2A1基因之變異發生在第五十個外顯子(exon),相對於此基因之傳訊RNA (mRNA)上第3665核苷的位置 (參考序列為NCBI NM_001844)。此一由核苷G 轉換為A之變異造成第二型膠原蛋白之第1170個胺基酸的改變(參考序列為NCBI NP_001835)。在第二個家族中,亦發現如在第一個家族中所偵測到之位於第五十個外顯子的突變點。而第三個家族則帶有一個位於第三十三個外顯子,由核苷G 轉換為A之COL2A1基因的突變。這個突變位於此基因之傳訊RNA上第2306核苷的位置 (參考序列為 NCBI NM_001844),導致第二型膠原蛋白之第717個胺基酸由甘胺酸轉換為絲胺酸(參考序列為NCBI NP_001835)。此外,我們發現在COL2A1基因啟動子(promoter) 上–767的位置,具有一個由核苷C轉換為A之單一核苷的多型性變異(SNP)。而偶發性股骨頭缺血性壞死(sporadic ANFH)的病人與正常人的對照組中,帶有此一單一核苷的多型性變異的比率,在統計學上具有顯著差異。
綜合以上結果,我們推論COL2A1基因序列之變異與遺傳性及偶發性的「股骨頭缺血性壞死」有關連。本研究之發現不但有助於了解此一骨頭疾病的致病機轉,未來甚可藉由篩檢COL2A1基因序列變異之有無,提供罹患「股骨頭缺血性壞死」的病人更及早的診斷,以設計更有效的預防或治療的方式。
This thesis describes a linkage study for mapping the disease locus and gene cloning for avascular necrosis of the femoral head (ANFH).
First, the exclusion study was aimed at testing a linkage between three candidate genes: protein C, protein S and plasminogen activator inhibitor (PAI) and familial ANFH Pedigree I. Both two-point and multipoint linkage analysis showed a LOD score less than –2, which excluded the existence of linkage between the family and three genes related to thrombophilia and hypofibrinolysis.
Secondly, a whole genome scan was conducted in two phases to map the ANFH disease gene locus. Candidate regions were initially identified by genotyping with a marker set of moderate density in Pedigree I. A significant two-point LOD score of 3.45 at q=0 was obtained for marker D12S85 and multiple-point linkage analysis revealed significant LOD scores (LOD >3) in a 33-cM interval spanning the markers D12S345 and D12S326. Regions showing significant LOD score were then inspected with high-density markers for Pedigree I and II. Replicated linkage analysis gave a combined maximum two-point LOD score of 5.22 (q=0.10) for the marker D12S368. A maximum combined multipoint LOD score of 6.43 was produced between D12S1663 and D12S85 under an age-dependent penetrance model. Thus, the ANFH disease locus was mapped to a 15-cM region between D12S1663 and D12S1632 on chromosome 12q13.
Thirdly, a positional candidate gene approach was applied in order to isolate the gene(s) associated with idiopathic ANFH. Re-sequencing of the type II collagen (COL2A1) gene demonstrated a glycine with serine mutation in the G-X-Y repeat of type II collagen, in all affected individuals in three pedigrees. In the Pedigree I, a 3665G >A mutation in exon 50 of the COL2A1 gene (RefSeq NM_001844) and the substitution resulted in a Gly1170Ser codon change (RefSeq NP_001835). A second pedigree was shown to harbor the same mutation but the mutant allele existed in a different haplotype background. In a third pedigree, a 2306G>A mutation occurred in exon 33 of the gene (RefSeq NM_001844), causing glycine to serine change at codon 717 (RefSeq NP_001835). Furthermore, a single nucleotide polymorphism (SNP) at position –767(C>A) of COL2A1 in the promoter region was detected, which displayed significant difference in allele frequency between sporadic ANFH patients and healthy controls (p= 0.0005).
In conclusion, the data from the genetic analysis of familial and sporadic forms of ANFH indicate that sequence variations in the COL2A1 gene are associated with ANFH. This finding will provide new insight to study the molecular mechanism (s) of ANFH pathogenesis and, possibly, lead to better management of ANFH patients.
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