ClinVar Genomic variation as it relates to human health

The p.Arg496Cys variant in SMAD4 has been reported in at least 12 individuals with Myhre syndrome, and was confirmed as a de novo occurrence in at least four of these individuals (Kenis 2014, Caputo 2014, Michot 2014, Geisheker 2017, Artemis 2019, Meerschaut 2019, Lin 2020, Broad Rare Genomes Project). Additionally, four of these individuals are reported to have neoplasia, including three with endometrial cancer (Lin 2020). This variant has also been reported in ClinVar (Variation ID 88673) and has been identified in 1/10078 of Ashkenazi Jewish and 1/113728 European chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/). Structural analysis (Caputo 2014) and in vitro functional studies (Piccolo 2014, Li 2020) provide evidence that the p.Arg496Cys variant may impact protein function. However, these types of assays may not accurately represent biological function. In addition, computational prediction tools and conservation analysis support that the p.Arg496Cys variant may impact the protein. In summary, this variant meets criteria to be classified as pathogenic for Myhre syndrome in an autosomal dominant manner based upon case counts, de novo occurrence, location at a critical residue, functional evidence, and predicted impact on protein. ACMG/AMP Criteria applied: PS2_VeryStrong, PS4_Moderate, PS3_Supporting, PP3.

This variant was determined to be pathogenic according to ACMG Guidelines, 2015 [PMID:25741868].

The same variant was also reported as de novo in one or more affected individuals with a consistent phenotype from multiple, unrelated families (PMID: 24424121, 31654632, PS2_VS). The variant has been observed in multiple (>3) similarly affected unrelated individuals(PMID: 24424121, 31654632, 31595668, 30968316, PS4_S). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.961, PP3_P). It is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.000008, PM2_M). The variantwas confirmed as de novo by parental testing. Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

ACMG classification criteria: PS2 very strong, PS3 supporting, PS4 moderate, PP3

Based on the classification scheme VCGS_Germline_v1.1.1, this variant is classified as 5 - Pathogenic. Following criteria are met: 0101 - Gain-of-function is a known mechanism of disease for this gene. Missense variants result in gain of function effects associated with Myhre syndrome (PMID 22158539; PMID 31837202; PMID 31595668). (N) 0102 - Loss-of-function is a known mechanism of disease for this gene. Premature termination codon variants result in loss of function associated with hereditary haemorrhagic telangiectasia syndrome or juvenile polyposis (OMIM; ClinVar; PMID 31837202). (N) 0107 - This gene is known to be associated with autosomal dominant disease. (N) 0200 - Variant is predicted to result in a missense amino acid change from an arginine to a cysteine (exon 12). (N) 0251 - Variant is heterozygous. (N) 0302 - Variant is present in gnomAD <0.001 for a dominant condition (2 heterozygotes, 0 homozygotes). (P) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools and is very highly conserved with a major amino acid change. (P) 0602 - Variant is located in a hotspot region or cluster of pathogenic variants (MH2 domain; PDB). (P) 0801 - Strong previous evidence of pathogenicity in unrelated individuals. This variant has previously been reported pathogenic in multiple individuals with Myhre syndrome (ClinVar; Decipher; PMID 31837202; PMID 31595668). (P) 1208 - Inheritance information for this variant is not currently available. (N) Legend: (P) - Pathogenic, (N) - Neutral, (B) - Benign

Published functional studies demonstrate a damaging effect showing elevated SMAD4 levels and impaired microfibril deposition causing an extracellular matrix defect (Piccolo et al., 2014); In silico analysis supports a deleterious effect; Not observed at a significant frequency in large population cohorts (Lek et al., 2016); This variant is associated with the following publications: (PMID: 24424121, 24398790, 24715504, 27302097, 28051901, 24841914, 24580733, 28867141, 31654632, 31595668, 31837202, 31447099, 33428109, 30787465, 31785789, 17873119, 18823382, 15235019)

