OBJECTIVE: To establish a simple functional assay to determine the pathogenicity of IHH VUS and confirm that variants in the C-terminal domain affect protein function.
METHODS: In vitro studies were performed for 9 IHH heterozygous variants, to test their effect on secretion and IHH intracellular processing by western blot of cells expressing each variant.
RESULTS: IHH secretion was significantly reduced in all mutants, regardless of the location. Similarly, intracellular levels of N-terminal and C-terminal IHH peptides were severely reduced in comparison with the control. Two variants present at a relatively high frequency in the general population also reduced secretion but to a lesser degree in the heterozygous state.
CONCLUSIONS: These studies provide the first evidence that variants in the C-terminal domain affect the secretion capacity of IHH and thus, reduce availability of IHH ligand, resulting in short stature and mild skeletal defects. The secretion assay permits a relatively easy test to determine the pathogenicity of IHH variants. All studied variants affected secretion and interestingly, more frequent population variants appear to have a deleterious effect and thus contribute to height variation.
目的:建立一种简单的功能测定来确定IHHVUS的致病性,并确认C端结构域中的变体影响蛋白质功能。
方法:对9个IHH杂合变体进行了体外研究,通过表达每个变体的细胞的蛋白质印迹来测试它们对分泌和IHH细胞内加工的影响。
结果:所有突变体的IHH分泌均显著减少,不管位置。同样,与对照相比,N-末端和C-末端IHH肽的细胞内水平显著降低。两种变体在一般人群中以相对较高的频率出现,也减少了分泌,但在杂合状态下程度较低。
结论:这些研究提供了第一个证据,表明C末端结构域的变体会影响IHH的分泌能力,因此,降低IHH配体的可用性,导致身材矮小和轻度骨骼缺陷。分泌测定允许相对容易的测试来确定IHH变体的致病性。所有研究的变异都会影响分泌,有趣的是,更频繁的群体变异似乎具有有害作用,从而导致身高变异.