ClinVar Genomic variation as it relates to human health

The RIT1 c.221C>G variant is predicted to result in the amino acid substitution p.Ala74Gly. Using an alternate transcript, this variant is also referred to as c.170C>G (p.Ala57Gly). This variant has been reported to be causative for Noonan syndrome (see for example - Aoki et al. 2013. PubMed ID: 23791108; Bertola et al. 2014. PubMed ID: 25124994; Koenighofer et al. 2016. PubMed ID: 25959749). Functional studies demonstrate this variant affects protein function (Fang et al. 2016. PubMed ID: 27226556). This variant has not been reported in a large population database (http://gnomad.broadinstitute.org), indicating this variant is rare. This variant is interpreted as pathogenic.

The p.A57G variant (also known as c.170C>G), located in coding exon 3 of the RIT1 gene, results from a C to G substitution at nucleotide position 170. The alanine at codon 57 is replaced by glycine, an amino acid with similar properties. This variant is also known as p.A74G (c.221C>G). This variant has been described in Noonan syndrome cohorts and pediatric cardiomyopathy cohorts, and it has been detected as a de novo and inherited variant in multiple unrelated individuals who meet clinical diagnostic criteria for Noonan syndrome (Aoki Y et al. Am J Hum Genet, 2013 Jul;93:173-80, Bertola DR et al. Am J Med Genet A, 2014 Nov;164A:2952-7; Chen PC et al. Proc Natl Acad Sci U S A, 2014 Aug;111:11473-8; Iglesias A et al. Genet Med, 2014 Dec;16:922-31; Cavé H et al. Eur J Hum Genet, 2016 08;24:1124-31; Kouz K et al. Genet Med, 2016 12;18:1226-1234; Yaoita M et al. Hum Genet, 2016 Feb;135:209-22; Popp B et al. Eur J Hum Genet, 2017 12;25:1364-1376; Shoji Y et al. Endocr J, 2019 Nov;66:983-994; Li X et al. Clin Genet, 2019 10;96:290-299; Chen H et al. Orphanet J Rare Dis, 2019 02;14:29; Aly SA et al. Mol Genet Genomic Med, 2020 07;8:e1253; Rupp S et al. Clin Res Cardiol, 2019 Mar;108:282-289). Experimental studies show this variant impacts protein function and may contribute to the pathogenesis of Noonan syndrome, at least in part, through enhancing ERK1/2 pathway signaling (Aoki Y et al. Am J Hum Genet, 2013 Jul;93:173-80; Chen PC et al. Proc Natl Acad Sci U S A, 2014 Aug;111:11473-8; Fang Z et al. J Biol Chem, 2016 07;291:15641-52; Koenighofer M et al. Clin Genet, 2016 Mar;89:359-66; Meyer Zum Büschenfelde U et al. PLoS Genet, 2018 05;14:e1007370). In addition, a heterozygous A57G knock-in mouse model recapitulated the Noonan syndrome phenotypic spectrum, including the fibrotic cardiac hypertrophy that appears to be a Noonan syndrome phenotype most frequently associated with RIT1 gene mutations (Takahara S et al. EBioMedicine, 2019 Apr;42:43-53). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

The p.Ala74Gly variant in RIT1 (reported as p.Ala57Gly based on amino acid seque nce predicted from NM_006912.5) has been identified in more than 10 individuals with Noonan syndrome and occurred de novo in four of these individuals (Aoki 201 3, Chen 2014, Bertola 2014, Iglesias 2014, Koenighofer 2015, LMM data). It has n ot been identified in large population studies. Pathogenic variants in RIT1 clus ter in the Switch I domain of the protein, where this variant is located (Aoki 2 013, Chen 2014). In summary, this variant meets our criteria to be classified a s pathogenic for Noonan syndrome in an autosomal dominant manner based upon its de novo and over-represented occurrences in affected individuals compared to the general population.

The heterozygous p.Ala74Gly variant in RIT1 was identified by our study in one individual with Noonan syndrome. Trio exome analysis showed this variant to be de novo. This variant was absent from large population studies. Computational prediction tools and conservation analyses do not provide strong support for or against an impact to the protein. This variant has been reported in ClinVar with an alternate name, p.Ala57Gly (Variation ID: 60506). There are at least five individuals with Noonan syndrome and de novo inheritance of this variant in ClinVar and the literature (PMID: 25959749, 26714497, 23791108). In summary, the p.Ala74Gly variant is pathogenic based off of our findings, multiple de novo reports in ClinVar, and the literature. ACMG/AMP Criteria applied: PM2, PS2, PM6_Strong (Richards 2015).

Variant summary: RIT1 c.170C>G (p.Ala57Gly) results in a non-conservative amino acid change located in the Small GTP-binding protein domain (IPR005225; also called as the G2 domain, Chen_2014) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 252130 control chromosomes (gnomAD and publication data). c.170C>G has been reported in the literature in multiple individuals affected with Noonan Syndrome or a related phenotype, including de novo occurrences (Aoki_2013, Bertola_2014, Chen_2014, Fang_2016, Koenighofer_2016). These data indicate that the variant is very likely to be associated with disease. Functional studies report this variant activated RAS-ERK pathway, exhibited more rapid nucleotide exchange on GTPase assay, revealed abnormalities of eye development, emphasizing the importance of RIT1 for spatial and temporal organization of the growing organism in zebrafish, exhibited decreased viability, edema, subcutaneous hemorrhage and AKT activation in mice (Chen_2014, Fang_2016, Koenighofer_2016, Takahara_2019). Four ClinVar submitters (evaluation after 2014) cite the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

Published functional studies demonstrate that A57G enhanced ELK1 trans-activation in a luciferase assay above wild-type levels (Aoki et al., 2013); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 25959749, 30105547, 23791108, 25049390, 25124994, 24901346, 24803665, 27226556, 30732632, 28991257, 30898653, 29158550, 31292302, 31219622, 32396283, 32368696, 34643321)

The missense variant c.221C>G, p.(Ala74Gly) in RIT1 was identified in a boy with clinically suspected Noonan syndrome and osteogenesis imperfecta due to a de novo mutation in COL1A1. The variant in RIT1 was shown to be de novo and had been reported in several other patients with Noonan syndrome.

The variant has been reported as de novo multiple (>3) similarly affected unrelated individuals(PMID: 23791108, 25959749, 26714497, PS2_VS, PS4_S). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.62, 3CNET: 0.822, PP3_P). A missense variant is a common mechanism associated with Noonan syndrome 8 (PP2_P). It is not observed in the gnomAD v2.1.1 dataset (PM2_M). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

This sequence change replaces alanine, which is neutral and non-polar, with glycine, which is neutral and non-polar, at codon 57 of the RIT1 protein (p.Ala57Gly). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with Noonan syndrome (PMID: 23791108, 24901346, 25049390, 25124994, 25959749, 26714497, 26757980, 27101134). In at least one individual the variant was observed to be de novo. ClinVar contains an entry for this variant (Variation ID: 60506). Advanced modeling performed at Invitae incorporating data from internal and/or published experimental studies (Invitae) did not meet the statistical confidence thresholds required to predict the impact of this variant on RIT1 function. Experimental studies have shown that this missense change affects RIT1 function (PMID: 25049390, 25959749, 27226556). For these reasons, this variant has been classified as Pathogenic.