RB1 c.13A>C, p.(Thr5Pro)

Reasoning

RB1 NM_000321.3:c.13A>C (p.Thr5Pro) is missense and has REVEL 0.127, below the governing generic fallback BP4 threshold of 0.250. BP4 supporting is met. SpliceAI max delta 0.00 was not separately counted because the governing fallback assigns missense variants to the REVEL path only; BayesDel was not used because no verified pipeline calibration is available.

Rule

Generic ACMG fallback, missense computational path: REVEL only; BP4 supporting if REVEL <0.250. Do not double-count SpliceAI or use uncalibrated BayesDel.

Evidence considered

• No applicable RB1 CSPEC/VCEP material or gene-specific PP3/BP4 lookup was retrieved; the case framework is generic ACMG fallback.
• REVEL score is 0.127. The governing generic fallback specifies REVEL <0.250 for BP4 supporting in missense variants. Calibration of individual computational predictors for PP3/BP4 is described by Pejaver et al., PMID:36413997.
• SpliceAI max delta is 0.00 but is not independently applied to this missense variant under the governing mechanism-specific rule. BayesDel score -0.136964 is not used because the pipeline has no verified published BayesDel calibration.

Sources

Reasoning

The bundle reports no statistically significant cancer hotspot at RB1 T5 and contains no validated RB1-specific domain or residue-clustering evidence establishing residue 5 as a critical functional region. The available evidence does not support PM1.

Rule

PM1 requires a missense change in a mutational hotspot or well-established critical functional domain, without benign variation; the retrieved evidence does not establish either condition for residue 5.

Evidence considered

• The Cancer Hotspots query for RB1 T5 returned no result; the case evidence states that the variant does not lie in a statistically significant hotspot.
• No RB1 VCEP/CSPEC or other validated case source establishes residue 5 as a critical functional domain or pathogenic missense cluster.

Still missing

• No curated RB1 domain annotation or residue-level pathogenic missense-clustering analysis for residue 5 was retrieved.

Reasoning

PM2 is not met because the variant is present in a large population database. gnomAD v4.1 reports 59/1,497,328 alleles (AF 3.94035e-05), including seven Ashkenazi Jewish alleles; therefore the variant is not absent from population databases. Its absence from gnomAD v2.1 and gnomAD-Canada v1.0 is superseded by the documented gnomAD v4.1 presence for this criterion.

Rule

Generic ACMG/AMP PM2: absent from, or extremely rare in, population databases screened for the disorder; a documented presence in gnomAD v4.1 means PM2 is not met under this assessment.

Evidence considered

• gnomAD v4.1 directly identifies variant 13-48303925-A-C and reports AC 59, AN 1,497,328, AF 3.940352414434245e-05, and zero homozygotes.
• The highest listed subgroup is Ashkenazi Jewish with AC 7, AN 27,082, AF 0.00025847426334834944, and zero homozygotes.
• Although the variant is absent from gnomAD v2.1 and gnomAD-Canada v1.0, PM2 requires absence from population databases and is contradicted by the gnomAD v4.1 observation.

Sources

Reasoning

RB1 NM_000321.3:c.13A>C is a missense variant, so the governing generic ACMG fallback requires the REVEL path alone. Its REVEL score (0.127) is below the PP3 threshold and instead falls in the benign-supporting range; SpliceAI and BayesDel were not used to duplicate or substitute missense computational evidence.

Rule

Generic ACMG fallback, missense computational path: REVEL only; PP3 supporting if REVEL >0.750. Do not combine or substitute SpliceAI/BayesDel predictions for the missense path.

Evidence considered

• No applicable RB1 CSPEC/VCEP material or gene-specific PP3/BP4 lookup was retrieved; the case framework is generic ACMG fallback.
• REVEL score is 0.127. The governing generic fallback specifies REVEL <0.250 for BP4 supporting and REVEL >0.750 for PP3 supporting in missense variants. Calibration of individual computational predictors for PP3/BP4 is described by Pejaver et al., PMID:36413997.

Sources

Reasoning

PP5 is not met because ClinVar has no exact-variant expert-panel Pathogenic or Likely pathogenic assertion for RB1 c.13A>C (p.Thr5Pro).

Rule

PP5 may be applied only for an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic.

Evidence considered

• The exact ClinVar record (Variation ID 527924) has four single-submitter uncertain-significance submissions and zero expert-panel submissions.
• Ordinary laboratory submissions and aggregate ClinVar labels were not used for PP5.

