本文關(guān)鍵詞：廣西β地中海貧血胚胎植入前遺傳學(xué)診斷技術(shù)平臺(tái)建立及臨床應(yīng)用研究　出處：《廣西醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： β珠蛋白基因 β地中海貧血 短串聯(lián)重復(fù)序列 多態(tài)信息量 胚胎植入前遺傳學(xué)診斷 植入前遺傳學(xué)單體型分析 β地中海貧血 短串聯(lián)重復(fù)序列 二代測(cè)序 胚胎植入前遺傳學(xué)診斷 植入前遺傳學(xué)單體型分析 β地中海貧血 短串聯(lián)重復(fù)

【摘要】：第一部分廣西人群β珠蛋白基因15個(gè)STR基因座的遺傳多態(tài)性研究目的研究與β珠蛋白(β-globin,HBB)基因緊密連鎖的15個(gè)短串聯(lián)重復(fù)序列(short tandem repeat,STR)基因座在廣西人群中的遺傳多態(tài)性,為建立β地中海貧血胚胎植入前遺傳學(xué)診斷(preimplantation genetic diagnosis,PGD)的單體型分析技術(shù)(preimplantation genetic haplotyping,PGH)提供實(shí)驗(yàn)依據(jù)。方法采集廣西地區(qū)150名無關(guān)個(gè)體的外周靜脈血,提取外周血基因組DNA,通過熒光PCR技術(shù)擴(kuò)增與β珠蛋白基因緊密連鎖(距離HBB基因1Mb)的15個(gè)STR基因座(HBB4506、D11S988、HBB4677、D11S2362、HBB5089、D11S1243、HBB5138、HBB5178、HBB5205、D11S1760、HBB5576、HBB5655、HBB5820、HBB5859、D11S1338),擴(kuò)增產(chǎn)物使用ABI3500Dx遺傳分析儀進(jìn)行毛細(xì)管電泳,結(jié)果用Genemaper 4.1軟件進(jìn)行分析。最后利用Microsoft Excel軟件計(jì)算15個(gè)HBB-STR基因座的等位基因頻率分布以及多態(tài)信息量(polymorphism information content,PIC)等。結(jié)果在這15個(gè)HBB-STR基因座(HBB4506、D11S988、HBB4677、D11S2362、HBB5089、D11S1243、HBB5138、HBB5178、HBB5205、D11S1760、HBB5576、HBB5655、HBB5820、HBB5859、D11S1338)上分別檢出18、24、22、11、18、27、13、13、16、23、22、16、17、18和7個(gè)等位基因,等位基因頻率從0.003333到0.383333不等。這15個(gè)HBB-STR基因座的多態(tài)信息量(PIC)分別為0.8457、0.8983、0.8931、0.7592、0.8698、0.9046、0.7940、0.7925、0.7708、0.9102、0.8583、0.8467、0.7438、0.8366和0.7704。這15個(gè)基因座的PIC均0.7000,平均值為0.8329,其中PIC0.8500的HBB-STR基因座有6個(gè)。結(jié)論本研究檢測(cè)的這15個(gè)HBB-STR基因座在廣西人群中的多態(tài)信息量(PIC)較高,具有高度的遺傳多態(tài)性,在建立β地中海貧血胚胎植入前遺傳學(xué)診斷(PGD)的單體型分析(PGH)技術(shù)中具有較高的應(yīng)用價(jià)值,為后續(xù)工作的開展提供了實(shí)驗(yàn)依據(jù)。第二部分β地中海貧血胚胎植入前遺傳學(xué)診斷中不同診斷方法的比較目的對(duì)β地貧PGD中兩種遺傳學(xué)診斷方法——反向點(diǎn)雜交(reverse dot blot,RDB)結(jié)合STR單體型分析技術(shù)與二代測(cè)序(Next generation sequencing,NGS)技術(shù),從診斷時(shí)間、確診率、費(fèi)用等方面進(jìn)行對(duì)比研究,建立最適宜的遺傳學(xué)診斷技術(shù)應(yīng)用于廣西地區(qū)β地貧PGD。方法選擇雙方均為β地中海貧血基因突變攜帶者、有PGD指征且同意參加本研究的的夫婦。患者夫婦及另外三名家系成員(女方母親、男方母親和男方父親)一同抽取外周靜脈血進(jìn)行15個(gè)STR位點(diǎn)的家系連鎖分析�；颊呓�(jīng)過常規(guī)的藥物促排卵、取卵、取精、卵胞漿內(nèi)單精子注射(Intracytoplasmic sperm injection,ICSI)、胚胎培養(yǎng)等程序,將所得胚胎全部進(jìn)行囊胚培養(yǎng),形成囊胚后,取囊胚期數(shù)個(gè)(5-8個(gè))滋養(yǎng)外胚層細(xì)胞活檢,活檢后即將所有囊胚行玻璃化冷凍。