RB1 c.*1G>C, p.?

Reasoning

The variant is extremely rare in population databases: gnomAD v4.1 reports 1/1,609,556 alleles (AF 6.212893493609418e-07), zero homozygotes, and a maximum subpopulation AF of 8.501174862365979e-07; it is absent from gnomAD v2.1 and gnomAD-Canada. This is below the generic non-VCEP rare-variant PM2 threshold of 0.1% and supports PM2 at supporting strength. Human review remains appropriate because the variant is in the 3' UTR and its disease mechanism/clinical relevance are not established by population data alone.

Rule

PM2: apply at supporting strength for an absent or extremely rare variant in appropriate population databases; operational threshold used here is AF <0.1% in the generic non-VCEP setting.

Evidence considered

• Generic non-VCEP PM2 operating threshold: AF <0.1% supports PM2; the gnomAD v4.1 AF of 6.212893493609418e-07 is below this threshold.
• The variant is absent from gnomAD v2.1 and gnomAD-Canada v1.0, and the gnomAD v4.1 record shows zero homozygotes.
• Because this is a 3' UTR variant with no established clinical or functional disease mechanism in the case bundle, rarity is treated as population evidence only and not as evidence of pathogenicity by itself.

Still missing

• Population rarity does not establish that this 3' UTR variant affects RB1 disease risk or penetrance.
• The gnomAD v4.1 finding is a singleton, so the exact allele frequency is subject to sampling uncertainty.

Sources

Reasoning

Under the generic ACMG/AMP fallback calibration (no RB1-specific VCEP PP3/BP4 lookup material available), non-missense variants are assessed by SpliceAI alone. This 3' UTR variant (NM_000321.3:c.*1G>C) has a SpliceAI max delta score of 0.007-0.01, well below the <0.1 threshold, indicating no predicted splicing impact and supporting a benign computational assessment. BP4 (supporting) is met.

Rule

generic_acmg_pp3_bp4_spliceai_non_missense

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai') scores: DS_AG=0.003, DS_AL=0.007, DS_DG=0.0, DS_DL=0.0, max_delta_score=0.007; evidence_sentence 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01)'.
• Generic fallback rule (generic_acmg_classification_rules.md, PMID:25741868 base framework, SVI-recommended SpliceAI thresholds also reflected in VCEP CSPECs such as ATM's per Walker et al. 2023): SpliceAI max delta < 0.1 -> BP4 supporting for non-missense variants; observed max delta (~0.007-0.01) clearly falls under this threshold.
• No RB1-specific VCEP PP3/BP4 lookup spreadsheet was available to pre-assign a code (vcep_materials.json: no cspec url available), so the generic calibration governs.

Sources

Reasoning

NM_000321.3:c.*1G>C is a 3' UTR variant (first nucleotide after the stop codon), not a missense substitution, so REVEL/BayesDel are not the applicable predictor and generic-fallback rules direct use of SpliceAI only. SpliceAI predicts no significant splice impact (max delta score = 0.007-0.01; DS_AG=0.003, DS_AL=0.007, DS_DG=0.0, DS_DL=0.0), well below the >0.2 threshold required for PP3. RB1 has no ClinGen VCEP CSPEC on record for this case (cspec.found=false, framework_mode=generic_acmg), so the generic ACMG/AMP fallback calibration applies rather than any gene-specific PP3/BP4 lookup material.

Rule

generic_acmg_pp3_bp4_spliceai_non_missense

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai') reports max delta score 0.007 (DS_AG=0.003, DS_AL=0.007, DS_DG=0.0, DS_DL=0.0; evidence_sentence 'SpliceAI predicts no significant splice impact for this variant, max delta score = 0.01').
• Generic fallback rule (generic_acmg_classification_rules.md, based on Richards et al. 2015 PMID:25741868, SVI-recommended SpliceAI thresholds): SpliceAI max delta > 0.2 -> PP3 supporting; < 0.1 -> BP4 supporting; observed value (~0.007-0.01) is well below the BP4 threshold and far below the PP3 threshold.
• No gene-specific RB1 VCEP CSPEC or PP3/BP4 lookup spreadsheet was found in vcep_materials.json (note: 'no cspec url available'), so no VCEP-specific pre-assigned code overrides the generic calibration.
• REVEL and BayesDel scores were both unavailable (found=false, score=null) in prefetch.json and evidence.json; in any case this variant is not a missense substitution so the REVEL/BayesDel missense path is not applicable to it.

