chr3-12604195-A-T

Position:

• chr3-12604195-A-T

Variant summary

Our verdict is Pathogenic. Variant got 20 ACMG points: 20P and 0B. PM5_Strong PS3 PM2 PM6_Strong PS4_Supporting PP2 PP3 PP1 PM1

This summary comes from the ClinGen Evidence Repository: The c.775T>A (p.Ser259Thr) 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; APHP-Robert Debré Hospital internal data; GTR ID's: 28338). The p.Ser259Thr variant has been identified in at least 8 independent occurrences in patients with a RASopathy (PS4_Supporting; GeneDx, Partners LMM, Institute of Human Genetics, Otto von Guericke University Magdeburg internal data; GTR ID's: 26957, 21766, 506381 PMID:21784453; ClinVar SCV000061360.5; SCV000209017.10). The p.Ser259Thr variant in RAF1 has been reported to segregate with clinical features of a RASopathy in at least 3 family members (PP1; GeneDx, Institute of Human Genetics, Otto von Guericke University Magdeburg internal data; GTR ID's: 26957, 506381; ClinVar SCV000209017.9). In vitro functional studies provide some evidence that the p.Ser259Thr variant may impact protein function (PS3; PMID:21784453, 20052757). At least 2 other pathogenic missense variants have been previously identified at this codon of RAF1 which may indicate that this residue is critical to the function of the protein (PM5_Strong; ClinVar 40603, 40602, 228288). This variant was absent from large population studies (PM2; ExAC, http://exac.broadinstitute.org). 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). 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.Ser259Thr variant may impact 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): PM6_Strong, PS4_Supporting, PP1, PM5_Strong, PM2, PM1, PP2, PP3, PS3. LINK:https://erepo.genome.network/evrepo/ui/classification/CA261617/MONDO:0021060/004

• Frequency: Genomes: not found (cov: 33)
• Consequence: RAF1 NM_002880.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:8
• Conservation: PhyloP100: 9.32

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 20 ACMG points.

• PS3: For more information check the summary or visit ClinGen Evidence Repository.
• PS4: 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.
• PM5: For more information check the summary or visit ClinGen Evidence Repository.
• PM6: For more information check the summary or visit ClinGen Evidence Repository.
• PP1: For more information check the summary or visit ClinGen Evidence Repository.
• PP2: For more information check the summary or visit ClinGen Evidence Repository.
• PP3: 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.775T>A	p.Ser259Thr	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.775T>A	p.Ser259Thr	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

Submissions summary: Pathogenic:8

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Mayo Clinic Laboratories, Mayo Clinic	Jun 23, 2019	PM6_Strong, PS3, PS4_Supporting, PM1, PM2, PM5_Strong, PP2, PP3, PP1 -
Pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Jan 10, 2022	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Mar 16, 2023	Not observed at significant frequency in large population cohorts (gnomAD); Published functional studies demonstrate increased activation of MEK and ERK (Kobayashi et al., 2010; Lee et al., 2011); Missense variants in this gene are often considered pathogenic (HGMD); In silico analysis supports that this missense variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 20052757, 24957944, 9689060, 15520807, 17603483, 17603482, 29493581, 19020799, 33318624, 21784453) -

Noonan syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Sep 09, 2010	The Ser259Thr variant in RAF1 has been previously identified in two individuals with clinical features of Noonan syndrome (Ko 2008, LMM unpublished data). In ad dition, two different amino acid changes at this location (Ser259Phe, Ser259Tyr) have been identified in individuals with Noonan spectrum features, suggesting a lterations at this amino acid are not tolerated (Pandit 2007, Kobayashi 2009; LM M unpublished data). Ser259 is also known to be of critical importance to the r egulation of the RAF1 protein (Kobayashi 2009). Therefore, it is likely that thi s variant is pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Genetics and Molecular Pathology, SA Pathology	Jun 21, 2022	The RAF1 c.775T>A variant is classified as a Pathogenic variant (PS4, PS3, PM5, PP3) This variant is a single nucleotide change in exon 7/18 of the RAF1 gene, which is predicted to change the amino acid serine at position 259 in the protein to threonine. The variant has been reported multiple times in patients with Noonan syndrome and with clinical features of a RASopathy (PMID: 19020799, 21784453, 33318624, 29493581). This variant is in dbSNP (rs3730271) but is absent from population databases (PS4). Functional studies have shown that this variant may impact protein function by increasing activation of MEK and ERK (PMID: 21784453) (PS3). Missense variants in the same residue (p.S259C, p.S259F, p.S259P, p.A259Y) have been previously reported as pathogenic in association with Noonan spectrum disorders (PM5).The variant has been reported in the ClinVar and HGMD as pathogenic/ disease causing. Computational predictions support a deleterious effect on the gene or gene product (PP3). -