利用多重置換擴(kuò)增(Multiple displacement amplification,MDA)法對(duì)滋養(yǎng)外胚層細(xì)胞進(jìn)行全基因組擴(kuò)增(Whole genome amplification,WGA)。MDA擴(kuò)增產(chǎn)物一部分采用反向點(diǎn)雜交(RDB)結(jié)合15個(gè)短串聯(lián)重復(fù)序列(STR)位點(diǎn)的單體型分析(PGH)技術(shù)進(jìn)行遺傳學(xué)診斷(A組);另一部分MDA擴(kuò)增產(chǎn)物采用二代測(cè)序(NGS)方法進(jìn)行診斷(B組)。比較兩種方法的檢出率、診斷符合率、所需時(shí)間和費(fèi)用等。待診斷結(jié)果出來后選擇最合適的囊胚進(jìn)行解凍移植。結(jié)果納入本研究的患者獲卵24個(gè),胚胎13個(gè),囊胚培養(yǎng)后形成6枚囊胚(囊胚評(píng)分分別為4BB,4BC,3CC,3CC,3BC,3CC),每個(gè)囊胚取滋養(yǎng)外胚層細(xì)胞活檢,6例樣本MDA法擴(kuò)增均擴(kuò)增成功,擴(kuò)增成功率100%。A組中,囊胚1地貧基因型診斷結(jié)果為β-28/βCD41-42;囊胚2地貧基因型診斷結(jié)果為βCD41-42/βN;囊胚3地貧基因型診斷結(jié)果為βN/βN;囊胚4的地貧基因型診斷結(jié)果為β-28/βN/βN;囊胚5和6的地貧基因型診斷結(jié)果均為β-28/βN。B組的地貧基因型診斷結(jié)果與A組完全一致,B組同時(shí)對(duì)囊胚2-6進(jìn)行染色體拷貝數(shù)檢測(cè),診斷出囊胚2為整倍體,囊胚3-6均為非整倍體。A組所需診斷時(shí)間為2-3天,結(jié)果分析所需時(shí)間約為1個(gè)工作日;B組所需診斷時(shí)間為2-3天,結(jié)果外送嘉寶仁和公司分析,所需時(shí)間為7個(gè)工作日。A組費(fèi)用為2000元/囊胚,而B組費(fèi)用為3500元/囊胚。最后選擇囊胚2解凍移植,遺憾的是未獲得臨床妊娠。結(jié)論與二代測(cè)序(NGS)技術(shù)相比,反向點(diǎn)雜交結(jié)合STR單體型分析技術(shù)也能在β地中海貧血PGD中有效、準(zhǔn)確地診斷出植入前囊胚的地貧基因型,能夠應(yīng)用于β地貧PGD臨床。雖然目前二代測(cè)序價(jià)格昂貴、結(jié)果分析人員要求高,臨床推廣應(yīng)用受限,但NGS是PGD遺傳學(xué)診斷技術(shù)的發(fā)展方向和未來趨勢(shì)。然而考慮到目前各方面條件的限制以及在廣西地區(qū)開展β地貧PGD的急切需求,RDB結(jié)合15個(gè)STR位點(diǎn)單體型分析的診斷方法更加適合目前的廣西地區(qū)β地貧PGD,值得推廣應(yīng)用。第三部分MDA結(jié)合RDB及STR單體型分析在β地貧PGD中的臨床應(yīng)用研究目的研究多重置換擴(kuò)增(MDA)結(jié)合反向點(diǎn)雜交(RDB)及短串聯(lián)重復(fù)序列(STR)單體型分析技術(shù)在β地貧PGD中的臨床應(yīng)用。方法納入8對(duì)雙方均為β地中海貧血基因突變攜帶者且要求進(jìn)行β地貧PGD治療的夫婦。攜帶者夫婦及其雙方父母(每個(gè)家系一共6名成員)均抽取外周靜脈血進(jìn)行15個(gè)STR位點(diǎn)的家系連鎖分析�；颊呓�(jīng)過常規(guī)的藥物促排卵、取卵、取精、卵胞漿內(nèi)單精子注射(ICSI)、胚胎培養(yǎng)等程序,將所得胚胎全部進(jìn)行囊胚培養(yǎng),形成囊胚后,取囊胚期數(shù)個(gè)(5-8個(gè))滋養(yǎng)外胚層細(xì)胞活檢,活檢后即將所有囊胚進(jìn)行玻璃化冷凍。利用多重置換擴(kuò)增(MDA)法對(duì)取得的滋養(yǎng)外胚層細(xì)胞進(jìn)行全基因組擴(kuò)增(WGA)。MDA擴(kuò)增產(chǎn)物采用反向點(diǎn)雜交(RDB)結(jié)合15個(gè)短串聯(lián)重復(fù)序列(STR)位點(diǎn)的單體型分析(PGH)技術(shù)進(jìn)行遺傳學(xué)診斷。待診斷結(jié)果出來后,結(jié)合致病基因遺傳學(xué)診斷結(jié)果和囊胚評(píng)分,選擇最佳囊胚進(jìn)行解凍移植,妊娠后孕18-22周通過羊水產(chǎn)前診斷進(jìn)一步驗(yàn)證PGD診斷結(jié)果。結(jié)果這8對(duì)夫婦,β地中海貧血基因突變類型分別為CD41-42/N、CD17/N、-28/N或IVS-II-654/N。