RB1 c.2439dup, p.(Lys814Ter)

Reasoning

NM_000321.2:c.2439dup is a validated coding-exon 23 nonsense variant producing NP_000312.2:p.(Lys814Ter), truncating RB1 from 929 amino acids to approximately 813 residues. RB1 is a gene in which germline loss of function is an established disease mechanism, and this premature termination is well upstream of the terminal exons, so the transcript is predicted to undergo nonsense-mediated decay rather than escape it. The RefSeq transcript is the selected/canonical RB1 transcript, and the variant is not a canonical splice alteration or a repeat-region event. Under the generic ClinGen SVI PVS1 framework (PMC6185798), these findings support full, un-downgraded PVS1; no named NMD-escape, non-critical-exon, distal-region, or population-LoF downgrade rule applies.

Rule

ClinGen SVI generic PVS1 framework (PMC6185798): full strength for a disease-relevant nonsense variant predicted to undergo NMD; no downgrade rule triggered.

Evidence considered

• VariantValidator maps NM_000321.2:c.2439dup to RB1 coding exon 23 and confirms NP_000312.2:p.(K814*); the selected RefSeq transcript is NM_000321.2.
• Mutalyzer predicts a premature stop at p.(Lys814Ter), with the predicted protein ending at residue 813 versus the 929-residue reference protein, a loss of 116 amino acids.
• The gene-level PVS1 eligibility assessment supports RB1 germline loss of function as a disease mechanism and identifies no RB1-specific CSPEC/VCEP specification, so the generic ClinGen SVI framework applies.
• The generic ClinGen SVI PVS1 framework supports full strength for a disease-relevant nonsense variant predicted to undergo NMD; downgrade considerations include NMD escape, non-critical exons/distal regions, and biologically irrelevant exons, none of which is supported here.
• A reviewed hereditary-retinoblastoma cohort paper describes nonsense-mediated decay as the mechanism that degrades RB1 transcripts containing premature termination codons, providing gene-level context for the predicted NMD mechanism (PMID:22205104).

Sources

Reasoning

No applicable RB1-specific VCEP or CSPEC population rule was available, so the supplied generic PM2 threshold applies. The variant is absent from gnomAD v2.1 and gnomAD v4.1, with no observed allele frequency, satisfying the generic PM2 supporting threshold of <=0.0001 (ClinGen SVI recommendation, PMID:25741868). The non-cancer subset checks were also absent, and no governing specification required substituting those subsets for the default all-comers sources.

Rule

Generic PM2: absent or extremely rare in population databases, operationalized here as allele frequency <=0.0001 at supporting strength.

Evidence considered

• gnomAD v2.1 reports the variant as absent, with no observed allele count or frequency.
• gnomAD v4.1 reports the variant as absent, with no observed allele count or frequency.
• The supplied generic PM2 threshold is allele frequency <=0.0001 at supporting strength under the ClinGen SVI recommendation (PMID:25741868).
• No RB1-specific VCEP or CSPEC population specification was available; therefore the default all-comers gnomAD sources were used. The separately checked gnomAD v2.1 non-cancer and v3.1 non-cancer records were also absent.

Sources

Reasoning

ClinVar returned no exact record for NM_000321.2:c.2439dupT and therefore no exact-variant expert-panel Pathogenic or Likely pathogenic classification is available. OncoKB's variant-specific Likely Oncogenic label is not a ClinVar expert-panel assertion and cannot trigger PP5 under the specified rule. PP5 is not met.

Rule

PP5 applies only to an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification; ordinary laboratory submissions, aggregate labels, non-expert assertions, and non-ClinVar sources do not qualify.

Evidence considered

• ClinVar search found no exact record for NM_000321.2:c.2439dupT; the case evidence states that the variant is absent from ClinVar.
• The available OncoKB Likely Oncogenic label is not an exact-variant ClinVar expert-panel classification and is excluded from PP5.

