NM_000321.2:c.2236G>T

RB1 · NP_000312.2:p.(Glu746Ter) · NM_000321.2

GRCh37: chr13:49039158 G>T · GRCh38: chr13:48465022 G>T

Gene: RB1 Transcript: NM_000321.2

Final call

VUS

PVS1 very strong PM2 supporting

Variant details

Gene

RB1

Transcript

NM_000321.2

Protein

NP_000312.2:p.(Glu746Ter)

gnomAD AF

8.417593443704819e-07 (v4.1)

ClinVar

Pathogenic

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

NM_000321.2:c.2236G>T (p.Glu746Ter) is a nonsense variant in exon 22 of RB1, introducing a premature termination codon predicted to trigger nonsense-mediated decay in a gene with an established loss-of-function disease mechanism for retinoblastoma.

PVS1 is met at very strong strength.

This variant is extremely rare in population databases, absent from gnomAD v2.1 and observed in only 1 of 1,187,988 alleles in gnomAD v4.1 (AF = 8.42e-7), satisfying PM2 at supporting strength.

This variant has been reported in ClinVar as Pathogenic by two clinical laboratories (Variation ID 1069507, 2-star review status) and is listed in association with retinoblastoma.

This variant has been observed in the somatic context in COSMIC (COSV57314725, n = 6), consistent with a pathogenic role, though somatic data alone do not independently satisfy germline ACMG criteria.

No variant-specific functional studies, de novo observations, co-segregation data, or statistical case-frequency data were identified in the reviewed literature to satisfy PS3, PS2, PS4, or PP1.

Under the generic ACMG/AMP 2015 combination rules (PMID:25741868), one very strong criterion (PVS1) plus one supporting criterion (PM2) does not reach the threshold for Likely Pathogenic or Pathogenic classification. The combination of PVS1 and PM2 alone results in a variant of uncertain significance (VUS) by strict scoring.

Note: In clinical practice, a PVS1-level nonsense variant in RB1 with ClinVar Pathogenic consensus and extreme population rarity would typically be classified as at least Likely Pathogenic. The strict ACMG/AMP 2015 scoring framework requires additional supporting criteria to reach Likely Pathogenic (e.g., 1 Very Strong + 1 Moderate, or 1 Strong + 2 Supporting). ClinVar submissions from two clinical laboratories and somatic COSMIC observations provide orthogonal clinical support not captured by the generic scoring matrix.

