The study was performed in accordance with the Declaration of Helsinki and with the approval of the Faculty of Health Sciences Human Research Ethics Committee, University of Cape Town (HREC: 449/2016). Written informed consent was obtained from the parents and/or the patient prior to their involvement into the study, including permission to publish photographs.

This study was conducted in Cape Town, South Africa, and included 26 participants (20 children and six adults); among them, 20 were unrelated. Patients were recruited through the University of Cape Town (UCT) affiliated Hospitals, namely Red Cross War Memorial Children's Hospital (RCWMCH) and Groote Schuur Hospital (GSH). Patients were selected retrospectively and prospectively using the Van der Burgt scoring system for clinical diagnosis of NS (Van der Burgt, 2007). All pediatric and adult patients were assessed by trained clinical geneticists familiar with RASopathies. For each proband, family history suggestive of NS was systematically assessed by means of three or more generations pedigree. Phenotypic data recorded included all clinical characteristics, with emphasis on antenatal features, NS-specific dysmorphology assessment, investigation of bleeding diathesis and cardiovascular abnormalities on Electrocardiograms (ECG) and echocardiograms.

Genomic DNA of each selected patient was isolated from peripheral blood leukocytes at the National Health Laboratory Service (NHLS)-Molecular Genetics Laboratory, GSH, following the manufacturer's instructions, the standard Maxwell 16 protocol (Maxwell®16 Blood DNA purification kit, Promega, Madison, WI 53711, USA).

Of the 26 DNA samples isolated, 16 DNA samples from unrelated patients were genotyped. Targeted Sequencing was performed with the Ion Torrent platform, at the sequencing laboratory of the Division of Human Genetics of UCT, using the Ion PGM™ system (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Pre-designed primers for Ion AmpliSeq Noonan Research Panel were used (Life Technologies, Carlsbad, CA). The primers amplify exons and intron/exon boundaries of 14 genes known to be associated with NS and related conditions, including A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, RIT1, SHOC2, SOS1, and SPRED1. This multigene panel is predicted to cover 100% of the targeted regions, in 268 amplicons (Nelen et al., 2014). Sequencing data analysis, including quality assessment, read alignment, variants identification, variant annotation, visualization, and prioritization was primarily performed using the bioinformatics pipeline of the Ion Torrent Suite and the Ion Reporter cloud-based software (Thermo Fisher Scientific, Waltham, Massachusetts, USA). From the usable reads, 99% could be mapped to the human reference genome used (Homo sapiens, hg19, build 37.2). Further manual analysis was executed for variant prioritization and interpretation based on the variant call format (VCF) file generated by the Ion Reporter software. In this step, variants were prioritized using their minor allele frequency (MAF < 0.01) based on 1,000 genomes and 5,000 exomes projects, their zygosity, their function, their location within the gene, and their pathogenicity according to ClinVar. A parallel analysis of sequencing data was performed based on the binary alignment map (BAM) file generated by the Ion Reporter software. Picard package with option to SortSAM, MarkDuplicates and FixMateInformation on a per-sample basis were used to sort coordinate, mark polymerase chain reaction (PCR) duplicate reads and verify mate-pair information, respectively (Mckenna et al., 2010). The variant calling was done using Samtools, bcftools (Li et al., 2009) and Varscan 2 (Koboldt et al., 2012) with the reference Human genome (Hg19; build 37.2). The conservation and deleteriousness of the variant were investigated using ANNOVAR which interrogated the following tools: SIFT, PolyPhen 2 HVAR, Polyphen2 HDIV, MutationTaster, MutationAssessor, Likelihood ratio test (LRT), FATHMM, MetaSVM, MetaLR, GERP++, PhyloP, VEST3, DANN, CADD, PROVEAN, Fathmm-MKL, Integrated_fitCons, SiPhy_29way, PhastCons (Wang et al., 2010). The second level of variant filtration to avoid false positives or false negatives was conducted on annotated VCF files using an in-house python script to select and retain only deleterious disease-causing variants that have functional prediction using the 19 tools interrogated by ANNOVAR. The in-house python script uses two approaches to select deleterious variant (i) free hypothesis: cast of the vote of the annotated variant filter for “Deleterious or damaging disease-causing (D)” among annotation prediction tools based on a defined cut-off (~50%); (ii) non free hypothesis: provide a list of known genes of the study with another level of prediction cut-off (~25%) (Lebeko et al., 2017). The cut-off for both hypothesis is defined as follow; (i) free hypothesis: select only variant which 10 tools interrogated by ANNOVAR predicted presence of a “D”; (ii) no free hypothesis: select only the gene which 5 tools interrogated by ANNOVAR predicted presence of a “D” using the list of 14 NS genes analyzed. Existing online databases for previously reported NS variants and published literature on NS-associated variants were consulted for each candidate variant.

All variants identified and predicted to be pathogenic were subsequently confirmed with Sanger capillary direct cycle sequencing and capillary electrophoresis using standard protocols. Depending on availability of family members, segregation studies were performed: the proband's parents were screened to ascertain the origin of the variant. In addition, and where possible, other available family members clinically affected or not, were screened for the identified variant, using Sanger direct cycle sequencing.

