ClinVar Genomic variation as it relates to human health

The c.268A>G (p.Met90Val) variant in RIT1 was absent from both versions of gnomAD (PM2_Supporting; gnomad.broadinstitute.org). The p.Met90Val variant has been observed in at least 7 probands with a RASopathy or clinical features of a RASopathy (PS4_Moderate; PMID: 27109146, 30712878, 30293990, 32304219, Hopital Robert Debre internal data ClinVar SCV001438552.1, GeneDx internal data ClinVar SCV000808482.1). Of note, the majority of these were fetal cases. It has been reported as a de novo occurrence with parental confirmation in 3 probands and without confirmation in 3 probands (PS2_VeryStrong). Computational prediction tools and conservation analysis suggest that the p.Met90Val variant may impact the protein (PP3). ERK1/2 phosphorylation assays in HEK293T cells showed elevated and prolonged ERK1/2 phosphorylation indicating that this variant impacts protein function (PMID:29734338; PS3_Supporting). In summary, this variant meets criteria to be classified as pathogenic for RASopathies in an autosomal dominant manner. Rasopathy-specific ACMG/AMP criteria applied: PS2_VeryStrong, PS4_Moderate, PM2_Supporting, PP3, PS3_supporting (Version 2.1; 09/27/24).

Variant summary: RIT1 c.268A>G (p.Met90Val) results in a conservative amino acid change located in the Small GTP-binding protein domain of the encoded protein sequence. Three of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251366 control chromosomes. c.268A>G has been reported in the literature as a de-novo occurrence in multiple fetal cases affected with Noonan Syndrome And Related Conditions (example, Milosavljevic_2016, Quinan-Jones_2019, Becher_2020, Stuurman_2019, Petrovski_2019). These data indicate that the variant is very likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function. The most pronounced variant effect results in increased activation of ERK signaling supporting a gain of function outcome (Buschenfelde_2018). Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This variant, also known in the literature as c.268A>G (p.Met90Val) based on transcript NM_006912.6, has been previously reported as a de novo heterozygous change in at least two patients with Noonan syndrome (PMID: 27109146, 30293990). Missense variants at the same amino acid residue (p.Met90Ile) and (p.Met90Thr) and at neighboring amino acid residues (p.Tyr89His) and (p.Gly95Ala) have been reported in individuals with Noonan Syndrome (PMID: 23791108, 24939608). Functional studies have demonstrated that this variant leads to increased ERK1/2 phosphorylation compared to wild-type RIT1 (PMID: 29734338). It is absent from the gnomAD population database and thus is presumed to be rare. The c.319A>G (p.Met107Val) variant affects a highly conserved amino acid and is predicted by multiple in silico tools to have a deleterious effect on protein function. Based on the available evidence, the c.319A>G (p.Met107Val) variant is classified as Pathogenic.

Published functional studies have shown p.(M90V) results in increased ERK1/2 activation in response to serum activation compared to wild type (Meyer et al., 2018).; Not observed in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 29565699, 30293990, 31324109, 30712878, 34306696, 27109146, 35595454, 36274670, 29734338, 32304219)

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0101 - Gain of function is a known mechanism of disease in this gene and is associated with Noonan syndrome 8, (MIM#615355). (I) 0107 - This gene is associated with autosomal dominant disease. (I) 0200 - Variant is predicted to result in a missense amino acid change from methionine to valine. (I) 0251 - This variant is heterozygous. (I) 0301 - Variant is absent from gnomAD (both v2 and v3). (SP) 0602 - Variant is located in a hotspot region or cluster of pathogenic variants (DECIPHER). (SP) 0701 - Other missense variants comparable to the one identified in this case have very strong previous evidence for pathogenicity. p.(Met90Ile) and p.(Met90Leu) have been reported in at least ten individuals with Noonan Syndrome (PMID: 27109146; ClinVar, internal VCGS cohort). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. It has been reported de novo in at least three individuals with Noonan Syndrome and consistently classified as pathogenic by diagnostic laboratories in ClinVar (PMID: 27109146, 34306696). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

For these reasons, this variant has been classified as Pathogenic. This variant disrupts the p.Met90 amino acid residue in RIT1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 23791108, 24939608, 25959749, 7109146, 27101134). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may create or strengthen a splice site, but this prediction has not been confirmed by published transcriptional studies. This variant has been reported to affect RIT1 protein function (PMID: 29734338). This variant has been observed in individual(s) with Noonan syndrome (PMID: 27109146). In at least one individual the variant was observed to be de novo. ClinVar contains an entry for this variant (Variation ID: 561681). This variant is not present in population databases (ExAC no frequency). This sequence change replaces methionine with valine at codon 90 of the RIT1 protein (p.Met90Val). The methionine residue is highly conserved and there is a small physicochemical difference between methionine and valine.

The RIT1 c.268A>G, p.Met90Val variant is reported de novo in the literature in multiple individuals affected with Noonan syndrome (Bercher 2020, Miceikaite 2021, Milosavljevic 2016). This variant is also reported in ClinVar (Variation ID: 561681) and is absent from the Genome Aggregation Database, indicating it is not a common polymorphism. Additionally, another variant at this codon (p.Met90Ile) has been reported in individuals with Noonan syndrome and is considered pathogenic (Aoki 2013, Gos 2014). Computational analyses predict that this variant is deleterious (REVEL: 0.732). Based on available information, this variant is considered to be pathogenic. References: Aoki Y et al. Gain-of-function mutations in RIT1 cause Noonan syndrome, a RAS/MAPK pathway syndrome. Am J Hum Genet. 2013 Jul 11;93(1):173-80. PMID: 23791108. Becher N et al. Implementation of exome sequencing in fetal diagnostics-Data and experiences from a tertiary center in Denmark. Acta Obstet Gynecol Scand. 2020 Jun;99(6):783-790. PMID: 32304219. Gos M et al. Contribution of RIT1 mutations to the pathogenesis of Noonan syndrome: four new cases and further evidence of heterogeneity. Am J Med Genet A. 2014 Sep;164A(9):2310-6. PMID: 24939608. Miceikaite I et al. Prenatal cases with rare RIT1 variants causing severe fetal hydrops and death. Clin Case Rep. 2021 Jul 21;9(7):e04507.PMID: 34306696. Milosavljevic D et al. Two cases of RIT1 associated Noonan syndrome: Further delineation of the clinical phenotype and review of the literature. Am J Med Genet A. 2016 Jul;170(7):1874-80. PMID: 27109146.