染色体微阵列分析在胎儿多囊性肾发育不良的产前遗传学诊断中的应用研究

发布时间：2018-07-02 23:52

本文选题：多囊性肾发育不良 + 染色体微阵列分析　；参考：《南京大学》2016年博士论文

【摘要】：研究目的：本研究旨在探索拷贝数的变异(Copy number variants, CNVs)与多囊性肾发育不良(Multicystic dysplastic kidney, MCDK)之间的关系,研究染色体微阵列技术(Chromosomal microarray analysis, CMA)在胎儿多囊性肾发育不良产前遗传学诊断中的应用价值。研究方法：本研究纳入2007年7月至2015年12月于南京大学医学院附属鼓楼医院妇产科行超声结构筛查发现胎儿多囊性肾发育不良的共37例为实验组。其中,33例为单纯的单侧/双侧多囊性肾发育不良；4例为包含了多囊性肾发育不良为表型的多发畸形。本研究对染色体微阵列分析技术引入之前即出生的新生儿/儿童进行电话随访,召回15例新生儿/儿童,进行了生长发育指标的测量、血压的测量、尿常规、血肌酐的检测,并对新生儿/儿童及其父母行肾脏超声的检查,随访其患侧肾脏的自然发展进程,验证其父母是否亦具有肾脏表型,同时,留取新生儿/儿童及其父母的血样。染色体微阵列分析技术引入之后,收录了因胎儿多囊性肾发育不良兼之孕妇本人血清学筛查高风险而行羊水穿刺的22例羊水标本。从上述37例在胎儿期超声下即诊断为多囊性肾发育不良的新生儿/儿童的外周血和胎儿的羊水中,提取DNA,在Affymetrix CytoScan平台上,用Affymetrix HD芯片进行染色体微阵列分析。对所检出的拷贝数的变异,进行数据库及文献的检索,判读病理性CNVs。以在南京大学医学院附属鼓楼医院妇产科因除了先天性肾脏和尿路畸形(Congenitial anomalies of kidney and urinary tract, CAKUT)以外的其他先天异常而进行羊水穿刺或外周血采血行CMA检测的461例病例为对照组1；以于南京大学医学院附属鼓楼医院妇产科分娩的124例正常新生儿脐血为对照组2。比较多囊性肾发育不良实验组和对照组1中各CNV的检出频率并进行统计学分析,筛选出只在多囊性肾发育不良组中出现的临床意义不明的CNV和在多囊性肾发育不良组中出现频率更高的CNV。对于频数最高的CNV片段,采用实时荧光定量PCR (Quantitative Real-time PCR, qPCR)的方法,检测其在对照组2中的检测频率,使用统计学方法比较其在多囊性肾发育不良组和对照组2中的频率高低。在本研究中所判读的CNV都经过实时荧光定量PCR或者多重连接探针扩增技术(Multiplex ligation-dependent probe amplification,MLPA)进行二次确认。研究结果：在多囊性肾发育不良组中,病理性CNV的检出率为13.5%(5/37)。其中,在单纯的单侧多囊性肾发育不良病例中,检出了4例病理性CNV——2例17q12缺失(胎儿18：17q12(34,823,294-36,316,144)×1；胎儿21：17q12(34,822,465-36,404,104)×1)、1例22q11.2缺失(胎儿10：22q11.21(21,033,586-21,800,797)×1)和1例22q11.2重复(胎儿16：22q11.21(18,648,855-21,800,471)×3)。本研究首次报道一例因携带5号染色体长臂部分片段缺失而确诊为Cri-du-chat综合征的胎儿,携带第二个较大的CNV——1q31.33q44重复。该重复含有多个基因(CCSAP、DSTYK、REN、AGT)的剂量变异即可致病且参与肾脏发育的过程并和肾脏疾病的发生相关。此外,本研究还检出了8个临床意义不明的变异,与对照组1中的461例病例进行对比,只出现于多囊性肾发育不良病例中。通过统计学比较,我们还筛选出5个在多囊性肾发育不良组中频率更高,与对照组1相比具有统计学意义的5个片段。并选出其中频数最高的14q11.2缺失,在对照组2中进行进一步验证,证实与对照组2中124例正常新生儿相比,14q11.2缺失在多囊性肾发育不良组中,出现频率仍较高。并且提出14q11.2缺失是通过所涵盖的T细胞受体基因参与了肾脏发育和肾脏疾病的发生结论：部分多囊性肾发育不良的发生与拷贝数的变异相关。对产前超声诊断为多囊性肾发育不良的胎儿应当进行羊水穿刺,行染色体微阵列分析。在多囊性肾发育不良中发现的病理性拷贝数变异具有很大的异质性,可能带来的表型多种多样,各种表型外显率也有所不同,增加了产前遗传咨询的难度,需要进一步的研究。
[Abstract]:Objective: the purpose of this study was to explore the relationship between Copy number variants (CNVs) and polycystic renal dysplasia (Multicystic dysplastic kidney, MCDK) and to study the application price of chromosome microarray (Chromosomal microarray analysis, CMA) in prenatal genetic diagnosis of fetal polycystic kidney dysplasia. Methods: in this study, 37 cases of fetal polycystic kidney dysplasia were found in the obstetrics and Gynecology Department of the Affiliated Drum Tower Hospital of Nanjing University Medical College from July 2007 to December 2015. Among them, 33 cases were simple unilateral / bilateral polycystic kidney dysplasia, and 4 cases included polycystic kidney dysplasia. Phenotypic multiple malformation. This study was followed up by telephone follow-up of newborn babies / children before chromosome microarray analysis. 15 neonates / children were recalled, measurements of growth and development, measurement of blood pressure, urine routine, blood creatinine, and renal ultrasound examination of new children / children and their parents were followed up. The natural development of the kidneys was developed to verify whether their parents had kidney phenotypes and to retain the blood samples of the newborn / children and their parents. After the introduction of chromosomal microarray analysis, 22 amniotic fluid specimens were collected for amniocentesis due to the high risk of fetal polycystic kidney dysplasia. DNA was extracted from the 37 cases of the newborn / children's peripheral blood and fetal amniotic fluid diagnosed as polycystic kidney dysplasia in the fetal phase. On the Affymetrix CytoScan platform, the chromosome microarray analysis was performed on the Affymetrix HD chip. The variation