發(fā)布時(shí)間：2018-07-02 23:52

  本文選題：多囊性腎發(fā)育不良 + 染色體微陣列分析��； 參考：《南京大學(xué)》2016年博士論文

【摘要】：研究目的：本研究旨在探索拷貝數(shù)的變異(Copy number variants, CNVs)與多囊性腎發(fā)育不良(Multicystic dysplastic kidney, MCDK)之間的關(guān)系,研究染色體微陣列技術(shù)(Chromosomal microarray analysis, CMA)在胎兒多囊性腎發(fā)育不良產(chǎn)前遺傳學(xué)診斷中的應(yīng)用價(jià)值。研究方法：本研究納入2007年7月至2015年12月于南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院婦產(chǎn)科行超聲結(jié)構(gòu)篩查發(fā)現(xiàn)胎兒多囊性腎發(fā)育不良的共37例為實(shí)驗(yàn)組。其中,33例為單純的單側(cè)/雙側(cè)多囊性腎發(fā)育不良；4例為包含了多囊性腎發(fā)育不良為表型的多發(fā)畸形。本研究對(duì)染色體微陣列分析技術(shù)引入之前即出生的新生兒/兒童進(jìn)行電話隨訪,召回15例新生兒/兒童,進(jìn)行了生長(zhǎng)發(fā)育指標(biāo)的測(cè)量、血壓的測(cè)量、尿常規(guī)、血肌酐的檢測(cè),并對(duì)新生兒/兒童及其父母行腎臟超聲的檢查,隨訪其患側(cè)腎臟的自然發(fā)展進(jìn)程,驗(yàn)證其父母是否亦具有腎臟表型,同時(shí),留取新生兒/兒童及其父母的血樣。染色體微陣列分析技術(shù)引入之后,收錄了因胎兒多囊性腎發(fā)育不良兼之孕婦本人血清學(xué)篩查高風(fēng)險(xiǎn)而行羊水穿刺的22例羊水標(biāo)本。從上述37例在胎兒期超聲下即診斷為多囊性腎發(fā)育不良的新生兒/兒童的外周血和胎兒的羊水中,提取DNA,在Affymetrix CytoScan平臺(tái)上,用Affymetrix HD芯片進(jìn)行染色體微陣列分析。對(duì)所檢出的拷貝數(shù)的變異,進(jìn)行數(shù)據(jù)庫及文獻(xiàn)的檢索,判讀病理性CNVs。以在南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院婦產(chǎn)科因除了先天性腎臟和尿路畸形(Congenitial anomalies of kidney and urinary tract, CAKUT)以外的其他先天異常而進(jìn)行羊水穿刺或外周血采血行CMA檢測(cè)的461例病例為對(duì)照組1；以于南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院婦產(chǎn)科分娩的124例正常新生兒臍血為對(duì)照組2。比較多囊性腎發(fā)育不良實(shí)驗(yàn)組和對(duì)照組1中各CNV的檢出頻率并進(jìn)行統(tǒng)計(jì)學(xué)分析,篩選出只在多囊性腎發(fā)育不良組中出現(xiàn)的臨床意義不明的CNV和在多囊性腎發(fā)育不良組中出現(xiàn)頻率更高的CNV。對(duì)于頻數(shù)最高的CNV片段,采用實(shí)時(shí)熒光定量PCR (Quantitative Real-time PCR, qPCR)的方法,檢測(cè)其在對(duì)照組2中的檢測(cè)頻率,使用統(tǒng)計(jì)學(xué)方法比較其在多囊性腎發(fā)育不良組和對(duì)照組2中的頻率高低。在本研究中所判讀的CNV都經(jīng)過實(shí)時(shí)熒光定量PCR或者多重連接探針擴(kuò)增技術(shù)(Multiplex ligation-dependent probe amplification,MLPA)進(jìn)行二次確認(rèn)。研究結(jié)果：在多囊性腎發(fā)育不良組中,病理性CNV的檢出率為13.5%(5/37)。其中,在單純的單側(cè)多囊性腎發(fā)育不良病例中,檢出了4例病理性CNV——2例17q12缺失(胎兒18：17q12(34,823,294-36,316,144)×1；胎兒21：17q12(34,822,465-36,404,104)×1)、1例22q11.2缺失(胎兒10：22q11.21(21,033,586-21,800,797)×1)和1例22q11.2重復(fù)(胎兒16：22q11.21(18,648,855-21,800,471)×3)。本研究首次報(bào)道一例因攜帶5號(hào)染色體長(zhǎng)臂部分片段缺失而確診為Cri-du-chat綜合征的胎兒,攜帶第二個(gè)較大的CNV——1q31.33q44重復(fù)。該重復(fù)含有多個(gè)基因(CCSAP、DSTYK、REN、AGT)的劑量變異即可致病且參與腎臟發(fā)育的過程并和腎臟疾病的發(fā)生相關(guān)。此外,本研究還檢出了8個(gè)臨床意義不明的變異,與對(duì)照組1中的461例病例進(jìn)行對(duì)比,只出現(xiàn)于多囊性腎發(fā)育不良病例中。通過統(tǒng)計(jì)學(xué)比較,我們還篩選出5個(gè)在多囊性腎發(fā)育不良組中頻率更高,與對(duì)照組1相比具有統(tǒng)計(jì)學(xué)意義的5個(gè)片段。并選出其中頻數(shù)最高的14q11.2缺失,在對(duì)照組2中進(jìn)行進(jìn)一步驗(yàn)證,證實(shí)與對(duì)照組2中124例正常新生兒相比,14q11.2缺失在多囊性腎發(fā)育不良組中,出現(xiàn)頻率仍較高。并且提出14q11.2缺失是通過所涵蓋的T細(xì)胞受體基因參與了腎臟發(fā)育和腎臟疾病的發(fā)生結(jié)論：部分多囊性腎發(fā)育不良的發(fā)生與拷貝數(shù)的變異相關(guān)。對(duì)產(chǎn)前超聲診斷為多囊性腎發(fā)育不良的胎兒應(yīng)當(dāng)進(jìn)行羊水穿刺,行染色體微陣列分析。在多囊性腎發(fā)育不良中發(fā)現(xiàn)的病理性拷貝數(shù)變異具有很大的異質(zhì)性,可能帶來的表型多種多樣,各種表型外顯率也有所不同,增加了產(chǎn)前遺傳咨詢的難度,需要進(jìn)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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R714.5

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