Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany.
Hum Mol Genet. 2013 Apr 15;22(8):1643-53. doi: 10.1093/hmg/ddt014. Epub 2013 Jan 17.
Costello syndrome is a congenital disorder comprising a characteristic face, severe feeding difficulties, skeletal, cardiac and skin abnormalities, intellectual disability and predisposition to malignancies. It is caused by heterozygous germline HRAS mutations mostly affecting Gly(12) or Gly(13), which impair HRAS-GTPase activity and result in increased downstream signal flow independent of incoming signals. Functional analyses of rarer HRAS mutations identified in individuals with attenuated Costello syndrome phenotypes revealed altered GDP/GTP nucleotide affinities (p.K117R) and inefficient effector binding (p.E37dup). Thus, both phenotypic and functional variability associated with HRAS mutations are evident. Here, we report on a novel heterozygous HRAS germline mutation (c.187_207dup, p.E63_D69dup) in a girl presenting with a phenotype at the milder end of the Costello syndrome spectrum. The p.E63_D69dup mutation impaired co-precipitation of recombinant HRAS with NF1 GTPase-activating protein (GAP) suggesting constitutive HRAS(E63_D69dup) activation due to GAP insensitivity. Indeed, we identified strongly augmented active HRAS(E63_D69dup) that co-precipitated with effectors RAF1, RAL guanine nucleotide dissociation stimulator and phospholipase C1. However, we could not pull down active HRAS(E63_D69dup) using the target protein PIK3CA, indicating a compromised association between active HRAS(E63_D69dup) and PIK3CA. Accordingly, overexpression of HRAS(E63_D69dup) increased steady-state phosphorylation of MEK1/2 and ERK1/2 downstream of RAF, whereas AKT phosphorylation downstream of phosphoinositide 3-kinase (PI3K) was not enhanced. By analyzing signaling dynamics, we found that HRAS(E63_D69dup) has impaired reagibility to stimuli resulting in reduced and disrupted capacity to transduce incoming signals to the RAF-MAPK and PI3K-AKT cascade, respectively. We suggest that disrupted HRAS reagibility, as we demonstrate for the p.E63_D69dup mutation, is a previously unappreciated molecular pathomechanism underlying Costello syndrome.
神经纤维瘤病 1 型相关 Ras 相关区域家族成员 A（HRAS）综合征是一种先天性疾病，其特征为特殊面容、严重的喂养困难、骨骼、心脏和皮肤异常、智力障碍和易患恶性肿瘤。它是由杂合的种系 HRAS 突变引起的，这些突变主要影响 Gly(12)或 Gly(13)，从而损害 HRAS-GTP 酶的活性，并导致下游信号流的增加，而与传入信号无关。对具有减轻的神经纤维瘤病 1 型相关 Ras 相关区域家族成员 A（HRAS）综合征表型的个体中发现的更罕见的 HRAS 突变的功能分析表明，改变了 GDP/GTP 核苷酸亲和力（p.K117R）和低效的效应物结合（p.E37dup）。因此，与 HRAS 突变相关的表型和功能的可变性都是明显的。在这里，我们报道了一个患有更温和的神经纤维瘤病 1 型相关 Ras 相关区域家族成员 A（HRAS）综合征表型的女孩中的一个新的杂合 HRAS 种系突变（c.187_207dup，p.E63_D69dup）。p.E63_D69dup 突变干扰了重组 HRAS 与神经纤维瘤病 1 型 GTP 酶激活蛋白（NF1 GAP）的共沉淀，表明由于 GAP 不敏感而导致 HRAS（E63_D69dup）的组成型激活。事实上，我们鉴定出与效应物 RAF1、Ral 鸟嘌呤核苷酸解离刺激因子和磷脂酶 C1 共沉淀的强烈增强的活性 HRAS（E63_D69dup）。然而，我们不能使用靶蛋白 PIK3CA 下拉活性 HRAS（E63_D69dup），表明活性 HRAS（E63_D69dup）与 PIK3CA 之间的关联受损。因此，HRAS（E63_D69dup）的过表达增加了 RAF 下游 MEK1/2 和 ERK1/2 的稳态磷酸化，而磷酸肌醇 3-激酶（PI3K）下游的 AKT 磷酸化没有增强。通过分析信号动力学，我们发现 HRAS（E63_D69dup）对刺激的反应性受损，导致其向 RAF-MAPK 和 PI3K-AKT 级联分别转导传入信号的能力降低和中断。我们认为，正如我们对 p.E63_D69dup 突变所证明的那样，HRAS 反应性的破坏是神经纤维瘤病 1 型相关 Ras 相关区域家族成员 A（HRAS）综合征的一种以前未被认识到的分子发病机制。
