粒線體 DNA 變異與嬰兒猝死症候群相關性之研究
Mitochondrial DNA Variations in Sudden Infant Death Syndrome
中文摘要 論文名稱： 粒線體 DNA 變異與嬰兒猝死症候群相關性之研究 研究所名稱：台北醫學大學生物醫學技術研究所 研究生姓名：黃純英 畢業時間：九十四學年度第二學期 指導教授：高淑慧助理教授 生物醫學技術研究所嬰兒猝死症候群（Sudden Infant Death Syndrome, SIDS）至目前為止，確切的原 因仍不明，回溯許多文獻報告，SIDS 的發生可能與粒線體基因（mitochondrial
DNA, mtDNA）突變有關，本篇研究即針對嬰兒猝死症候群（SIDS）、非嬰兒猝
死症候群（non-SIDS）及正常對照組（live health control）探討粒線體 DNA 變異 與 SIDS 之間的相關性。另外也探討 DNA 修補機制有關的酵素 hOGG1 基因多型 性與 SIDS 之間的相關性。本研究中我們收集 7 位 SIDS 及 19 位 non-SIDS 的個 案。死亡個案採取血液、骨骼肌及心肌組織，正常對照組則採取正常嬰兒的唾液 檢體（buccal epithelial sample），利用聚合?鏈鎖反應（polymerase chain reaction,
PCR）、Long-extension PCR、Primer-shift PCR 及即時定量 PCR（Real-time
Quantitative PCR ）技術，分析 mtDNA hypervariable region I（HVR I）、HVR II
變異情形、mtDNA 斷損突變（large-scale deletion）及相對 mtDNA 拷貝數（relative
amount of mtDNA copy number）。實驗結果發現，SIDS、non-SIDS 及 live health
control 三組之 mtDNA HVR I、HVR II DNA 序列變異情形，包括鹼基的取代（DNA
substitution）種類、位置及數目並無顯著差異。於本研究中檢測有三種 mtDNA deletion 於 SIDS 及 non-SIDS 檢體中，分別為 4977 bp、5335 bp 及 7599 bp deletion。live health control 的 buccal epithelial sample 中僅發現一位有 7599 bp deletion。4977 bp deletion 僅出現在一位 non-SIDS 死於先天性心臟畸形的心肌樣 品中。比較 SIDS 於不同組織的 mtDNA deletion 發生率，發現骨骼肌的 5335 bp deletion 發生率高出心肌及血液有 1.5 及 2 倍，另骨骼肌的 7599 bp deletion 發生 率高出血液及心肌有 1.6 倍。比較 SIDS 與 non-SIDS 之間 5335 bp 及 7599 bp deletion 發生率的差異，發現 SIDS 血液 5335 bp deletion 高出 non-SIDS 血液 4 倍， SIDS 骨骼肌 5335 bp deletion 高出 non-SIDS 骨骼肌 1.8 倍。SIDS 血液中 7599 bp deletion 高出 non-SIDS 血液有 2 倍。SIDS victims 於血液及骨骼肌的 multiple deletion 發生率高出 non-SIDS 血液及骨骼肌有 4 倍及 2 倍。利用費雪精準檢定 (Fishers＇ Exact Test)比較 SIDS 與 non-SIDS 之間於血液、骨骼肌及心肌的 mtDNA deletion，發現並沒有顯著性差異(p>0.05)，其中骨骼肌 5335 bp deletion 之
p 值為 0.093，趨近於 0.05。統計 SIDS 與 non-SIDS 於血液、骨骼肌及心肌 multiple deletion 的比率，兩組之間亦沒有顯著性差異，其中骨骼肌之 p 值為 0.085，趨近 於 0.05。SIDS victims 骨骼肌與心肌的相對 mtDNA copy number 均高於 non-SIDS victims 約 0.4 倍，但 SIDS victims 其血液的相對 mtDNA copy number 反而比 non-SIDS victims 低。比較 SIDS 與 non-SIDS victims 在血液、心肌及骨骼肌中相 對 mtDNA copy number，並無顯著性差異，p 值分別為 0.25、0.28 及 0.14。SIDS 的骨骼肌及心肌比 non-SIDS 有較高的 deletion 發生率及較高的相對 mtDNA 數 目，此現象可能係因帶有 mtDNA 缺損的粒線體因生物能量不足，所產生的補償 機制，使粒線體增殖，mtDNA copy number 相對的升高。而 SIDS group 血液的 mtDNA deletion 發生率高於 non-SIDS，但相對 mtDNA 數目卻比 non-SIDS group 低，此種不同於骨骼肌及心肌代償作用的結果，是否與 mtDNA deletion 引發 apoptosis 作用於骨髓幹細胞有關，仍有待進一步研究。另外，分析 SIDS、non-SIDS 及 live health control 的 hOGG1 基因型，SIDS victims 的 1245G allelic frequency 為 0.786，較 non-SIDS 的 0.611 及正常對照組的 0.450 高，比較 SIDS 與 live health control 及 non-SIDS 與 live health control 三種 hOGG1 基因型，發現並沒有顯著 性差異，p 值分別為 0.062 及 0.246。雖然 SIDS 及 non-SIDS 與 live health control 之唾液檢體來源不同，但整體而言 SIDS 及 non-SIDS 之斷損突變發生率比 live health control 高出許多倍。mtDNA 斷損除了使細胞內的生物能量產生危機外， 也可能會促進細胞 apoptosis。過去的研究指出許多 SIDS 在腦幹的病理檢查發現 有 apoptosis，而這些 apoptosis 被認為與缺氧有關。綜合實驗結果，我們的結論 認為 mtDNA deletion 本身並非直接與 SIDS 的死因有關係，但或許可能當嬰兒處 於發育階段的脆弱時期，由於 mtDNA deletion，嬰兒易受到能量不足或缺氧的間 接的作用下，導致嬰兒有猝死發生的傾向。
英文摘要
The Sudden Infant Death Syndrome (SIDS) is one kind of leading cause of
