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Abstract：
Due to advances in sequencing technologies, identification of genetic variants is rapid. However, the functional consequences of most genomic variants remain unknown. Consequently, variants of uncertain significance (VUS) that appear in clinical DNA diagnostic reports lack sufficient data for interpretation. Algorithms exist to aid prediction of a variant\'s likelihood of pathogenicity, but these predictions usually lack empiric evidence. To examine the feasibility of generating functional evidence in vitro for a given variant\'s role in disease, a panel of 29 coding sequence variants in the G6PC gene was assessed. G6PC encodes glucose-6 phosphatase enzyme, and reduction in its function causes the rare metabolic disease glycogen storage disease type 1a (GSD1a). Variants were heterologously expressed as fusion proteins in a hepatocyte-derived cell line and examined for effects on steady-state protein levels, biosynthetic processing, and intracellular distribution. The screen revealed variant effects on protein levels, N-linked glycosylation status, and cellular distribution. Of the eight VUS tested, seven behaved similar to wild-type protein while one VUS, p.Cys109Tyr, exhibited features consistent with pathogenicity for all molecular phenotypes assayed, including significantly reduced protein levels, alteration in protein glycosylation status, and abnormally diffuse protein localization pattern, and has recently been reported in a patient with GSD1a. Our results show that such a screen can add empiric evidence to existing databases to aid in diagnostics, and also provides further classification for molecular phenotypes that could be used in future therapeutic screening approaches for small molecule or gene editing strategies directed at specific variants.
摘要：
由于测序技术的进步，遗传变异的鉴定是迅速的。然而,大多数基因组变异的功能后果仍然未知.因此,临床DNA诊断报告中出现的意义不确定的变异(VUS)缺乏足够的解释数据.存在有助于预测变异体致病性可能性的算法，但是这些预测通常缺乏经验证据。为了研究在体外为给定的变异体在疾病中的作用产生功能证据的可行性，对G6PC基因中29种编码序列变体进行了评估.G6PC编码葡萄糖-6磷酸酶，其功能的降低导致罕见的代谢疾病糖原贮积病1a型（GSD1a）。变体在肝细胞来源的细胞系中异源表达为融合蛋白，并检查其对稳态蛋白水平的影响。生物合成加工，和细胞内分布。屏幕显示了蛋白质水平的变异效应，N-连接糖基化状态，细胞分布。在测试的八个VUS中，七个表现类似于野生型蛋白，而一个VUS，p.Cys109Tyr,表现出与所测定的所有分子表型的致病性一致的特征，包括显著降低的蛋白质水平，蛋白质糖基化状态的改变，和异常扩散的蛋白质定位模式，最近报道了一名GSD1a患者。我们的结果表明，这样的屏幕可以为现有的数据库添加经验证据，以帮助诊断，并且还提供了分子表型的进一步分类，可用于针对特定变体的小分子或基因编辑策略的未来治疗性筛选方法。
