3-12604189-G-C

Position:

• chr3-12604189-G-C

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>G​(p.Pro261Ala) 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 Likely pathogenic (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. P261L) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 33)
• Consequence: RAF1 NM_002880.4 missense
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:8
• 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 Pathogenic. Variant got 17 ACMG points.

• PM1: In a compositionally_biased_region Polar residues (size 47) in uniprot entity RAF1_HUMAN there are 14 pathogenic changes around while only 0 benign (100%) 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 chr3-12604188-G-A is described in Lovd as [Pathogenic].
• PP2: Missense variant in gene, where missense usually causes diseases (based on misZ statistic), RAF1. . Gene score misZ 2.4628 (greater than the threshold 3.09). Trascript score misZ 3.4185 (greater than threshold 3.09). GenCC has associacion of gene with Noonan syndrome 5, dilated cardiomyopathy 1NN, Noonan syndrome with multiple lentigines, Noonan syndrome, cardiofaciocutaneous syndrome, familial isolated dilated cardiomyopathy, LEOPARD syndrome 2, Costello syndrome.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.87
• PP5: Variant 3-12604189-G-C is Pathogenic according to our data. 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. Variant chr3-12604189-G-C is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
RAF1	NM_002880.4	c.781C>G	p.Pro261Ala	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.781C>G	p.Pro261Ala	missense_variant	7/17	1	NM_002880.4	ENSP00000251849.4

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 33

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:8

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

not provided Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Eurofins Ntd Llc (ga)	Sep 17, 2014	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Jun 27, 2016	The P261A variant has been published previously in association with RAF1-related disorders (Razzaque et al., 2007; Stevenson et al., 2011; Croonen et al., 2013). The variant was not observed in approximately 6,500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project, indicating it is not a common benign variant in these populations. P261A is a semi-conservative amino acid substitution, which may impact secondary protein structure as these residues differ in some properties. This substitution occurs at a position that is conserved across species, and in silico analysis predicts this variant is probably damaging to the protein structure/function. Functional studies have shown that P261A leads to increased activity of the RAF1 protein (Razzaque et al., 2007; Molzan et al., 2010). Missense variants in the same residue (P261T/S/H/L/R) and in nearby residues (R256S, S257L, S259P/T/F, T260I/R, N262I, N262K, V263A/D) have been reported in the Human Gene Mutation Database in association with RAF1-related disorders (Stenson et al., 2014), supporting the functional importance of this region of the protein. Therefore, we consider this variant to be pathogenic. -

LEOPARD syndrome 2;C1969057:Noonan syndrome 5;C4014656:Dilated cardiomyopathy 1NN Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Fulgent Genetics, Fulgent Genetics

Oct 31, 2018

- -

Noonan syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine

Oct 27, 2014

The p.Pro261Ala variant in RAF1 has been identified in three individuals with cl inical features of Noonan syndrome (Razzaque 2007, Croonen 2013, LMM unpublished data), and was absent from large population studies (http://evs.gs.washington.e du/EVS/; dbSNP rs121434594). Different pathogenic amino acid changes (p.Pro261Le u, p.Pro261Ser, p.Pro261Thr) at this location have been identified in individual s with clinical features of Noonan syndrome, two of which (p.Pro261Leu, p.Pro261 Thr) were reported to have occurred de novo in one individual each (Razzaque 200 7, Pandit 2007, LMM unpublished data), suggesting that changes at this position are not tolerated. Studies have shown that the p.Pro261Ala variant impacts prote in function by increasing its kinase activity (Razzaque 2007). However, these in vitro assays may not accurately represent biological function. Individuals wit h 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 No onan syndrome (Razzaque 2007, Pandit 2007). In summary, the p.Pro261Ala variant meets our criteria to be classified as pathogenic (http://personalizedmedicine.p artners.org/Laboratory-For-Molecular-Medicine/) based upon number of cases, abse nce from controls, evidence of other pathogenic variants in the same codon, and functional evidence. -

Primary familial hypertrophic cardiomyopathy Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Blueprint Genetics

Jan 27, 2015

- -

Noonan syndrome 5 Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Victorian Clinical Genetics Services, Murdoch Childrens Research Institute

Oct 09, 2024

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 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 with 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 alanine. (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 high 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. This variant has been classified as pathogenic and likely pathogenic by multiple clinical laboratories in ClinVar. This variant has also been observed in multiple unrelated heterozygous individuals with Noonan syndrome, some of whom had hypertrophic cardiomyopathy (PMID: 23885229, 20052757, 33128510, 17603482). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign -

RASopathy Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Jul 29, 2022

For these reasons, this variant has been classified as Pathogenic. 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, 20683980, 21784453, 26266034). 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: 17603482, 20052757, 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: 40605). This missense change has been observed in individual(s) with Noonan syndrome (PMID: 17603482, 20052757, 23885229). 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 proline, which is neutral and non-polar, with alanine, which is neutral and non-polar, at codon 261 of the RAF1 protein (p.Pro261Ala). -

RAF1-related disorder Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

PreventionGenetics, part of Exact Sciences

Dec 18, 2023

The RAF1 c.781C>G variant is predicted to result in the amino acid substitution p.Pro261Ala. This variant has been reported in multiple individuals with Noonan syndrome (see for example - Razzaque et al. 2007. PubMed ID: 17603482; Maher et al. 2018. PubMed ID: 30355600; Table S2, Leach et al. 2018. PubMed ID: 29907801). Functional studies found this variant leads to increased RAF1 kinase, consistent with a gain-of-function mechanism (Razzaque et al. 2007. PubMed ID: 17603482). This variant has not been reported in a large population database (http://gnomad.broadinstitute.org), indicating this variant is rare. Additionally, alternate missense variants affecting this amino acid (p.Pro261Thr, p.Pro261Ser, p.Pro261His, p.Pro261Arg, p.Pro261Leu) have been reported as pathogenic (Human Gene Mutation Database). This variant is interpreted as pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.99
BayesDel_addAF	Pathogenic	0.41	D
BayesDel_noAF	Pathogenic	0.35
CADD	Pathogenic	29
DANN	Uncertain	0.99
DEOGEN2	Pathogenic	0.88	D;.;T
Eigen	Pathogenic	0.87
Eigen_PC	Pathogenic	0.81
FATHMM_MKL	Uncertain	0.94	D
LIST_S2	Pathogenic	1.0	D;D;D
M_CAP	Uncertain	0.18	D
MetaRNN	Pathogenic	0.87	D;D;D
MetaSVM	Uncertain	0.38	D
MutationAssessor	Pathogenic	3.0	M;M;.
PrimateAI	Uncertain	0.76	T
PROVEAN	Pathogenic	-4.7	D;D;D
REVEL	Pathogenic	0.83
Sift	Uncertain	0.0040	D;D;D
Sift4G	Benign	0.071	T;D;D
Polyphen		1.0	D;.;.
Vest4		0.80
MutPred		0.68		Gain of glycosylation at S257 (P = 0.0137);Gain of glycosylation at S257 (P = 0.0137);.;
MVP		0.91
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		0.76
gMVP		0.64

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;