STR-HLA配型試驗於胚胎著床前基因診斷(PGD)之運用

Abstract

胚胎著床前基因診斷(PGD)配合HLA配型試驗進行雙重篩檢胚胎，是近年來新興之基因檢測技術，其目的是幫助具有急迫致病之遺傳疾病家族，渴望生一個不具此遺傳疾患的小孩，且同時可以臍帶血或骨髓移植治療罹病之兄姐；礙於時間之急迫及檢體DNA之微量，我們必須研發出更快速準確之技術方可成功。統之HLA配型試驗無法對微量細胞之DNA做檢測，本研究是利用檢測遍佈HLA之短序列重複片段 (STR)來進行連鎖分析 (Linkage analysis)，因不同族群會有不同之HLA-STR基因頻率及盛行率，所以當務之急先篩選17個STR標誌檢測台灣族群之盛行率，隨機選取台灣族群100個檢體進行HLA-STR分析，統計其異質性比率 (Heterozygosity rate) 及對偶基因頻率 (Allele frequency)，利用異質性比率較高之至少八個STR標誌進行單一淋巴細胞之檢測得到良好的分析結果，將此17個STR標誌依其異質性高低及PCR的條件予以分組，分成四組 (Panel A-D)，方便於進行胚胎著床前基因診斷(PGD)時可快速精確之應用。於單一細胞之放大過程中經常發生Allele drop-out (ADO) 的情形，即只有一股被放大的困擾，本研究同時利用HLA-STR來檢測ADO的比率，而微量細胞放大技術之研發亦可改善ADO的情形，本實驗使用全基因體放大技術 (WGA)，可大量放大此單一細胞DNA，並可重複檢測不會互相干擾，且目前胚胎著床前基因診斷(PGD)之胚胎切片技術除了單一胚葉細胞之抽取外，另可以進行囊胚期切片技術抽取較多細胞，所以比較單一淋巴細胞與五顆淋巴細胞在進行全基因體放大技術 (WGA) 後，利用HLA-STR檢測其ADO比率各為34.0%、20.9%，有明顯減少之情況；以傳統放大技術Nested PCR進行單一淋巴細胞與五顆淋巴細胞之倍增，再利用HLA-STR測其ADO比率各為45.4%、27.2%，根據這些結果顯示以全基因放大技術 (WGA) 進行HLA-STR配型試驗於微量細胞之檢測是可行的，且囊胚期切片技術抽取較多的細胞數 (約五顆細胞) 可大大增加檢測之準確度，是未來發展之趨勢，利用此技術平台之建立，可實際應用於胚胎著床前基因檢測，達到最準確之檢測結果。

Preimplantation HLA matching has recently emerged as a tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder . DNA in single blastomeres removed from 8-cell embryos by embryo biopsy following in vitro fertilization (IVF) was analyzed for short tandem repeats (STR) in the HLA region to select and transfer only those embryos that were HLA matching to affected siblings .n this study , the allele frequency and the prevalence rate of seventeen short tandem repeats (STR) makers which are located in HLA complex region (6p21.3) in 100 Taiwan individuals were analyzed . The selected STR makers displayed high heterozygosity, broad distribution of alleles and identifiable allelic size ranges ; thus , obtained the accurate result for a single lymphocyte . he allele drop-out (ADO) remains a significant problem for single cell PCR and diagnosis of single-gene disorders . Biopsy is done by removing one blastomere from a cleavage-stage embryo having 6-8 cells , and can also be done at the blastocyte stage , involving the removal of multiple trophectoderm cells . In the second part of this study , we further evaluate the performance of the newly developed whole genome amplification (WGA) in DNA amplification and compared with the traditional Nested PCR strategy . The ADO rates of whole genome amplification (WGA) for a single lymphocyte and five lymphocytes were 34.0% and 20.9% , accordingly . The ADO rates of Nested PCR for a single lymphocyte and five lymphocytes were 45.4% and 27.2% , accordingly . Our data suggested that diagnostic accuracy for DNA analysis in minimal cell would be benefit significantly from the biopsy of larger number of cells (from one cell to 5 cells) and the performance could be further improved by using whole genome amplification (WGA) technique .