Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is caused by aberrant RAS-MAPK signaling and is genetically heterogeneous, which explains, in part, the marked clinical variability documented for this Mendelian trait. Recently, we and others identified SOS1 as a major gene underlying NS. Here, we explored further the spectrum of SOS1 mutations and their associated phenotypic features. Mutation scanning of the entire SOS1 coding sequence allowed the identification of 33 different variants deemed to be of pathological significance, including 16 novel missense changes and in-frame indels. Various mutation clusters destabilizing or altering orientation of regions of the protein predicted to contribute structurally to the maintenance of autoinhibition were identified. Two previously unappreciated clusters predicted to enhance SOS1's recruitment to the plasma membrane, thus promoting a spatial reorientation of domains contributing to inhibition, were also recognized. Genotype-phenotype analysis confirmed our previous observations, establishing a high frequency of ectodermal anomalies and a low prevalence of cognitive impairment and reduced growth. Finally, mutation analysis performed on cohorts of individuals with nonsyndromic pulmonic stenosis, atrial septal defects, and ventricular septal defects excluded a major contribution of germline SOS1 lesions to the isolated occurrence of these cardiac anomalies.

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population.

Uncovering frequent motives of action by which variants impair 3' splice site (3'ss) recognition and selection is essential to improve our understanding of this complex process. Through several mini-gene experiments, we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3(NF1):c.1722-11T>G, although expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG-exclusion zone (AGEZ) between the authentic 3'ss AG and the branch point. Evaluation of 90 additional noncanonical intronic NF1 3'ss mutations confirmed that 63% of all mutations and 89% (49/55) of the single-nucleotide variants upstream of positions -3 interrupt the AGEZ. Of these AGEZ-interrupting mutations, 24/49 lead to exon skipping suggesting that absence of AG in this region is necessary for accurate 3'ss selection already in the initial steps of splicing. The analysis of 91 noncanonical NF1 3'ss mutations also shows that 90% either introduce a novel AG in the AGEZ, cause a Y>R transversion at position -3 or remove ≥2 Ys in the AGEZ. We confirm in a validation cohort that these three motives distinguish spliceogenic from splice-neutral variants with 85% accuracy and, therefore, are generally applicable to select among variants of unknown significance those likely to affect splicing.

The RASopathies constitute a family of autosomal-dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal-regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease-causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS-causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.