rs3730271

Positions:

• chr3-12604195-A-C
• chr3-12604195-A-G
• chr3-12604195-A-T

Variant summary

Our verdict is Pathogenic. Variant got 12 ACMG points: 12P and 0B. PM5_Strong PS2 PM2 PS4_Supporting PP2

This summary comes from the ClinGen Evidence Repository: The c.775T>G (p.Ser259Ala) variant in RAF1 was absent from large population studies (PM2; gnomAD, http://gnomad.broadinstitute.org). It has been reported as a confirmed de novo occurrence in 2 probands (PS2_VeryStrong; SCV000808501.1). The variant has been identified in 1 proband with Noonan syndrome (PS4_Supporting; SCV000710868.2). At least 2 other pathogenic missense variants have been previously identified at the Ser259 codon of RAF1 which may indicate that this residue is critical to the function of the protein (PM5_Strong; ClinVar 40601, 44633). A functional assay has been performed on c.775T>G (p.Ser259Ala), but given that it is not currently an approved assay outlined by the RASopathy VCEP, it has not been assessed for PS3 at this time (PMID:23391722). Finally, the variant is located in RAF1, 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). Of note, this variant has also been observed in association with somatic malignancies; however, analysis and classification of somatic variation is currently not used to inform germline classifications for the RASopathies. 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): PS2_VeryStrong, PS4_Supporting, PM5_Strong, PM2, PP2. LINK:https://erepo.genome.network/evrepo/ui/classification/CA351512423/MONDO:0021060/004

• Frequency: Genomes: not found (cov: 33)
• Consequence: RAF1 NM_002880.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:3 U:1
• 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 12 ACMG points.

• PS2: For more information check the summary or visit ClinGen Evidence Repository.
• PS4: 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.
• 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.775T>G	p.Ser259Ala	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>G	p.Ser259Ala	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:3 Uncertain:1

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories	Apr 29, 2022	The RAF1 c.775T>G; p.Ser259Ala variant (rs3730271), to our knowledge, is not reported in the literature but is reported as pathogenic by an FDA recognized database in ClinVar (Variation ID: 504514). This variant is also absent from the Genome Aggregation Database, indicating it is not a common polymorphism. The serine at codon 259 is highly conserved and occurs in the 14-3-3 binding site, but computational analyses are uncertain whether this variant is neutral or deleterious (REVEL: 0.601). However, functional studies of the variant in a mouse model show an increased ERK activation and increased production of lymphatic endothelial cells (Deng Blood 2013, Deng J Clin Invest 2013). Additionally, several other missense variants at this codon (p.Ser257Pro, p.Ser259Phe, p.Ser259Thr, p.Ser259Tyr) have been reported in individuals with Noonan syndrome (Croonen 2013, Hakami 2016, Ko 2008, Pandit 2007). Based on available information, this variant is classified as pathogenic. References: Croonen EA et al. Prenatal diagnostic testing of the Noonan syndrome genes in fetuses with abnormal ultrasound findings. Eur J Hum Genet. 2013 Sep;21(9):936-42. PMID: 23321623. Deng Y et al. Endothelial RAF1/ERK activation regulates arterial morphogenesis. Blood. 2013 May 9;121(19):3988-96, S1-9. PMID: 23529931. Deng Y et al. Endothelial ERK signaling controls lymphatic fate specification. J Clin Invest. 2013 Mar;123(3):1202-15. PMID: 23391722. Hakami F et al. Retrospective study of prenatal ultrasound findings in newborns with a Noonan spectrum disorder. Prenat Diagn. 2016 May;36(5):418-23. PMID: 26918529. Ko JM et al. PTPN11, SOS1, KRAS, and RAF1 gene analysis, and genotype-phenotype correlation in Korean patients with Noonan syndrome. J Hum Genet. 2008;53(11-12):999-1006. PMID: 19020799. Pandit B et al. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet. 2007 Aug;39(8):1007-12. PMID: 17603483. -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Nov 06, 2017	The S259A variant in the RAF1 gene has not been reported previously as a germline pathogenic variant nor as a benign variant, to our knowledge. However, variants at the same residue (S259P, S259T, S259F) have been reported in individuals with Noonan syndrome, or prenatal findings suggestive of Noonan syndrome (Pandit et al., 2007; Ko JM et al., 2008; Croonen et al., 2013). In addition, missense variants in nearby residues (S257L, T260R, P261S, etc.) have been reported in the Human Gene Mutation Database in association with Noonan syndrome (Stenson et al., 2014), supporting the functional importance of this region of the protein. The S259A variant is not observed in large population cohorts (Lek et al., 2016). The S259A variant is a non-conservative amino acid substitution, which is likely to impact secondary protein structure as these residues differ in polarity, charge, size and/or other properties. In-silico analyses, including protein predictors and evolutionary conservation, support a deleterious effect. Also, it has been shown that expression of S259A leads to ERK activation, and induces SOX18 and PROX1 expression. This leads to increased commitment of venous endothelial cells to the lymphatic fate, resulting in excessive production of lymphatic endothelial cells and lymphanogiectasia in mice (Deng et al., 2013; Pandit et al., 2007). We interpret S259A as a pathogenic variant. -

