RB1 c.2165A>G, p.(Lys722Arg)

Reasoning

PM2 is met at supporting strength. The exact variant is reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0. No population allele count or frequency is reported in the available records. This supports rarity in population databases, while the absence of ancestry-specific coverage and maximum credible allele-frequency analysis limits the assessment to supporting strength.

Rule

Generic ACMG/AMP PM2: absent from or extremely rare in population databases; applied here at supporting strength because no RB1-specific specification was retrieved and population coverage/threshold details are incomplete.

Evidence considered

• The consolidated evidence bundle reports the exact variant NM_000321.3:c.2165A>G (NP_000312.2:p.(Lys722Arg)) as absent from gnomAD v2.1.
• The consolidated evidence bundle reports the exact variant as absent from gnomAD v4.1.
• The consolidated evidence bundle reports the exact variant as absent from gnomAD-Canada v1.0.
• Generic ACMG/AMP PM2 supports rarity when a variant is absent from or extremely rare in population databases; this assessment uses supporting strength because the bundle lacks coverage, allele-number, ancestry-stratified, prevalence, penetrance, and maximum credible allele-frequency details.

Still missing

• No allele-number, allele-count, coverage, or per-ancestry data are provided for the queried databases.
• No disease prevalence, penetrance, inheritance-model, or maximum credible allele-frequency calculation is available.
• The absence result alone does not establish pathogenicity or exclude technical/capture limitations.

Sources

Reasoning

RB1 has no retrieved VCEP/CSPEC or gene-specific PP3/BP4 specification, so the generic missense-path framework applies. NM_000321.3:c.2165A>G causes p.(Lys722Arg), a missense substitution. Its REVEL score of 0.703 is in the generic framework gray zone and does not meet PP3. SpliceAI is not independently applied to this missense variant under the generic framework.

Rule

Generic ACMG/AMP non-VCEP missense computational-evidence rule: REVEL only; score 0.250-0.750 is gray zone and supports neither PP3 nor BP4.

Evidence considered

• No RB1 VCEP/CSPEC or local gene-specific framework was retrieved; the case is designated generic_acmg fallback.
• The variant is NM_000321.3:c.2165A>G, NP_000312.2:p.(Lys722Arg), a missense substitution. REVEL is 0.703.
• The generic non-VCEP missense rule uses REVEL only and assigns neither PP3 nor BP4 for REVEL 0.250-0.750. Independently, the ClinGen SVI calibrated REVEL thresholds are PP3 at >=0.773 and BP4 at <=0.290 (Pejaver et al., PMID:36413997); 0.703 meets neither calibrated threshold.
• SpliceAI maximum delta is 0.001, but the generic framework explicitly does not use a missense variant's splice score to adjudicate its protein-level PP3/BP4 path.

Sources

Reasoning

No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion is present, so PP5 cannot be applied.

Rule

PP5 applies only to an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification.

Evidence considered

• The exact ClinVar record has two clinical-laboratory submissions, both Uncertain significance; neither submission is an expert-panel assertion.
• Aggregate ClinVar review status and ordinary laboratory submissions were not used to trigger PP5.

Still missing

• An exact-variant ClinVar expert-panel assertion of Pathogenic or Likely pathogenic would be required for PP5 under this review's rule.

Sources

Reasoning

BA1 is not met. The variant is reported as absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; therefore there is no population allele frequency approaching the generic BA1 threshold of greater than 5%.

Rule

Generic ACMG/AMP BA1: allele frequency greater than 5% in population databases; no applicable RB1-specific VCEP framework was retrieved.

Evidence considered

• The consolidated evidence bundle reports the exact variant NM_000321.3:c.2165A>G (NP_000312.2:p.(Lys722Arg)) as absent from gnomAD v2.1.
• The consolidated evidence bundle reports the exact variant as absent from gnomAD v4.1.
• The consolidated evidence bundle reports the exact variant as absent from gnomAD-Canada v1.0.
• Under generic ACMG/AMP population evidence, BA1 requires an allele frequency greater than 5%; absence from the queried population databases does not satisfy that benign frequency threshold.

Still missing

• The case bundle provides absence results but no database-specific allele-number, allele-count, or coverage metrics.

Sources

Reasoning

BS1 is not met. The variant is absent from the available gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0 records, so no allele frequency greater than expected for retinoblastoma or RB1-related disease is demonstrated.

Rule

Generic ACMG/AMP BS1: allele frequency is greater than expected for the disorder; interpretation should account for disease prevalence, penetrance, inheritance, and ancestry when frequency is observed.

Evidence considered

• The exact variant is reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0.
• Because no nonzero population frequency is reported, the evidence does not support BS1, which requires a frequency greater than expected for the disorder.

Still missing

• No disease prevalence, penetrance, genetic model, or ancestry-specific maximum credible allele frequency calculation is supplied.
• The case bundle provides absence results but no database-specific allele-number, allele-count, or coverage metrics.

