Gasparini P, Arbustini E, Restagno G, Zelante L, Stanziale P, Gatta L, Sbaiz L, Sedita A M, Banchieri N, Sapone L, Fiorucci G C, Brinson E, Shulse E, Rappaport E, Fortina P
Servizio di Genetica Medica, IRCCS Casa Sollievo della Sofferenza, Foggia, Italy.
J Med Screen. 1999;6(2):67-9. doi: 10.1136/jms.6.2.67.
Molecular biological testing for genetic diseases has grown rapidly, but speed, accuracy, specificity, sensitivity, throughput, and cost become more important as large scale screening is considered. This is a pilot study of an assay for the simultaneous detection of up to 31 cystic fibrosis mutations in a multicentre population based screening of 4476 Italian newborns.
The assay is a polymerase chain reaction, followed by an oligonucleotide ligation assay (PCR/OLA) and finally a sequence coded separation. It allows the detection of up to 31 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Guthrie spots were used as a source of template DNA.
144 carriers were detected during the analysis of 4476 samples, which translates into a carrier frequency of 1/31.1. Forty two carriers were detected from 1341 samples in Pavia (1/31.9), 53 from 1574 in Turin (1/29.7), and 49 from 1561 in San Giovanni Rotondo (1/31.8). Fifteen different mutations were detected, the most common being delta F508 (0.625). Other common mutations included G542X (16 of 144), which was particularly common in southern Italy (14 of 49), N1303K (8 of 144), and R117H (8 of 144), detected only in the northern centres.
PCR/OLA is a robust, accurate, user friendly method for cystic fibrosis screening of newborns using blood spots in a semiautomated way at a low cost per mutation (0.8 Euro).
针对遗传疾病的分子生物学检测发展迅速,但在考虑大规模筛查时,速度、准确性、特异性、灵敏度、通量和成本变得更为重要。这是一项关于一种检测方法的试点研究,该方法可在对4476名意大利新生儿进行的多中心人群筛查中同时检测多达31种囊性纤维化突变。
该检测方法是先进行聚合酶链反应,接着进行寡核苷酸连接分析(PCR/OLA),最后进行序列编码分离。它能够检测囊性纤维化跨膜传导调节因子(CFTR)基因中的多达31种突变。干血斑用作模板DNA的来源。
在分析的4476份样本中检测到144名携带者,携带者频率为1/31.1。在帕维亚,从1341份样本中检测到42名携带者(1/31.9),在都灵,从1574份样本中检测到53名(1/29.7),在圣乔瓦尼罗通多,从1561份样本中检测到49名(1/31.8)。检测到15种不同的突变,最常见的是ΔF508(0.625)。其他常见突变包括G542X(144例中有16例),在意大利南部尤为常见(49例中有14例),N1303K(144例中有8例),以及R117H(144例中有8例),仅在北部中心检测到。
PCR/OLA是一种稳健、准确、用户友好的方法,可用于以半自动化方式利用血斑对新生儿进行囊性纤维化筛查,每个突变的成本较低(0.8欧元)。