of the number of copies detected, the retrieval of database and literature, and the interpretation of pathology. Sex CNVs. in the Department of gynaecology and obstetrics, affiliated to the Drum Tower Hospital, Nanjing University Medical College, 461 cases of amniocentesis or peripheral blood collecting in addition to other congenital abnormalities other than congenital kidney and urinary tract malformation (Congenitial anomalies of kidney and urinary tract, CAKUT) were 1 of the control group, which was attached to the Medical College of Nanjing University. The umbilical cord blood of 124 normal newborns in the obstetrics and Gynecology of Drum Tower Hospital was the control group 2. compared the detection frequency of CNV in the experimental group of polycystic kidney dysplasia and the control group 1, and the statistical analysis was carried out. The CNV and the frequency of unexplained clinical significance in the polycystic kidney dysplasia group and the frequency of the polycystic kidney dysplasia group were screened out. The higher rate of CNV. for the highest frequency CNV fragments, using real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qPCR) method to detect the frequency of detection in the control group 2, using statistical methods to compare the frequency of its rate in the polycystic kidney dysplasia group and the control group 2. The CNV read in this study is in real time. Two times confirmed by fluorescence quantitative PCR or multiple connection probe amplification (Multiplex ligation-dependent probe amplification, MLPA). The results of the study: in the polycystic kidney dysplasia group, the detection rate of pathological CNV was 13.5% (5/37). Among them, 4 cases of pathological CNV were detected in simple unilateral polycystic renal dysplasia. 2 cases of 17q12 deletion (fetal 18:17q12 (34823294-36316144) x 1, fetal 21:17q12 (34822465-36404104) x 1), 1 cases of 22q11.2 deletion (fetal 10:22q11.21 (21033586-21800797) x 1) and 1 cases of 22q11.2 repetition (fetal 16:22q11.21 (18648855-21800471) * 3). The fetus of Cri-du-chat syndrome, which is diagnosed with partial deletion, carries second larger CNV - 1q31.33q44 repeats. The repeated dose variation of multiple genes (CCSAP, DSTYK, REN, AGT) can be pathogenic and involved in the process of kidney development and associated with the occurrence of renal diseases. In addition, 8 clinical implications are also detected in this study. The variation, compared with the 461 cases in the control group 1, only appeared in the cases of polycystic kidney dysplasia. Through statistical comparison, we also screened 5 higher frequencies in the polycystic kidney dysplasia group and 5 segments with statistical significance compared with the control group 1, and selected the highest frequency of 14q11.2 deletion in the control group. Further verification in group 2 confirmed that 14q11.2 deletion was still high in the polycystic kidney dysplasia group compared with 124 normal newborns in the control group 2, and that the 14q11.2 deletion was involved in the conclusion of renal development and renal disease through the T cell receptor gene covered by the group: partial polycystic kidney dysplasia. An amniocentesis for prenatal ultrasound diagnosis of polycystic kidney dysplasia should be carried out by amniocentesis and chromosomal microarray analysis. The pathological copy number variation found in polycystic kidney dysplasia has a large heterogeneity, a variety of phenotypes may be brought, and the phenotype of various phenotypes may also be detected. In addition, the difficulty of prenatal genetic counseling is increased, and further research is needed.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.5

【相似文献】