Sources

Reasoning

RB1 has no retrieved VCEP/CSPEC or gene-specific PP3/BP4 specification, so the generic missense-path framework applies. NM_000321.3:c.2165A>G causes p.(Lys722Arg), a missense substitution. Its REVEL score of 0.703 is in the generic framework gray zone and does not meet BP4. The very low SpliceAI score does not establish a benign protein-level prediction for this missense variant and is not separately counted.

Rule

Generic ACMG/AMP non-VCEP missense computational-evidence rule: REVEL only; score 0.250-0.750 is gray zone and supports neither PP3 nor BP4.

Evidence considered

• No RB1 VCEP/CSPEC or local gene-specific framework was retrieved; the case is designated generic_acmg fallback.
• The variant is NM_000321.3:c.2165A>G, NP_000312.2:p.(Lys722Arg), a missense substitution. REVEL is 0.703.
• The generic non-VCEP missense rule uses REVEL only and assigns neither PP3 nor BP4 for REVEL 0.250-0.750. Independently, the ClinGen SVI calibrated REVEL thresholds are PP3 at >=0.773 and BP4 at <=0.290 (Pejaver et al., PMID:36413997); 0.703 meets neither calibrated threshold.
• SpliceAI maximum delta is 0.001, but the generic framework explicitly does not use a missense variant's splice score to adjudicate its protein-level PP3/BP4 path.

Sources

Reasoning

No exact-variant ClinVar expert-panel Benign or Likely benign assertion is present, so BP6 cannot be applied.

Rule

BP6 applies only to an exact-variant ClinVar expert-panel Benign or Likely benign classification.

Evidence considered

• The exact ClinVar record has two clinical-laboratory submissions, both Uncertain significance; neither submission is an expert-panel assertion.
• Aggregate ClinVar review status and ordinary laboratory submissions were not used to trigger BP6.

Still missing

• An exact-variant ClinVar expert-panel assertion of Benign or Likely benign would be required for BP6 under this review's rule.

Sources

Reasoning

PS1 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

No proband-level de novo observation or parental testing results are present in the case-evidence bundle. Neither confirmed maternity and paternity nor absence of the variant in both tested parents can be established.

Rule

Generic ACMG/AMP 2015 PS2: de novo occurrence with confirmed maternity and paternity; parental absence and phenotype consistency are required for application.

Still missing

• Proband phenotype and family record
• Documented parental maternity and paternity
• Variant testing results for both biological parents
• Confirmation that the variant is absent from both parents

Reasoning

No applicable RB1-specific VCEP framework was retrieved, so generic ACMG/AMP principles apply. The case bundle contains no validated functional assay for NM_000321.3:c.2165A>G (NP_000312.2:p.(Lys722Arg)); there are no assay results, assay controls, biological-relevance details, or reproducible loss-of-function measurements to support PS3. SpliceAI is a computational splice predictor, not a functional assay, and the ClinVar uncertain-significance classification cannot be used as functional evidence.

Evidence considered

• The consolidated bundle reports no retrieved RB1-specific VCEP or gene-specific functional-evidence rule.
• The extracted literature records reviewed for this group do not report this variant or functional assay data: PMID:25356965 is an ACMG policy statement without RB1-specific functional data; PMID:15604628 provides hereditary-retinoblastoma background only; and PMID:27854360 lists RB1 in a secondary-findings policy table without variant-specific functional data.
• The bundle reports SpliceAI max delta score 0.001, but this is an in-silico splice prediction and does not establish PS3.

Still missing

• A validated assay directly measuring the effect of p.Lys722Arg on RB1 protein function is absent.
• Assay controls, replicate data, quantitative results, and evidence that the assay models the relevant RB1 biology are absent.

Sources

Reasoning

No exact-variant case-control, cohort enrichment, or statistically interpretable affected-versus-control prevalence data were provided for RB1 c.2165A>G (p.Lys722Arg).

Rule

Generic ACMG/AMP PS4: prevalence of the variant in affected individuals is significantly increased compared with controls.

Evidence considered

• The supplied literature extractions for the PS4-triaged papers do not mention RB1 c.2165A>G or p.Lys722Arg and contain no case-control or cohort enrichment data for this variant.
• No applicable RB1 VCEP/ClinGen specification was retrieved; generic ACMG/AMP PS4 requires a statistically significant increase in variant prevalence among affected individuals compared with controls.

Still missing

• A study reporting the exact variant in affected individuals and an appropriate control or population comparison, with sufficient data to assess enrichment, is required.

Sources

Reasoning

PM1 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

PM5 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

No suspected de novo occurrence is documented in the case-evidence bundle. There are no parental testing results, parentage confirmation, or proband phenotype data to support a de novo interpretation without confirmed parental testing.

Rule

Generic ACMG/AMP 2015 PM6: assumed de novo occurrence without confirmation of maternity and/or paternity; use only when the de novo observation is otherwise documented but parentage or parental testing is incomplete.

Still missing

• Proband phenotype and family record
• Documented parental maternity and paternity
• Variant testing results for both biological parents
• Clinical documentation supporting a suspected de novo event

Reasoning

No affected relatives, unaffected relatives, segregation results, number of informative meioses, or phenotype-consistent family observations are provided. PP1 cannot be applied from the variant's ClinVar record, population absence, or general RB1 disease descriptions.