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	Met	Nonsense variant NM_000321.2:c.2236G>T (p.Glu746Ter) in exon 22 of 27 introduces a premature termination codon at position 746 of 929 amino acids, predicted to trigger nonsense-mediated decay. RB1 has an established loss-of-function disease mechanism for retinoblastoma. Under the ClinGen SVI PVS1 framework (PMC6185798), this nonsense variant qualifies for PVS1 at very strong strength.	pvs1_generic_framework pvs1_gene_context pvs1_variant_assessment
PS1	Not assessed	No data available on whether an alternative nucleotide change at c.2236 (e.g., c.2236G>A producing the same p.Glu746Ter) has been established as pathogenic. Without this comparator, PS1 cannot be assessed.
PS2	Not met	No confirmed de novo observation of NM_000321.2:c.2236G>T was identified in the literature reviewed. PS2 requires a specific de novo report with confirmed maternity and paternity.
PS3	Not met	No variant-specific functional data was identified for NM_000321.2:c.2236G>T (p.Glu746Ter). The OncoKB-curated literature describes RB1 loss-of-function mechanisms at the gene level (protein truncation testing, RB1 deletion in cell lines, resistance studies) but none directly tested this specific variant or systematically characterized the region spanning codon 746.
PS4	Not met	The variant has been reported in ClinVar as Pathogenic by two clinical laboratories, and one submission notes a bilateral retinoblastoma case, but no systematic case-control or case-frequency data are available to establish statistically significant enrichment in affected individuals.	clinvar
PS5	N/A	PS5 applies to a novel missense variant at a codon where a different missense change is established as pathogenic. This is a nonsense variant, not a missense variant.
PM1	Not met	Residue Glu746 is located in the C-terminal region of RB1, but this position is not within a statistically significant mutational hotspot (cancerhotspots.org negative). No evidence from the available literature characterizes codon 746 as lying within a well-defined critical functional domain meeting PM1 criteria.
PM2	Met	This variant is absent from gnomAD v2.1 and is extremely rare in gnomAD v4.1 (1/1,187,988 alleles; AF 8.42e-7), well below the 0.1% threshold for PM2. The single observation is in the East Asian subpopulation (AF 3.84e-5).	gnomad_v2 gnomad_v4 gnomad_canada
PM5	Not met	This variant produces a premature termination codon (p.Glu746Ter), not a missense change. Automated PM5 candidate harvesting was unable to identify same-residue pathogenic missense comparator variants for this nonsense substitution.	pm5_candidates
PM3	N/A	Skipped per directive: trivially not_applicable.
PM4	N/A	Skipped per directive: trivially not_applicable.
PM6	Not met	No confirmed de novo observation of NM_000321.2:c.2236G>T with confirmed maternity and paternity was identified in the available literature.
PP1	Not met	No co-segregation data for NM_000321.2:c.2236G>T in affected families was identified in the available literature. One ClinVar submission notes a bilateral case and pedigree but without co-segregation details across multiple affected family members.	clinvar
PP2	N/A	PP2 applies to missense variants in genes with a low rate of benign missense variation where missense is a common disease mechanism. NM_000321.2:c.2236G>T is a nonsense variant, not a missense variant.
PP3	N/A	For nonsense variants where PVS1 is applied at very strong strength, PP3 (in silico prediction of deleterious effect) is not independently applied. The predicted protein truncation effect is already captured by PVS1. SpliceAI max delta score of 0.27 does not add independent evidence.	spliceai bayesdel
PP4	Not met	No detailed patient phenotype data specific to NM_000321.2:c.2236G>T is available to assess whether the clinical presentation is highly specific for RB1-related disease. The variant is reported in ClinVar in association with retinoblastoma, but detailed phenotypic data are not available.	clinvar
PP5	Not met	ClinVar reports this variant as Pathogenic (2 clinical laboratories, 2-star review status: criteria provided, multiple submitters, no conflicts). The PP5 rule in effect requires a 3-star expert panel review for application at supporting strength. With 2 stars, PP5 is not met under the governing evidence policy.	clinvar
BA1	Not met	The maximum population allele frequency for this variant is 3.84e-5 (East Asian, gnomAD v4.1), which is far below the BA1 threshold of 1%.	gnomad_v4
BS1	Not met	The maximum population allele frequency is 3.84e-5, well below the BS1 threshold of 0.3%.	gnomad_v4
BS2	Not met	No homozygous observations of this variant are reported in gnomAD or any other population database.	gnomad_v2 gnomad_v4
BS3	Not met	No functional studies demonstrating a benign effect for NM_000321.2:c.2236G>T (p.Glu746Ter) were identified. The available literature describes RB1 loss-of-function as a disease mechanism, which is consistent with a deleterious effect of this nonsense variant.
BS4	Not met	No segregation data are available to assess lack of co-segregation with disease.
BP1	N/A	BP1 applies to missense variants in genes where truncating variants are the primary disease mechanism. This is a nonsense (truncating) variant, not a missense variant.
BP2	Not met	No data are available indicating that this variant has been observed in trans with a known pathogenic RB1 variant in a healthy individual.
BP3	N/A	Skipped per directive: trivially not_applicable.
BP4	N/A	BP4 applies when multiple lines of in silico evidence suggest no impact. For a nonsense variant with PVS1 applied, in silico predictors are not independently assessed for benign effect.
BP5	Not met	This variant is not reported as benign in ClinVar or any other reputable database. It is reported as Pathogenic by two clinical laboratories.	clinvar
BP6	Not met	ClinVar reports this variant as Pathogenic, not Benign or Likely Benign. BP6 does not apply.	clinvar
BP7	N/A	BP7 applies to synonymous variants with no predicted splice impact. NM_000321.2:c.2236G>T is a nonsense variant, not a synonymous variant.

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