二代測(cè)序在胚胎植入前染色體疾病診斷中的臨床應(yīng)用
發(fā)布時(shí)間:2018-01-24 23:00
本文關(guān)鍵詞: 拷貝數(shù)變異 比較基因組雜交芯片 植入前遺傳學(xué)檢測(cè) 二代測(cè)序 染色體疾病 出處:《中國(guó)人民解放軍醫(yī)學(xué)院》2015年博士論文 論文類型:學(xué)位論文
【摘要】:研究背景染色體異常包括倍數(shù)改變、非整倍性改變、結(jié)構(gòu)改變和微小片段重復(fù)和缺失(≤10 Mb)等。染色體異常在臨床中與胚胎種植失敗、早期妊娠丟失、孕期引產(chǎn)甚至缺陷兒出生息息相關(guān),因此有效地開(kāi)展植入前遺傳學(xué)檢測(cè)(preimplantation genetic test, PGT)對(duì)保證不孕夫婦擁有健康的后代有重要意義。近十年,大多生殖中心采用芯片技術(shù),如比較基因組雜交芯片(array comparative genomic hybr i d i zat i on,aCGH)和單核苷酸多態(tài)性芯片(single nucleotide polymorphism array,SNP array),進(jìn)行PGT。近5年,二代測(cè)序(next generation sequencing, NGS),如拷貝數(shù)變異測(cè)序(copy number variation sequencing, CNV-Seq),是一種新興的前沿技術(shù),其臨床應(yīng)用逐漸開(kāi)展,并且在輔助生殖臨床工作中已經(jīng)證明對(duì)胚胎植入前遺傳學(xué)篩查(preimplantation genetic screening, PGS)和胚胎植入前遺傳學(xué)診斷(preimplantation genetic diagnosis, PGD)有重要作用。PGS主要篩查染色體非整倍性改變。同時(shí),臨床工作中由于一些小的片段的重復(fù)和缺失(≤10 Mb)導(dǎo)致產(chǎn)前診斷中發(fā)現(xiàn)異常進(jìn)而流產(chǎn)引起了我們的注意。目前,已經(jīng)有研究證明CNV-Seq在基因組水平診斷染色體疾病的價(jià)值,其在PGD和PGS中的價(jià)值也有探索,但很少有關(guān)于CNV-Seq在細(xì)胞水平的重復(fù)性、分辨率以及與染色體疾病相關(guān)的微小重復(fù)、缺失的檢測(cè)的報(bào)道。因此我們確立了本研究:探索CNV-Seq在PGT領(lǐng)域全面檢測(cè)染色體疾病中的意義。研究目的1.證實(shí)CNV-Seq在基因組水平診斷染色體疾病的價(jià)值及在33 pg稀釋基因組和單細(xì)胞水平診斷的穩(wěn)定性;2.明確CNV-Seq在細(xì)胞水平的重復(fù)性、診斷異常時(shí)拷貝數(shù)(copy number, CN) cut-off點(diǎn)和細(xì)胞水平的分辨率;3.建立胚胎植入前活檢的細(xì)胞模型;4.以CNV-Seq為主要診斷手段對(duì)尋求遺傳學(xué)診斷的不孕夫婦的胚胎進(jìn)行染色體疾病的全面檢測(cè)。研究方法1.在基因組水平,比較CNV-Seq和aCGH在檢測(cè)6個(gè)CNVs (6.52 Mb-93.02 Mb)的一致性,證實(shí)CNV-Seq在基因組水平診斷染色體疾病的價(jià)值。隨后進(jìn)一步降低模板量到33 pg稀釋基因組和單細(xì)胞,進(jìn)行全基因組擴(kuò)增(whole genome amplification, WGA),用產(chǎn)物作為測(cè)序文庫(kù)構(gòu)建的底物,探索33 pg稀釋基因組和單細(xì)胞水平診斷的穩(wěn)定性:2.選用上一部分在三個(gè)水平都準(zhǔn)確檢測(cè)到的6.52 Mb缺失和14.76 Mb的重復(fù)(aCGH診斷)作為研究樣本,用CNV-Seq為診斷手段在基因組水平進(jìn)行3次重復(fù),而后分別進(jìn)行15次五細(xì)胞和單細(xì)胞WGA產(chǎn)物重復(fù)測(cè)序,統(tǒng)計(jì)不同水平的兩個(gè)已知異常區(qū)域拷貝數(shù)值(copy number value, CN value),各個(gè)水平差異以均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)表示,采用SPSS 17.0軟件進(jìn)行分析,三組之間用單向ANOVA,每?jī)山M之間采用LSD-t檢驗(yàn)進(jìn)行分析,以α=0.05為檢驗(yàn)水準(zhǔn),明確CNV-Seq診斷異常的CN值的cut-off點(diǎn):3.選擇5份凍存的外周血,包含6個(gè)從0.56 Mb到5.78 Mb大小不同的CNVs(基因組水平CNV-Seq已經(jīng)明確診斷)作為細(xì)胞來(lái)源,分別用五細(xì)胞和單細(xì)胞為底物進(jìn)行WGA,并對(duì)其WGA產(chǎn)物進(jìn)行不同水平的三次重復(fù)測(cè)序,統(tǒng)計(jì)結(jié)果,進(jìn)一步探索CNV-Seq在細(xì)胞水平的分辨率;4.選擇中國(guó)人民解放軍總醫(yī)院生殖中心的有PGT指針且同意納入研究的7對(duì)夫婦的胚胎為觀察對(duì)象,以CNV-Seq為主要診斷手段進(jìn)行診斷,檢測(cè)包括染色體非整倍體、非平衡易位和微小CNVs ( 10 Mb的重復(fù)和缺失),從而指導(dǎo)選擇平衡胚胎進(jìn)行移植。結(jié)果1.基因組水平在診斷6個(gè)CNVs時(shí),CNV-Seq與aCGH診斷高度一致,同時(shí)異常區(qū)域上升和下降趨勢(shì)顯著,并且能夠提供精確的位置信息。而用33pg稀釋基因組和單細(xì)胞WGA產(chǎn)物后,CNV-Seq也成功檢測(cè)到異常區(qū)域;2. CNV-Seq對(duì)6.52 Mb缺失和14.76 Mb復(fù)制的檢測(cè),在3次基因組水平和30次細(xì)胞水平的重復(fù)中,均成功地檢測(cè)到異常區(qū)域。兩個(gè)異常區(qū)域在基因組水平、五細(xì)胞水平和單細(xì)胞水平的CN值的Mean±SD分別1.01±0.03與2.96±0.03(基因組DNA),1.16±0.06與2.75±0.10(五細(xì)胞),和1.13±0.10與2.69±0.13(單細(xì)胞)。