NM_000321.2:c.743G>C

RB1 · NP_000312.2:p.(Gly248Ala) · NM_000321.2

GRCh37: chr13:48936975 G>C · GRCh38: chr13:48362839 G>C

Gene: RB1 Transcript: NM_000321.2

Final call

VUS

PM2 moderate BP4 supporting benign

Variant details

Gene

RB1

Transcript

NM_000321.2

Protein

NP_000312.2:p.(Gly248Ala)

gnomAD AF

ClinVar

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

PM2 (moderate): The variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada, with an allele frequency of 0% in all queried population databases, supporting moderate evidence for pathogenicity.

BP1 (supporting benign): RB1 is a gene in which >80% of pathogenic germline variants are truncating, and missense variants account for only 9.2% of pathogenic variants (PMID:42461076). This missense variant occurs in a gene where the primary disease mechanism is loss of function through null alleles.

BP4 (supporting benign): Multiple lines of computational evidence — REVEL score 0.272, BayesDel score -0.129, and SpliceAI max delta 0.01 — converge on a non-deleterious prediction, providing supporting evidence against pathogenicity.

Classification: Uncertain Significance (VUS). There is conflicting evidence: PM2 (moderate pathogenicity) from complete absence in population databases is offset by BP1 (supporting benign) and BP4 (supporting benign). The two supporting benign criteria do not reach the 'likely benign' threshold in the presence of a moderate pathogenic criterion. Additional evidence — particularly functional studies, segregation data, or clinical case reports — is needed to resolve this variant's clinical significance.

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	Missense variant (c.743G>C, p.Gly248Ala); does not fall into the generic PVS1 null-variant buckets of nonsense, frameshift, or canonical ±1,2 splice consensus variants per ClinGen SVI PVS1 recommendations (PMC6185798).	pvs1_generic_framework pvs1_variant_assessment
PS1	Not met	No known pathogenic variant affecting the same amino acid (Gly248) via a different nucleotide change has been identified. The variant is absent from ClinVar and no literature reports a pathogenic comparator at this residue.	clinvar pm5_candidates
PS2	Not met	No de novo data available; both paternal and maternal confirmation were not assessed. No family-based or parent-of-origin testing data are present for this case.
PS3	Not met	No variant-specific functional data available. OncoKB reports Unknown Oncogenic Effect with no reviewed functional evidence for G248A. No publication identified with experimental characterization of this variant or a systematically characterized range that includes residue 248.	oncokb
PS4	Not met	No case-control data or statistical enrichment data available for this variant in affected individuals versus controls.
PS5	N/A	PS5 is only applicable for recessive disorders (biallelic variant in trans with a known pathogenic variant). RB1-associated disease follows autosomal dominant inheritance.
PM1	Not met	Residue Gly248 is located in the N-terminal region of RB1, outside the well-characterized Pocket A and Pocket B domains. Cancerhotspots.org does not identify this residue as a statistically significant hotspot. Published RB1 variant landscape data (PMID:42461076) shows that pathogenic missense variants are concentrated in Pocket B, not the N-terminal region. The N-terminal domain accumulates primarily nonsense, not missense, pathogenic variants.
PM2	Met	This variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, with a population allele frequency of 0%. This meets the PM2 threshold for absence from large population databases (AF < 0.1% under non-VCEP generic ACMG rules).	gnomad_v2 gnomad_v4 gnomad_canada
PM5	N/A	No comparator variants at the same amino acid residue (Gly248) with a different amino acid change were identified. PM5 candidate harvesting returned zero same-residue candidates, and ClinVar contains no entries for any variant at residue 248.	pm5_candidates clinvar
PM6	Not met	No de novo data available for this variant. No family-based studies or trio analyses have been performed. No literature reports a de novo occurrence of c.743G>C.
PP1	Not met	No co-segregation data available. No family pedigree or segregation analysis has been performed for this variant.
PP2	Not met	RB1 is a tumor suppressor gene where truncating variants (nonsense, frameshift, splice-site, large deletions) account for >80% of pathogenic germline variants (PMID:42461076). Missense variants comprise only 9.2% of pathogenic germline variants and are not a common disease mechanism for RB1. The variant does not meet the PP2 criterion requiring missense variants to be a common mechanism of disease in a gene with a low rate of benign missense variation.
PP3	Not met	Multiple in silico predictors do not support a deleterious effect. REVEL score is 0.272 (below the commonly used 0.5 threshold for pathogenicity). BayesDel score is -0.129 (negative, indicating benign). SpliceAI max delta score is 0.01 (no predicted splicing impact). The weight of computational evidence does not support PP3.	revel bayesdel spliceai
PP4	Not met	No patient phenotype or family history data are available. The variant was assessed in isolation without clinical context; PP4 requires a highly specific phenotype or family history for the disease.
PP5	Not met	This variant is absent from ClinVar. PP5 requires a reputable source (e.g., ClinVar expert panel) to have classified the variant as pathogenic; no such classification exists.	clinvar
BA1	Not met	This variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada. The allele frequency of 0% does not meet the BA1 threshold of >1%.	gnomad_v2 gnomad_v4
BS1	Not met	This variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada. The allele frequency of 0% does not meet the BS1 threshold of >0.3% under generic non-VCEP rules.	gnomad_v2 gnomad_v4
BS2	Not met	No observation data in healthy adult controls are available. BS2 requires the variant to be observed in a healthy adult individual for a disorder with full penetrance expected at an early age, which cannot be assessed without such data.
BS3	Not met	No well-established functional studies demonstrating no deleterious effect are available for this variant. OncoKB reports Unknown Oncogenic Effect. No published experimental data characterize the functional consequence of p.Gly248Ala.	oncokb
BS4	Not met	No segregation data available. BS4 requires lack of co-segregation with disease in affected family members; no family studies have been performed for this variant.
BP1	Not assessed	RB1 is a gene in which >80% of pathogenic germline variants are truncating (nonsense, frameshift, splice-site, large deletions). Missense variants account for only 9.2% of pathogenic variants (PMID:42461076). The variant c.743G>C is a missense change in a gene where the primary disease mechanism is loss of function through truncating variants, satisfying BP1 at supporting benign strength.
BP2	Not met	No data on whether this variant has been observed in trans with a known pathogenic variant. BP2 requires observation in trans with a pathogenic variant in a recessive disorder or in cis with a pathogenic variant in a dominant disorder, which cannot be assessed without phase data.
BP4	Met	Multiple lines of computational evidence suggest no deleterious effect. REVEL score is 0.272 (benign range, below 0.5 threshold). BayesDel score is -0.129 (negative, benign prediction). SpliceAI max delta score is 0.01 (no splicing impact, well below 0.2). Three independent in silico predictors concur on a benign or non-deleterious prediction, satisfying BP4 at supporting benign strength.	revel bayesdel spliceai
BP5	Not met	This variant is absent from ClinVar. BP5 requires a reputable source to have classified the variant as benign; no such classification exists.	clinvar
BP6	Not met	This variant is absent from ClinVar. BP6 requires a reputable source to have classified the variant as benign; no such classification exists.	clinvar
BP7	N/A	This is a missense variant (c.743G>C, p.Gly248Ala), not a synonymous variant. BP7 applies only to synonymous variants with no predicted splice impact.

Disclaimer: The content and results provided by LYFE Sciences are for research and educational purposes only and must not be used as a substitute for professional medical judgment, diagnosis, or treatment. Always consult a qualified healthcare professional before making any clinical decisions.