Abstract：
BACKGROUND: Heterozygous Indian Hedgehog gene (IHH) variants are associated with brachydactyly type A1 (BDA1). However, in recent years, numerous variants have been identified in patients with short stature and more variable forms of brachydactyly. Many are located in the C-terminal domain of IHH (IHH-C), which lacks signaling activity but is critical for auto-cleavage and activation of the N-terminal (IHH-N) peptide. The absence of functional studies of IHH variants, particularly for those located in IHH-C, has led to these variants being classified as variants of uncertain significance (VUS).
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.
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.
摘要：
背景：杂合印度Hedgehog基因（IHH）变体与短指A1型（BDA1）相关。然而,近年来,在身材矮小和短指形式更多的患者中已经发现了许多变体。许多位于IHH（IHH-C）的C末端结构域，其缺乏信号传导活性,但对于N-末端(IHH-N)肽的自切割和活化是关键的。缺乏IHH变体的功能研究，特别是对于那些位于IHH-C的人来说，导致这些变体被归类为不确定显著性变体(VUS)。
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.
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.
