NM_005359.5:c.1082G>A

SMAD4 · NP_005350.1:p.(Arg361His) · NM_005359.5

GRCh37: chr18:48591919 G>A · GRCh38: chr18:51065549 G>A

Gene: SMAD4 Transcript: NM_005359.5

Final call

Pathogenic

PS3 strong PS4 moderate PM1 moderate PM2 supporting PM5 supporting PP3 supporting PP4 supporting PP5 supporting

Variant details

Gene

SMAD4

Transcript

NM_005359.5

Protein

NP_005350.1:p.(Arg361His)

gnomAD AF

ClinVar

Pathogenic

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

NM_005359.5:c.1082G>A (p.Arg361His) is a missense variant in exon 9 of SMAD4, located within the MH2 domain mutation cluster region (codons 330-370) critical for Smad protein heterodimerization and TGF-beta signaling.

This variant is absent from gnomAD-Canada v1.0 population data (PM2_Supporting).

Multiple in silico predictors support a deleterious effect, including a REVEL score of 0.955 and BayesDel score of 0.578 (PP3).

Residue Arg361 lies within the MH2 domain mutation cluster region (MCR, codons 330-370), a well-established mutational hot spot containing the majority of SMAD4 missense mutations in human tumors. Cancerhotspots.org confirms this residue as a statistically significant hotspot (PM1).

Functional studies demonstrate that R361H disrupts Smad2-Smad4 heterodimer formation in vitro (PMID:11274206) and abolishes TGF-beta-dependent transcriptional activity in reporter assays (PMID:17132729). Both findings are consistent with loss of SMAD4 tumor-suppressor function (PS3).

Different pathogenic missense changes at the same residue have been reported: R361C in juvenile polyposis syndrome and R361S/R361W in colorectal and duodenal cancers (PM5_Supporting).

The variant has been reported as Pathogenic by 5 independent clinical laboratories and as Likely pathogenic by 1 laboratory in ClinVar (Variation ID 24832, 2-star review status) in individuals with juvenile polyposis syndrome, JPS/HHT overlap syndrome, and hereditary cancer-predisposing syndrome (PS4_Moderate, PP5, PP4).

The variant has been observed in colorectal cancer in the published literature (PMID:15014009 Table 2). COSMIC reports this variant in 261 somatic cancer samples, consistent with its role as a recurrent pathogenic alteration.