Rule

Generic ACMG/AMP 2015 PP1: co-segregation with disease in multiple affected family members; strength requires sufficient informative meioses and phenotype-consistent segregation.

Still missing

• Pedigree and affected-relative information
• Genotypes of informative relatives
• Number of informative meioses
• Phenotype consistency among relatives

Reasoning

PP2 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

The record contains condition labels but no phenotype, diagnostic, or family-history information for an individual carrying the exact variant; therefore phenotype specificity cannot be evaluated.

Rule

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

Evidence considered

• ClinVar lists Hereditary cancer-predisposing syndrome and Retinoblastoma as record-level conditions, but the two submissions have empty condition labels and provide no individual phenotype details.
• No applicable RB1 VCEP/ClinGen specification was retrieved; generic ACMG/AMP PP4 requires a patient's phenotype or family history to be highly specific for a disease with a single genetic etiology.

Still missing

• Individual-level phenotype and diagnostic context for an exact-variant carrier are required.
• Evidence that the observed phenotype is highly specific for an RB1-associated disorder is required.

Sources

Reasoning

BS2 cannot be assessed from the available evidence. No unaffected adult cohort, confirmed healthy homozygote or compound-heterozygote observation, or phenotype/penetrance follow-up is provided for this variant.

Rule

Generic ACMG/AMP BS2: observed in a healthy adult individual for a fully penetrant dominant disorder, or in a healthy adult homozygote/compound heterozygote for a recessive disorder, with appropriate phenotype and age considerations.

Evidence considered

• The population records in the case bundle report the variant as absent, but do not provide observations in healthy adults or any homozygote/compound-heterozygote data.
• Absence from population databases cannot be used as evidence that the variant occurs in healthy adults without disease.

Still missing

• No healthy-adult observation data are available.
• No homozygote or compound-heterozygote counts are available.
• No information is available on age, phenotype ascertainment, penetrance, or disease status of any carriers.

Reasoning

No applicable RB1-specific VCEP framework was retrieved, so generic ACMG/AMP principles apply. The case bundle contains no well-established, biologically relevant assay demonstrating that NM_000321.3:c.2165A>G (NP_000312.2:p.(Lys722Arg)) has a normal functional effect. In the absence of validated assay methodology, appropriate controls, and quantitative benign results, BS3 cannot be applied. The ClinVar uncertain-significance classification and computational splice prediction do not establish BS3.

Evidence considered

• The consolidated bundle reports no retrieved RB1-specific VCEP or gene-specific functional-evidence rule.
• The extracted literature records reviewed for this group do not report this variant or benign functional assay data: PMID:25356965 is an ACMG policy statement without RB1-specific functional data; PMID:15604628 provides hereditary-retinoblastoma background only; and PMID:27854360 lists RB1 in a secondary-findings policy table without variant-specific functional data.
• The bundle reports SpliceAI max delta score 0.001, but this computational result is not a validated normal-function assay and cannot establish BS3.

Still missing

• A validated, biologically relevant assay showing normal RB1 function for p.Lys722Arg is absent.
• Assay controls, replicate data, predefined normal-function thresholds, and evidence of assay validity are absent.

Sources

Reasoning

No unaffected relatives, family genotypes, segregation analysis, or adequately assessed phenotype information is available. Non-segregation cannot be evaluated, and no benign evidence can be assigned.

Rule

Generic ACMG/AMP 2015 BS4: lack of segregation in affected family members, or presence of the variant in unaffected relatives when disease status and penetrance are adequately established.

Still missing

• Pedigree and unaffected-relative information
• Genotypes of relevant unaffected relatives
• Age and clinical assessment sufficient to establish unaffected status
• Phenotype and penetrance context for interpreting non-segregation

Reasoning

BP1 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

BP2 could be relevant in an autosomal-dominant disorder if this variant were demonstrated in trans with a pathogenic variant, but the case bundle provides no parental testing, family genotypes, affected-proband data, or phase information. Therefore BP2 cannot be assessed from the available evidence and is not applied.

Rule

Generic ACMG/AMP BP2: for a dominant disorder, apply only when the variant is observed in trans with a pathogenic variant; phase and the relevant second variant must be documented.

Evidence considered

• PMID:27854360 provides the reviewed inheritance context for retinoblastoma as autosomal dominant but does not report this variant or any allelic phase observation.
• The consolidated case-evidence bundle contains no affected-proband observations, pathogenic variant in trans, pathogenic variant in cis, or segregation/phasing data for NM_000321.3:c.2165A>G (p.Lys722Arg).

Still missing

• No family or proband genotype data establishing a second pathogenic variant.
• No validated cis/trans phase information.

Sources

Reasoning

No individual-level phenotype or molecular diagnostic information was provided to establish an alternate molecular basis that fully explains a carrier's phenotype.

Rule

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

Evidence considered

• No applicable RB1 VCEP/ClinGen specification was retrieved; generic ACMG/AMP BP5 requires an alternate molecular basis for disease in an affected individual.

Still missing

• Individual-level phenotype and a confirmed alternative molecular diagnosis that fully explains that phenotype are required.