Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome. Homozygosity for three different variants—c.187C>T (p.Arg63^∗), c.299T>C (p.Leu100Pro), and c.1142_1143delTT (p.Leu381Hisfs^∗95)—were identified in four subjects from three families. All variants severely affected protein stability, causing accelerated degradation, and variably perturbed SPRED2 functional behavior. When overexpressed in cells, all variants were unable to negatively modulate EGF-promoted RAF1, MEK, and ERK phosphorylation, and time-course experiments in primary fibroblasts (p.Leu100Pro and p.Leu381Hisfs^∗95) documented an increased and prolonged activation of the MAPK cascade in response to EGF stimulation. Morpholino-mediated knockdown of spred2a and spred2b in zebrafish induced defects in convergence and extension cell movements indicating upregulated RAS-MAPK signaling, which were rescued by expressing wild-type SPRED2 but not the SPRED2^{Leu381Hisfs∗95} protein. The clinical phenotype of the four affected individuals included developmental delay, intellectual disability, cardiac defects, short stature, skeletal anomalies, and a typical facial gestalt as major features, without the occurrence of the distinctive skin signs characterizing Legius syndrome. These features, in part, characterize the phenotype of Spred2^−/− mice. Our findings identify the second recessive form of Noonan syndrome and document pleiotropic consequences of SPRED2 loss of function in development.

通过 RAS 和丝裂原相关蛋白激酶 (MAPK) 级联的上调信号流是 RASopathies 的统一机制主题，RASopathies 是影响发育和生长的疾病家族。超过 20 个基因的致病变异与 RASopathies 有因果关系，其中大多数在促进信号增强方面起主导作用。在这里，我们报告 SPRED2 功能丧失与 Noonan 综合征的隐性表型有因果关系。三种不同变体的纯合度——c.187C>T (p.Arg63 ^∗ )、c.299T>C (p.Leu100Pro) 和 c.1142_1143delTT (p.Leu381Hisfs ^∗95)——在来自三个家庭的四名受试者中被鉴定出来。所有变体都严重影响了蛋白质的稳定性，导致降解加速，并不同程度地扰乱了 SPRED2 的功能行为。当在细胞中过度表达时，所有变体都无法负调节 EGF 促进的 RAF1、MEK 和 ERK 磷酸化，并且原代成纤维细胞（p.Leu100Pro 和 p.Leu381Hisfs ^∗ 95）的时间进程实验记录了响应 EGF 刺激的 MAPK 级联。Morpholino 介导的斑马鱼spred2a和spred2b敲低诱导融合和延伸细胞运动缺陷，表明 RAS-MAPK 信号上调，通过表达野生型 SPRED2 而不是 SPRED2 ^{Leu381Hisfs 挽救}了这些^{信号* 95}蛋白质。四个受影响个体的临床表型包括发育迟缓、智力障碍、心脏缺陷、身材矮小、骨骼异常和典型的面部完形作为主要特征，没有出现表征 Legius 综合征的独特皮肤体征。这些特征部分表征了Spred2 ^-/-小鼠的表型。我们的研究结果确定了 Noonan 综合征的第二种隐性形式，并记录了 SPRED2 在发育过程中功能丧失的多效性后果。