Sources

Reasoning

BA1 is not met. The variant is far below the generic stand-alone benign frequency threshold of 5%: gnomAD v4.1 total AF is 3.94035e-05 (0.00394%), with maximum group FAF 7.074e-05 (0.007074%) and highest listed subpopulation AF 0.000258474 (0.02585% in Ashkenazi Jewish individuals).

Rule

Generic ACMG/AMP BA1: stand-alone benign when allele frequency is greater than 5%; no RB1-specific VCEP framework was available, so generic ACMG/AMP criteria apply.

Evidence considered

• gnomAD v4.1 reports 59/1,497,328 alleles, total AF 3.94035e-05, and zero homozygotes.
• The highest reported subgroup frequency is Ashkenazi Jewish AF 0.000258474 (7/27,082 alleles); this remains well below 5%.
• The variant is absent from gnomAD v2.1 and gnomAD-Canada v1.0; absence in those datasets does not change the BA1 threshold assessment.

Sources

Reasoning

BP6 is not met because ClinVar has no exact-variant expert-panel Benign or Likely benign assertion for RB1 c.13A>C (p.Thr5Pro).

Rule

BP6 may be applied only for an exact-variant ClinVar expert-panel classification of Benign or Likely benign.

Evidence considered

• The exact ClinVar record (Variation ID 527924) has four single-submitter uncertain-significance submissions and zero expert-panel submissions.
• Ordinary laboratory submissions and aggregate ClinVar labels were not used for BP6.

Sources

Reasoning

The case variant is NP_000312.2:p.(Thr5Pro). The bundle provides no validated pathogenic or likely pathogenic report for a different nucleotide change producing the same amino-acid change. ClinVar variation 527924 contains only uncertain-significance submissions and therefore cannot establish PS1.

Rule

PS1 requires a previously established pathogenic variant resulting in the same amino-acid change; a VUS record does not satisfy the criterion.

Evidence considered

• Normalization identifies the consequence as NP_000312.2:p.(Thr5Pro).
• ClinVar variation 527924 has four single-submitter uncertain-significance submissions and no expert-panel submission; those records do not establish PS1.

Still missing

• A validated pathogenic or likely pathogenic comparator producing RB1 p.Thr5Pro is not available in the case evidence.

Sources

Reasoning

No proband-specific de novo observation or documented parental testing is provided in the case-evidence bundle. The generic ACMG/AMP PS2 framework therefore cannot be applied.

Rule

Generic ACMG/AMP PS2: de novo occurrence in a patient with the disease and no family history, with maternity and paternity confirmed; strength depends on confirmation and phenotype/parental evidence.

Evidence considered

• The case-evidence bundle identifies the variant as NM_000321.3:c.13A>C (RB1; p.(Thr5Pro)) but contains no proband phenotype, family history, trio analysis, parental genotypes, or de novo testing results.
• No applicable RB1 ClinGen/VCEP or gene-specific framework was available; generic ACMG/AMP criteria were used as fallback.
• The available ClinVar submissions are all uncertain-significance submissions and do not provide usable criterion-lead evidence for de novo status.

Still missing

• Proband-level clinical record linking the variant to the phenotype
• Confirmed maternity and paternity
• Validated testing results showing the variant absent in both biological parents
• Assessment for parental mosaicism and technical limitations

Reasoning

No variant-specific functional assay demonstrating a damaging effect was identified in the consolidated case-evidence bundle. The available ClinVar submissions do not provide validated functional criterion leads, and OncoKB explicitly reports no variant-specific reviewed functional evidence. Therefore PS3 cannot be assessed.

Rule

Generic ACMG/AMP PS3: well-established functional studies showing a damaging effect may support PS3; calibrate strength to assay validity, controls, replication, and biological relevance. No applicable RB1-specific specification was retrieved.

Evidence considered

• ClinVar variation 527924 contains four single-submitter uncertain-significance submissions and no validated functional criterion lead or expert-panel functional assessment.
• OncoKB reports no variant-specific reviewed functional evidence for RB1 p.T5P and provides only gene-level context.
• The generic ACMG/AMP framework was used because no RB1-specific VCEP/CSPEC framework was available; PS3 requires well-established functional evidence showing a damaging effect, with strength dependent on assay validity, controls, replication, and biological relevance.

