粒線體基因中 D-loop 的高度變異區之分析

粒線體基因中 D-loop 的高度變異區之分析

 粒線體基因中 D-loop 的高度變異區之分析

 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 : 羅梅真 畢業時間 : 92 學 年度第 2 學期 指導教授 : 李宏謨 台北醫學大學 教授 曾嶔元 馬偕醫院 病理科主任

 台北醫學大學 兼任副教授

 粒線體 DNA 是細胞內獨立於核染色體外的 DNA 分子。相較於核染色體 DNA ，它具有母 系遺傳，呈環狀結構，拷貝份數多，及變異性大等特點。因此適合族群演化及刑事法醫 鑑識。過去的研究發現粒線體 DNA 在控制區，即 D 環上有較高的變異率， D 環的長度 約 1.4 Kb ，其上有高變異區（ hypervariable region ）呈現叢集現象。這些叢集區共分為 H VR-I, HVR-II, HVR-III （高度變異區 I, II, III ）。在許多報告中其叢集的起始點及終止點 不同。本實驗的研究計劃即是定義高變異（ hypervariable region ）區的區間。利用族群基 因變異統計計算出 HVR-I 之起點與終點為 nucleotide position (np) 16051-16362 其鑑別率最 好。基因歧異度 (allelic diversity) h =0.992 。 HVRII 之起點與終點為 np 52-309CC 其鑑別 率最好， h =0.983 。 HVR-III 的 h 值為 0.864 ，而且我們也算出 HVR-IV 的 h 值為 0.614

。

 當 D 環上有單點異質點 (Heteroplasmy) 存在時，尤其在親屬鑑定上容易產生爭議。因此 本研究想知道若有異質點存在時其親屬相似度（ likelihood ratio ）數值如何？我們想要建 立一個可信賴的數值 , 以判定當粒線體 DNA 在一個群體中有一點核苷酸不一樣時 , 其可 能為親屬之關係。由公式計算出本實驗中的一個家族（外婆、媽媽、兩個女兒、一個兒 子），他們在 np 204 存在異質點，其 likelihood ratio 為 1.78×105 。。我們在外婆及二女 兒的 DNA 序列看不出來具有異質點存在，事實上經由 dHPLC 證明他們都是異質點。因 此我們可以計算親屬相似度及 dHPLC 來輔助法醫鑑定工

Analysis of hypervariable regions in the D-loop of mitochondrial genome

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Analysis of hypervariable regions in the D-loop of mitochondrial genome Author: Mei-chen Lo

Thesis advised by: Horng-Mo Lee, Ph.D., Chin- Yuan Tzen M.D., Ph.D.

The displacement loop (D-loop) of the mitochondrial DNA (mtDNA), approximately 1.4 kb in length, is a noncoding control region. mtDNA is maternally inherited, and exhibits high degree of homoplasmy. Heter oplasmy has been observed to be an intermediate condition in which new mutations are in the process of se gregation to homoplasmy through genetic drift after relatively few generations. The control region consists of three hypervariable regions; HVR-I HVR-II and HVR-III. However, the specific ranges of these HVRs ﹑ have never been defined. We analyzed the mtDNA HVR-I from 1762 unrelated individuals and precisely d efined that the HVR-I. Ranged from nucleotide position 16051 to 16399 for HVR-I with genetic diversity was 0.941-0.999. Ranged from nucleotid position 52 to 309CC for HVR-II with genetic diversity was 0.95 0-0.993. The values for HVR-III with genetic diversity was 0.864. In this material, HVR-IV was investigat ed, the values for HVR-IV with genetic diversity was 0.614.

The presence of a heteroplasmic site may complicate sequence analysis for forensic purpose when two sam

ples were compared. We analyzed the hypervariable region of the displacement loop (D-loop) in a family

with five individuals, i.e., grandmother, mother, one son and two daughters. The result showed a heteroplas

mic site at the np 204, which located in hypervariable region II. The nucleotide at this position was predom

inately cytosine in some samples and predominately thymine in others. Using Bayesian inference to assess

the significance of the mother-offspring pairs, the likelihood ratio was 1.78×105. The ratio is lower than pr

evious report. We conclude that dHPLC analysis is a sensitive and specific method to detect heteroplasmic

mtDNA mutation. The chromatogram shows a heteroplasmy at np 204 in this family. This study demonstra

ted that heteroplasmy is a common occurrence in tissue from normal individuals, and should be considered

in forensic cases where two samples appear to differ at a single nucleotide position by direct sequencing.