Abstract

Context

Severe forms of Growth Hormone Insensitivity (GHI) are characterized by extreme short stature, dysmorphism and metabolic anomalies.

Objective

Identification of the genetic cause of growth failure in 3 ‘classical’ GHI subjects.

Design

A novel intronic GHR variant was identified, and in vitro splicing assays confirmed aberrant splicing. A 6Ω pseudoexon GHR vector and patient fibroblast analysis assessed the consequences of the novel pseudoexon inclusion and the impact on GHR function.

Results

We identified a novel homozygous intronic GHR variant (g.5:42700940T>G, c.618 + 836T> G), 44bp downstream of the previously recognized intronic 6Ψ GHR pseudoexon mutation in the index patient. Two siblings also harbored the novel intronic 6Ω pseudoexon GHR variant in compound heterozygosity with the known GHR c.181C>T (R43X) mutation. In vitro splicing analysis confirmed inclusion of a 151bp mutant 6Ω pseudoexon not identified in wild-type constructs. Inclusion of the 6Ω pseudoexon causes a frameshift resulting in a non-functional truncated GHR lacking the transmembrane and intracellular domains. The truncated 6Ω pseudoexon protein demonstrated extracellular accumulation and diminished activation of STAT5B signaling following growth hormone stimulation.

Conclusion

Novel GHR 6Ω pseudoexon inclusion results in loss of GHR function consistent with a severe GHI phenotype. This represents a novel mechanism of Laron syndrome and is the first deep intronic variant identified causing severe postnatal growth failure. The 2 kindreds originate from the same town in Campania, Southern Italy, implying common ancestry. Our findings highlight the importance of studying variation in deep intronic regions as a cause of monogenic disorders.

中文翻译：

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生长激素受体 (Ghr) 6ω 假外显子激活：严重生长激素不敏感 (Ghi) 的新原因

摘要

语境

生长激素不敏感 (GHI) 的严重形式的特征是身材极度矮小、畸形和代谢异常。

客观的

确定 3 名“经典”GHI 受试者生长障碍的遗传原因。

设计

鉴定了一种新的内含子GHR变体，体外剪接试验证实了异常剪接。6Ω 假外显子GHR载体和患者成纤维细胞分析评估了新型假外显子包含的后果和对 GHR 功能的影响。

结果

我们发现了一种新的纯合内含子GHR变异（g.5:42700940T>G，c.618 + 836T>G），位于索引患者先前识别的内含子 6Ψ GHR假外显子突变下游 44bp 。两个兄弟姐妹还在复合杂合性中携带了新型内含子 6Ω 假外显子GHR变体，具有已知的GHR c.181C>T (R43X) 突变。体外剪接分析证实包含在野生型构建体中未鉴定的 151bp 突变 6Ω 假外显子。包含 6Ω 假外显子会导致移码，导致无功能的截断 GHR，缺少跨膜和细胞内结构域。在生长激素刺激后，截短的 6Ω 假外显子蛋白表现出细胞外积聚和 STAT5B 信号激活减弱。

结论

新的GHR 6Ω 假外显子包含导致与严重 GHI 表型一致的 GHR 功能丧失。这代表了 Laron 综合征的一种新机制，并且是第一个发现的导致严重出生后生长障碍的深度内含子变异。这两个家族起源于意大利南部坎帕尼亚的同一个城镇，暗示着共同的血统。我们的研究结果强调了研究深层内含子区域变异作为单基因疾病原因的重要性。