ClinVar Genomic variation as it relates to human health

The c.199G>A (p.Asp67Asn) variant in MAP2K1 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 17704260). In vitro functional studies provide some evidence that the p.Asp67Asn variant may impact protein function (PS3; PMID 25049390). This variant was absent from large population studies (PM2; ExAC, http://exac.broadinstitute.org). The variant is located in the MAP2K1 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.Asp67Asn 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, PS3, PM2, PP2, PP3.

The MAP2K1 c.199G>A (p.Asp67Asn) variant is a missense variant that has been reported in a heterozygous state in at least four unrelated individuals with a diagnosis of either Noonan syndrome or cardiofaciocutaneous syndrome (Nava et al. 2007; Chen et al. 2014). In two cases, the variant was confirmed to have occurred de novo. The p.Asp67Asn variant is absent from the Genome Aggregation Database in a region of good sequencing coverage and is presumed to be rare. Expression of Asp67Asn MAP2K1 in HEK293T cells resulted in constitutive ERK activation (Chen et al. 2014), a functional effect consistent with the known disease mechanism. Based on the collective evidence, the p.Asp67Asn variant is classified as pathogenic for MAP2K1-related RASopathy.

The p.Asp67Asn variant in MAP2K1 has been identified in 9 individuals with clini cal features of Noonan syndrome and/or Cardio-facio-cutaneous syndrome including 2 de novo occurences (Nava 2007, LMM unpublished data). This variant was absent from large population studies. In vitro functional studies provide some evidenc e that the p.Asp67Asn variant may impact protein function (Estep 2007). However, these types of assays may not accurately represent biological function. This va riant has also been reported in tumor samples from 2 individuals with colon canc er (Murugan 2009, Choi 2012). In summary, this variant meets our criteria to be classified as pathogenic for RASopathies in an autosomal dominant manner (http:/ /www.partners.org/personalizedmedicince/LMM) based upon de novo occurrences, abs ence from controls, and functional evidence.

Same nucleotide change resulting in same amino acid change has been previously reported as pathogenic/likely pathogeic with strong evidence (ClinVar ID: VCV000040781.11, PS1). The missense variant is located in a mutational hot spot and/or well-established functional domain in which established pathogenic variants have been reported (PM1). It is not observed in the gnomAD v2.1.1 dataset (PM2). The variant was observed as assumed (i.e. paternity and maternity not confirmed) de novoo (3billion dataset, PM6). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.697, 3Cnet: 0.969, PP3). Patient's phenotype is considered compatible with Cardiofaciocutaneous syndrome 3(3billion dataset, PP4).Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

The p.Asp67Asn variant in the MAP2K1 gene has been previously reported in at least 4 individuals with cardiofaciocutaneous syndrome or Noonan syndrome (Nava et al., 2007; Chen et al., 2014) and confirmed de novo in at least 2 individuals (Nava et al., 2007). This variant was absent from large population databases, including the Genome Aggregation Database (http://gnomad.broadinstitute.org/). The MAP2K1 gene has fewer missense variants in the general population than expected. A low rate of missense variation may suggest that this gene is intolerant to missense variation. Functional studies of this variant demonstrated constitutive ERK activation in human embryonic kidney cells of the and are supportive of a deleterious effect to the protein (Chen et al., 2014). Computational tools predict that the p.Asp67Asn variant is deleterious; however, the accuracy of in silico algorithms is limited. These data were assessed using the ACMG/AMP variant interpretation guidelines. In summary, there is sufficient evidence to classify the p.Asp67Asn variant as pathogenic for cardiofaciocutaneous syndrome in an autosomal dominant manner based on the information above. [ACMG evidence codes used: PM6; PS3; PM2; PP2; PP3]

The c.199G>A (p.D67N) alteration is located in exon 2 (coding exon 2) of the MAP2K1 gene. This alteration results from a G to A substitution at nucleotide position 199, causing the aspartic acid (D) at amino acid position 67 to be replaced by an asparagine (N). This variant was not reported in population-based cohorts in the Genome Aggregation Database (gnomAD). This mutation was reported as a de novo occurrence in multiple individuals with clinical findings consistent with a RASopathy (Nava, 2007; Firth, 2009; DECIPHER v.9.32) as well as in a childhood epilepsy cohort (Costain, 2019). Additional cases with a RASopathy phenotype have been reported, for which inheritance was not reported (Chen, 2014; Leung, 2018). This amino acid position is highly conserved in available vertebrate species. This missense alteration is located in a region that has a low rate of benign missense variation (Lek, 2016; Firth, 2009). Functional analysis showed that D67N-mutant protein resulted in constitutive ERK activation in HEK293T cells (Chen, 2014) The in silico prediction for this alteration is inconclusive. Based on the available evidence, this alteration is classified as pathogenic.