This missense variant replaces arginine with cysteine at codon 496 of the SMAD4 protein. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Experimental functional studies have provided evidence that this variant may impact protein function. In vitro studies have shown an increase in SMAD4 protein levels and the phosphorylated SMAD2/total SMAD2 protein ratio (PMID: 24398790), a significant increase in gene expression compared to wild type in transcriptional activation assays (PMID: 31654632), and decreased proliferation, and elevated expression of cellular senescence and inflammatory markers in a cell based assay (PMID: 33428109). In silico structural analyses have also suggested an impact to the stability of the SMAD heterotrimer and/or proper SMAD4 ubiquitination (PMID: 24715504). This variant has been reported in numerous individuals affected with Myhre syndrome (PMID: 22158539, 24398790, 24424121, 24715504, 24841914, 26633542, 28628100, 30968316, 31595668, 31837202). Three individuals were also affected with endometrial cancer (PMID: 31837202). This variant has been identified in 2/251432 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Although this variant is considered Pathogenic for autosomal dominant Myhre Syndrome, the available evidence is insufficient to determine the role of this variant in cancer predisposition conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

This sequence change replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 496 of the SMAD4 protein (p.Arg496Cys). This variant is present in population databases (rs397518413, gnomAD 0.01%). This missense change has been observed in individual(s) with Myhre syndrome (PMID: 24424121, 24715504). In at least one individual the variant was observed to be de novo. ClinVar contains an entry for this variant (Variation ID: 88673). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on SMAD4 protein function. Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on SMAD4 function (PMID: 24398790). For these reasons, this variant has been classified as Pathogenic.

The p.R496C pathogenic mutation (also known as c.1486C>T), located in coding exon 11 of the SMAD4 gene, results from a C to T substitution at nucleotide position 1486. The arginine at codon 496 is replaced by cysteine, an amino acid with highly dissimilar properties. This alteration has been reported in several individuals with a de novo clinical diagnosis of Myhre syndrome (Le Goff C et al. Clin. Genet. 2014 Jun; 85(6):503-13; Kenis C et al. Otol. Neurotol. 2014 Oct; 35(9):e253-5; Michot C et al. Eur. J. Hum. Genet. 2014 Nov; 22(11):1272-7). The p.R496C alteration is located in the MH2 domain, which is required for SMAD oligomerization and TGF-&beta; / bone morphogenic protein (BMP) signal transduction. In silico structural analyses suggest that conformational changes promoted by the replacement of arginine at position 496 impacts the stability of the SMAD heterotrimer and proper SMAD4 ubiquitination (Caputo V et al. Am. J. Med. Genet. 2014 Jul; 164A(7):1835-40). Functional consequences of this alteration have been investigated; skin fibroblasts exhibited increased SMAD4 proteins compared with wildtype controls which only showed detectable SMAD4 protein following TGF-&beta; stimulation, increased phosphorylated SMAD2 proteins, matrix metalloproteinase (MMP) overexpression, and impaired microfibril deposition within the extracellular domain (Piccolo P et al. Eur. J. Hum. Genet. 2014 Aug; 22(8):988-94). This variant was not reported in population based cohorts in the following databases: Database of Single Nucleotide Polymorphisms (dbSNP), NHLBI Exome Sequencing Project (ESP), and 1000 Genomes Project. In the ESP, this variant was not observed in 6503 samples (13006 alleles) with coverage at this position. This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the majority of available evidence to date, this variant is classified as a pathogenic mutation associated with Myhre syndrome.

SMAD4: PS2, PS4, PP2, PP3

Converted during submission to Pathogenic.

Variant confirmed as disease-causing by referring clinical team

The SMAD4 c.1486C>T variant is predicted to result in the amino acid substitution p.Arg496Cys. This variant has been reported in multiple patients with Myhre syndrome and is a recurrent de novo finding (Caputo et al. 2014. PubMed ID: 24715504; Piccolo et al. 2014. PubMed ID: 24398790). Functional in vitro studies showed an increase in SMAD4 protein in fibroblasts with the p.Arg496Cys variant and altered expression of downstream transcriptional target genes (Piccolo et al. 2014. PubMed ID: 24398790). This variant is reported in 0.0099% of alleles in individuals of Ashkenazi Jewish descent in gnomAD (http://gnomad.broadinstitute.org/variant/18-48604664-C-T). It is interpreted as pathogenic or likely pathogenic in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/88673/). This variant is interpreted as pathogenic.