FQ-PCR法對孕婦SMN基因篩查并應(yīng)用于產(chǎn)前診斷的研究
發(fā)布時間：2018-08-02 08:06
【摘要】：研究目的:通過對孕婦人群進(jìn)行SMN1基因外顯子7和外顯子8改變情況的篩查研究,建立一種準(zhǔn)確、高通量、快速、簡便的針對該基因位置的產(chǎn)前篩查及診斷方法,初步制定指導(dǎo)臨床的標(biāo)準(zhǔn)化實(shí)施方案,預(yù)防和阻斷脊肌萎縮癥(SMA)患兒的出生,提高出生人口的質(zhì)量。實(shí)驗方法:本研究主要采用FQ-PCR技術(shù),以2014年12月~2016年10月在天津市中心婦產(chǎn)科醫(yī)院進(jìn)行產(chǎn)前檢查孕中期孕婦162例為研究對象,對SMN1基因外顯子7和外顯子8缺失情況進(jìn)行篩查,經(jīng)篩查基因型為SMN1雜合缺失孕婦的配偶外周血中DNA進(jìn)行檢測,當(dāng)夫婦雙方均為SMN1雜合缺失時對其胎兒采集羊水進(jìn)行產(chǎn)前診斷。實(shí)驗具體分為二部分,實(shí)驗一為對5個有SMA確診病例的家系進(jìn)行FQ-PCR法檢測其成員SMN1基因的第7、8外顯子缺失情況,并使用基因組測序聯(lián)合MLPA進(jìn)行驗證,確認(rèn)FQ-PCR技術(shù)檢測該基因改變的可行性。實(shí)驗二為實(shí)驗的主體部分,主要為對162例孕中期婦女進(jìn)行FQ-PCR技術(shù)下的SMN1基因外顯子7和外顯子8缺失情況篩查,將篩查后確認(rèn)為SMN1外顯子7雜合缺失者判定為SMA致病基因攜帶者。對攜帶者孕婦的配偶進(jìn)行此項檢查,當(dāng)攜帶者孕婦的配偶也為SMA致病基因攜帶者時,對其胎兒進(jìn)行羊膜腔穿刺后羊水采樣,并提取羊水中胎兒DNA進(jìn)行SMN基因缺失情況檢測,最終結(jié)合羊水培養(yǎng)后核型檢查進(jìn)行產(chǎn)前診斷。實(shí)驗結(jié)果:實(shí)驗一(1)DNA提取的質(zhì)量用離心柱法方法提取外周血,提取DNA的濃度在22.7ng/μl~61.4 ng/μl(36.8±12.1 ng/μl),OD260/OD280在1.75~1.89之間。(2)FQ-PCR與基因組測序聯(lián)合MLPA方法對5個核心家系外周血DNA進(jìn)行檢測的結(jié)果實(shí)驗結(jié)果顯示5例臨床診斷為SMA的患兒經(jīng)FQ-PCR方法檢測后4例為SMN1的外顯子7、8均為純合缺失,1例為SMN1外顯子7純合缺失,外顯子8雜合缺失。對所有患兒雙親進(jìn)行該項檢測后發(fā)現(xiàn),4名SMN1的外顯子7純合缺失伴外顯子8純合缺失的患兒父母均為相同基因型,即存在SMN1的第7外顯子和第8外顯子均為雜合缺失的情況。對SMN1外顯子7純合缺失,外顯子8雜合缺失患兒的雙親進(jìn)行檢測后發(fā)現(xiàn),患兒的母親基因型為SMN1的外顯子7雜合缺失伴外顯子8雜合缺失,患兒的父親僅表現(xiàn)為SMN1基因第7外顯子的雜合缺失,外顯子8顯示為不含缺失改變。與經(jīng)基因組測序聯(lián)合MLPA法進(jìn)行檢測后結(jié)果一致。實(shí)驗二(1)孕婦及經(jīng)陽性攜帶者配偶外周血DNA和胎兒羊水DNA質(zhì)量外周血DNA濃度在6.8 ng/μl~194.1 ng/μl(67.3±33.1 ng/μl),OD260/OD280在1.75~1.89之間。羊水DNA的濃度分別為22.9 ng/μl和27.1 ng/μl,OD260/OD280為1.81和1.83。(2)FQ-PCR法對孕中期孕婦外周血DNA檢測結(jié)果在162例孕婦中共篩查出含有SMN1外顯子7雜合缺失的攜帶者6例。(3)FQ-PCR法對SMA陽性攜帶者配偶外周血DNA檢測結(jié)果6例篩查出SMN1外顯子7雜合缺失攜帶者中4例表現(xiàn)為外顯子7和外顯子8均為正常型,2例表現(xiàn)為外顯子7和外顯子8均雜合缺失。(4)對夫婦雙方均為SMA攜帶者的高危胎兒羊水DNA檢測結(jié)果排除母血污染前提下,2名胎兒中1名為純合型(即該基因外顯子7和外顯子8均表現(xiàn)為純合缺失),另一名胎兒基因型為與父母一樣的SMN1基因為外顯子7雜合缺失伴外顯子8雜合缺失(即為SMA致病基因攜帶者)。與基因組測序聯(lián)合MLPA法結(jié)果一致。(5)依據(jù)羊水培養(yǎng)后核型檢測結(jié)合SMN缺失情況對胎兒以及攜帶者家庭進(jìn)行產(chǎn)前診斷和遺傳咨詢。結(jié)論:(1)SMN1基因第7外顯子與SMA的發(fā)生密切相關(guān),可以依據(jù)人群中SMN1基因外顯子7的缺失情況對SMA患者,SMA致病基因攜帶者以及正常人群予以區(qū)分。(2)FQ-PCR法作為一種檢測手段,可以應(yīng)用于SMA患者的初步診斷以及大規(guī)模人群的攜帶者篩查。與其他檢測方法相比,FQ-PCR法具有方法簡單、反應(yīng)迅速、成本低廉、檢測結(jié)果準(zhǔn)確可靠的優(yōu)勢。(3)鑒于SMA危害嚴(yán)重,在人群中有著較高的攜帶率,對產(chǎn)前人群進(jìn)行SMA攜帶者的篩查,并對夫婦雙方均為攜帶者的家庭進(jìn)行產(chǎn)前診斷和生育指導(dǎo)是非常重要的。
[Abstract]:Objective: to establish an accurate, high throughput, rapid and simple method of prenatal screening and diagnosis for the location of the gene by screening the changes of exon 7 and exon 8 of SMN1 gene in pregnant women, and to establish a standardized clinical case to prevent and block the birth of children with spinal muscular atrophy (SMA). To improve the quality of the birth population. Experimental methods: This study mainly used FQ-PCR technology in October December 2014 ~2016 year in Tianjin Central Obstetrics and Gynecology Hospital in the mid-term pregnancy test of pregnant women 162 cases as the research object, the SMN1 gene exon 7 and exon 8 deletion of the screening, the screening genotype for SMN1 heterozygous missing pregnant women DNA was detected in the peripheral blood of the spouses, and the prenatal diagnosis of amniotic fluid was carried out when both couples were SMN1 heterozygous. The