Sources

Reasoning

No exact-variant ClinVar record was found; therefore there is no ClinVar expert-panel Pathogenic or Likely pathogenic assertion that could support PP5.

Rule

PP5 is considered only when an exact-variant ClinVar expert-panel assertion is Pathogenic or Likely pathogenic; ordinary laboratory submissions and aggregate labels do not qualify.

Evidence considered

• ClinVar exact-variant searches for NM_000321.3:c.*1G>C and its genomic representation returned no result.

Sources

Reasoning

The variant is far below a population frequency threshold that would support BA1. gnomAD v4.1 reports 1/1,609,556 alleles (AF 6.212893493609418e-07; maximum reported subpopulation AF 8.501174862365979e-07), and gnomAD v2.1 and gnomAD-Canada report no observation. This is not compatible with a benign allele frequency sufficient for BA1.

Rule

BA1: apply only when the variant allele frequency is sufficiently high to establish benignity; not met here.

Evidence considered

• Generic ACMG/AMP population interpretation: BA1 requires a clearly high population frequency; the observed gnomAD v4.1 AF of 6.212893493609418e-07 and maximum subpopulation AF of 8.501174862365979e-07 are many orders of magnitude below that threshold.
• The gnomAD v4.1 observation is a singleton with zero homozygotes; gnomAD v2.1 and gnomAD-Canada show no observation.

Sources

Reasoning

The observed population frequency is not greater than expected for a rare, highly penetrant RB1 disorder. gnomAD v4.1 reports AF 6.212893493609418e-07 from one allele in 1,609,556 alleles, with a maximum subpopulation AF of 8.501174862365979e-07; these data do not support an excess frequency incompatible with disease causality.

Rule

BS1: apply when population frequency exceeds the maximum expected frequency for the disorder; not met.

Evidence considered

• Generic ACMG/AMP BS1 requires an allele frequency greater than expected for the disorder; the observed frequency is an ultra-rare singleton and is not elevated.
• No ancestry-specific enrichment is present: the only observed allele is in the European (non-Finnish) group, and all other listed gnomAD v4.1 groups have zero observed alleles.

Sources

Reasoning

No exact-variant ClinVar record was found; therefore there is no ClinVar expert-panel Benign or Likely benign assertion that could support BP6.

Rule

BP6 is considered only when an exact-variant ClinVar expert-panel assertion is Benign or Likely benign; ordinary laboratory submissions and aggregate labels do not qualify.

Evidence considered

• ClinVar exact-variant searches for NM_000321.3:c.*1G>C and its genomic representation returned no result.

Sources

Reasoning

No proband-level de novo observation or parental testing result is present in the case-evidence bundle. PS2 cannot be assessed without confirmed maternity and paternity and evidence that the variant is absent from both tested parents, with appropriate consideration of parental age, mosaicism, and phenotype.

Rule

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

Evidence considered

• The consolidated case-evidence bundle contains no family history, proband segregation record, or parental testing result relevant to de novo assessment.
• The variant is documented as NM_000321.3:c.*1G>C in RB1, but variant identity alone does not establish de novo occurrence.

Still missing

• Proband clinical record and variant confirmation are not provided.
• Maternal and paternal testing results are not provided.
• Confirmation of maternity and paternity is not provided.
• Assessment for parental mosaicism is not provided.

Reasoning

No functional/validated assay data (e.g., minigene splicing assay, reporter assay, luciferase/mRNA stability assay, or other biologically relevant functional study) for NM_000321.3:c.*1G>C was identified in this case's evidence bundle. No CSPEC/VCEP RB1 functional framework is present (cspec.found=false, vcep_materials empty), so a generic ACMG/AMP fallback applies. Literature search surfaced 0 PMIDs specific to this variant (literature_pass.json: total_pmids=0), paper_extracts.json is empty, and fulltext_map.json is empty; the RB1-related PVS1 gene-context papers concern unrelated RB1 truncating/splice-donor variants (e.g., c.539+1G>A) in different patients, not this 3' UTR variant, and none report functional assay results for c.*1G>C itself. ClinVar and OncoKB both returned no result for this variant. There is no validated assay evidence to support PS3.

