ClinVar Genomic variation as it relates to human health

The c.1455G>C (p.Leu485Phe) variant in BRAF is absent from gnomAD (PM2). It has been detected in at least 4 patients with clinical features of a RASopathy, 1 of which was reported as a de novo case with parentage confirmation (PS4_Moderate; PS2; 19206169, 16474404, SCV000197149.4). In vitro functional studies provide some evidence that the p.L485F variant may impact protein function (PS3; PMID: 18413255). The variant is located in the BRAF gene, which has been defined by the ClinGen RASopathy Expert Panel as a gene with a low rate of benign missense variants and pathogenic missense variants are common (PP2; PMID: 29493581). Additionally, computational prediction tools and conservation analysis suggest that this variant may affect the protein (PP3). In summary, this variant meets criteria to be classified as pathogenic for RASopathies in an autosomal dominant manner. Rasopathy-specific ACMG/AMP criteria applied (PMID:29493581): PS4_Moderate, PS2, PS3, PM2, PP2, PP3.

Variant summary: BRAF c.1455G>C (p.Leu485Phe) results in a non-conservative amino acid change located in the Protein kinase domain (IPR000719) 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 251326 control chromosomes. c.1455G>C has been widely reported in the literature in individuals affected with Cardiofaciocutaneous Syndrome (example, Niihori_2006, Sarkozy_2009, Rodiguez-Viciana_2008). These data indicate that the variant is likely to be associated with disease. At least two publications report experimental evidence evaluating an impact on protein function. The most pronounced variant effect results in an activated ERK pathway (Niihori_2006) and an increased kinase activity compared to wild-type in a MEK-ERK MBP phosphorylation assay (Rodriguez-Viciana_2008), both findings consistent with the established gain of function mechanism of disease. Multiple clinical diagnostic laboratories and an expert panel (ClinGen RASopathy Variant Curation Expert Panel) have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All submitters classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change (ClinVar ID: 177844 - c.1575G>T;p.(Leu525Phe)) PS1.Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (PMID: 18413255; 18953432) - PS3_moderate.The c.1455G>C;p.(Leu485Phe) missense variant has been observed in affected individual(s) and ClinVar contains an entry for this variant (ClinVar ID: 13975; PMID: 19206169; PMID: 18953432; PMID: 18039235; PMID: 16474404) - PS4. The variant is located in a mutational hot spot and/or critical and well-established functional domain (Pkinase_Tyr) - PM1. This variant is not present in population databases (rs180177036- gnomAD; ABraOM no frequency - http://abraom.ib.usp.br/) - PM2. Pathogenic missense variant in this residue have been reported (ClinVar ID: 40370) - PM5. The variant was assumed de novo, but without confirmation of paternity and maternity (PMID: 19206169) - PM6. Missense variant in BRAF that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease - PP2. Multiple lines of computational evidence support a deleterious effect on the gene or gene product - PP3. In summary, the currently available evidence indicates that the variant is pathogenic.

Published functional studies demonstrate a damaging effect with the variant causing a gain-of-function to the MAPK pathway (Niihori et al., 2006; Hu et al., 2015); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Missense variants in this gene are often considered pathogenic (HGMD); This variant is associated with the following publications: (PMID: 16439621, 18039235, 28972961, 24803665, 18413255, 23093928, 24458522, 25348715, 26150740, 26744778, 20301365, 28468735, 28524057, 22369373, 33753861, 32005694, 29493581, 19206169, 25194980, 16474404)