Still missing

• A validated, biologically relevant functional assay for RB1 NM_000321.3:c.13A>C (p.Thr5Pro) with appropriate positive and negative controls is not available.
• No replicated or quantitatively calibrated assay result was identified to support PS3 strength calibration.

Sources

Reasoning

No variant-specific affected-case series with an appropriate control comparison, case-control odds ratio, or statistically significant enrichment is available for RB1 c.13A>C (p.Thr5Pro). PS4 cannot be applied.

Rule

Generic ACMG/AMP PS4: prevalence of the variant in affected individuals is significantly increased compared with controls; requires documented variant-specific enrichment evidence.

Evidence considered

• No applicable RB1 ClinGen/VCEP or gene-specific framework was available; generic ACMG/AMP was used as fallback.
• The available literature extractions do not report RB1 c.13A>C or p.(Thr5Pro) in a case-control or enrichment study.

Still missing

• Variant-specific counts in well-phenotyped RB1-related disease cases
• An ancestry-matched control dataset and statistical enrichment analysis
• Assessment of ascertainment, relatedness, and phenotype definition

Reasoning

No applicable RB1-specific VCEP framework was retrieved, so the generic ACMG/AMP framework was used. The available case bundle contains no affected-proband observation, co-occurring pathogenic or likely pathogenic variant, parental testing, or phase information showing this variant in trans with a pathogenic variant. PM3 therefore cannot be assessed.

Rule

Generic ACMG/AMP PM3: pathogenic or likely pathogenic variants observed in trans with the variant in affected individuals support a recessive disorder interpretation; phase and affected-proband evidence are required.

Evidence considered

• The case bundle identifies the variant as NM_000321.3:c.13A>C (NP_000312.2:p.(Thr5Pro)) in RB1.
• The case bundle reports no CSPEC/VCEP framework and no family, phase, affected-proband, or allelic observations relevant to PM3.
• ClinVar contains only uncertain-significance submissions and does not provide usable criterion-lead evidence for this assessment.

Still missing

• Affected-proband data are not available.
• No pathogenic or likely pathogenic variant in the same individual is documented for phase comparison.
• No parental testing or segregation/phasing data establish a trans relationship.

Reasoning

The case-specific PM5 artifact contains zero same-residue candidates and states that classic same-residue PM5 semantics could not be confirmed safely. No independently validated pathogenic or likely pathogenic missense comparator at RB1 codon 5 is available in the bundle.

Rule

PM5 requires a novel missense change at a residue where a different missense change is established as pathogenic or likely pathogenic under the applicable framework.

Evidence considered

• The PM5 candidate artifact lists zero total candidates and zero same-residue candidates, and instructs that PM5 should not be applied from that artifact alone.

Still missing

• A validated pathogenic or likely pathogenic missense comparator affecting RB1 codon 5 is not available.
• No RB1-specific same-residue PM5 rule was retrieved.

Sources

Reasoning

No suspected de novo occurrence without confirmed maternity and paternity is documented. PM6 cannot be applied without a specific proband and parental-testing observation.

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; requires a documented case observation.

Evidence considered

• The case-evidence bundle provides no family segregation data, parental genotypes, trio testing, or proband phenotype for NM_000321.3:c.13A>C.
• No variant-specific publication in the extracted literature reports this variant in a de novo context.

Still missing

• Specific proband case report or clinical record
• Evidence that the variant was absent from tested parent samples
• Maternity/paternity confirmation status
• Phenotype consistency with an RB1-related disorder

Reasoning

No affected relatives, informative meioses, cosegregation results, or family genotypes are provided. Segregation evidence is therefore unavailable and PP1 cannot be applied.

Rule

Generic ACMG/AMP PP1: cosegregation with disease in multiple affected family members; strength may increase with multiple informative meioses, but only documented variant-specific segregation is eligible.

Evidence considered

• The case-evidence bundle contains no pedigree, family history, affected-relative testing, unaffected-relative testing, or meiosis count for this variant.
• The extracted publications are general RB1/cancer-genetics or ACMG policy sources and do not report segregation of NM_000321.3:c.13A>C.

Still missing

• Pedigree with affected and unaffected relatives
• Genotypes for informative family members
• Phenotype assessment for each tested relative
• Number of informative meioses and segregation analysis

Reasoning

No applicable RB1-specific framework or validated gene-level analysis was retrieved showing both that benign RB1 missense variation is uncommon and that disease-causing RB1 variants are predominantly missense. Variant-level ClinVar, population, hotspot, and predictor data cannot establish PP2.

