Muñoz-Pujol Gerard, Ugarteburu Olatz, Segur-Bailach Eulàlia, Moliner Sonia, Jurado Susana, Garrabou Glòria, Guitart-Mampel Mariona, García-Villoria Judit, Artuch Rafael, Fons Carme, Ribes Antonia, Tort Frederic
Secció d'Errors Congènits del Metabolisme-IBC, Servei de Bioquímica i Genètica Molecular, Hospital Clínic de Barcelona, IDIBAPS, CIBERER, Barcelona, Spain.
Inherited Metabolic diseases and Muscle Disorder's lab, Cellex-IDIBAPS, Faculty of Medicine and Health Sciences, University of Barcelona, Internal Medicine Service-Hospital Clinic of Barcelona and CIBERER, Barcelona, Spain.
J Inherit Metab Dis. 2023 Nov;46(6):1029-1042. doi: 10.1002/jimd.12681. Epub 2023 Oct 3.
The determination of the functional impact of variants of uncertain significance (VUS) is one of the major bottlenecks in the diagnostic workflow of inherited genetic diseases. To face this problem, we set up a CRISPR/Cas9-based strategy for knock-in cellular model generation, focusing on inherited metabolic disorders (IMDs). We selected variants in seven IMD-associated genes, including seven reported disease-causing variants and four benign/likely benign variants. Overall, 11 knock-in cell models were generated via homology-directed repair in HAP1 haploid cells using CRISPR/Cas9. The functional impact of the variants was determined by analyzing the characteristic biochemical alterations of each disorder. Functional studies performed in knock-in cell models showed that our approach accurately distinguished the functional effect of pathogenic from non-pathogenic variants in a reliable manner in a wide range of IMDs. Our study provides a generic approach to assess the functional impact of genetic variants to improve IMD diagnosis and this tool could emerge as a promising alternative to invasive tests, such as muscular or skin biopsies. Although the study has been performed only in IMDs, this strategy is generic and could be applied to other genetic disorders.
意义未明变异（VUS）功能影响的确定是遗传性疾病诊断流程中的主要瓶颈之一。为解决这一问题，我们建立了一种基于CRISPR/Cas9的敲入细胞模型构建策略，重点关注遗传性代谢疾病（IMD）。我们在7个与IMD相关的基因中选择了变异，包括7个已报道的致病变异和4个良性/可能良性变异。总体而言，使用CRISPR/Cas9通过同源定向修复在HAP1单倍体细胞中生成了11个敲入细胞模型。通过分析每种疾病的特征性生化改变来确定变异的功能影响。在敲入细胞模型中进行的功能研究表明，我们的方法能够在广泛的IMD中以可靠的方式准确区分致病变异和非致病变异的功能效应。我们的研究提供了一种评估基因变异功能影响以改善IMD诊断的通用方法，并且该工具可能成为侵入性检测（如肌肉或皮肤活检）的有前景的替代方法。尽管该研究仅在IMD中进行，但这种策略具有通用性，可应用于其他遗传疾病。