每個(gè)家系6名成員檢測(cè)15個(gè)STR位點(diǎn)后結(jié)果顯示每個(gè)家系均有7個(gè)或以上(HBB基因上游或下游均有2個(gè)以上)的STR位點(diǎn)可以提供診斷信息,單體型構(gòu)建成功�；颊呓�(jīng)過常規(guī)藥物促排卵,取卵、卵胞漿內(nèi)單精子注射(ICSI)后共獲得147枚胚胎,形成86枚囊胚,囊胚形成率58.50%。其中82枚囊胚擴(kuò)增成功,擴(kuò)增成功為95.35%。80枚囊胚獲得了明確診斷,其中24枚囊胚為正常純合子;38枚囊胚診斷為雜合子;18枚囊胚為不能移植的異常純合子或雙重雜合子。8例患者中一共4例患者進(jìn)行了囊胚解凍移植(例1、例2、例3和例6),均為單囊胚移植。其中3例(例1、例2和例3)獲得妊娠,1例(例6)未妊娠。妊娠率為75%(3/4)。例1和例2患者移植正常純合子囊胚(基因型βN/βN),中孕期羊水產(chǎn)前診斷結(jié)果與PGD診斷結(jié)果相符。例3患者移植雜合子囊胚(基因型為βCD41-42/βN),移植后獲得妊娠,但目前還未行產(chǎn)前診斷驗(yàn)證PGD診斷結(jié)果。結(jié)論本研究建立的β地貧PGD方法——即經(jīng)多重置換擴(kuò)增(MDA)后,反向點(diǎn)雜交(RDB)結(jié)合短串聯(lián)重復(fù)序列(STR)單體型連鎖分析,經(jīng)驗(yàn)證有效、準(zhǔn)確,可以在廣西地區(qū)β地貧PGD臨床中推廣應(yīng)用。我們期待著更大樣本量的研究證實(shí)。
[Abstract]:The first part of the Guangxi population of beta globin gene 15 STR loci genetic polymorphism to study with beta globin (beta -globin, HBB) gene is closely linked to the 15 short tandem repeat (short tandem, repeat, STR) loci in Guangxi population genetic polymorphism for beta thalassemia embryos preimplantation genetic diagnosis (preimplantation genetic diagnosis, PGD) haplotype analysis technology (preimplantation genetic haplotyping, PGH) to provide experimental basis. Methods to collect the Guangxi area in 150 unrelated individuals of peripheral blood genomic DNA from peripheral blood, amplified by fluorescent PCR and beta globin gene (HBB gene linked distance 1Mb) of the 15 STR loci (HBB4506, D11S988, HBB4677, D11S2362, HBB5089, D11S1243, HBB5138, HBB5178, HBB5205, D11S1760, HBB5576, HBB5655, HBB5820, HBB5859, D11S1338), the PCR products By capillary electrophoresis with ABI3500Dx genetic analyzer. The results were analyzed using Genemaper 4.1 software. The final calculation of 15 HBB-STR allele frequency distribution and polymorphism information content using Microsoft Excel software (polymorphism information, content, PIC). Results in the 15 HBB-STR loci (HBB4506, D11S988, HBB4677, D11S2362, HBB5089 D11S1243, HBB5138, HBB5178, HBB5205, D11S1760, HBB5576, HBB5655, HBB5820, HBB5859, D11S1338, 18,24,22,11,18,27,13,13,16,23,22,16,17,18) were detected and 7 alleles, allele frequencies from 0.003333 to 0.383333 dollars. The polymorphic information content of 15 HBB-STR loci (PIC) were 0.8457,0.8983,0.8931,0.7592,0.8698,0.9046,0.7940,0.7925,0.7708,0.9102,0.8583,0.8467,0.7438,0.8366 and 0.7704. 