Kooper Angelique J A, Faas Brigitte H W, Feuth Ton, Creemers Johan W T, Zondervan Hans H, Boekkooi Peter F, Quartero Rik W P, Rijnders Robbert J P, van der Burgt Ineke, van Kessel Ad Geurts, Smits Arie P T
Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
J Mol Diagn. 2009 Jan;11(1):17-24. doi: 10.2353/jmoldx.2009.070140. Epub 2008 Dec 12.
The objective of this study was to examine the suitability of multiplex ligation-dependent probe amplification (MLPA) in chorionic villus samples as a replacement for traditional karyotyping for the detection of (an)euploidies of chromosomes 21, 18, 13, X, and Y. Chorionic villus samples were diagnosed by traditional karyotyping using short-term cultures (STC) and long-term cultures (LTC), and by MLPA using kit P095. DNA was extracted after digestion of whole villi with proteinase K and/or trypsin and collagenase. Different cell-dissociation procedures were tested to obtain MLPA results representative of the cytotrophoblast layer and the mesenchymal core. Over 95% of the MLPA results were in concordance with the traditional karyotyping of STC and LTC. Traditional karyotyping revealed seven mosaics. After digestion of whole villi with proteinase K, only abnormal cell lines confined to the STC gave rise to abnormal MLPA results. In one sample, the complete discrepancy between STC and LTC was resolved after enzymatic dissociation of cells from the cytotrophoblast layer and the mesenchymal core. MLPA in chorionic villus samples was found to be a reliable test for the detection of (an)euploidies of chromosomes 21, 18, 13, X, and Y. Whole villi digestion with proteinase K resulted in the over-representation of cytotrophoblasts in the DNA pool. To obtain MLPA results representative for STC and LTC, enzymatic dissociation of cells from the cytotrophoblast layer and mesenchymal core is required.
本研究的目的是检验多重连接依赖探针扩增技术（MLPA）在绒毛膜绒毛样本中作为传统核型分析的替代方法用于检测21、18、13、X和Y染色体非整倍体的适用性。绒毛膜绒毛样本通过使用短期培养（STC）和长期培养（LTC）的传统核型分析以及使用试剂盒P095的MLPA进行诊断。用蛋白酶K和/或胰蛋白酶及胶原酶消化整个绒毛后提取DNA。测试了不同的细胞解离程序以获得代表细胞滋养层和间充质核心的MLPA结果。超过95%的MLPA结果与STC和LTC的传统核型分析结果一致。传统核型分析发现了7个嵌合体。用蛋白酶K消化整个绒毛后，仅局限于STC的异常细胞系产生了异常的MLPA结果。在一个样本中，通过从细胞滋养层和间充质核心进行酶解细胞后，解决了STC和LTC之间的完全差异。发现绒毛膜绒毛样本中的MLPA是检测21、18、13、X和Y染色体非整倍体的可靠检测方法。用蛋白酶K消化整个绒毛导致DNA池中细胞滋养层细胞过度富集。为了获得代表STC和LTC的MLPA结果，需要从细胞滋养层和间充质核心进行酶解细胞。