Sources

Reasoning

No applicable RB1-specific VCEP or CSPEC population rule was available, so the supplied generic ACMG/AMP threshold applies. The variant is absent from gnomAD v2.1 and gnomAD v4.1, with no observed allele frequency, which is far below the generic BA1 stand-alone threshold of >=0.05 (ACMG/AMP 2015, PMID:25741868). The non-cancer subset checks were also absent, but they do not replace the default all-comers sources because no governing specification requires them.

Rule

Generic BA1: allele frequency >=0.05; absence from gnomAD does not meet this threshold.

Evidence considered

• gnomAD v2.1 reports the variant as absent, with no observed allele count or frequency.
• gnomAD v4.1 reports the variant as absent, with no observed allele count or frequency.
• The generic BA1 stand-alone threshold is allele frequency >=0.05 under ACMG/AMP 2015 (PMID:25741868).
• No RB1-specific VCEP or CSPEC population specification was available; therefore the default all-comers gnomAD sources were used. The separately checked gnomAD v2.1 non-cancer and v3.1 non-cancer records were also absent.

Sources

Reasoning

No applicable RB1-specific VCEP or CSPEC population rule was available, so the supplied generic BS1 threshold applies. The variant is absent from gnomAD v2.1 and gnomAD v4.1, with no observed allele frequency, and therefore does not reach the generic BS1 threshold of >=0.01 (generic SVI default; Whiffin et al. 2017, PMID:28518168). The non-cancer subset checks were also absent, but no governing specification required those subsets.

Rule

Generic BS1: allele frequency >=0.01; absence from gnomAD does not meet this threshold.

Evidence considered

• gnomAD v2.1 reports the variant as absent, with no observed allele count or frequency.
• gnomAD v4.1 reports the variant as absent, with no observed allele count or frequency.
• The supplied generic BS1 threshold is allele frequency >=0.01, with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168).
• No RB1-specific VCEP or CSPEC population specification was available; therefore the default all-comers gnomAD sources were used. The separately checked gnomAD v2.1 non-cancer and v3.1 non-cancer records were also absent.

Sources

Reasoning

ClinVar returned no exact record for NM_000321.2:c.2439dupT and therefore no exact-variant expert-panel Benign or Likely benign classification is available. No ordinary laboratory, aggregate, or non-expert assertion may trigger BP6. BP6 is not met.

Rule

BP6 applies only to an exact-variant ClinVar expert-panel Benign or Likely benign classification; ordinary laboratory submissions, aggregate labels, and non-expert assertions do not qualify.

Evidence considered

• ClinVar search found no exact record for NM_000321.2:c.2439dupT; the case evidence states that the variant is absent from ClinVar.
• No qualifying exact-variant ClinVar expert-panel Benign or Likely benign assertion is present.

Sources

Reasoning

No proband-level parental testing or other evidence establishes that NM_000321.2:c.2439dupT arose de novo. The available case materials provide variant normalization and population/literature retrieval results but no parent genotypes, maternity/paternity confirmation, or parental phenotype information.

Rule

Generic ACMG/AMP PS2: de novo occurrence in a patient with the disease and no family history, with maternity and paternity confirmed; stronger application requires additional de novo evidence and phenotype consistency.

Evidence considered

• The case materials identify NM_000321.2:c.2439dupT in RB1 but contain no proband-level parental testing results.
• No source reviewed for this case reports de novo occurrence of this exact variant in the proband.

Still missing

• Parental genotypes for both biological parents are missing.
• Maternity and paternity confirmation are missing.
• Proband phenotype and disease-consistency details are missing.

Reasoning

No applicable RB1-specific VCEP specification was available, so generic ACMG/AMP functional-evidence principles were used. The case contains no validated assay performed on NM_000321.2:c.2439dup (NP_000312.2:p.(Lys814Ter)); the available papers describe other RB1 variants or experimental RB1 loss and do not test this variant. Therefore, there is insufficient variant-specific functional evidence to apply PS3 at any strength.

