Variant classification summary

NM_005359.5:c.1067C>T

SMAD4 · NP_005350.1:p.(Pro356Leu) · NM_005359.5

GRCh37: chr18:48591904 C>T · GRCh38: chr18:51065534 C>T

Gene: SMAD4 Transcript: NM_005359.5

Final call

VUS

PM1 moderate PM2 moderate PP3 supporting

Variant details

Gene

SMAD4

Transcript

NM_005359.5

Protein

NP_005350.1:p.(Pro356Leu)

gnomAD AF

ClinVar

Uncertain significance

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_005359.5:c.1067C>T (p.Pro356Leu) is a missense variant in exon 9 of SMAD4. It is absent from gnomAD v2.1, v4.1, and gnomAD-Canada, supporting rarity in the general population.

2

Residue Pro356 is located in the SMAD4 MH2 domain (aa 324-552), a well-established critical functional domain that mediates SMAD oligomerization and transcriptional activation. This residue additionally lies within a statistically significant mutational hotspot.

3

Multiple in silico predictors support a deleterious effect: REVEL score 0.968 (damaging), BayesDel score 0.541 (damaging). SpliceAI predicts no significant splice impact (max delta 0.03).

4

This variant has been reported in ClinVar as Uncertain Significance by a single clinical laboratory (Ambry Genetics; ClinVar ID 1782219). It has been observed in 40 somatic cancer samples (COSMIC COSV61685209) and is classified as Likely Oncogenic by OncoKB.

5

PVS1 is not applicable (missense variant). In silico evidence meets PP3 (supporting). Population absence meets PM2 (moderate). MH2 domain location and hotspot status meet PM1 (moderate). Verified criteria: PM1 (moderate) + PM2 (moderate) + PP3 (supporting). Under ACMG/AMP 2015 combination rules, 2 moderate + 1 supporting is insufficient for Likely Pathogenic (requires ≥3 moderate, or 2 moderate + ≥2 supporting). No benign criteria are met. PS3 (functional) and PP1 (segregation) were not assessed because variant-specific evidence could not be verified in accessible full-text publications. Overall classification: Uncertain Significance (VUS).

6

Gaps: Variant-specific germline functional studies (PS3) and co-segregation data (PP1) were referenced in exploratory literature review (PMID:20101697, PMID:15031030) but could not be verified — abstracts do not mention this variant and full texts are unavailable. Resolution of these gaps could upgrade the classification to Likely Pathogenic.

Final determination: Generic ACMG/AMP 2015 fallback rules do not meet benign, likely benign, likely pathogenic, or pathogenic combination thresholds, so the variant is classified as Variant of Uncertain Significance.

