FQ-PCR法对孕妇SMN基因筛查并应用于产前诊断的研究

发布时间：2018-08-02 08:06

【摘要】：研究目的:通过对孕妇人群进行SMN1基因外显子7和外显子8改变情况的筛查研究,建立一种准确、高通量、快速、简便的针对该基因位置的产前筛查及诊断方法,初步制定指导临床的标准化实施方案,预防和阻断脊肌萎缩症(SMA)患儿的出生,提高出生人口的质量。实验方法:本研究主要采用FQ-PCR技术,以2014年12月~2016年10月在天津市中心妇产科医院进行产前检查孕中期孕妇162例为研究对象,对SMN1基因外显子7和外显子8缺失情况进行筛查,经筛查基因型为SMN1杂合缺失孕妇的配偶外周血中DNA进行检测,当夫妇双方均为SMN1杂合缺失时对其胎儿采集羊水进行产前诊断。实验具体分为二部分,实验一为对5个有SMA确诊病例的家系进行FQ-PCR法检测其成员SMN1基因的第7、8外显子缺失情况,并使用基因组测序联合MLPA进行验证,确认FQ-PCR技术检测该基因改变的可行性。实验二为实验的主体部分,主要为对162例孕中期妇女进行FQ-PCR技术下的SMN1基因外显子7和外显子8缺失情况筛查,将筛查后确认为SMN1外显子7杂合缺失者判定为SMA致病基因携带者。对携带者孕妇的配偶进行此项检查,当携带者孕妇的配偶也为SMA致病基因携带者时,对其胎儿进行羊膜腔穿刺后羊水采样,并提取羊水中胎儿DNA进行SMN基因缺失情况检测,最终结合羊水培养后核型检查进行产前诊断。实验结果:实验一(1)DNA提取的质量用离心柱法方法提取外周血,提取DNA的浓度在22.7ng/μl~61.4 ng/μl(36.8±12.1 ng/μl),OD260/OD280在1.75~1.89之间。(2)FQ-PCR与基因组测序联合MLPA方法对5个核心家系外周血DNA进行检测的结果实验结果显示5例临床诊断为SMA的患儿经FQ-PCR方法检测后4例为SMN1的外显子7、8均为纯合缺失,1例为SMN1外显子7纯合缺失,外显子8杂合缺失。对所有患儿双亲进行该项检测后发现,4名SMN1的外显子7纯合缺失伴外显子8纯合缺失的患儿父母均为相同基因型,即存在SMN1的第7外显子和第8外显子均为杂合缺失的情况。对SMN1外显子7纯合缺失,外显子8杂合缺失患儿的双亲进行检测后发现,患儿的母亲基因型为SMN1的外显子7杂合缺失伴外显子8杂合缺失,患儿的父亲仅表现为SMN1基因第7外显子的杂合缺失,外显子8显示为不含缺失改变。与经基因组测序联合MLPA法进行检测后结果一致。实验二(1)孕妇及经阳性携带者配偶外周血DNA和胎儿羊水DNA质量外周血DNA浓度在6.8 ng/μl~194.1 ng/μl(67.3±33.1 ng/μl),OD260/OD280在1.75~1.89之间。羊水DNA的浓度分别为22.9 ng/μl和27.1 ng/μl,OD260/OD280为1.81和1.83。(2)FQ-PCR法对孕中期孕妇外周血DNA检测结果在162例孕妇中共筛查出含有SMN1外显子7杂合缺失的携带者6例。(3)FQ-PCR法对SMA阳性携带者配偶外周血DNA检测结果6例筛查出SMN1外显子7杂合缺失携带者中4例表现为外显子7和外显子8均为正常型,2例表现为外显子7和外显子8均杂合缺失。(4)对夫妇双方均为SMA携带者的高危胎儿羊水DNA检测结果排除母血污染前提下,2名胎儿中1名为纯合型(即该基因外显子7和外显子8均表现为纯合缺失),另一名胎儿基因型为与父母一样的SMN1基因为外显子7杂合缺失伴外显子8杂合缺失(即为SMA致病基因携带者)。与基因组测序联合MLPA法结果一致。(5)依据羊水培养后核型检测结合SMN缺失情况对胎儿以及携带者家庭进行产前诊断和遗传咨询。结论:(1)SMN1基因第7外显子与SMA的发生密切相关,可以依据人群中SMN1基因外显子7的缺失情况对SMA患者,SMA致病基因携带者以及正常人群予以区分。(2)FQ-PCR法作为一种检测手段,可以应用于SMA患者的初步诊断以及大规模人群的携带者筛查。与其他检测方法相比,FQ-PCR法具有方法简单、反应迅速、成本低廉、检测结果准确可靠的优势。(3)鉴于SMA危害严重,在人群中有着较高的携带率,对产前人群进行SMA携带者的筛查,并对夫妇双方均为携带者的家庭进行产前诊断和生育指导是非常重要的。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.5;R440

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