Evidence considered

• No RB1-specific VCEP or CSPEC specification was available; generic ACMG/AMP principles therefore apply.
• PMID:14769601 tested germline RB1 mutations using protein truncation testing but did not mention or assay c.2439dup/p.Lys814Ter.
• PMID:22205104 evaluated clinical outcomes associated with germline RB1 mutation classes but did not test the queried variant in a functional assay.
• PMID:26607597 studied conditional Rb1 loss in mouse germinal-center B cells, not the queried human variant.
• PMID:30206110 reported acquired truncating RB1 variants in a treatment cohort, not the queried variant or a direct assay of its function.
• PMID:31138663 used experimentally induced RB1 mutations and deletions in cancer cell lines, not the queried variant.

Still missing

• A validated assay of the queried variant, with appropriate controls and a disease-relevant readout, is unavailable.

Sources

Reasoning

No case-control enrichment analysis, affected/unaffected cohort counts, odds ratio, p-value, or other phenotype-prevalence statistic for NM_000321.2:c.2439dup was provided. The available literature is not variant-specific for this alteration and does not supply PS4-usable case-control data. Therefore PS4 cannot be assessed.

Rule

Generic ACMG/AMP PS4 requires statistically significant increased prevalence of the variant in affected individuals compared with controls; no applicable RB1-specific rule was available.

Evidence considered

• The available literature extracts explicitly state that the queried variant is not mentioned and provide no case-control enrichment statistic for it.
• No PS4-compatible case-control dataset or phenotype prevalence comparison was identified in the available evidence.

Still missing

• Variant-specific affected-case and control counts or a validated case-control enrichment statistic are unavailable.

Reasoning

No proband-level report establishes that NM_000321.2:c.2439dupT is apparently de novo without parental testing. The reviewed literature extraction contains no report of this exact variant and no informative family or meiosis data.

Rule

Generic ACMG/AMP PM6: assumed de novo, without confirmation of paternity and maternity; this is weaker than PS2 and requires a documented affected proband with a consistent phenotype.

Evidence considered

• The available paper extracts state that the queried variant is not mentioned in the reviewed publications.
• No case-level apparent de novo observation, family history, or meiosis count is available.

Still missing

• A proband-level case report or cohort record is missing.
• Parental testing or a documented reason for unavailable parental testing is missing.
• Phenotype consistency and family-history information are missing.

Reasoning

No affected relatives, informative meioses, or genotype-phenotype co-segregation data are provided for NM_000321.2:c.2439dupT. Therefore, neither supporting nor stronger segregation evidence can be assigned.

Rule

Generic ACMG/AMP PP1: co-segregation with disease in multiple affected family members; strength depends on the number and informativeness of meioses and any applicable validated statistical framework.

Evidence considered

• The case materials contain no family pedigree, relative testing results, or count of informative meioses.
• The reviewed publications do not provide segregation data for this exact variant.

Still missing

• Affected-relative genotypes and phenotypes are missing.
• Unaffected-relative genotypes and phenotypes are missing.
• The number of informative meioses is unavailable.

Reasoning

No individual-level clinical phenotype or phenotype-to-disease specificity assessment was supplied for the variant carrier. The available papers are not variant-specific and describe RB1 or retinoblastoma at a general cohort or experimental level, which cannot establish that this patient's phenotype is highly specific for an RB1-related disorder. Therefore PP4 cannot be assessed.

Rule

Generic ACMG/AMP PP4 may be applied when the patient's phenotype or family history is highly specific for a disease with a single-gene etiology; no applicable RB1-specific rule was available.

Evidence considered

• No individual-level phenotype, diagnostic features, or phenotype specificity assessment was available for this variant case.
• General RB1 and retinoblastoma descriptions in the available literature do not substitute for a highly specific patient phenotype.

Still missing

• A documented patient phenotype and assessment of its specificity for an RB1-related disorder are unavailable.