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	N/A	PVS1 applies only to null variants (nonsense, frameshift, canonical splice ±1,2, exon deletion). NM_005359.5:c.1067C>T is a missense variant (p.Pro356Leu) in exon 9; variant_bucket classified as 'other' by generic PVS1 framework.	pvs1_gene_context pvs1_variant_assessment
PS1	Not assessed	No evidence of a different nucleotide change producing the same amino acid substitution (Pro356Leu) was identified. No comparator variants at the nucleotide level were found in ClinVar or literature for this position.
PS2	Not met	No de novo occurrence with confirmed maternity and paternity has been reported for NM_005359.5:c.1067C>T. ClinVar submission SCV002718105 (Ambry Genetics) does not report de novo status. Exploratory literature search identified no de novo reports.	clinvar
PS3	Not assessed	OncoKB classifies this variant as Likely Oncogenic based on somatic curated evidence, suggesting functional data exist. Exploratory literature review referenced PMID:20101697 for functional characterization, but the abstract does not mention c.1067C>T or p.Pro356Leu and no full text is available for verification. Without verified variant-specific functional study data, germline PS3 cannot be applied. COSMIC reports 40 somatic occurrences (COSV61685209) but these are somatic, not germline functional evidence.	oncokb
PS4	Not met	ClinVar contains a single submission (Ambry Genetics, SCV002718105) classifying this variant as Uncertain Significance. No multiple independent proband counts or case-control data are available. Variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada, but this supports rarity (PM2) rather than independently establishing enrichment in affected individuals.	clinvar gnomad_v2 gnomad_v4
PS5	Not assessed	PS5 is an alternative to PM5 when multiple pathogenic variants at the same codon exist but the specific PM5 comparator cannot be confirmed. No comparator variants at codon 356 were identified. PM5 candidate harvesting returned zero candidates.	pm5_candidates
PM1	Met	Residue Pro356 is located in the SMAD4 MH2 domain (aa 324-552), a well-established critical functional domain that mediates SMAD oligomerization and transcriptional activation. Pathogenic missense variants cluster in the MH2 domain in JP-HHT. This residue additionally lies within a statistically significant hotspot identified by the cancer hotspots pipeline.	pvs1_gene_context
PM2	Met	Variant is absent from gnomAD v2.1 (exomes), gnomAD v4.1 (exomes/genomes), and gnomAD-Canada v1.0. Allele frequency is 0.0 across all population databases, well below the <0.1% threshold for PM2 under generic ACMG/AMP rules applied to autosomal dominant disorders.	gnomad_v2 gnomad_v4 gnomad_canada
PM5	N/A	No alternative pathogenic missense variant at Pro356 with a different amino acid change was identified. PM5 candidate harvesting returned zero candidates at this residue in ClinVar. Unable to confirm classic same-residue PM5 semantics.	pm5_candidates
PM6	Not met	No presumed de novo occurrence (without confirmed maternity/paternity) has been reported for this variant. ClinVar submission does not annotate de novo status, and no published case report identifies a de novo observation.	clinvar
PP1	Not assessed	Exploratory literature review suggested co-segregation of c.1067C>T with JP-HHT in a family (PMID:15031030, Gallione et al. 2004), but the PubMed abstract does not mention this specific variant. No full text is available for verification. Segregation evidence cannot be applied without confirmation that the variant is specifically identified.
PP2	Not assessed	PP2 applies to genes with a low rate of benign missense variation where missense variants are a common mechanism of disease. SMAD4 has established pathogenic missense variants in the MH2 domain, and loss of function is the disease mechanism. However, formal missense constraint metrics (e.g., gnomAD missense Z-score, missense depletion metrics) were not retrieved for this case, and PP2 is not routinely applied under generic ACMG/AMP without explicit gene-level constraint data.	pvs1_gene_context
PP3	Met	Multiple in silico predictors support a deleterious effect: REVEL score 0.968 (well above 0.932 threshold for damaging prediction), BayesDel score 0.541 (above 0.27 threshold). SpliceAI predicts no significant splice impact (max delta score 0.03), but this does not offset the strong missense pathogenicity predictions.	revel bayesdel spliceai
PP4	Not assessed	No patient-specific phenotypic or family history data were provided for this case. PP4 requires that the patient's phenotype or family history is highly specific for the disease with a single genetic etiology.
PP5	Not met	ClinVar classification is Uncertain Significance (single submitter, Ambry Genetics SCV002718105), not Pathogenic or Likely Pathogenic. No reputable source has classified this variant as pathogenic with evidence unavailable for independent review. The eight ClinVar-associated PMIDs are all general guideline or technical standards papers that do not mention this variant.	clinvar
BA1	Not met	Variant is absent from gnomAD v2.1 and v4.1. Allele frequency does not exceed the BA1 threshold of >1% in any population database.	gnomad_v2 gnomad_v4
BS1	Not met	Variant is absent from all population databases. Allele frequency does not exceed the BS1 threshold of >0.3% under generic ACMG/AMP non-VCEP rules.	gnomad_v2 gnomad_v4
BS2	Not met	BS2 requires observation in a healthy adult individual (homozygous for recessive, or hemizygous/heterozygous for fully penetrant dominant) with complete penetrance expected at an early age. No such observation for this variant in a healthy adult has been reported. Variant is absent from gnomAD, which includes ostensibly healthy population controls.	gnomad_v2 gnomad_v4
BS3	Not met	No well-established functional studies demonstrate a benign effect of p.Pro356Leu on protein function. OncoKB classifies this variant as Likely Oncogenic (somatic), which contradicts a benign functional interpretation. REVEL score 0.968 and BayesDel 0.541 both predict damaging effects.	oncokb revel bayesdel
BS4	Not met	No evidence of non-segregation with disease in affected family members. BS4 requires that the variant does not segregate with disease, which would contradict pathogenic interpretation. No such observation exists.
BP1	N/A	BP1 applies only to genes where truncating variants are the sole cause of disease. SMAD4 has established pathogenic missense variants, particularly in the MH2 domain, in JPS and JP-HHT.	pvs1_gene_context
BP2	Not met	BP2 requires observation in trans with a pathogenic variant for a fully penetrant dominant disorder, or in cis with a pathogenic variant for a recessive disorder. SMAD4-associated disorders are autosomal dominant. No biallelic SMAD4 observations have been reported, as this would likely be lethal.
BP4	Not met	Multiple in silico predictors support a damaging effect: REVEL 0.968 (damaging) and BayesDel 0.541 (damaging). SpliceAI max delta 0.03 indicates no splice impact, but this does not offset the strong missense pathogenicity predictions. BP4 is contradicted by the preponderance of in silico evidence.	revel bayesdel spliceai
BP5	Not assessed	BP5 requires identification of the variant in a case with an alternate molecular basis for disease. No such case has been reported or is available for assessment.
BP6	Not met	BP6 requires classification as Benign or Likely Benign by a reputable source without access to the underlying evidence. ClinVar classification is Uncertain Significance (single submitter, Ambry Genetics), not Benign or Likely Benign.	clinvar
BP7	N/A	BP7 applies to synonymous (silent) variants for which splicing prediction algorithms predict no impact. NM_005359.5:c.1067C>T is a missense variant (p.Pro356Leu), not synonymous. SpliceAI max delta score is 0.03, consistent with no splice impact, but the variant class is missense.	spliceai

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