Yu Seok-Ho, Kubaski Francyne, Arno Gavin, Phinney Whitney, Wood Tim C, Flanagan-Steet Heather, Pollard Laura M, Steet Richard
Research Division, Greenwood Genetic Center, Greenwood, SC, USA.
Biochemical Diagnostic Laboratory, Greenwood Genetic Center, Greenwood, SC, USA.
NPJ Genom Med. 2024 Dec 19;9(1):68. doi: 10.1038/s41525-024-00457-1.
With the expansion of newborn screening efforts for MPS disorders, the number of identified variants of uncertain significance in IDUA continues to increase. To better define functional consequences of identified IDUA variants, we developed a HEK293-based expression platform that can be used to determine the relative specific activity of variant α-iduronidases by combining a fluorescence-based activity assay and semi-quantitative western blotting. We employed the current platform to characterize over thirty different IDUA variants, including known benign and pathogenic variants, as well as multiple variants of uncertain significance identified through newborn screening. This analysis allowed the stratification of variant enzymes based on their relative specific activity, and uncovered distinct effects of the different variants on enzyme folding, processing, and stability. While relative specific activity serves as a useful first-level test for enzyme function, our observations reinforce the need for secondary analyses of enzyme function to fully assess variant pathogenicity.
随着黏多糖贮积症（MPS）疾病新生儿筛查工作的扩大，在艾杜糖醛酸酶（IDUA）中鉴定出的意义未明的变异体数量持续增加。为了更好地定义已鉴定的IDUA变异体的功能后果，我们开发了一个基于人胚肾293（HEK293）细胞的表达平台，该平台可通过结合基于荧光的活性测定和半定量蛋白质免疫印迹法来确定变异型α-艾杜糖醛酸酶的相对比活性。我们利用当前平台对三十多种不同的IDUA变异体进行了表征，包括已知的良性和致病性变异体，以及通过新生儿筛查鉴定出的多个意义未明的变异体。该分析允许根据变异酶的相对比活性对其进行分层，并揭示了不同变异体对酶折叠、加工和稳定性的不同影响。虽然相对比活性作为酶功能的有用一级测试，但我们的观察结果强化了对酶功能进行二级分析以全面评估变异体致病性的必要性。
