發(fā)布時(shí)間：2018-05-30 22:30

  本文選題：Alport綜合征 + X連鎖　； 參考：《北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2017年05期

【摘要】：目的:探討多重競爭性熒光PCR在X連鎖Alport綜合征分子診斷中的應(yīng)用。方法:選擇20例在北京大學(xué)第一醫(yī)院確診且未進(jìn)行基因診斷的X連鎖Alport綜合征患者為研究對(duì)象,同時(shí)選擇2例經(jīng)多重連接依賴性探針擴(kuò)增技術(shù)檢測到COL4A5基因大片段缺失突變的患者作為陽性對(duì)照和1例經(jīng)腎活檢組織電子顯微鏡檢查證實(shí)非Alport綜合征的男性作為正常對(duì)照。首先應(yīng)用多重競爭性熒光PCR技術(shù)擴(kuò)增COL4A5基因53個(gè)外顯子和4個(gè)參照基因,對(duì)于檢測到COL4A5基因缺失第1外顯子者,進(jìn)而應(yīng)用相同技術(shù)擴(kuò)增COL4A5基因外顯子1~4、COL4A6基因外顯子1~4、兩基因共用啟動(dòng)子以及3個(gè)參照基因;對(duì)于檢測到拷貝數(shù)缺失者,應(yīng)用瓊脂糖凝膠電泳鑒定擴(kuò)增后的PCR產(chǎn)物或直接測序。結(jié)果:兩例陽性對(duì)照應(yīng)用多重競爭性熒光PCR技術(shù)檢測到的COL4A5基因缺失突變與應(yīng)用多重連接依賴性探針擴(kuò)增技術(shù)檢測到的COL4A5基因缺失突變一致。20例患者中6例(30%)明確了基因型,其中2例患者具有累及COL4A5和COL4A6兩個(gè)基因5'端的大片段缺失,2例患者具有累及COL4A5基因30個(gè)外顯子以上的大片段缺失,1例患者具有累及COL4A5基因至少1個(gè)外顯子的大片段缺失,1例患者具有COL4A5基因缺失13個(gè)堿基的小的缺失突變,未檢測到重復(fù)突變。結(jié)論:多重競爭性熒光PCR技術(shù)可用于檢測X連鎖Alport綜合征大片段缺失突變,是對(duì)該病分子診斷檢測方法的重要補(bǔ)充。
[Abstract]:Objective: to investigate the application of multiple competitive fluorescent PCR in molecular diagnosis of X-linked Alport syndrome. Methods: twenty patients with X-linked Alport syndrome diagnosed in the first Hospital of Peking University without gene diagnosis were selected as subjects. At the same time, two patients with large deletion mutations of COL4A5 gene were selected as positive control and one male with non Alport syndrome confirmed by electron microscopy of renal biopsy tissue as normal control. At first, 53 exons and 4 reference genes of COL4A5 gene were amplified by multiplex competitive fluorescence PCR. For those with COL4A5 gene missing exon 1, Then the same technique was used to amplify exon 1n4COL4A6 of COL4A5 gene, the two genes shared promoter and three reference genes, and the amplified PCR products or direct sequencing were identified by agarose gel electrophoresis for those with copy number deletion detected. Results: the deletion mutations of COL4A5 gene detected by multiplex competitive fluorescent PCR in two positive controls were identical to those of COL4A5 gene deletion detected by multiplex conjugate dependent probe amplification. The genotypes were identified in 6 out of 20 patients. Of these, 2 patients had large deletions at the 5'end of the COL4A5 and COL4A6 genes and 2 patients had large deletions involving more than 30 exons of the COL4A5 gene. One patient had a large area involving at least one exon of the COL4A5 gene. One patient with segment deletion had a small deletion mutation with 13 bases of COL4A5 gene deletion. No repeated mutations were detected. Conclusion: multiplex competitive fluorescent PCR technique can be used to detect large fragment deletion mutations in X-linked Alport syndrome, which is an important supplement to the molecular diagnostic methods of X-linked Alport syndrome.
【作者單位】： 北京大學(xué)第一醫(yī)院兒科;
【基金】：國家十二五科技支撐計(jì)劃(2012BAI03B02) 國家重點(diǎn)研發(fā)計(jì)劃(2016YFC0901505) 國家自然科學(xué)基金(81070545) 北京市自然科學(xué)基金(7102148) 兒科遺傳性疾病分子診斷與研究北京市重點(diǎn)實(shí)驗(yàn)室(Z141107004414036)資助~~
【分類號(hào)】：R440;R692

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1 丁潔,郭順華,俞禮霞,楊霽云;用PCR SSCP方法檢測中國人Alport綜合征COL4A5基因突變[J];中華兒科雜志;1999年08期

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本文編號(hào)：1957104