MAP2K1 NM_02755.3 p.Asp67Asn (c.199G>A): This variant has been reported in the literature in at least 5 individuals with Noonan syndrome or Cardio-Facio-Cutaneous syndrome (Nava 2007 PMID:17704260; Chen 2014 PMID:25049390; Carcavilla 2015 PMID:25194980) including 2 de novo cases (Nava 2007 PMID:17704260) and is not present in large control databases. This variant is present in ClinVar, with several labs classifying this variant as pathogenic (Variation ID: 40781). Evolutionary conservation and computational predictive tools suggest that this variant may impact the protein. In addition, in vitro (Chen 2014 PMID:25049390; Estep 2014 PMID: 18060073) and in vivo (Jindal 2016 PMID:2804985) functional studies suggest a deleterious effect of this variant. In summary, this variant is classified as pathogenic based on presence of affected individuals (including de novo occurences), absence from controls, and functional studies.

This variant was identified as de novo (maternity and paternity confirmed).

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (PMID: 25049390) - PS3_moderate. The c.199G>A;p.(Asp67Asn) missense change has been observed in affected individual(s) and ClinVar contains an entry for this variant (ClinVar ID: 40781; PMID: 17704260; PMID: 20301365; PMID: 25049390)-PS4. This variant is not present in population databases (rs727504317- gnomAD; ABraOM no frequency - http://abraom.ib.usp.br.) - PM2. The variant was assumed de novo, but without confirmation of paternity and maternity (PMID: 17704260) - PM6_strong. Missense variant in MAP2K1 that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease - PP2. Multiple lines of computational evidence support a deleterious effect on the gene or gene product - PP3. In summary, the currently available evidence indicates that the variant is pathogenic

Published functional studies demonstrate that expression in human embryonic kidney cells caused constitutive ERK activation; under the same conditions, wild type cells had little effect (Chen et al., 2014); Not observed 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); This variant is associated with the following publications: (PMID: 25049390, 17704260, 28049852, 24803665, 30763456, 31487502, 33448881, 31785789)

MAP2K1 NM_02755 p.Asp67Asn (c.199G>A): This variant has been reported in the literature in at least 5 individuals with Noonan syndrome or Cardio-Facio-Cutaneous syndrome (Nava 2007 PMID:17704260; Chen 2014 PMID:25049390; Carcavilla 2015 PMID:25194980) including 2 de novo cases (Nava 2007 PMID:17704260) and is not present in large control databases. This variant is present in ClinVar, with several labs classifying this variant as pathogenic (Variation ID: 40781). Evolutionary conservation and computational predictive tools suggest that this variant may impact the protein. In addition, in vitro (Chen 2014 PMID:25049390; Estep 2014 PMID: 18060073) and in vivo (Jindal 2016 PMID:2804985) functional studies suggest a deleterious effect of this variant. In summary, this variant is classified as pathogenic based on presence of affected individuals (including de novo occurences), absence from controls, and functional studies.

This sequence change replaces aspartic acid, which is acidic and polar, with asparagine, which is neutral and polar, at codon 67 of the MAP2K1 protein (p.Asp67Asn). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with Noonan syndrome or cardio-facio-cutaneous syndrome (PMID: 17704260, 25049390). In at least one individual the variant was observed to be de novo. ClinVar contains an entry for this variant (Variation ID: 40781). 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 not expected to disrupt MAP2K1 protein function with a negative predictive value of 80%. Experimental studies have shown that this missense change affects MAP2K1 function (PMID: 25049390). For these reasons, this variant has been classified as Pathogenic.

Criteria applied: PS2, PS3, PM2, PP2, PP3