rs121434594

chr3-12604189-G-Achr3-12604189-G-Cchr3-12604189-G-T

Variant summary

Our verdict is Pathogenic. Variant got 17 ACMG points: 17P and 0B. PM1 PM2 PM5 PP2 PP3_Moderate PP5_Very_Strong

The NM_002880.4(RAF1):c.781C>T(p.Pro261Ser) variant causes a missense change involving the alteration of a conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. P261A) has been classified as Likely pathogenic.

• Frequency: Genomes: not found (cov: 33)
• Consequence: RAF1 NM_002880.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:14
• 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]

ACMG classification

Verdict is Pathogenic. Variant got 17 ACMG points.

• PM1: In a hotspot region, there are 9 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 0 benign, 7 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 chr3-12604189-G-C is described in ClinVar as [Likely_pathogenic]. Clinvar id is 40605.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars.
• PP2: Missense variant where missense usually causes diseases, RAF1
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.871
• PP5: Variant 3-12604189-G-A is Pathogenic according to our data. Variant chr3-12604189-G-A is described in ClinVar as [Pathogenic]. Clinvar id is 13958.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-12604189-G-A is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
RAF1	NM_002880.4	c.781C>T	p.Pro261Ser	missense_variant	7/17	ENST00000251849.9

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
RAF1	ENST00000251849.9	c.781C>T	p.Pro261Ser	missense_variant	7/17	1	NM_002880.4	P3

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 33

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:14

Revision: reviewed by expert panel

Submissions by phenotype

Noonan syndrome Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Mar 22, 2019	The p.Pro261Ser variant in RAF1 has been identified in >10 individuals with clinical features of Noonan syndrome (Razzaque 2007, Pandit 2007, Longoni 2010, LMM unpublished data), reported de novo in 2 individuals and reported to segregate in 4 relatives with clinical features of Noonan syndrome from 2 families (Razzaque 2007, Pandit 2007). In addition, this variant was absent from large population studies and is classified as pathogenic by the ClinGen RASopathy variant curation expert panel. Individuals with pathogenic variants in exon 7 or 17 in RAF1 are reported to also have a higher incidence of hypertrophic cardiomyopathy (HCM 80-95%) than typically seen in Noonan syndrome (Razzaque 2007, Pandit 2007). Studies have shown that the Pro261Ser variant impacts protein function by increasing its kinase activity (Razzaque 2007, Pandit 2007). However, these in vitro assays may not accurately represent biological function. In summary, this variant meets criteria to be classified as pathogenic for RASopathies in an autosomal dominant manner. ACMG/AMP criteria applied: PP2, PM1, PM2, PS3, PM6_Strong. -
Likely pathogenic, no assertion criteria provided	literature only	Database of Curated Mutations (DoCM)	May 13, 2016	- -
Pathogenic, reviewed by expert panel	curation	ClinGen RASopathy Variant Curation Expert Panel	Apr 03, 2017	The c.781C>T (p.Pro261Ser) variant in RAF1 has been reported in the literature in at least 2 unconfirmed de novo occurrences in patients with clinical features of a RASopathy (PM6_Strong; PMID 17603482, 20683980). In vitro functional studies provide some evidence that the p.Pro261Leu variant may impact protein function (PS3; PMID 17603483, 17603482). 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). 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). In summary, this variant meets criteria to be classified as pathogenic for RASopathies in an autosomal dominant manner. ACMG/AMP criteria applied: PP2, PM1, PM2, PS3, PM6_Strong. -

not provided Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Institute of Medical Genetics and Applied Genomics, University Hospital Tübingen	Oct 23, 2020	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Mar 14, 2023	Published functional studies demonstrate that P261S increases kinase activity and MEK and ERK activation (Pandit et al., 2007; Razzaque et al., 2007); 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; The majority of missense variants in this gene are considered pathogenic (HGMD); Reported in ClinVar as pathogenic by the ClinGen RASopathy Variant Curation Expert Panel (ClinVar Variant ID# 13958; ClinVar); This variant is associated with the following publications: (PMID: 30055033, 17603483, 22585553, 17603482, 20683980, 22781091, 24803665, 31219622, 33673806, 24957944, 9689060, 15520807, 29493581, 19020799, 34429303) -

RASopathy Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Sep 11, 2018	Variant summary: RAF1 c.781C>T (p.Pro261Ser) results in a non-conservative amino acid change in 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 31494 control chromosomes (gnomAD and publications). c.781C>T has been reported in the literature in multiple individuals affected with Noonan Syndrome and Related Conditions (Pandit_2007, Razzaque_2007). These data indicate that the variant is very likely to be associated with disease. Multiple functional assessments found the variant to cause an increase in kinase activity. Five ClinVar submissions from clinical diagnostic laboratories (evaluation after 2014) cite the variant as likely pathogenic/pathogenic. In addition, other mutations at this position, Pro261Ala and Pro261Thr, have been reported as pathogenic and associated with Noonan syndrome, indicating the location is a mutational hotspot and critical for gene function. Based on the evidence outlined above, the variant was classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Invitae	Jan 24, 2024	This sequence change replaces proline, which is neutral and non-polar, with serine, which is neutral and polar, at codon 261 of the RAF1 protein (p.Pro261Ser). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with Noonan syndrome (PMID: 17603482, 17603483, 20683980). ClinVar contains an entry for this variant (Variation ID: 13958). 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 with a positive predictive value of 95%. Experimental studies have shown that this missense change affects RAF1 function (PMID: 17603482, 17603483). This variant disrupts the p.Pro261 amino acid residue in RAF1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 17603483, 20052757, 21784453). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic. -

