rs869025501

Positions:

• chr3-12604191-G-A
• chr3-12604191-G-C
• chr3-12604191-G-T

Variant summary

Our verdict is Uncertain significance. Variant got 9 ACMG points: 9P and 0B. PS3 PM2 PP2 PM1

This summary comes from the ClinGen Evidence Repository: The c.779C>T (p.Thr260Ile) variant in RAF1 was absent from large population databases (PM2; gnomad.broadinstitute.org). It occurs in the CR2 domain of the protein, which has been identified as a region important for protein function (PM1, 29493581). In vitro functional assays indicate that this variant may impact protein function (PS3; PMID:20679480). 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 variants are common (PP2; PMID:29493581). Computational prediction tools and conservation analyses do not provide strong support for or against an impact to the protein. This variant has been observed in multiple probands who lacked consistent RASopathy phenotypes; however, many probands presented with cardiomyopathy (PMID:17603483; Blueprint Genetics internal data, SCV000264163.2; Invitae internal data, SCV000824563.1; Ambry internal data, SCV000740058.2; GeneDx internal data, SCV000582743.4). Although criteria suggest that this variant is likely pathogenic given its location and in vitro functional studies, the lack of clear clinical presentations of a RASopathy phenotype supports that this variant is of uncertain significance at this time. RASopathy-specific ACMG/AMP criteria applied: PS3, PM1, PM2, PP2. LINK:https://erepo.genome.network/evrepo/ui/classification/CA351736/MONDO:0021060/004

• Frequency: Genomes: not found (cov: 32)
• Consequence: RAF1 NM_002880.4 missense
• Clinical Significance: Uncertain significance reviewed by expert panel P:5 U:1
• Conservation: PhyloP100: 10.0

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]

RAF1 (HGNC:):

ACMG classification

Verdict is Uncertain_significance. Variant got 9 ACMG points.

• PS3: For more information check the summary or visit ClinGen Evidence Repository.
• PM1: For more information check the summary or visit ClinGen Evidence Repository.
• PM2: For more information check the summary or visit ClinGen Evidence Repository.
• PP2: For more information check the summary or visit ClinGen Evidence Repository.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
RAF1	NM_002880.4	c.779C>T	p.Thr260Ile	missense_variant	7/17	ENST00000251849.9	NP_002871.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
RAF1	ENST00000251849.9	c.779C>T	p.Thr260Ile	missense_variant	7/17	1	NM_002880.4	ENSP00000251849.4

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 32

ClinVar

Significance: Uncertain significance

Submissions summary: Pathogenic:5 Uncertain:1

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Jul 31, 2023	Not observed at significant frequency in large population cohorts (gnomAD); Missense variants in this gene are often considered pathogenic (HGMD); Published functional studies suggest a damaging effect as the p.(T260I) variant has decreased affinity for C-RAF-PSer, increased enzymatic activity and decreases p.S259 phosphorylation suggestive of an overactive Ras-RAF-MAPK pathway (Molzan et al., 2010); 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: 24803665, 33686258, 17603483, 33673806, 29493581, 24957944, 9689060, 15520807, 17603482, 19020799, 20679480) -
Likely pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Nov 27, 2019	- -

RASopathy Pathogenic:1 Uncertain:1

Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Oct 17, 2022	Experimental studies have shown that this missense change affects RAF1 function (PMID: 20679480). For these reasons, this variant has been classified as Pathogenic. This variant disrupts the p.Thr260 amino acid residue in RAF1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 17603483; Invitae). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. 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: 222774). This missense change has been observed in individual(s) with clinical features of Noonan syndrome (PMID: 17603483; Invitae). 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 threonine, which is neutral and polar, with isoleucine, which is neutral and non-polar, at codon 260 of the RAF1 protein (p.Thr260Ile). -
Uncertain significance, reviewed by expert panel	curation	ClinGen RASopathy Variant Curation Expert Panel	Mar 09, 2020	The c.779C>T (p.Thr260Ile) variant in RAF1 was absent from large population databases (PM2; gnomad.broadinstitute.org). It occurs in the CR2 domain of the protein, which has been identified as a region important for protein function (PM1, 29493581). In vitro functional assays indicate that this variant may impact protein function (PS3; PMID: 20679480). 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 variants are common (PP2; PMID: 29493581). Computational prediction tools and conservation analyses do not provide strong support for or against an impact to the protein. This variant has been observed in multiple probands who lacked consistent RASopathy phenotypes; however, many probands presented with cardiomyopathy (PMID: 17603483; Blueprint Genetics internal data, SCV000264163.2; Invitae internal data, SCV000824563.1; Ambry internal data, SCV000740058.2; GeneDx internal data, SCV000582743.4). Although criteria suggest that this variant is likely pathogenic given its location and in vitro functional studies, the lack of clear clinical presentations of a RASopathy phenotype supports that this variant is of uncertain significance at this time. RASopathy-specific ACMG/AMP criteria applied: PS3, PM1, PM2, PP2. -

Primary familial hypertrophic cardiomyopathy Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Blueprint Genetics

Feb 03, 2015

- -

Cardiovascular phenotype Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Oct 31, 2016

The p.T260I variant (also known as c.779C>T), located in coding exon 6 of the RAF1 gene, results from a C to T substitution at nucleotide position 779. The threonine at codon 260 is replaced by isoleucine, an amino acid with similar properties. This alteration is located in the consensus recognition site for 14-3-3, which is a hotspot for Noonan syndrome (NS) mutations. Mutations in this region have been associated with a high prevalence of hypertrophic cardiomyopathy (HCM), and this variant has been previously reported in an HCM cohort (Pandit B et al. Nat. Genet. 2007;39:1007-12). Functional studies have demonstrated that this alteration leads to decreased phosphorylation of an invariant phosphorylated serine in the 14-3-3 recognition site, impaired 14-3-3 binding affinity, and increased RAF1 catalytic activity (Molzan M et al. Mol. Cell. Biol. 2010;30:4698-711). Furthermore, a likely pathogenic alteration affecting the same codon (p.T260R) has been reported in an individual with NS (Pandit B et al. Nat. Genet. 2007;39:1007-12). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the majority of available evidence to date, this variant is likely to be pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.97
BayesDel_addAF	Pathogenic	0.38	D
BayesDel_noAF	Pathogenic	0.31
CADD	Pathogenic	30
DANN	Uncertain	1.0
DEOGEN2	Uncertain	0.65	D;.;T
Eigen	Pathogenic	0.74
Eigen_PC	Pathogenic	0.73
FATHMM_MKL	Uncertain	0.93	D
LIST_S2	Uncertain	0.97	D;D;D
M_CAP	Uncertain	0.16	D
MetaRNN	Uncertain	0.70	D;D;D
MetaSVM	Uncertain	0.096	D
MutationAssessor	Uncertain	2.8	M;M;.
PrimateAI	Uncertain	0.78	T
PROVEAN	Uncertain	-2.4	N;D;D
REVEL	Uncertain	0.62
Sift	Uncertain	0.0040	D;D;D
Sift4G	Uncertain	0.031	D;D;T
Polyphen		0.96	D;.;.
Vest4		0.70
MutPred		0.44		Gain of glycosylation at S257 (P = 0.0137);Gain of glycosylation at S257 (P = 0.0137);.;
MVP		0.92
MPC		0.89
ClinPred		0.90	D
GERP RS		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.85
gMVP		0.68

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: rs869025501; hg19: chr3-12645690; COSMIC: COSV52574554; COSMIC: COSV52574554;