ClinVar Genomic variation as it relates to human health

The P261A variant has been published previously in association with RAF1-related disorders (Razzaque et al., 2007; Stevenson et al., 2011; Croonen et al., 2013). The variant was not observed in approximately 6,500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project, indicating it is not a common benign variant in these populations. P261A is a semi-conservative amino acid substitution, which may impact secondary protein structure as these residues differ in some properties. This substitution occurs at a position that is conserved across species, and in silico analysis predicts this variant is probably damaging to the protein structure/function. Functional studies have shown that P261A leads to increased activity of the RAF1 protein (Razzaque et al., 2007; Molzan et al., 2010). Missense variants in the same residue (P261T/S/H/L/R) and in nearby residues (R256S, S257L, S259P/T/F, T260I/R, N262I, N262K, V263A/D) have been reported in the Human Gene Mutation Database in association with RAF1-related disorders (Stenson et al., 2014), supporting the functional importance of this region of the protein. Therefore, we consider this variant to be pathogenic.

For these reasons, this variant has been classified as Pathogenic. This variant disrupts the p.Pro261 amino acid residue in RAF1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 17603483, 20683980, 21784453, 26266034). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. Experimental studies have shown that this missense change affects RAF1 function (PMID: 17603482, 20052757, 20679480). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt RAF1 protein function. ClinVar contains an entry for this variant (Variation ID: 40605). This missense change has been observed in individual(s) with Noonan syndrome (PMID: 17603482, 20052757, 23885229). In at least one individual the variant was observed to be de novo. This variant is not present in population databases (gnomAD no frequency). This sequence change replaces proline, which is neutral and non-polar, with alanine, which is neutral and non-polar, at codon 261 of the RAF1 protein (p.Pro261Ala).

The p.Pro261Ala variant in RAF1 has been identified in three individuals with cl inical features of Noonan syndrome (Razzaque 2007, Croonen 2013, LMM unpublished data), and was absent from large population studies (http://evs.gs.washington.e du/EVS/; dbSNP rs121434594). Different pathogenic amino acid changes (p.Pro261Le u, p.Pro261Ser, p.Pro261Thr) at this location have been identified in individual s with clinical features of Noonan syndrome, two of which (p.Pro261Leu, p.Pro261 Thr) were reported to have occurred de novo in one individual each (Razzaque 200 7, Pandit 2007, LMM unpublished data), suggesting that changes at this position are not tolerated. Studies have shown that the p.Pro261Ala variant impacts prote in function by increasing its kinase activity (Razzaque 2007). However, these in vitro assays may not accurately represent biological function. Individuals wit h pathogenic variants in exon 7 or 17 in RAF1 are reported to also have a higher incidence of hypertrophic cardiomyopathy (HCM 80-95%) than typically seen in No onan syndrome (Razzaque 2007, Pandit 2007). In summary, the p.Pro261Ala variant meets our criteria to be classified as pathogenic (http://personalizedmedicine.p artners.org/Laboratory-For-Molecular-Medicine/) based upon number of cases, abse nce from controls, evidence of other pathogenic variants in the same codon, and functional evidence.

The RAF1 c.781C>G variant is predicted to result in the amino acid substitution p.Pro261Ala. This variant has been reported in multiple individuals with Noonan syndrome (see for example - Razzaque et al. 2007. PubMed ID: 17603482; Maher et al. 2018. PubMed ID: 30355600; Table S2, Leach et al. 2018. PubMed ID: 29907801). Functional studies found this variant leads to increased RAF1 kinase, consistent with a gain-of-function mechanism (Razzaque et al. 2007. PubMed ID: 17603482). This variant has not been reported in a large population database (http://gnomad.broadinstitute.org), indicating this variant is rare. Additionally, alternate missense variants affecting this amino acid (p.Pro261Thr, p.Pro261Ser, p.Pro261His, p.Pro261Arg, p.Pro261Leu) have been reported as pathogenic (Human Gene Mutation Database). This variant is interpreted as pathogenic.