15 gene the seat of PIC was 0.7000, the average value is 0.8329, PIC0.85 The HBB-STR locus was 00 with 6. The 15 HBB-STR loci detected the conclusions of this study in Guangxi population of polymorphism information content (PIC) high and has high genetic polymorphism, in the establishment of beta thalassemia preimplantation genetic diagnosis (PGD) haplotype analysis (PGH) has a high application value in the technology, provides the experimental basis for the follow-up work. The aim of the second part of beta thalassemia preimplantation genetic diagnosis in different diagnosis of beta thalassemia PGD two genetic diagnosis methods -- reverse dot blot (reverse dot, blot, RDB) with the combination of technology and the two generation sequencing analysis of STR haplotype (Next generation sequencing, NGS) technology, from the time of diagnosis, diagnosis rate, cost and other aspects of comparative study, establish the most suitable application of genetic diagnosis technology for PGD. beta thalassemia in Guangxi area to choose both beta Thalassemia gene mutation carriers, PGD indications and agreed to participate in the study. The couple with the couple and three other family members (the woman's mother, his mother and his father) linkage analysis with venous blood of 15 loci of STR. Patients after routine ovulation drugs, take eggs, sperm and intracytoplasmic sperm injection (Intracytoplasmic sperm, injection, ICSI), embryo culture and other procedures, the embryo blastocyst culture, blastocyst formation, and blastocyst number (5-8) trophectodermal cell biopsy biopsy after all the blastocyst vitrification for use. Multiple displacement amplification (Multiple displacement, amplification, MDA) method on trophoblast cells were derived from outside the whole genome amplification (Whole genome amplification, WGA.MDA) amplified part of using reverse dot blot (RDB) combined with 15 short string 鑱旈噸澶嶅簭鍒，

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