RASopathy Pathogenic:1

Pathogenic, reviewed by expert panel

curation

ClinGen RASopathy Variant Curation Expert Panel

Jun 25, 2020

The c.775T>G (p.Ser259Ala) variant in RAF1 was absent from large population studies (PM2; gnomAD, http://gnomad.broadinstitute.org). It has been reported as a confirmed de novo occurrence in 2 probands (PS2_VeryStrong; SCV000808501.1). The variant has been identified in 1 proband with Noonan syndrome (PS4_Supporting; SCV000710868.2). At least 2 other pathogenic missense variants have been previously identified at the Ser259 codon of RAF1 which may indicate that this residue is critical to the function of the protein (PM5_Strong; ClinVar 40601, 44633). A functional assay has been performed on c.775T>G (p.Ser259Ala), but given that it is not currently an approved assay outlined by the RASopathy VCEP, it has not been assessed for PS3 at this time (PMID:23391722). Finally, the variant is located in RAF1, 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). Of note, this variant has also been observed in association with somatic malignancies; however, analysis and classification of somatic variation is currently not used to inform germline classifications for the RASopathies. 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): PS2_VeryStrong, PS4_Supporting, PM5_Strong, PM2, PP2. -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine

Apr 26, 2016

Variant classified as Uncertain Significance - Favor Pathogenic. The p.Ser259Ala variant in RAF1 has not been previously reported in individuals with clinical f eatures of Noonan syndrome or in large population studies. However, different di sease-causing amino acid changes at this location (p.Ser259Pro, p.Ser259Phe, p.S er259Thr) have been previously associated with Noonan spectrum disorders, sugges ting a change at this position may not be tolerated (Pandit 2007, Kobayashi 2009 , LMM unpublished data). In vitro functional studies provide some evidence that the p.Ser259Ala variant may impact protein function (Deng 2013a, Deng 2013b). Ho wever, these types of assays may not accurately represent biological function. C omputational prediction tools and conservation analysis do not provide strong su pport for or against an impact to the protein. In summary, while there is some s uspicion for a pathogenic role, the clinical significance of the p.Ser259Ala var iant is uncertain. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.87
BayesDel_addAF	Pathogenic	0.19	D
BayesDel_noAF	Uncertain	0.030
CADD	Pathogenic	28
DANN	Uncertain	1.0
DEOGEN2	Uncertain	0.62	D;.;T
Eigen	Pathogenic	0.71
Eigen_PC	Pathogenic	0.67
FATHMM_MKL	Uncertain	0.93	D
LIST_S2	Uncertain	0.97	D;D;D
M_CAP	Uncertain	0.22	D
MetaRNN	Uncertain	0.67	D;D;D
MetaSVM	Uncertain	0.33	D
MutationAssessor	Uncertain	2.9	M;M;.
PrimateAI	Uncertain	0.79	T
PROVEAN	Benign	-1.9	N;N;N
REVEL	Uncertain	0.60
Sift	Uncertain	0.0010	D;D;D
Sift4G	Uncertain	0.0070	D;D;D
Polyphen		0.94	P;.;.
Vest4		0.66
MutPred		0.44		Gain of glycosylation at S257 (P = 0.0137);Gain of glycosylation at S257 (P = 0.0137);.;
MVP		0.83
MPC		0.73
ClinPred		0.93	D
GERP RS		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.40
gMVP		0.67

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; COSMIC: COSV52586258; COSMIC: COSV52586258;