SPSS 17.0軟件分析后,三組差異有統(tǒng)計(jì)學(xué)意義(p0.05),五細(xì)胞和基因組水平的差異有統(tǒng)計(jì)學(xué)意義(p0.05),單細(xì)胞和基因組水平的差異有統(tǒng)計(jì)學(xué)意義(p0.05),但兩個(gè)細(xì)胞水平之間的差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05):3.探索CNV-Seq在細(xì)胞水平的分辨率部分,針對(duì)6個(gè)從0.56 Mb到5.78 Mb大小不同的CNVs,分離單細(xì)胞和五細(xì)胞,每個(gè)水平和每個(gè)CNV分別重復(fù)3次,共36次目標(biāo)觀測(cè)檢測(cè),除4次0.56 Mb和1次2.32 Mb高度重復(fù)區(qū)域之外,其余異常區(qū)域成功檢出;4. 在臨床研究中,7對(duì)夫婦共有35個(gè)囊胚進(jìn)行活檢后的遺傳學(xué)診斷,其中18個(gè)為染色體非整倍性改變,1個(gè)為非整倍體合并4.98 Mb缺失(5q35.2-qter deletion,與Sotos syndrome相關(guān)),1個(gè)為6.66 Mb缺失(7pter-p22.1 deletion,與7pterminal deletion syndrome相關(guān)),14個(gè)沒(méi)有明確致病性的CNVs(可進(jìn)行胚胎移植)和1個(gè)因?yàn)閃GA擴(kuò)增失敗沒(méi)有得到結(jié)果。其中7對(duì)夫婦中,有5對(duì)至少有1個(gè)可供移植的胚胎。結(jié)論1. CNV-Seq在診斷染色體疾病方面與aCGH有較高的一致性,且在細(xì)胞水平診斷有很好重復(fù)性和穩(wěn)定性;2.成功構(gòu)建了胚胎活檢模型;3. CNV-Seq在細(xì)胞水平診斷異常時(shí)CN值cut-off點(diǎn)可能為2.6(2.6為重復(fù))和1.3(1.3為缺失);4. CNV-Seq有較高的分辨率(1.5Mb);5. CNV-Seq在PGT中有較強(qiáng)的診斷能力,涵蓋對(duì)非整倍體、非平衡易位和微小CNVs (≤10 Mb)相關(guān)的染色體疾病,可盡可能減少致病性胚胎的移植。
[Abstract]:In recent 10 years , most of the reproductive centers use chip technology , such as comparative genomic hybridization chip ( array comparative genomic hybri zat i on , aCGH ) and single nucleotide polymorphism array ( PGD ) . This study was carried out to investigate the significance of CNV - Seq in the diagnosis of chromosomal diseases in the field of PGT . The results showed that CNV - Seq was used as a substrate for the diagnosis of chromosomal diseases . Results 1 . In the diagnosis of 6 CNVs , CNV - Seq was highly consistent with aCGH diagnosis , while the abnormal regional ascending and descending trend was significant , while the abnormal region was detected successfully with 33 pg of diluted genome and single cell WGA product . There was significant difference between the two abnormal regions ( p . 05 ) , and the difference between the five cell level and the single cell level was statistically significant ( p . 05 ) , but the difference between the two cell levels was statistically significant ( p . 05 ) , but the difference between the two cell levels was statistically significant ( p . 05 ) . Of the 7 couples , there were 5 pairs of at least one embryo available for transplantation . Conclusion 1 . CNV - Seq has high consistency with aCGH in diagnosis of chromosomal diseases , and has good repeatability and stability in the diagnosis of cell level ; 2 . The embryo biopsy model is successfully constructed ; 3 . The CN value cut - off point may be 2.6 ( 2.6 repeats ) and 1.3 ( 1.3 is missing ) at the time of the cell level diagnosis of the CNV - Seq . CNV - Seq has a high resolution ( 1.5Mb ) ; 5 . CNV - Seq has a strong diagnostic capacity in PGT , covering chromosomal diseases associated with non - euploid , non - equilibrium translocation and minute CNVs ( 鈮,
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