粒線體基因中 D-loop 的高度變異區之分析 Analysis of hypervariable regions in the D-loop of mitochondrial genome

粒線體基因中 D-loop 的高度變異區之分析

Analysis of hypervariable regions in the D-loop of mitochondrial

genome

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 HVR-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 英文摘要

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. Heteroplasmy has been observed to be an intermediate condition in which new mutations are in the process of segregation 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 defined 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 0.950-0.993. The values for HVR-III with genetic diversity was 0.864. In this material, HVR-IV was investigated, 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 samples 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 heteroplasmic site at the np 204, which located in hypervariable region II. The nucleotide at this position was predominately 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 previous 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 demonstrated 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.