PDF(Pubmed)
Abstract：
The goal of this study is to demonstrate the utility of a growth assay to quantify the functional impact of single nucleotide variants (SNVs) in SLC2A1, the gene responsible for Glut1DS.
The functional impact of 40 SNVs in SLC2A1 was quantitatively determined in HAP1 cells in which SLC2A1 is required for growth. Donor libraries were introduced into the endogenous SLC2A1 gene in HAP1-Lig4KO cells using CRISPR/Cas9. Cell populations were harvested and sequenced to quantify the effect of variants on growth and generate a functional score. Quantitative functional scores were compared to 3-OMG uptake, SLC2A1 cell surface expression, CADD score, and clinical data, including CSF/blood glucose ratio.
Nonsense variants (N = 3) were reduced in cell culture over time resulting in negative scores (mean score: -1.15 ± 0.17), whereas synonymous variants (N = 10) were not depleted (mean score: 0.25 ± 0.12) (P < 2e-16). Missense variants (N = 27) yielded a range of functional scores including slightly negative scores, supporting a partial function and intermediate phenotype. Several variants with normal results on either cell surface expression (p.N34S and p.W65R) or 3-OMG uptake (p.W65R) had negative functional scores. There is a moderate but significant correlation between our functional scores and CADD scores.
Cell growth is useful to quantitatively determine the functional effects of SLC2A1 variants. Nonsense variants were reliably distinguished from benign variants in this in vitro functional assay. For facilitating early diagnosis and therapeutic intervention, future work is needed to determine the functional effect of every possible variant in SLC2A1.
The functional impact of 40 SNVs in SLC2A1 was quantitatively determined in HAP1 cells in which SLC2A1 is required for growth. Donor libraries were introduced into the endogenous SLC2A1 gene in HAP1-Lig4KO cells using CRISPR/Cas9. Cell populations were harvested and sequenced to quantify the effect of variants on growth and generate a functional score. Quantitative functional scores were compared to 3-OMG uptake, SLC2A1 cell surface expression, CADD score, and clinical data, including CSF/blood glucose ratio.
Nonsense variants (N = 3) were reduced in cell culture over time resulting in negative scores (mean score: -1.15 ± 0.17), whereas synonymous variants (N = 10) were not depleted (mean score: 0.25 ± 0.12) (P < 2e-16). Missense variants (N = 27) yielded a range of functional scores including slightly negative scores, supporting a partial function and intermediate phenotype. Several variants with normal results on either cell surface expression (p.N34S and p.W65R) or 3-OMG uptake (p.W65R) had negative functional scores. There is a moderate but significant correlation between our functional scores and CADD scores.
Cell growth is useful to quantitatively determine the functional effects of SLC2A1 variants. Nonsense variants were reliably distinguished from benign variants in this in vitro functional assay. For facilitating early diagnosis and therapeutic intervention, future work is needed to determine the functional effect of every possible variant in SLC2A1.
摘要：
目的：这项研究的目的是证明生长测定的实用性，以量化SLC2A1中负责Glut1DS的基因的单核苷酸变体（SNV）的功能影响。
方法：在生长需要SLC2A1的HAP1细胞中定量测定了SLC2A1中40种SNV的功能影响。使用CRISPR/Cas9将供体文库引入HAP1-Lig4KO细胞中的内源性SLC2A1基因中。收获细胞群并测序以定量变体对生长的影响并产生功能评分。将定量功能评分与3-OMG摄取进行比较，SLC2A1细胞表面表达，CADD得分,和临床数据，包括CSF/血糖比。
结果：随着时间的推移，细胞培养物中的无义变体（N=3）减少，导致阴性评分（平均评分：-1.15±0.17），而同义变体（N=10）未耗尽（平均得分：0.25±0.12）（P<2e-16）。错义变体（N=27）产生了一系列功能评分，包括略负的评分，支持部分功能和中间表型。在任一细胞表面表达上具有正常结果的几种变体（p。N34S和p.W65R）或3-OMG摄取（p。W65R)的功能评分为阴性。我们的功能评分和CADD评分之间存在中等但显着的相关性。
结论：细胞生长可用于定量确定SLC2A1变体的功能作用。在该体外功能测定中,无义变体可靠地与良性变体区分开。为了促进早期诊断和治疗干预，需要未来的工作来确定SLC2A1中每个可能变体的功能效果。
方法：在生长需要SLC2A1的HAP1细胞中定量测定了SLC2A1中40种SNV的功能影响。使用CRISPR/Cas9将供体文库引入HAP1-Lig4KO细胞中的内源性SLC2A1基因中。收获细胞群并测序以定量变体对生长的影响并产生功能评分。将定量功能评分与3-OMG摄取进行比较，SLC2A1细胞表面表达，CADD得分,和临床数据，包括CSF/血糖比。
结果：随着时间的推移，细胞培养物中的无义变体（N=3）减少，导致阴性评分（平均评分：-1.15±0.17），而同义变体（N=10）未耗尽（平均得分：0.25±0.12）（P<2e-16）。错义变体（N=27）产生了一系列功能评分，包括略负的评分，支持部分功能和中间表型。在任一细胞表面表达上具有正常结果的几种变体（p。N34S和p.W65R）或3-OMG摄取（p。W65R)的功能评分为阴性。我们的功能评分和CADD评分之间存在中等但显着的相关性。
结论：细胞生长可用于定量确定SLC2A1变体的功能作用。在该体外功能测定中,无义变体可靠地与良性变体区分开。为了促进早期诊断和治疗干预，需要未来的工作来确定SLC2A1中每个可能变体的功能效果。
