rs397516826

chr3-12604202-C-Achr3-12604202-C-Gchr3-12604202-C-T

Variant summary

Our verdict is Pathogenic. Variant got 23 ACMG points: 23P and 0B. PS1_Very_Strong PM1 PM2 PM5 PP2 PP5_Very_Strong

The NM_002880.4(RAF1):c.768G>T(p.Arg256Ser) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. Variant has been reported in ClinVar as Pathogenic (★★★). Another nucleotide change resulting in same amino acid change has been previously reported as Likely pathogenicin ClinVar. Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R256T) has been classified as Uncertain significance.

• Frequency: Genomes: not found (cov: 32)
• Consequence: RAF1 NM_002880.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:6
• Conservation: PhyloP100: -0.0880

Genes affected

RAF1 (HGNC:9829): (Raf-1 proto-oncogene, serine/threonine kinase) This gene is the cellular homolog of viral raf gene (v-raf). The encoded protein is a MAP kinase kinase kinase (MAP3K), which functions downstream of the Ras family of membrane associated GTPases to which it binds directly. Once activated, the cellular RAF1 protein can phosphorylate to activate the dual specificity protein kinases MEK1 and MEK2, which in turn phosphorylate to activate the serine/threonine specific protein kinases, ERK1 and ERK2. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, apoptosis, cell differentiation and cell migration. Mutations in this gene are associated with Noonan syndrome 5 and LEOPARD syndrome 2. [provided by RefSeq, Jul 2008]

ACMG classification

Verdict is Pathogenic. Variant got 23 ACMG points.

• PS1: Transcript NM_002880.4 (RAF1) is affected with MISSENSE_VARIANT having same AA change as one Pathogenic present in ClinVar as 44632
• PM1: In a hotspot region, there are 8 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 0 benign, 6 uncertain in NM_002880.4
• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr3-12604204-T-C is described in ClinVar as [Likely_pathogenic]. Clinvar id is 44631.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars.
• PP2: Missense variant where missense usually causes diseases, RAF1
• PP5: Variant 3-12604202-C-A is Pathogenic according to our data. Variant chr3-12604202-C-A is described in ClinVar as [Pathogenic]. Clinvar id is 40599.Status of the report is reviewed_by_expert_panel, 3 stars.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
RAF1	NM_002880.4	c.768G>T	p.Arg256Ser	missense_variant	7/17	ENST00000251849.9

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
RAF1	ENST00000251849.9	c.768G>T	p.Arg256Ser	missense_variant	7/17	1	NM_002880.4	P3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:6

Revision: reviewed by expert panel

Submissions by phenotype

RASopathy Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	May 02, 2022	Variant summary: RAF1 c.768G>T (p.Arg256Ser) results in a non-conservative amino acid change in the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251458 control chromosomes (gnomAD). c.768G>T has been reported in the literature in individuals affected with Noonan Syndrome (Pandit_2007, Digilio_2011, Shoji_2019). These data indicate that the variant is likely to be associated with disease. Experimental evidence evaluating an impact on protein function demonstrated the catalytic activity of the variant was significantly increased compared to wild-type, binding of 14-3-3 proteins to C-RAF was impaired and subcellular localization was altered (Molzan_2010). Two ClinVar submitters including an expert panel (ClinGen RASopathy Variant Curation Expert Panel) (evaluation after 2014) cite the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic. -
Pathogenic, reviewed by expert panel	curation	ClinGen RASopathy Variant Curation Expert Panel	Apr 03, 2017	The c.768G>T (p.Arg256Ser) variant in RAF1 has been reported in the literature in 1 individual with clinical features of Noonan syndrome and one individual with clinical features of Noonan syndrome with multiple lentigines. This variant was absent from large population studies (PM2; ExAC, http://exac.broadinstitute.org). The variant is located in the RAF1 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). Computational prediction tools and conservation analysis suggest that the p.Arg256Ser variant may impact the protein (PP3). In vitro functional studies provide some evidence that the p.Arg256Ser variant may impact protein function (PS3; 20679480). Furthermore, the variant is in a location that has been defined by the ClinGen RASopathy Expert Panel to be a mutational hotspot or domain of RAF1 (PM1; PMID 29493581). 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): PP2, PP3, PM1, PM2, PS3. -
Pathogenic, criteria provided, single submitter	clinical testing	Invitae	Jan 31, 2022	This variant disrupts the p.Arg256 amino acid residue in RAF1. Other variant(s) that disrupt this residue have been determined to be pathogenic (Invitae). 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: 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: 40599). This missense change has been observed in individuals with Noonan syndrome (PMID: 17603483; Invitae). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces arginine, which is basic and polar, with serine, which is neutral and polar, at codon 256 of the RAF1 protein (p.Arg256Ser). For these reasons, this variant has been classified as Pathogenic. -