Rule

PP2 requires a gene in which missense variation is a common disease mechanism and benign missense variation is uncommon; variant-level evidence alone is insufficient.

Evidence considered

• The case framework is generic ACMG/AMP because no RB1 CSPEC/VCEP was retrieved.
• The available case evidence is variant-level and does not establish a gene-wide missense disease mechanism or low benign-missense rate.

Still missing

• Validated RB1 gene-level missense constraint and disease-causing missense enrichment data are not available.
• The case evidence does not establish the gene-level requirements for PP2.

Reasoning

The bundle contains no proband phenotype, diagnostic findings, age at onset, laterality, family history, or other clinical information linking this variant to an RB1-related disorder. PP4 cannot be assessed.

Rule

Generic ACMG/AMP PP4: the patient's phenotype or family history is highly specific for a disease with a single genetic etiology; requires documented patient-level phenotype evidence.

Evidence considered

• No applicable RB1 ClinGen/VCEP or gene-specific framework was available; generic ACMG/AMP was used as fallback.
• The ClinVar record lists disease labels but supplies no usable patient-level phenotype evidence for this criterion.
• The available literature extractions do not report RB1 c.13A>C or p.(Thr5Pro) in a phenotyped individual.

Still missing

• Proband-level phenotype and diagnostic records
• Assessment of whether the phenotype is highly specific for an RB1-related disorder
• Evaluation of alternative molecular explanations for the phenotype

Reasoning

BS1 cannot be adjudicated confidently from the available evidence. The observed gnomAD v4.1 AF is rare (3.94035e-05 overall; group maximum FAF 7.074e-05), but the case bundle provides no validated RB1-specific maximum credible allele frequency or penetrance-adjusted disease-frequency model for this missense variant. The Ashkenazi Jewish subgroup AF is higher (0.000258474) and creates a possible prevalence/penetrance threshold artifact, but is not by itself sufficient to establish that the frequency is greater than expected for RB1-associated disease.

Rule

Generic ACMG/AMP BS1: allele frequency greater than expected for the disorder; requires a defensible disorder-specific expectation and consideration of penetrance and ancestry. No such RB1-specific threshold was available.

Evidence considered

• gnomAD v4.1 reports total AF 3.94035e-05 (59/1,497,328 alleles), group maximum FAF 7.074e-05, and Ashkenazi Jewish AF 0.000258474 (7/27,082 alleles).
• The variant is absent from gnomAD v2.1 and gnomAD-Canada v1.0.
• No applicable RB1-specific VCEP specification or validated penetrance-adjusted frequency threshold is present in the case bundle; therefore a borderline prevalence comparison should not be converted into BS1 automatically.

Still missing

• A validated RB1-specific maximum credible allele frequency or disease-prevalence/penetrance model is unavailable.
• Variant-specific penetrance and the relevance of the observed ancestry-specific frequency are unknown.

Sources

Reasoning

BS2 cannot be established from population allele counts alone. gnomAD v4.1 contains 59 alleles and zero observed homozygotes, but the bundle does not provide phenotype-confirmed healthy adult carriers, age-of-onset follow-up, or a validated number of unaffected heterozygotes sufficient for a highly penetrant RB1-associated condition. The absence of homozygotes is not evidence for BS2 for this autosomal-dominant cancer-predisposition context.

Rule

Generic ACMG/AMP BS2: observed in healthy adults for a fully penetrant dominant disorder, or in healthy adults in numbers expected for recessive/X-linked disease; population allele and homozygote counts without health-status and penetrance evidence are insufficient.

Evidence considered

• gnomAD v4.1 reports 59 total alleles and zero homozygotes; subgroup homozygote counts are also zero in the reported populations.
• gnomAD allele observations do not document that the carriers are healthy adults without RB1-associated disease or that they have passed the relevant risk period.
• The variant is absent from gnomAD v2.1 and gnomAD-Canada v1.0.

Still missing

• No phenotype-confirmed healthy carrier observations or age/health-status data are available.
• No validated RB1 penetrance model or carrier-count threshold for BS2 is available.

Sources

Reasoning

No variant-specific functional assay demonstrating a normal or non-damaging effect was identified in the consolidated case-evidence bundle. The available ClinVar submissions do not provide validated functional criterion leads, and OncoKB explicitly reports no variant-specific reviewed functional evidence. Therefore BS3 cannot be assessed.