postneonatal infant death which also rises a difficult problem between the legal and medical systems. SIDS is defined as the sudden death of an infant less than 12 month old that remains unexplained after a complete clinical review, autopsy and death scene investigation. Despite the fact that many hypotheses have been proposed, the causes of SIDS are still uncertain. Although a number of coding region mtDNA mutations involving SIDS have been reported, the role of mtDNA variations in SIDS victims is still not known. The purpose of this study was to investigate whether the mtDNA variations or large-scale deletion exert any effect on the etiology of SIDS. Moreover, the biologic significance of the hOGG1 1245C"G polymorphism for SIDS has not yet been elucidated. The polymorphism of the hOGG1 gene was assessed by using a PCR-restriction fragement length polymorphism (RFLP) method. Seven SIDS and nineteen non-SIDS victims were enrolled. We determined the relative amount of
mtDNA copy number and the occurrence of mtDNA deletion in blood, skeletal muscle and cardiac muscle from both SIDS and non-SIDS group using real-time quantitative PCR, primer-shift PCR analysis and DNA sequencing. Buccal epithelial samples from twenty age-matched live health control subjects were also examined. In this study we also explore the DNA sequence variation of D-loop hypervariable region I(HVR I) and HVR II. D-loop sequence of all subjects were sequenced and compared with the Cambridage sequence. The mean number of substitutions in HVR I and HVR II between SIDS, non-SIDS and live health control were no significant difference. Three types of mtDNA deletions were found in this study such as 4977, 5335 and 7599 bp deletion. The breakpoints of deletion were identified by sequencing method. In live health control group only one subject was found with 7599 bp
deletion from buccal epithelial sample. Only one specimen was found with 4977 bp mtDNA deletion from cardiac muscle in congenital heart malformation subject. The frequencies of occurrence of 5335 and 7599 bp deletions in SIDS and non-SIDS were much higher than live health control group. There were no statistically significant difference associated with the frequencies of occurrence of 5335 bp and 7599 bp mtDNA deletions between SIDS and non-SIDS victims was found. The frequency of occurrence of 5335 and 7599 bp mtDNA deletion in blood from SIDS were four and two fold to non-SIDS, respectively. The frequency of occurrence of 5335 bp mtDNA deletion in skeletal muscle was 1.8 fold to non-SIDS. No significant correlation between the relative amount of mtDNA copy number and the frequencies of occurrence of mtDNA deletions in SIDS and non-SIDS. The allelic frequency of hOGG1 1245G was 78.6 % in SIDS, 61.1 % in non-SIDS and 45.0 % in live health control. The genotypic frequencies were no statistically significance between SIDS and live health control(p=0.062) and between non-SIDS and live health
control(p=0.246). These defects in mtDNA may result in impaired production of ATP or sensitized cells to apoptosis. We proposed that mtDNA deletions in themselves do not cause SIDS but may cause energy deficiency or hypoxia in stressful situation during a vulnerable developmental stage. So the primitive results of mitochondrial DNA deletion might predispose infant to death in critical situations.