experiment was divided into two parts. The experiment one was to detect the deletion of the exon 7,8 of the SMN1 gene of the family members of 5 families with SMA confirmed cases, and the genome sequencing combined with MLPA into MLPA. The main part of the experiment was to screen the deletion of exon 7 and exon 8 of SMN1 gene of 162 pregnant women in the middle of pregnancy, which was confirmed to be a carrier of SMA pathogeny gene with SMN1 exon 7 after screening in 162 cases of pregnant women. The spouse of the pregnant woman carried out this examination. When the spouse of the pregnant woman was the carrier of the SMA disease gene, the amniotic fluid sampling was performed on the fetus after amniocentesis, and the fetal DNA in amniotic fluid was extracted to detect the deletion of the SMN gene. Finally, the prenatal diagnosis was carried out after the karyotype examination of the amniotic fluid culture. The experimental results: a (1) DNA extraction experiment The quality of peripheral blood was extracted by centrifuge column method, the concentration of DNA was extracted from 22.7ng/ l~61.4 ng/ Mu L (36.8 + 12.1 ng/ Mu L), OD260/OD280 at 1.75~1.89. (2) FQ-PCR and genome sequencing combined MLPA method for the detection of peripheral blood DNA in 5 core families The exon 7,8 of 4 cases of SMN1 was homozygous deletion, 1 cases were SMN1 exon homozygous deletion and exon 8 heterozygous deletion. The parents of 4 children with SMN1 exon 7 homozygous deletion and exon 8 homozygous deletion were all the same genotypes, that is, seventh exons and eighth exons of SMN1. The homozygous deletion of the SMN1 exon 7 and the parents of the exon 8 heterozygous missing children found that the mother genotype of the child was SMN1 exon 7 heterozygous with exon 8 heterozygosity, and the child's father was only the heterozygous deletion of the SMN1 gene seventh exon, and the exon 8 showed no deficiency. The results were in agreement with the results of the test after the genome sequencing combined with MLPA. Experiment two (1) the DNA concentration in peripheral blood of pregnant women and positive carriers' spouses was 6.8 ng/ mu l~194.1 ng/ Mu L (67.3 + 33.1 ng/ L) and OD260/OD280 in 1.75~1.89. The concentration of amniotic fluid was 22.9 and 27.1 micron respectively. OD260/OD280 1.81 and 1.83. (2) FQ-PCR method for DNA detection of peripheral blood in pregnant women of pregnant women in 162 cases of pregnant women, 6 cases were screened with SMN1 exon 7 heterozygosity. (3) FQ-PCR method was used to detect DNA in peripheral blood of spouses of SMA positive carriers in 6 cases, 6 cases were screened out of SMN1 exon 7 heterozygous deletion carriers, and 4 cases were exon 7 8 of the exon and exon 8 were normal, 2 cases were exon 7 and exon 8 heterozygous deletion. (4) the DNA detection of high risk fetal amniotic fluid for both couples and couples excluded from maternal blood pollution precondition, 1 of the 2 fetuses were homozygous (i. e., exon 7 and exon 8 all showed homozygous deletion), and the other fetal genotypes were The SMN1 gene, like the parents, is the exon 7 heterozygous deletion with the exon 8 heterozygous deletion (that is, the carrier of the SMA pathogenicity gene). The result is consistent with the