Rule

Generic ACMG/AMP BS3: well-established functional studies showing no damaging effect may support BS3; calibrate strength to assay validity, controls, replication, and biological relevance. No applicable RB1-specific specification was retrieved.

Evidence considered

• ClinVar variation 527924 contains four single-submitter uncertain-significance submissions and no validated functional criterion lead or expert-panel functional assessment.
• OncoKB reports no variant-specific reviewed functional evidence for RB1 p.T5P and provides only gene-level context.
• The generic ACMG/AMP framework was used because no RB1-specific VCEP/CSPEC framework was available; BS3 requires well-established functional evidence showing no damaging effect, with strength dependent on assay validity, controls, replication, and biological relevance.

Still missing

• A validated, biologically relevant functional assay for RB1 NM_000321.3:c.13A>C (p.Thr5Pro) with appropriate positive and negative controls is not available.
• No replicated or quantitatively calibrated assay result was identified to support BS3 strength calibration.

Sources

Reasoning

No unaffected relatives known to carry the variant, no non-segregation analysis, and no phenotype/genotype data from informative family members are provided. BS4 cannot be assessed.

Rule

Generic ACMG/AMP BS4: lack of segregation in affected members of a family; requires appropriate informative relatives and consideration of penetrance, phenotype definition, and family structure.

Evidence considered

• The case-evidence bundle contains no family pedigree or genotype-phenotype data showing non-segregation of NM_000321.3:c.13A>C.
• Population frequency and ClinVar uncertainty do not constitute BS4 non-segregation evidence and were not used for this criterion.

Still missing

• Documented unaffected relatives with reliable age-appropriate phenotype assessment
• Variant genotypes for those relatives
• Informative family structure and expected penetrance
• Evaluation of phenocopies, reduced penetrance, and age-dependent expression

Reasoning

The bundle supports RB1 loss-of-function biology in general but does not provide a validated gene-specific analysis demonstrating that RB1 disease is caused primarily by truncating variants and that missense variants are not a recognized disease mechanism. BP1 cannot be inferred from the current evidence.

Rule

BP1 may be considered for a missense variant in a gene for which primarily truncating variants cause disease; reliable gene-level evidence that missense variants are not a recognized disease mechanism is required.

Evidence considered

• The case framework is generic ACMG/AMP because no RB1 CSPEC/VCEP was retrieved.
• General RB1 loss-of-function evidence does not by itself establish the BP1 gene-level exclusion of missense disease mechanism.

Still missing

• A validated RB1-specific missense-versus-truncating disease-variant analysis excluding missense as a disease mechanism is not available.

Reasoning

No applicable RB1-specific VCEP framework was retrieved, so the generic ACMG/AMP framework was used. The available case bundle contains no documented co-occurring pathogenic or likely pathogenic variant, phase determination, or individual-level observation establishing the variant in trans with a pathogenic variant in a fully penetrant dominant disorder. BP2 therefore cannot be assessed.

Rule

Generic ACMG/AMP BP2: observation of the variant in trans with a pathogenic variant for a fully penetrant dominant disorder, or in cis with a pathogenic variant for a recessive disorder, supports a benign interpretation; phase and the applicable inheritance model must be established.

Evidence considered

• The case bundle identifies RB1 and reports no affected-proband, co-occurring-variant, phase, or inheritance data relevant to BP2.
• The available ClinVar submissions are uncertain-significance classifications and do not document a phase relationship with another pathogenic variant.

Still missing

• No individual-level allelic observations are available.
• No pathogenic or likely pathogenic variant is documented in cis or trans with this variant.
• No parental testing or molecular phasing is available.
• The inheritance and penetrance context for the specific observed allele combination is not documented.

Reasoning

No affected individual, phenotype, or independent molecular diagnosis is documented in the bundle. There is no evidence that an alternate molecular cause fully explains a phenotype in a carrier of this variant, so BP5 cannot be assessed.

Rule

Generic ACMG/AMP BP5: a variant is found in a case with an alternate molecular basis for disease; requires a documented alternate diagnosis that explains the phenotype.

Evidence considered

• No case-level phenotype or concurrent molecular diagnosis is provided for RB1 c.13A>C.
• ClinVar uncertainty and population-frequency data are not evidence for BP5 and were not used.

Still missing

• Proband-level phenotype and diagnostic records
• Results of testing for alternative molecular diagnoses
• Evidence that any alternate diagnosis fully explains the observed phenotype