RASopathy Pathogenic:2

Pathogenic, reviewed by expert panel	curation	ClinGen RASopathy Variant Curation Expert Panel	Apr 03, 2017	The c.775T>A (p.Ser259Thr) 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; APHP-Robert Debré Hospital internal data; GTR ID's: 28338). The p.Ser259Thr variant has been identified in at least 8 independent occurrences in patients with a RASopathy (PS4_Supporting; GeneDx, Partners LMM, Institute of Human Genetics, Otto von Guericke University Magdeburg internal data; GTR ID's: 26957, 21766, 506381 PMID: 21784453; ClinVar SCV000061360.5; SCV000209017.10). The p.Ser259Thr variant in RAF1 has been reported to segregate with clinical features of a RASopathy in at least 3 family members (PP1; GeneDx, Institute of Human Genetics, Otto von Guericke University Magdeburg internal data; GTR ID's: 26957, 506381; ClinVar SCV000209017.9). In vitro functional studies provide some evidence that the p.Ser259Thr variant may impact protein function (PS3; PMID: 21784453, 20052757). At least 2 other pathogenic missense variants have been previously identified at this codon of RAF1 which may indicate that this residue is critical to the function of the protein (PM5_Strong; ClinVar 40603, 40602, 228288). This variant was absent from large population studies (PM2; ExAC, http://exac.broadinstitute.org). 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). 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.Ser259Thr variant may impact 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): PM6_Strong, PS4_Supporting, PP1, PM5_Strong, PM2, PM1, PP2, PP3, PS3. -
Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Nov 19, 2022	For these reasons, this variant has been classified as Pathogenic. This variant disrupts the p.Ser259 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. Experimental studies have shown that this missense change affects RAF1 function (PMID: 21784453). 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: 40601). This missense change has been observed in individuals with Noonan syndrome (PMID: 19020799; Invitae). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces serine, which is neutral and polar, with threonine, which is neutral and polar, at codon 259 of the RAF1 protein (p.Ser259Thr). -

Noonan syndrome 1 Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Molecular Genetics, Centre for Human Genetics

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Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.92
BayesDel_addAF	Pathogenic	0.35	D
BayesDel_noAF	Pathogenic	0.26
CADD	Pathogenic	27
DANN	Uncertain	0.99
DEOGEN2	Uncertain	0.60	D;.;T
Eigen	Pathogenic	0.76
Eigen_PC	Pathogenic	0.71
FATHMM_MKL	Uncertain	0.93	D
LIST_S2	Pathogenic	0.98	D;D;D
M_CAP	Uncertain	0.21	D
MetaRNN	Pathogenic	0.79	D;D;D
MetaSVM	Uncertain	0.38	D
MutationAssessor	Uncertain	2.9	M;M;.
PrimateAI	Pathogenic	0.80	T
PROVEAN	Benign	-1.7	N;N;N
REVEL	Pathogenic	0.75
Sift	Uncertain	0.025	D;D;D
Sift4G	Benign	0.062	T;D;T
Polyphen		0.97	D;.;.
Vest4		0.60
MutPred		0.60		Gain of glycosylation at S257 (P = 0.0137);Gain of glycosylation at S257 (P = 0.0137);.;
MVP		0.92
MPC		1.2
ClinPred		0.95	D
GERP RS		5.7
Varity_R		0.29
gMVP		0.72

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: rs3730271; hg19: chr3-12645694;