Mullin Benjamin H, Pavlos Nathan J, Brown Suzanne J, Walsh John P, McKellar Ross A, Wilson Scott G, Ward Bryan K
Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, WA 6009, Australia.
School of Biomedical Sciences, University of Western Australia, Nedlands, WA 6009, Australia.
J Endocr Soc. 2022 Feb 18;6(5):bvac025. doi: 10.1210/jendso/bvac025. eCollection 2022 May 1.
In the clinic it is important to differentiate primary hyperparathyroidism (PHPT) from the more benign, inherited disorder, familial hypocalciuric hypercalcemia (FHH). Since the conditions may sometimes overlap biochemically, identification of calcium-sensing receptor ( gene variants causative of FHH (but not PHPT) is the most decisive diagnostic aid. When novel variants are identified, bioinformatics and functional assessment are required to establish pathogenicity.
We identified 3 novel transmembrane domain missense variants, Thr699Asn, Arg701Gly, and Thr808Pro, in 3 probands provisionally diagnosed with FHH and examined the variants using bioinformatics and functional analysis.
Bioinformatics assessment utilized wANNOVAR software. For functional characterization, each variant was cloned into a mammalian expression vector; wild-type and variant receptors were transfected into HEK293 cells, and their expression and cellular localization were assessed by Western blotting and confocal immunofluorescence, respectively. Receptor activation in HEK293 cells was determined using an IP-One ELISA assay following stimulation with Ca ions.
Bioinformatics analysis of the variants was unable to definitively assign pathogenicity. Compared with wild-type receptor, all variants demonstrated impaired expression of mature receptor reaching the cell surface and diminished activation at physiologically relevant Ca concentrations.
Three missense variants identified in probands provisionally diagnosed with FHH result in receptor inactivation and are therefore likely causative of FHH. Inactivation may be due to inadequate processing/trafficking of mature receptor and/or conformational changes induced by the variants affecting receptor signaling. This study demonstrates the value of functional studies in assessing genetic variants identified in hypercalcemic patients.
在临床中，区分原发性甲状旁腺功能亢进症（PHPT）与更良性的遗传性疾病——家族性低钙血症性高钙血症（FHH）非常重要。由于这两种病症在生化指标上有时会重叠，因此鉴定导致FHH（而非PHPT）的钙敏感受体（CaSR）基因变异是最具决定性的诊断辅助手段。当发现新的变异时，需要进行生物信息学和功能评估以确定其致病性。
我们在3名初步诊断为FHH的先证者中鉴定出3种新的跨膜结构域错义变异，即Thr699Asn、Arg701Gly和Thr808Pro，并使用生物信息学和功能分析方法对这些变异进行了研究。
生物信息学评估使用wANNOVAR软件。为了进行功能表征，将每个变异克隆到哺乳动物表达载体中；将野生型和变异型受体转染到HEK293细胞中，分别通过蛋白质免疫印迹法和共聚焦免疫荧光法评估其表达和细胞定位。在用钙离子刺激后，使用IP-One ELISA检测法测定HEK293细胞中的受体激活情况。
对这些变异的生物信息学分析无法明确确定其致病性。与野生型受体相比，所有变异均显示成熟受体到达细胞表面的表达受损，并且在生理相关的钙浓度下激活减少。
在初步诊断为FHH的先证者中鉴定出的3种错义变异导致受体失活，因此可能是FHH的病因。失活可能是由于成熟受体的加工/运输不足和/或变异诱导的影响受体信号传导的构象变化。本研究证明了功能研究在评估高钙血症患者中鉴定出的基因变异方面的价值。