not provided Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

GeneDx

Mar 19, 2019

Published functional studies demonstrate a damaging effect on protein localization and activity (Molzan et al., 2010); Identified in patients with Noonan syndrome and/or hypertrophic cardiomyopathy referred for genetic testing at GeneDx and in published literature (Pandit et al., 2007); Reported in ClinVar as pathogenic by another clinical laboratory and the ClinGen RASopathy Variant Curation Expert Panel (ClinVar Variant ID# 40599; SCV000616377.3, SCV000061363.5; Landrum et al., 2016); Not observed in large population cohorts (Lek et al., 2016); Located in the CR2 critical functional domain; The majority of missense variants in this gene are considered pathogenic (Stenson et al., 2014); In silico analysis, which includes protein predictors and evolutionary conservation, supports a deleterious effect -

Noonan syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine

Sep 19, 2007

- -

Cardiovascular phenotype Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Oct 18, 2023

The c.768G>T (p.R256S) alteration is located in exon 7 (coding exon 6) of the RAF1 gene. This alteration results from a G to T substitution at nucleotide position 768, causing the arginine (R) at amino acid position 256 to be replaced by a serine (S). This variant was not reported in population-based cohorts in the Genome Aggregation Database (gnomAD). This variant has been reported as heterozygous in two other individuals with clinical features consistent with RAF1-related RASopathy (Pandit, 2007; Molzan, 2010). Three other alterations at the same codon, c.766A>G (p.R256G), c.767G>A (p.R256K), and c.767G>C (p.R256T) have been detected in affected individuals (Burstein, 2021; Ambry internal data). This amino acid position is highly conserved in available vertebrate species. This variant is located in the conserved region 2 (CR2). The in silico prediction for this alteration is inconclusive. Based on the available evidence, this alteration is classified as pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.99
BayesDel_addAF	Pathogenic	0.34	D
BayesDel_noAF	Pathogenic	0.25
Cadd	Benign	23
Dann	Uncertain	1.0
DEOGEN2	Pathogenic	0.84	D;.;T
Eigen	Uncertain	0.34
Eigen_PC	Uncertain	0.24
FATHMM_MKL	Benign	0.48	N
LIST_S2	Uncertain	0.96	D;D;D
M_CAP	Uncertain	0.29	D
MetaRNN	Uncertain	0.73	D;D;D
MetaSVM	Uncertain	-0.011	T
MutationAssessor	Pathogenic	3.0	M;M;.
MutationTaster	Benign	1.0	D;D;D;D
PrimateAI	Uncertain	0.77	T
PROVEAN	Uncertain	-3.5	D;D;D
REVEL	Pathogenic	0.67
Sift	Uncertain	0.023	D;D;D
Sift4G	Uncertain	0.031	D;D;D
Polyphen		1.0	D;.;.
Vest4		0.78
MutPred		0.54		Gain of glycosylation at S252 (P = 0.0093);Gain of glycosylation at S252 (P = 0.0093);.;
MVP		0.98
MPC		1.4
ClinPred		0.97	D
GERP RS		2.5
Varity_R		0.84
gMVP		0.82

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.0		Details are displayed if max score is > 0.2

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs397516826; hg19: chr3-12645701;