Identification of variants in the calcium-sensing receptor (CASR) gene is an important means of distinguishing between familial hypocalciuric hypercalcaemia (FHH) and primary hyperparathyroidism. However, identification and bioinformatics analysis of genetic variants alone is now considered insufficient as definitive proof; additional functional assessment is required to diagnose FHH with certainty. We identified two novel variants, D433Y and C739Y, and one previously reported variant G509R in the CASR of four kindreds provisionally diagnosed with FHH and aimed to functionally characterise these variants to confirm the diagnosis. Variant receptors were cloned as FLAG-tagged constructs into the mammalian expression vector, pcDNA3.1. Wild type and variant receptor constructs were expressed in HEK293 cells and their expression assessed by Western blot analysis and their functionality analysed using an IP-One assay which measures myo-inositol 1-phosphate accumulation following CaSR activation. Western blot analysis showed that the D433Y receptor had diminished mature glycosylated receptor compared with wild type CaSR whereas the G509R receptor had a complete lack of mature receptor. The C739Y receptor was consistently overexpressed. Functional assessment showed the D433Y receptor to be mildly inactivating at physiological calcium concentrations whereas the G509R receptor was inactive at all calcium concentrations. By contrast, the C739Y variant was activating compared to wild type receptor which is inconsistent with it causing FHH. We conclude that functional assessment of CaSR variants using the IP-One assay was useful in the investigation of suspected FHH probands, confirming the D433Y and G509R variants as likely pathogenic/pathogenic, but dismissing the C739Y variant as causing FHH.

钙敏感受体（CASR）基因变异的鉴定是区分家族性低钙血症性高钙血症（FHH）和原发性甲状旁腺功能亢进症的重要手段。但是，现在仅凭遗传变异的鉴定和生物信息学分析就不足以作为确定的证据。要确定诊断FHH，还需要进行其他功能评估。我们在CASR中鉴定了两个新变体D433Y和C739Y，以及一个先前报道的变体G509R临时诊断为FHH的四亲中有四分之一的人，其目的是对这些变异进行功能鉴定以确认诊断。将变异受体作为FLAG标记的构建体克隆到哺乳动物表达载体pcDNA3.1中。野生型和变体受体构建体在HEK293细胞中表达，并通过蛋白质印迹分析评估其表达，并使用IP-One测定法分析其功能，该测定法测量CaSR激活后肌醇1-磷酸酯的积累。蛋白质印迹分析表明，与野生型CaSR相比，D433Y受体减少了成熟糖基化受体，而G509R受体却完全缺乏成熟受体。C739Y受体一直过表达。功能评估显示D433Y受体在生理钙浓度下轻度失活，而G509R受体在所有钙浓度下均无活性。相比之下，与野生型受体相比，C739Y变体被激活，而野生型受体与引起FHH的不一致。我们得出结论，使用IP-One分析对CaSR变体进行功能评估可用于调查可疑的FHH先证者，证实D433Y和G509R变体可能是致病性/致病性的，但认为C739Y变体可引起FHH。