Reasoning

The available evidence contains no documented healthy adult carriers, healthy homozygotes, or disease-relevant penetrance analysis for this specific variant. Population absence in gnomAD is relevant to rarity but does not establish the BS2 requirement of occurrence in unaffected individuals. Accordingly, BS2 cannot be assessed from the available evidence.

Rule

BS2 requires occurrence in healthy adults, typically including homozygotes for a fully penetrant dominant disorder or hemizygotes/homozygotes for a recessive disorder, as applicable; absence cannot satisfy BS2.

Evidence considered

• gnomAD v2.1 and v4.1 report the variant as absent, so they provide no unaffected carrier or homozygote observations for BS2.
• No case-specific study or population dataset documenting unaffected carriers or homozygotes was available in the reviewed evidence.

Still missing

• A sufficiently characterized unaffected population cohort with carrier and, if relevant, homozygote observations is needed for BS2.

Sources

Reasoning

No applicable RB1-specific VCEP specification was available, so generic ACMG/AMP functional-evidence principles were used. No validated assay demonstrated that NM_000321.2:c.2439dup (NP_000312.2:p.(Lys814Ter)) preserves normal RB1 function, and the reviewed publications did not perform a variant-specific benign functional assay with appropriate controls. Therefore, BS3 cannot be applied at any strength.

Evidence considered

• No RB1-specific VCEP or CSPEC specification was available; generic ACMG/AMP principles therefore apply.
• The reviewed functional publications did not test NM_000321.2:c.2439dup or NP_000312.2:p.(Lys814Ter) and provide no variant-specific evidence of preserved function.

Still missing

• A validated assay demonstrating preserved RB1 function for the queried variant, with appropriate controls and a disease-relevant readout, is unavailable.

Sources

Reasoning

No tested unaffected relatives or other informative non-segregation observations are provided. Absence of family data cannot establish that NM_000321.2:c.2439dupT fails to track with the relevant phenotype.

Rule

Generic ACMG/AMP BS4: lack of segregation in affected members or presence in unaffected members when disease is expected to be fully penetrant; reliable phenotype, age, and testing context are required.

Evidence considered

• No unaffected relative has a documented genotype and phenotype in the available case materials.
• No informative non-segregation observation for this exact variant is reported in the reviewed literature.

Still missing

• Unaffected relatives with reliable phenotyping are missing.
• Genotypes of unaffected relatives are missing.
• Age-related penetrance and surveillance information are missing.

Reasoning

No RB1-specific VCEP framework was available, so the generic ACMG/AMP BP2 definition was used. BP2 requires documented allelic context, such as the evaluated variant in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant. The available case evidence contains no second pathogenic or likely pathogenic variant, affected-proband genotype, phase determination, or family data establishing cis/trans configuration, so BP2 cannot be assessed.

Rule

Generic ACMG/AMP BP2: variant observed in trans with a pathogenic variant in a fully penetrant dominant disorder, or in cis with a pathogenic variant.

Evidence considered

• The case evidence does not document a second pathogenic or likely pathogenic variant in the same individual.
• No cis/trans phase, affected-proband genotype, or segregation information is provided.

Still missing

• Affected-proband genotype containing another pathogenic or likely pathogenic variant.
• Experimental or family-based phase information showing cis or trans configuration.
• Inheritance and phenotype details for any individual carrying both variants.

Reasoning

No evidence was provided that the individual has an alternate molecular explanation for the phenotype, and no relevant alternate pathogenic variant or established phenocopy was documented. The variant's absence from ClinVar and general RB1 literature do not evaluate BP5. Therefore BP5 cannot be assessed.

Rule

Generic BP5 requires a pathogenic variant in an alternate gene that explains the phenotype; no applicable RB1-specific rule was available.

Evidence considered

• The available case evidence does not document an alternate pathogenic variant, alternate molecular diagnosis, or phenocopy explaining the patient's presentation.
• No BP5-specific likelihood-ratio-style value or other governing threshold was supplied; no numeric comparison can be performed.

Still missing

• An evaluated alternate molecular diagnosis or pathogenic variant explaining the phenotype is unavailable.