The loss-of-function variants of the human asparagine synthetase (ASNS) gene cause asparagine synthetase deficiency (ASNSD). Diagnosis of ASNSD requires genetic tests because a specific biochemical diagnostic for ASNSD is not available. There are a few reports describing the functional evaluation of ASNS variants. Therefore, in vitro methods are needed to evaluate the detected variants in patients. In this report, five types of human ASNS proteins (wild-type and our reported four variants: p.Leu145Ser, p.Leu247Trp, p.Val489Asp, and p.Trp541Cysfs*5) were expressed in silkworm using a baculoviral expression system. An enzymatic activity assay of ASNS was performed, and the concentration of asparagine by ninhydrin and High Performance Liquid Chromatography methods using the purified recombinant proteins was measured. We established ASNS deficient HEK293 cells using the CRISPR/Cas9 method and evaluated the growth of cells without asparagine after transduction of ASNS variants with a lentiviral expression system. The four ASNS variants displayed significantly low enzymatic activity. The ASNS deficient HEK293 cells transduced with wild-type ASNS grew without asparagine, whereas cells transduced with the variants did not grow or showed significantly slower growth than cells transduced with wild-type ASNS. Herein, we established a method for evaluating the enzymatic activity of the recombinant human ASNS variants. The results of the cell-based assay corroborated the results of the enzymatic activity. These methods should enable the evaluation of the pathogenicity of ASNS variants.

中文翻译：

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人天冬酰胺合成酶四种变异体的体外功能分析

人类天冬酰胺合成酶 (ASNS) 基因的功能缺失变体导致天冬酰胺合成酶缺乏症 (ASNSD)。ASNSD 的诊断需要基因检测，因为没有针对 ASNSD 的特定生化诊断。有一些报告描述了 ASNS 变体的功能评估。因此，体外需要方法来评估在患者中检测到的变异。在本报告中，使用杆状病毒表达系统在家蚕中表达了五种人类 ASNS 蛋白（野生型和我们报告的四种变体：p.Leu145Ser、p.Leu247Trp、p.Val489Asp 和 p.Trp541Cysfs*5）。进行了 ASNS 的酶活性测定，并通过茚三酮和高效液相色谱法使用纯化的重组蛋白测量了天冬酰胺的浓度。我们使用 CRISPR/Cas9 方法建立了ASNS缺陷型 HEK293 细胞，并在用慢病毒表达系统转导 ASNS 变体后评估了不含天冬酰胺的细胞的生长。四种 ASNS 变体显示出显着低的酶活性。ASNS _用野生型 ASNS 转导的缺陷 HEK293 细胞在没有天冬酰胺的情况下生长，而用变体转导的细胞不生长或显示出比用野生型 ASNS 转导的细胞显着慢的生长。在此，我们建立了一种评估重组人 ASNS 变体的酶活性的方法。基于细胞的测定结果证实了酶活性的结果。这些方法应该能够评估 ASNS 变异的致病性。