Still missing

• No published functional assay (splicing minigene, mRNA stability/3'UTR reporter assay, protein function assay) for NM_000321.3:c.*1G>C was located.
• No RB1-specific VCEP/CSPEC functional criteria specification is available in this case bundle to define assay validation standards.
• SpliceAI predicts minimal splice impact (max delta 0.007) but this is a computational prediction, not a validated functional assay, and is out of scope for PS3/BS3.

Reasoning

No affected-case series, case-control dataset, or other variant-specific enrichment evidence was identified for NM_000321.3:c.*1G>C.

Rule

Generic ACMG/AMP PS4 requires a statistically increased prevalence of the variant in affected individuals compared with controls.

Evidence considered

• The consolidated literature pass reports no PMIDs and no criterion-screened publications for this exact variant.

Still missing

• Variant-specific affected and control allele counts, with phenotypic ascertainment and statistical comparison, are unavailable.

Reasoning

No applicable RB1 VCEP or gene-specific framework was available, so generic ACMG/AMP criteria were considered. PM3 requires an affected individual with a recessive disorder to carry the tested variant in trans with a pathogenic variant (with phase established or appropriately supported), with strength based on the number and quality of such observations. The case bundle contains no affected-proband observations, no second pathogenic allele, and no phase or inheritance data for NM_000321.3:c.*1G>C; therefore PM3 cannot be assessed.

Rule

Generic ACMG/AMP PM3: pathogenic variants in trans in affected individuals with a recessive disorder support PM3; strength depends on the number and quality of independent observations and the certainty of phase.

Evidence considered

• The case bundle identifies the variant as NM_000321.3:c.*1G>C in RB1 and reports no ClinVar record or literature PMIDs for this variant.
• The case bundle reports no applicable CSPEC/VCEP or local gene-specific framework; generic ACMG/AMP interpretation was therefore used.
• The case bundle provides no affected-proband, allelic, phase, or inheritance observations relevant to PM3.

Still missing

• Affected-proband observations involving NM_000321.3:c.*1G>C are unavailable.
• No pathogenic or likely pathogenic allele in trans has been documented.
• No phase testing or inheritance information is available.

Reasoning

No reliable but unconfirmed de novo observation is reported in the case-evidence bundle. PM6 cannot be assessed because neither parental genotype information nor a documented de novo event is available.

Rule

Generic ACMG/AMP PM6: assumed de novo occurrence, without confirmation of maternity and paternity, in a patient with the disease and no family history.

Evidence considered

• The consolidated case-evidence bundle reports no PMIDs and contains no proband or parental testing data relevant to a de novo assessment.
• Absence from ClinVar and population databases does not constitute evidence of de novo inheritance and was not used for PM6.

Still missing

• A reported de novo observation is not provided.
• Parental genotype results are not provided.
• Maternity and paternity confirmation are not provided.
• Phenotype consistency and family history are not provided.

Reasoning

No affected relatives, informative meioses, familial genotypes, or segregation counts are provided. PP1 cannot be assessed because there is no documented co-segregation of the variant with disease in the family.

Rule

Generic ACMG/AMP PP1: co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease; strength should be calibrated to the number and informativeness of meioses when such data are available.

Evidence considered

• The consolidated case-evidence bundle contains no family pedigree, affected-relative testing, or informative meiosis data.
• No segregation study or publication relevant to this variant is present in the bundle.

Still missing

• Family pedigree and phenotype data are not provided.
• Genotypes of affected relatives are not provided.
• Number of informative meioses is not provided.
• Phenotype specificity and clinical ascertainment of relatives are not provided.

Reasoning

No individual phenotype or phenotype-specific testing context is supplied for the proband, and no exact-variant publication was identified.

Rule

Generic ACMG/AMP PP4 requires a patient phenotype or family history highly specific for a disease with a single genetic etiology.

Evidence considered

• The case bundle identifies RB1 and NM_000321.3:c.*1G>C but contains no proband phenotype, family phenotype, or phenotype-specific testing indication.