Molecular dynamic (MD) simulations were conducted to assess the effect of novel variants on proteins function. The tertiary structure of the PTPN11 protein SHP-2 (PDB id: 2SHP) (Hof et al., 1998) and the MAP2K1 structure (UniProKD: A4QPA9) were generated using I-tasser homology webserver (Zhang, 2008). The CBL structure was generated by combining (PDB id: 1FBV) and homology model. Six independent MD simulations were conducted; (i) MAP2K1 mutant and wild-type (WT), SHP-2 mutant and WT, and CBL mutant and WT. All MD simulations were conducted with the GROMACS package, version 4.6.5 (Pronk et al., 2013) using OPLS force field (Jorgensen and Tirado-rives, 1988). The systems were simulated in cubic box and solvated in water TIP3P (Neira et al., 1996). The temperature and pressure were maintained at 300 K using the Parrinello-Donadio-Bussi V-rescale thermostat (Bussi et al., 2007) and a pressure of 1 bar using the Berendsen barostat (Berendsen et al., 1984). The short-range non-bonded interactions were modeled using Lennard Jones potentials. The long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) algorithm (Darden et al., 1993; Essmann et al., 1995). The LINCS algorithm was used to constrain all bond lengths (Hess et al., 1997) and the velocities were assigned according to the Maxwell-Boltzman distribution at 300 K.

A total of 26 patients were included in this study, mostly unrelated (n = 20; 77%). The majority were children (n = 20; 77%) with a median age at clinical diagnosis of 4.5 years (range: 1 month−51 years). Our cohort had preponderance of individuals of mixed-ancestry background (53.8%), followed by black Africans (30.8%). There was a slight predominance of males (sex ratio: 1.3; 15 males: 11 females).

Gross motor milestones were on par for most patients, with ability to walk before the age of 18 months in 61.5% (n = 16/26) of cases. Six (6/26; 23.1%) patients were unable to walk after 24 months. Speech delay was reported in 50% (13/26) of cases. Craniofacial features were widely variable, but more characteristic in infants (2–12 months; 4/26), with widely spaced eyes, epicanthic folds and ptosis found in 75% of cases (Table 1). Comparison of six key physical characteristics (short stature, ptosis, widely spaced eyes, epicanthic folds, low-set ears, and webbed neck) between ethnic groups showed that, features were less frequent in Caucasians (2/26). Black Africans (8/26) presented with the most consistent dysmorphic features, with epicanthic folds (87.7%), ptosis (75%), and low-set ears (75%) found to be the most common features (Table 2). At least one cardiovascular abnormality was identified in 65.4% (17/26) of patients. The most common CHD was pulmonary valve stenosis (PS), found in 34.6% (9/26) of our cohort. Hypertrophic Cardiomyopathy (HCM) was identified in 19.2% (5/26) and left axis deviation on ECG was found in 23.1% (6/26) of cases. The complete list of clinical features identified in our cohort of 26 patients can be found in the Table S1.

Following targeted NGS of 16 DNA samples from unrelated patients, in silico predictive algorithms supported the classification of seven heterozygous missense variants as pathogenic (7/16; Table 3; Figure 1). The novel variants were CBL c.2520T>G (p.Cys840Trp), PTPN11 c.1496C>T (p.Ser499Phe), and MAP2K1 c.200A>C (p.Asp67Ala). Analysis confirmed segregation of three novel variants with the disease in the respective families. Segregation analysis revealed a possible case of germline mosaicism in a family where the unaffected father does not carry the variant (MAP2K1; c.200A>C) found in his two affected children. These children were half-brothers, born from two separate mothers.

The two variants previously reported in ClinVar as pathogenic are MAP2K1 c.389A>G (p.Tyr130Cys) and PTPN11 c.1510A>G (p.Met504Val). Two identified CBL variants were predicted to be pathogenic by prediction tools, but were reported as benign: c.1858C>T (p. p.Leu620Phe) and as variant of uncertain significance: c.2345C>T (p.Pro782Leu) in ClinVar. Correlatively, CBL c.2345C>T failed to segregate with the disease in a large dominant family (Figure S1).

Molecular dynamic (MD) simulations (Figure 2) showed that: for CBL, substitution of the negatively charged and hydrophilic amino acid Cys840 with the non-polar and hydrophobic amino acid Trp840 could impact binding interactions, stability and the flexibility of the protein.

For MAP2K1, substitution of the negatively charged and hydrophilic amino acid Asp67 with the non-polar and hydrophobic amino acid Ala67 could impact binding interactions; for SHP-2, substitution of the polar and hydrophilic amino acid Ser499 with the non-polar and highly hydrophobic amino acid Phe499 in the active site of the protein could impact binding interactions and the stability of structure.

Correlations between the five variants that were predicted to be pathogenic, and NS related phenotypes are presented in Table 4. Comparisons showed that patients with variants in MAP2K1 were diagnosed earlier (mean age at diagnosis: 1 year) and presented with more typical clinical features of NS, followed by patients with variants in PTPN11 (mean age at diagnosis: 3.3 years; Figure 3). The patient with a variant in CBL was found to have more discreet clinical features (Table 5).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.