genome sequencing combined with the MLPA method. (5) the prenatal diagnosis and genetic counseling for the fetus and the family of the carrier are performed on the basis of the karyotype detection and SMN deletion in the amniotic fluid culture. Conclusion: (1) the SMN1 gene is the first one. 7 exon is closely related to the occurrence of SMA. According to the deletion of SMN1 exon 7 in the population, SMA patients, SMA carriers and normal people are distinguished. (2) FQ-PCR method as a detection method, can be applied to the preliminary diagnosis of SMA patients and the screening of carriers in large population. And other detection methods. In comparison, the FQ-PCR method has the advantages of simple method, quick reaction, low cost, and accurate and reliable results. (3) in view of the serious harm of SMA, it has a high carrying rate in the population, it is very important to carry out the screening of the SMA carriers for the prenatal population, and to carry out prenatal diagnosis and birth guidance to the families of the couples who are both carriers.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R714.5;R440
本文編號：2158755
[Abstract]:Objective: to establish an accurate, high throughput, rapid and simple method of prenatal screening and diagnosis for the location of the gene by screening the changes of exon 7 and exon 8 of SMN1 gene in pregnant women, and to establish a standardized clinical case to prevent and block the birth of children with spinal muscular atrophy (SMA). To improve the quality of the birth population. Experimental methods: This study mainly used FQ-PCR technology in October December 2014 ~2016 year in Tianjin Central Obstetrics and Gynecology Hospital in the mid-term pregnancy test of pregnant women 162 cases as the research object, the SMN1 gene exon 7 and exon 8 deletion of the screening, the screening genotype for SMN1 heterozygous missing pregnant women DNA was detected in the peripheral blood of the spouses, and the prenatal diagnosis of amniotic fluid was carried out when both couples were SMN1 heterozygous. The experiment was divided into two parts. The experiment one was to detect the deletion of the exon 7,8 of the SMN1 gene of the family members of 5 families with SMA confirmed cases, and the genome sequencing combined with MLPA into MLPA. The main part of the experiment was to screen the deletion of exon 7 and exon 8 of SMN1 gene of 162 pregnant women in the middle of pregnancy, which was confirmed to be a carrier of SMA pathogeny gene with SMN1 exon 7 after screening in 162 cases of pregnant women. The spouse of the pregnant woman carried out this examination. When the spouse of the pregnant woman was the carrier of the SMA disease gene, the amniotic fluid sampling was performed on the fetus after amniocentesis, and the fetal DNA in amniotic fluid was extracted to detect the deletion of the SMN gene. Finally, the prenatal diagnosis was carried out after the karyotype examination of the amniotic fluid culture. The experimental results: a (1) DNA extraction experiment The quality of peripheral blood was extracted by centrifuge column method, the concentration of DNA was extracted from 22.7ng/ l~61.4 ng/ Mu L (36.8 + 12.1 ng/ Mu L), OD260/OD280 at 1.75~1.89. (2) FQ-PCR and