The BRAF c.1455G>C variant is predicted to result in the amino acid substitution p.Leu485Phe. This variant has been reported in individuals with cardio-facio-cutaneous syndrome (CFCS) (see for example Niihori et al. 2006. PubMed ID: 16474404). Additionally, a different nucleotide substitution (c.1455G>T) resulting in the same missense variant (Leu485Phe) has been reported in individuals with CFCS (Table S1 - Mohan et al. 2022. PubMed ID: 34358384). Functional studies demonstrate this variant results in increased p-MEK/ERK levels, consistent with a gain-of-function mechanism, resulting in hyperactivation of the RAS pathway (Rodriguez-Viciana et al. 2008. PubMed ID: 18413255). Additionally, a different missense substitution (p.Leu485Ser) affecting this residue has been reported as pathogenic (Aizaki et al. 2011. PubMed ID: 20395089). 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 BRAF c.1455G>C; p.Leu485Phe variant (rs180177036) is reported in individuals with cardio-facio-cutaneous syndrome (Niihori 2006, Rodriguez-Viciana 2008, Sarkozy 2009). This variant is classified as pathogenic by an expert panel in the ClinVar database (Variation ID: 13975). It is absent from the Genome Aggregation Database, indicating it is not a common polymorphism. Computational analyses predict that this variant is deleterious (REVEL: 0.757). In support of these predictions, functional analyses have demonstrated this variant has an impact on BRAF function (Niihori 2006, Rodriguez-Viciana 2008). Based on available information, the p.Leu485Phe variant is considered to be pathogenic. References: Niihori T et al. Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet. 2006 Mar;38(3):294-6. PMID: 16474404. Rodriguez-Viciana P, Rauen KA. Biochemical characterization of novel germline BRAF and MEK mutations in cardio-facio-cutaneous syndrome. Methods Enzymol. 2008;438:277-89. doi: 10.1016/S0076-6879(07)38019-1. PMID: 18413255. Sarkozy A et al. Germline BRAF mutations in Noonan, LEOPARD, and cardiofaciocutaneous syndromes: molecular diversity and associated phenotypic spectrum. Hum Mutat. 2009 Apr;30(4):695-702. PMID: 19206169.

The c.1455G>C (p.L485F) alteration is located in coding exon 12 of the BRAF gene. This alteration results from a G to C substitution at nucleotide position 1455, causing the leucine (L) at amino acid position 485 to be replaced by a phenylalanine (F). This variant was not reported in population-based cohorts in the Genome Aggregation Database (gnomAD). This alteration was previously reported in multiple unrelated patients with cardio-facio-cutaneous (CFC) syndrome (Niihori, 2006; Rodriguez-Viciana, 2006; Yoon, 2007; Sarkozy, 2009). Parent-child trio confirmed the de novo occurrence of the c.1455G>C alteration in the patient reported by Sarkozy, et al. (2009). The patient reported by Rodriguez-Viciana et al. (2006) had the characteristic facies of CFC, developmental delay, hypotonia, hypertrophic cardiomyopathy, hair and skin anomalies, and other musculoskeletal, GI and ocular abnormalities. He was not reported to have short stature, seizures, or lymphadema. Another 7 year old boy with the p.L485F alteration had severe academic difficulty and multiple types of seizures, with onset at 2 weeks (Yoon, 2007). This amino acid position is highly conserved in available vertebrate species. Niihori et al. (2006) used luciferase assays to show that BRAF with the p.L485F mutation, when introduced into NIH3T3 cells, caused about a 2.5 fold increase in ELK1 transcription, indicating that it over-stimulated the ERK pathway compared to wild-type leading to increased kinase activity. Rodriguez-Viciana and Rauen (2008) introduced Flag-tagged BRAF p.L485F mutant into 293T cells and found about 1.8 fold increase in its kinase activity by two different methods namely a coupled MEK-ERK-MBP phosphorylation assay and western blotting by probing with antibodies that specifically detect phosphorylated ERK and MEK. This alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic.

This sequence change replaces leucine, which is neutral and non-polar, with phenylalanine, which is neutral and non-polar, at codon 485 of the BRAF protein (p.Leu485Phe). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with cardio-facio-cutaneous syndrome (PMID: 16474404, 19206169, 28524057). In at least one individual the variant was observed to be de novo. ClinVar contains an entry for this variant (Variation ID: 13975). 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 BRAF protein function with a positive predictive value of 80%. Experimental studies have shown that this missense change affects BRAF function (PMID: 18413255). For these reasons, this variant has been classified as Pathogenic.

The c.1455G>C (p.Leu485Phe) variant in BRAF has been previously identified in fo ur individuals with clinical features of a RASopathy (Niihori 2006, Rodriguez-Vi ciana 2006, LMM unpublished data). It was absent from large population studies ( http://evs.gs.washington.edu/EVS/; dbSNP rs180177036). In addition, a different variant with the same amino acid change (c.1455G>T) was identified by our labora tory in one individual with Cardio-facio-cutaneous syndrome (CFC). In vitro func tional studies provide some evidence that the p.Leu485Phe variant may impact pro tein function by increasing its kinase activity (Rodriguez-Viciana 2008). Howeve r, these types of assays may not accurately represent biological function. In s ummary, this variant meets our criteria to be classified as pathogenic (http://p cpgmwww.partners.org/personalizedmedicince/LMM).