molecular mechanism of abnormal growth hormone action

(1)在我们的患者中发现了GH-1基因的杂合错义突变R77C。该突变型GH不仅自身不能刺激酪氨酸磷酸化，而且作为拮抗剂抑制了野生型GH的活性。尽管患者的父亲的等位基因存在基因组突变，但他的血清中却没有表达突变的生长激素，这一问题至今仍未得到解决。为了排除基因组暗示的可能性，我们对其祖母的GH-1基因进行了基因型分析。由于没有突变，体细胞嵌合可能是更可能的原因。(2)我们报道了生长激素受体（GHR）基因内含子9的一种新的杂合供体剪接位点突变，这些突变发生在表现出部分生长激素不敏感和高血清GH结合蛋白（GHBP）水平的兄弟姐妹中。该突变导致剪接异常，产生由277个氨基酸组成的截断GHR (GHR-277)，缺失了GHR的大部分胞内结构域，包括box 1和box 2。在本研究中，我们在体外鉴定了GHR-277在COS-7或CHO细胞中的表达功能。Scatchard分析显示，GHR-277的结合位点数量是野生型全长GHR （GHR-fl）的两倍。表达ghr -277的细胞培养液中GHBP的水平比表达GHR-fl的细胞培养液中GHBP的水平高约3倍。与GHR-fl相比，配体诱导的内化明显减少。此外，在gh诱导的STAT5酪氨酸磷酸化中，当GHR-277和GHR-fl共转染时，GHR-277发挥了显性的负作用。这些体外实验结果可以很好地解释我们患者的临床特征。(3)在一位可能患有生长激素不敏感综合征的患者中，我们发现了一个杂合错义突变C422F。然而，该突变GHR的功能研究并不支持该突变是其生长失败的原因。

(1) A heterozygous missense mutation R77C in the GH-1 gene was identified in our patient. This mutant GH not only failed to stimulate tyrosine phosphorylation by itself, but also inhibited the activity of the wild-type GH as an antagonist. There are still unresolved problems why the patient's father did not express the mutant GH in his serum despite the presence of a genomic mutation in his allele. To exclude the possibility of genomic implinting, we investigated the genotype of GH-1 gene in his grand mother. Since there was no mutation, somatic mosaicism may be more likely cause.(2) We have reported a novel heterozygous donor splice site mutation in intron 9 of the GH receptor (GHR) gene in siblings who showed partial GH insensitivity and high serum GH-binding protein (GHBP) levels. This mutation caused the splicing abnormality and produced the truncated GHR consisting of 277 amino acids (GHR-277), which lacked most of the intracellular domain of GHR, including both box 1 and 2. in this study we have characterized the function of GHR-277 expression in COS-7 or CHO cells in vitro. Scatchard analysis revealed that GHR-277 possessed twice the number of binding sites compared to wild-type full-length GHR (GHR-fl). The GHBP level in culture medium of GHR-277-expressing cells was approximately 3 times higher than that in GHR-fl expressing cells. The ligand induced internalization was significantly reduced comapred with that of GHR-fl. Moreover, in GH-induced tyrosine phosphorylation of STAT5, GHR-277 exerted a dominant negative effect when GHR-277 and GHR-fl were cotransfected. These in vitro results would well explain the clinical characteristics in our patients.(3) In a patient with a possible GH insensitivity syndrome, we found a heterozygous missense mutation C422F.However, functional study of this mutant GHR did not support this mutation as a cause of his growth failure.