Final determination: Generic ACMG/AMP 2015 fallback rules support a Pathogenic classification based on the observed combination of very strong, strong, moderate, and supporting pathogenic criteria.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	Not met	NM_005359.5:c.1082G>A is a missense variant (p.Arg361His) and does not fall into the null-variant buckets of nonsense, frameshift, or canonical splice consensus variants recognized by generic PVS1 (PMC6185798). SMAD4 loss of function is the established disease mechanism for JPS/HHT, but missense variants are not captured by the default PVS1 framework.	pvs1_generic_framework pvs1_variant_assessment pvs1_gene_context
PS1	Not met	No alternative nucleotide change at codon 361 producing the same amino acid substitution (p.Arg361His) has been identified. The c.1082G>A variant represents the canonical nucleotide change for Arg361His.
PS2	Not met	No de novo occurrence data for this variant was identified in the case materials or reviewed literature.
PS3	Met	R361H was directly tested in two independent publications and demonstrated complete disruption of Smad2-Smad4 heterodimer formation and loss of TGF-beta-dependent transcriptional activity. In PMID:11274206, recombinant R361H Smad4 protein failed to form a heterodimer with Smad2 in gel filtration and electrophoretic mobility shift assays. In PMID:17132729, R361H abolished interaction with phosphorylated R-Smads and lost TGF-beta-responsive transcriptional activity in a reporter assay. Both findings are consistent with loss of SMAD4 tumor-suppressor function.	PMID:11274206 PMID:17132729 PMID:11779503 oncokb
PS4	Met	This variant has been reported as pathogenic by 5 independent clinical laboratories and as likely pathogenic by 1 laboratory in ClinVar (Variation ID 24832, 2-star review status). Reported clinical conditions include juvenile polyposis syndrome, JPS/HHT syndrome, and hereditary cancer-predisposing syndrome. The variant is listed in PMID:15014009 Table 2 as occurring in colorectal cancer. Multiple independent clinical observations support an increased prevalence in affected individuals.	clinvar PMID:15014009
PM1	Met	Residue Arg361 lies within the MH2 domain mutation cluster region (MCR, codons 330-370), a well-established mutational hot spot critical for Smad protein heterodimerization. PMID:15014009 characterized this MCR as containing 53% of all missense mutations in the MH2 domain. Cancerhotspots.org confirms this residue as a statistically significant hotspot. The loop-helix region of the MH2 domain is essential for Smad2-Smad4 heterocomplex formation and TGF-beta signaling.	PMID:15014009 PMID:11274206
PM2	Met	The variant is absent from gnomAD-Canada v1.0. gnomAD v2.1 and v4.1 data were not captured due to scrape timeout. Absence from population databases supports rarity consistent with a pathogenic variant.	gnomad_canada
PM5	Met	Different pathogenic missense changes at the same residue (Arg361) have been reported in the literature: R361C (CGC>TGC) in juvenile polyposis syndrome (refs 25-27 in PMID:15014009), R361S (CGC>AGC) in colorectal cancer, and R361W (CGG>TGG) in duodenal cancer. The automated PM5 candidate harvest did not identify ClinVar comparator variants, but the literature supports multiple pathogenic amino acid changes at this residue.	PMID:15014009
PM6	Not met	No de novo observation data was identified in the case materials or reviewed literature. PM6 requires specific de novo confirmation for this variant.
PP1	Not met	No segregation data was identified in the case materials or reviewed literature.
PP2	Not met	HCI prior data is not available for SMAD4, precluding calculation of the missense constraint z-score. Without gene-level constraint data, PP2 cannot be reliably applied.
PP3	Met	Multiple in silico predictors support a deleterious effect: REVEL score of 0.955 (strongly damaging, well above the 0.9 threshold) and BayesDel score of 0.578 (damaging). SpliceAI delta score of 0.08 predicts no significant splicing impact, consistent with a missense effect at the protein level.	revel bayesdel spliceai
PP4	Met	The variant has been reported in patients with clinical presentations highly specific for SMAD4-related disease, including juvenile polyposis syndrome, JPS/HHT overlap syndrome, and hereditary cancer-predisposing syndrome. The ClinVar conditions list includes these well-defined phenotypes with high specificity for SMAD4 pathogenic variants.	clinvar PMID:15014009
PP5	Met	The variant is classified as Pathogenic in ClinVar (Variation ID 24832) by 5 clinical laboratories and as Likely pathogenic by 1 laboratory, with a 2-star review status ('criteria provided, multiple submitters, no conflicts'). Multiple reputable clinical laboratories have independently reached the same classification.	clinvar
BA1	Not met	The variant is absent from gnomAD-Canada v1.0 and not observed at allele frequency greater than 1% in any population database. Does not meet BA1 threshold.	gnomad_canada
BS1	Not met	The variant is absent from gnomAD-Canada v1.0. No population frequency data supports an allele frequency above 0.3%. Does not meet BS1 threshold.	gnomad_canada
BS2	Not met	No evidence of this variant observed in healthy adults in the homozygous or hemizygous state, nor in a sufficiently large control cohort. Does not meet BS2 threshold.
BS3	Not met	All functional studies identified demonstrate a damaging effect: R361H disrupts Smad2-Smad4 heterodimer formation (PMID:11274206) and abolishes TGF-beta-dependent signaling (PMID:17132729). These findings support pathogenicity, not benign effect. BS3 is not met.	PMID:11274206 PMID:17132729
BS4	Not met	No segregation data demonstrating lack of cosegregation with disease was identified in the case materials or reviewed literature.
BP1	Not met	While SMAD4 disease is associated with truncating variants, missense variants in the MH2 domain mutation cluster region (codons 330-370) are a well-established pathogenic mechanism. BP1 specifically applies when a missense variant occurs in a gene where only truncating variants cause disease. This is not the case for SMAD4, as missense variants in the MH2 MCR are recurrently observed in JPS, colorectal cancer, and other SMAD4-related conditions.	PMID:15014009
BP2	Not met	No evidence of this variant observed in trans with a known pathogenic variant was identified in the case materials or reviewed literature.
BP4	Not met	Multiple in silico predictors conflict with a benign interpretation: REVEL score of 0.955 strongly predicts damaging effect, and BayesDel score of 0.578 is consistent with pathogenicity. These scores do not support benign computational evidence. BP4 is not met.	revel bayesdel
BP5	Not met	No evidence of an alternative molecular cause explaining the patient's phenotype was identified in the case materials or reviewed literature.
BP6	Not met	The variant is classified as Pathogenic in ClinVar by multiple clinical laboratories. No reputable source classifies this variant as benign or likely benign. BP6 is not met.	clinvar
BP7	N/A	NM_005359.5:c.1082G>A is a missense variant (p.Arg361His), not a synonymous variant. BP7 applies only to synonymous variants with no predicted splice impact.
BP3	N/A	Variant is a missense substitution, not an in-frame indel. BP3 applies to in-frame deletions/insertions.
PM3	N/A	SMAD4-related disorders (JPS, HHT) are autosomal dominant; PM3 (detected in trans with a pathogenic variant) is not applicable in this inheritance context.
PM4	N/A	Variant is a single-nucleotide substitution, not a protein-length-altering change (in-frame deletion/insertion, stop-loss). PM4 applies to non-repeat-region protein-length changes.
PS5	Not assessed	PS5 was not in the assessment list per the case instructions. This criterion is variably applied across frameworks.

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