Noonan syndrome (NS) is an autosomal-dominant disorder with variable expressivity and locus heterogeneity. Despite several RAS pathway genes were implicated in NS, 20–30% of patients remain without molecular diagnosis, suggesting the involvement of further genes or multiple mechanisms. Eight patients out of 60, negative for conventional NS mutation analysis, with heterogeneous NS phenotype were investigated by means of target resequencing of 26 RAS/MAPK pathway genes. A trio was further characterized by means of whole-exome sequencing. Protein modeling and in silico prediction of protein stability allowed to identify possible pathogenic RAS pathway variants in four NS patients. A new c.355T>C variant in LZTR1 was found in patient 43. Two patients co-inherited variants in LRP1 and LZTR1 (patient 53), or LRP1 and SOS1 genes (patient 67). The forth patient (56) carried a compound heterozygote of RASAL3 gene variants and also an A2ML1 variant. While these subclinical variants are singularly present in healthy parents, they co-segregate in patients, suggesting their addictive effect and supporting a digenic inheritance, as alternative model to a more common monogenic transmission. The ERK1/2 and SAPK/JNK activation state, assessed on immortalized lymphocytes from patients 53 and 67 showed highest phosphorylation levels compared to their asymptomatic parents. These findings together with the lack of their co-occurrence in the 1000Genomes database strengthen the hypothesis of digenic inheritance in a subset of NS patients. This study suggests caution in the exclusion of subclinical variants that might play a pathogenic role providing new insights for alternative hereditary mechanisms.

Noonan综合征（NS）是一种常染色体显性疾病，具有可变的表达能力和基因座异质性。尽管NS中牵涉到几种RAS通路基因，但仍有20-30％的患者未进行分子诊断，这提示进一步基因或多种机制的参与。60位中有8位常规NS突变分析阴性，异型NS表型的患者通过对26种RAS / MAPK途径基因进行靶标重新测序进行了研究。三重奏通过全外显子组测序进一步表征。蛋白质建模和蛋白质稳定性的计算机模拟预测可以确定4名NS患者的可能致病性RAS途径变异。新c.355T>用C变种LZTR1患者中发现43两个病人共遗传变异的LRP1和LZTR1（患者53），或LRP1和SOS1基因（患者67）。第四名患者（56）携带RASAL3基因变异的复合杂合子以及A2ML1变体。尽管这些亚临床变异体在健康的父母中单独存在，但它们在患者中共隔离，表明它们具有成瘾作用并支持双基因遗传，作为更常见的单基因传播的替代模型。与无症状父母相比，对来自患者53和67的永生淋巴细胞评估的ERK1 / 2和SAPK / JNK活化状态显示出最高的磷酸化水平。这些发现以及在1000Genomes数据库中缺少它们的共现，增强了部分NS患者中双基因遗传的假设。这项研究建议谨慎地排除可能起致病作用的亚临床变异体，为替代性遗传机制提供新见解。