Deficiency of succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (ALDH5A1), OMIM 271980, 610045), the second enzyme of GABA degradation, represents a rare autosomal-recessively inherited disorder which manifests metabolically as gamma-hydroxybutyric aciduria. The neurological phenotype includes intellectual disability, autism spectrum, epilepsy and sleep and behavior disturbances. Approximately 70 variants have been reported in the ALDH5A1 gene, half of them being missense variants. In this study, 34 missense variants, of which 22 novel, were evaluated by in silico analyses using PolyPhen2 and SIFT prediction tools. Subsequently, the effect of these variants on SSADH activity was studied by transient overexpression in HEK293 cells. These studies showed severe enzymatic activity impairment for 27 out of 34 alleles, normal activity for one allele and a broad range of residual activities (25 to 74%) for six alleles. To better evaluate the alleles that showed residual activity above 25%, we generated an SSADH-deficient HEK293-Flp-In cell line using CRISPR-Cas9, in which these alleles were stably expressed. This model proved essential in the classification as deficient for one out of the seven studied alleles.

For 8 out of 34 addressed alleles, there were discrepant results among the used prediction tools, and/or in correlating the results of the prediction tools with the functional data.

In case of diagnostic urgency of missense alleles, we propose the use of the transient transfection model for confirmation of their effect on the SSADH catalytic function, since this model resulted in fast and robust functional characterization for the majority of the tested variants. In selected cases, stable transfections can be considered and may prove valuable.

GABA降解的第二种酶-琥珀酸半醛脱氢酶（SSADH;醛脱氢酶5a1（ALDH5A1），OMIM 271980，610045）缺乏，代表一种罕见的常染色体隐性遗传疾病，在代谢上表现为γ-羟基丁酸尿症。神经表型包括智力障碍，自闭症谱图，癫痫病和睡眠以及行为障碍。在ALDH5A1中已经报道了大约70种变体基因，其中一半是错义变体。在这项研究中，使用PolyPhen2和SIFT预测工具通过计算机分析对34个错义变体进行了评估，其中22个是新颖的。随后，通过在HEK293细胞中瞬时过表达来研究这些变体对SSADH活性的影响。这些研究显示34个等位基因中有27个严重酶活性受损，一个等位基因具有正常活性，而六个等位基因具有广泛的残留活性（25％至74％）。为了更好地评估显示出25％以上残留活性的等位基因，我们使用CRISPR-Cas9生成了SSA​​DH缺陷型HEK293-Flp-In细胞系，其中这些等位基因得以稳定表达。该模型在分类中被证明是必不可少的，因为它对七个研究的等位基因中的一个是不足的。

在34个寻址的等位基因中，有8个使用的预测工具之间的结果不一致，并且/或者在将预测工具的结果与功能数据相关联时。

对于错义等位基因的诊断紧急性，我们建议使用瞬时转染模型来确认其对SSADH催化功能的影响，因为该模型可对大多数测试变体产生快速而稳定的功能表征。在某些情况下，可以考虑进行稳定的转染，并可能证明其有价值。