荧光素酶检测在三名患有视隔发育不良（SOD）女性中对KAL1变异体进行体外功能评估的新应用。
Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD).
作者信息
McCabe Mark J, Hu Youli, Gregory Louise C, Gaston-Massuet Carles, Alatzoglou Kyriaki S, Saldanha José W, Gualtieri Angelica, Thankamony Ajay, Hughes Ieuan, Townshend Sharron, Martinez-Barbera Juan-Pedro, Bouloux Pierre-Marc, Dattani Mehul T
机构信息
Section of Genetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Programme, UCL Institute of Child Health, London, UK; Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia; St Vincent's Clinical School, UNSW Australia, Sydney, NSW, Australia.
Centre for Neuroendocrinology, Royal Free Hospital and University College Medical School, University College London, London, UK; Department of Anaesthesiology, Nanjing Medical University First Affiliated Hospital, Jiangsu Province Hospital, Nanjing 210029, China.
出版信息
Mol Cell Endocrinol. 2015 Dec 5;417:63-72. doi: 10.1016/j.mce.2015.09.010. Epub 2015 Sep 14.
KAL1 is implicated in 5% of Kallmann syndrome cases, a disorder which genotypically overlaps with septo-optic dysplasia (SOD). To date, a reporter-based assay to assess the functional consequences of KAL1 mutations is lacking. We aimed to develop a luciferase assay for novel application to functional assessment of rare KAL1 mutations detected in a screen of 422 patients with SOD. Quantitative analysis was performed using L6-myoblasts stably expressing FGFR1, transfected with a luciferase-reporter vector containing elements of the FGF-responsive osteocalcin promoter. The two variants assayed [p.K185N, p.P291T], were detected in three females with SOD (presenting with optic nerve hypoplasia, midline and pituitary defects). Our novel assay revealed significant decreases in transcriptional activity [p.K185N: 21% (p < 0.01); p.P291T: 40% (p < 0.001)]. Our luciferase-reporter assay, developed for assessment of KAL1 mutations, determined that two variants in females with hypopituitarism/SOD are loss-of-function; demonstrating that this assay is suitable for quantitative assessment of mutations in this gene.
KAL1基因与5%的卡尔曼综合征病例有关，卡尔曼综合征是一种在基因型上与视隔发育不良（SOD）重叠的疾病。迄今为止，缺乏一种基于报告基因的检测方法来评估KAL1突变的功能后果。我们旨在开发一种荧光素酶检测方法，用于对在422例SOD患者筛查中检测到的罕见KAL1突变进行功能评估的新应用。使用稳定表达FGFR1的L6成肌细胞进行定量分析，这些细胞用含有FGF反应性骨钙素启动子元件的荧光素酶报告载体进行转染。检测的两个变体[p.K185N，p.P291T]在三名患有SOD的女性中被检测到（表现为视神经发育不全、中线和垂体缺陷）。我们的新检测方法显示转录活性显著降低[p.K185N：21%（p<0.01）；p.P291T：40%（p<0.001）]。我们为评估KAL1突变而开发的荧光素酶报告检测方法确定，患有垂体功能减退/SOD的女性中的两个变体是功能丧失型；证明该检测方法适用于对该基因中的突变进行定量评估。
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