Still missing

• A documented phenotype and testing context demonstrating high specificity for an RB1-associated condition are required.

Reasoning

One heterozygous observation is present in gnomAD v4.1 and no homozygotes are reported, but the available population database record does not establish that the carrier is a phenotyped healthy adult, does not provide age or disease-status follow-up, and does not demonstrate tolerance of the variant in a condition where disease would be expected to be fully penetrant. Therefore BS2 cannot be assigned from these data alone.

Rule

BS2: require documented unaffected healthy adults, or adequate phenotype evidence for unaffected homozygotes where relevant; population-database presence alone is insufficient.

Evidence considered

• gnomAD v4.1 contains one observed allele and zero homozygotes, but its aggregate population data do not provide the individual-level phenotype evidence required to establish BS2.
• No homozygote observation is available to support a recessive-disease BS2 assessment.

Still missing

• Confirmed healthy-adult phenotype, age, and relevant clinical follow-up for the gnomAD carrier are unavailable.
• No independent observation of unaffected carriers or homozygous individuals with adequate phenotype information is available.

Sources

Reasoning

No functional assay data demonstrating normal/wild-type-like activity for NM_000321.3:c.*1G>C was identified. As with PS3, no RB1 CSPEC/VCEP functional framework is present, no PMIDs specific to this variant were returned by the literature pass, and paper_extracts.json / fulltext_map.json contain no entries. No basis exists to conclude a validated assay shows no functional impact.

Still missing

• No published functional assay data (of any direction) for NM_000321.3:c.*1G>C was located in the case bundle.
• No RB1-specific VCEP/CSPEC functional criteria specification is available to define assay validation or strength calibration standards.

Reasoning

No unaffected relatives who are informative for the familial variant are documented. BS4 cannot be assessed because the case bundle provides no tested unaffected relatives, age-related penetrance information, or adequate phenotype evaluation demonstrating non-segregation.

Rule

Generic ACMG/AMP BS4: lack of segregation in affected members of a family, or presence of the variant in unaffected relatives, when the relatives are informative and the disease phenotype and age-related penetrance are adequately evaluated.

Evidence considered

• The consolidated case-evidence bundle contains no non-segregation observations or unaffected-relative genotype/phenotype data.
• Population frequency data and absence from ClinVar do not establish non-segregation and were not used for BS4.

Still missing

• Tested unaffected relatives are not identified.
• Familial variant genotypes in unaffected relatives are not provided.
• Age, surveillance, and phenotype assessment of relatives are not provided.
• Penetrance and phenocopy considerations are not provided.

Reasoning

No applicable RB1 VCEP or gene-specific framework was available, so generic ACMG/AMP criteria were considered. BP2 requires observation of the tested variant in trans with a pathogenic variant for a dominant disorder or in cis with a pathogenic variant for any inheritance pattern, in an appropriate individual or family context. The case bundle contains no individual or family observations, no second pathogenic variant, and no phase information; therefore BP2 cannot be assessed.

Rule

Generic ACMG/AMP BP2: observation of the variant in trans with a pathogenic variant for a dominant disorder or in cis with a pathogenic variant for any inheritance pattern supports BP2; phase and the pathogenicity of the comparator allele must be established.

Evidence considered

• The case bundle identifies the variant as NM_000321.3:c.*1G>C in RB1 and reports no ClinVar record or literature PMIDs for this variant.
• The case bundle reports no applicable CSPEC/VCEP or local gene-specific framework; generic ACMG/AMP interpretation was therefore used.
• The case bundle provides no family, co-occurring pathogenic-variant, phase, or inheritance observations relevant to BP2.

Still missing

• No individual or family observations containing the tested variant with another pathogenic variant are available.
• No pathogenic variant in trans or cis has been documented.
• No phase testing or inheritance information is available.

Reasoning

No proband-level molecular diagnosis or phenotype information is available to establish an alternate cause of the reported phenotype.

Rule

Generic ACMG/AMP BP5 requires a variant found in a case with an alternate molecular basis for disease.

Evidence considered

• No alternate molecular diagnosis or phenotype data are present in the consolidated case-evidence bundle.

Still missing

• A confirmed alternate molecular diagnosis and clinical evidence that it fully explains the phenotype are required.