genome sequencing combined MLPA method for the detection of peripheral blood DNA in 5 core families The exon 7,8 of 4 cases of SMN1 was homozygous deletion, 1 cases were SMN1 exon homozygous deletion and exon 8 heterozygous deletion. The parents of 4 children with SMN1 exon 7 homozygous deletion and exon 8 homozygous deletion were all the same genotypes, that is, seventh exons and eighth exons of SMN1. The homozygous deletion of the SMN1 exon 7 and the parents of the exon 8 heterozygous missing children found that the mother genotype of the child was SMN1 exon 7 heterozygous with exon 8 heterozygosity, and the child's father was only the heterozygous deletion of the SMN1 gene seventh exon, and the exon 8 showed no deficiency. The results were in agreement with the results of the test after the genome sequencing combined with MLPA. Experiment two (1) the DNA concentration in peripheral blood of pregnant women and positive carriers' spouses was 6.8 ng/ mu l~194.1 ng/ Mu L (67.3 + 33.1 ng/ L) and OD260/OD280 in 1.75~1.89. The concentration of amniotic fluid was 22.9 and 27.1 micron respectively. OD260/OD280 1.81 and 1.83. (2) FQ-PCR method for DNA detection of peripheral blood in pregnant women of pregnant women in 162 cases of pregnant women, 6 cases were screened with SMN1 exon 7 heterozygosity. (3) FQ-PCR method was used to detect DNA in peripheral blood of spouses of SMA positive carriers in 6 cases, 6 cases were screened out of SMN1 exon 7 heterozygous deletion carriers, and 4 cases were exon 7 8 of the exon and exon 8 were normal, 2 cases were exon 7 and exon 8 heterozygous deletion. (4) the DNA detection of high risk fetal amniotic fluid for both couples and couples excluded from maternal blood pollution precondition, 1 of the 2 fetuses were homozygous (i. e., exon 7 and exon 8 all showed homozygous deletion), and the other fetal genotypes were The SMN1 gene, like the parents, is the exon 7 heterozygous deletion with the exon 8 heterozygous deletion (that is, the carrier of the SMA pathogenicity gene). The result is consistent with the genome sequencing combined with the MLPA method. (5) the prenatal diagnosis and genetic counseling for the fetus and the family of the carrier are performed on the basis of the karyotype detection and SMN deletion in the amniotic fluid culture. Conclusion: (1) the SMN1 gene is the first one. 7 exon is closely related to the occurrence of SMA. According to the deletion of SMN1 exon 7 in the population, SMA patients, SMA carriers and normal people are distinguished. (2) FQ-PCR method as a detection method, can be applied to the preliminary diagnosis of SMA patients and the screening of carriers in large population. And other detection methods. In comparison, the FQ-PCR method has the advantages of simple method, quick reaction, low cost, and accurate and reliable results. (3) in view of the serious harm of SMA, it has a high carrying rate in the population, it is very important to carry out the screening of the SMA carriers for the prenatal population, and to carry out prenatal diagnosis and birth guidance to the families of the couples who are both carriers.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R714.5;R440
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