Noonan syndrome 5 Pathogenic:2

Pathogenic, no assertion criteria provided	literature only	OMIM	Aug 01, 2007	- -
Pathogenic, criteria provided, single submitter	clinical testing	Victorian Clinical Genetics Services, Murdoch Childrens Research Institute	May 07, 2021	0103 - Loss of function and gain of function are known mechanisms of disease in this gene. Gain of function is associated with Noonan (MIM#611553) and LEOPARD syndromes (MIM#2611554), while loss of function is associated non-HCM-associated variants (PMID: 17603483, 17603482). (I) 0107 - This gene is associated with autosomal dominant disease. (I) 0200 - Variant is predicted to result in a missense amino acid change from proline to serine. (I) 0251 - This variant is heterozygous. (I) 0301 - Variant is absent from gnomAD (both v2 and v3). (SP) 0502 - Missense variant with conflicting in silico predictions and uninformative conservation. (I) 0602 - Variant is located in a hotspot region or cluster of pathogenic variants (DECIPHER). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. It has been classified as Pathogenic by ClinGen's RASopathy expert panel (ClinVar). (SP) 1203 - This variant has been shown to be de novo in the proband (parental status confirmed) (by trio analysis). (SP) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign -

Inborn genetic diseases Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Apr 12, 2016

- -

Noonan syndrome 1 Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Molecular Genetics, Centre for Human Genetics

-

- -

Primary familial hypertrophic cardiomyopathy Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Blueprint Genetics

Sep 14, 2015

- -

Cardiovascular phenotype Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jun 02, 2022

The p.P261S pathogenic mutation (also known as c.781C>T), located in coding exon 6 of the RAF1 gene, results from a C to T substitution at nucleotide position 781. The proline at codon 261 is replaced by serine, an amino acid with similar properties. This alteration has been reported in a number of individuals with Noonan syndrome, including a de novo occurrence. This alteration was also reported to segregate with the disease in a few families (Pandit B et al. Nat. Genet., 2007 Aug;39:1007-12; Razzaque MA et al. Nat. Genet., 2007 Aug;39:1013-7; Longoni M et al. Am. J. Med. Genet. A, 2010 Sep;152A:2176-84; Digilio MC et al. Eur. J. Hum. Genet., 2013 Feb;21:200-4). In vitro studies have suggested a gain of function effect of this variant in the experimental system (Pandit B et al. Nat. Genet., 2007 Aug;39:1007-12; Razzaque MA et al. Nat. Genet., 2007 Aug;39:1013-7). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). In addition, this alteration is predicted to be deleterious by in silico analysis. Alterations affecting the same amino acid have also been described in affected individuals (Ratola A et al. Pediatr Rep, 2015 May;7:5955; Ezquieta B et al. Rev Esp Cardiol (Engl Ed), 2012 May;65:447-55). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Noonan syndrome and Noonan-related syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Genome Diagnostics Laboratory, The Hospital for Sick Children

Jun 01, 2020

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	1.0
BayesDel_addAF	Pathogenic	0.42	D
BayesDel_noAF	Pathogenic	0.36
Cadd	Pathogenic	30
Dann	Pathogenic	1.0
DEOGEN2	Pathogenic	0.87	D;.;T
Eigen	Pathogenic	0.87
Eigen_PC	Pathogenic	0.81
FATHMM_MKL	Uncertain	0.93	D
LIST_S2	Pathogenic	1.0	D;D;D
M_CAP	Uncertain	0.20	D
MetaRNN	Pathogenic	0.87	D;D;D
MetaSVM	Uncertain	0.38	D
MutationAssessor	Pathogenic	3.0	M;M;.
MutationTaster	Benign	1.0	A;A;A;A
PrimateAI	Uncertain	0.77	T
PROVEAN	Pathogenic	-4.7	D;D;D
REVEL	Pathogenic	0.82
Sift	Uncertain	0.0020	D;D;D
Sift4G	Uncertain	0.010	D;D;D
Polyphen		1.0	D;.;.
Vest4		0.77
MutPred		0.68		Loss of loop (P = 0.0128);Loss of loop (P = 0.0128);.;
MVP		0.93
MPC		1.3
ClinPred		0.99	D
GERP RS		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.83
gMVP		0.71

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: rs121434594; hg19: chr3-12645688; COSMIC: COSV52578629; COSMIC: COSV52578629;