【摘要】：目的:希特林缺陷病(Citrin Deficiency,CD)是由SLC25A13雙等位基因突變導(dǎo)致的常染色體隱性遺傳病,而希特林缺陷導(dǎo)致的新生兒肝內(nèi)膽汁淤積癥(Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency,NICCD)是目前最主要的兒科CD表型,其確診依賴SLC25A13基因突變分析。目前我國人群的SLC25A13基因突變特征尚未被充分認識,還有大量CD患者被漏診或誤診。本研究旨在研究我國SLC25A13基因突變譜及其地理分布特征,為不同區(qū)域CD患者的確診提供相應(yīng)的分子靶標。對象和方法:研究對象包括2013年3月初至2017年3月底我院兒科就診的304位高度疑診NICCD的患者及其父母;以及2005年至2013年2月底我院兒科已確診的119位NICCD患兒及其父母。本研究以外周血為標本來源,利用PCR-RFLP/LA-PCR、Sanger測序、c DNA克隆和Western Blot分析等技術(shù),開展NICCD分子診斷研究,并總結(jié)這12年來診斷的所有CD患者的SLC25A13基因突變譜。然后,以長江為界,將我國分南方、中部和北方三個區(qū)域,利用統(tǒng)計學(xué)SPSS17.0軟件卡方檢驗分析比較不同區(qū)域間的突變分布特征和等位基因異質(zhì)性。結(jié)果:(1)本研究確診新CD患者204位,發(fā)現(xiàn)SLC25A13新突變類型7種,即c.755-1GC(p.252fs269X)、c.1381GT(p.E461X)、c.845_c.848+1del G(p.D283fs X285)、c.493CT(p.Q165X)、c.933_c.933+1ins GCAG(p.A312fs X317)、c.1706_1707del TA(p.S331fs X363)和[c.329-154_c.468+2352del2646;c.468+2392_c.468+2393ins23](p.E110fs127X)。(2)截止2017年3月底,課題組共確診323位CD患者,其SLC25A13基因突變譜包含44種突變類型,其中有14種錯義突變,7種缺失突變,12種無義突變,4種剪接突變,3種插入突變,1種重復(fù)突變,1種致病性SNP,1種異常剪接和1種復(fù)雜突變。(3)突變c.475CT(p.Q159X)、c.775CT(p.Q259X)、c.851_854del4、c.1078CT(p.R360X)、IVS11+1GA、c.1364GT(p.R455L)、c.1399CT(p.R467X)和IVS16ins3kb在我國不同區(qū)域之間的地理分布差異在統(tǒng)計學(xué)上具有顯著性意義(P0.05)。(4)在58種SLC25A13基因型中,c.851_854del4/c.851_854del4、c.851_854del4/IVS16ins3kb、c.851_854del4/c.1399CT(p.R467X)和IVS16ins3kb/IVS11+1GA四種基因型的頻率在各地區(qū)之間的差異有顯著性意義(P0.05)。(5)北方人群SLC25A13等位基因異質(zhì)性高于南方地區(qū)。結(jié)論:本研究通過傳統(tǒng)DNA分析方法結(jié)合cDNA克隆和Western Blot分析等分子診斷技術(shù),共診斷了204位新的CD患者,同時發(fā)現(xiàn)了7種新的致病性突變,擴展了SLC25A13基因突變譜。課題組目前共診斷CD患者323名,建立了國內(nèi)外文獻中最大的CD患者隊列,其豐富的SLC25A13基因突變譜和獨特的地理分布特征為后續(xù)NICCD分子診斷提供了可靠依據(jù),同時為我國不同區(qū)域CD患者分子診斷靶標的確定提供了科學(xué)依據(jù)。
[Abstract]:Objective: Khitrin deficiency disease (Citrin defect CD) is an autosomal recessive disease caused by SLC25A13 double allele mutation, and (Neonatal Intrahepatic Cholestasis caused by Citrin deficiency of neonatal intrahepatic cholestasis caused by Khitrin deficiency is the most important pediatric CD phenotype. Its diagnosis depends on SLC25A13 gene mutation analysis. At present, the characteristics of SLC25A13 gene mutation in Chinese population have not been fully recognized, and a large number of patients with CD have been missed or misdiagnosed. The purpose of this study was to study the SLC25A13 gene mutation profile and its geographical distribution in China, and to provide molecular targets for the diagnosis of CD patients in different regions. Participants and methods: the subjects included 304 pediatric patients with suspected NICCD and their parents from March 2013 to the end of March 2017, and 119 children with NICCD and their parents who had been diagnosed in pediatrics from 2005 to February 2013. In this study, we used PCR-RFLP / LA-PCRGR Sanger sequencing DNA cloning and Western Blot analysis to study the molecular diagnosis of NICCD, and summarized the SLC25A13 gene mutation profiles of all CD patients diagnosed in the past 12 years. Then, taking the Yangtze River as the boundary, China is divided into three regions: south, middle and north, and the mutation distribution and allelic heterogeneity among different regions are analyzed and compared by statistical SPSS17.0 software chi-square test. 緇撴灉:(1)鏈爺絀剁‘璇婃柊CD鎮(zhèn)ｈ，

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