RET c.1783G>A, p.(Glu595Lys)

Reasoning

Variant is at extremely low frequency across all population datasets: gnomAD v4.1 total AF 1.86e-06 (0.00019%, 3/1,610,212 alleles), maximum subpopulation AF 0.00336% (AMR), grpmax FAF 5.57e-06, 0 homozygotes; gnomAD v2.1 total AF 4.14e-06 (0.00041%, 1/241,470 alleles), max 0.00296% (AMR), 0 homozygotes; absent from gnomAD-Canada v1.0. Max AF (0.00336%) is well below the operational non-VCEP PM2 threshold of <0.1%. Confidence tempered to medium because one ClinVar submission (SCV001414200, Labcorp) cautions that gnomAD data quality at this position may be poor; the raw frequencies are nonetheless consistent with ultra-rare, so PM2 (supporting) is applied.

Rule

Generic ACMG/AMP 2015 (PMID:25741868), PM2: absent in controls or at extremely low frequency in population databases (operational non-VCEP threshold: <0.1%). Max observed AF 0.00336% (<0.1%), 0 homozygotes -> met at supporting strength.

Evidence considered

• gnomad_v2
• gnomad_v4
• gnomad_canada
• clinvar

Still missing

• Optional: confirm gnomAD position-level quality metrics (the Labcorp submission cites possible poor data quality at this position); does not change PM2 outcome at supporting strength.

Sources

Reasoning

Generic ACMG/AMP 2015 (PMID:25741868) applies (no RET CSPEC/VCEP retrieved; cspec.found=false, final_classification_framework.found=false). PS3 requires well-established functional studies demonstrating a deleterious effect on protein function or splicing. No such evidence exists for RET p.Glu595Lys in any retrieved source: (1) the All of Us Research Program ClinVar submission (SCV005430350, variation 966506) explicitly states 'to our knowledge, functional studies have not been reported for this variant'; (2) OncoKB reports 'Unknown Oncogenic Effect' with no variant-specific reviewed functional evidence (is_variant_specific=false, not_reviewed=true); (3) the literature pass retrieved no functional-assay papers for this variant; (4) the only somatic observation (COSMIC COSV60693777, n=1; recorded in evidence.json but not registered in source_registry.json) is an occurrence count, not a functional assay result, and cannot substitute for PS3; (5) in-silico predictors (REVEL 0.47, BayesDel 0.197, SpliceAI max delta 0.00) are not functional studies and are outside PS3's evidence class. Absence of functional evidence is affirmatively supported by multiple independent sources, so PS3 is not met rather than merely unassessable.

Rule

ACMG/AMP 2015 (PMID:25741868) PS3: Well-established functional studies show a deleterious effect (Strong, Pathogenic). Applied under generic ACMG fallback because no RET-specific CSPEC/VCEP framework was available in the workspace.

Evidence considered

• ClinVar variation 966506: 5 submissions all Uncertain significance; SCV005430350 (All of Us) states functional studies have not been reported for this variant
• OncoKB RET E595K: Unknown Oncogenic Effect, no variant-specific reviewed functional evidence
• COSMIC COSV60693777 somatic count n=1 (from evidence.json; not a source_registry key) considered and excluded as non-functional evidence
• Literature pass: no functional-theme papers for this variant retrieved (only guideline/case-observation papers)
• PMID:25741868: defines PS3 evidence requirements (well-established functional studies, assay validity/biological relevance)

Still missing

• No published functional assay data for p.Glu595Lys (e.g., RET kinase autophosphorylation/activation assays, NIH3T3 transformation, PC12 differentiation, or receptor signaling readouts); a targeted search of RET functional assay literature could be pursued to confirm absence
• No validated functional assay with appropriate positive/negative controls (e.g., known MEN2-activating mutants like p.M918T or p.C634R as comparators) was identified for this variant

Sources

Reasoning

Generic ACMG PM1 requires location in a mutational hot spot and/or a critical, well-established functional domain without benign variation. p.Glu595 is not in a mutational hotspot: CancerHotspots.org returned no result for RET E595 (evidence.json results.hotspots, search_status=no_result), OncoKB reports 'Unknown Oncogenic Effect' with no variant-specific functional evidence, and COSMIC shows only a single somatic occurrence (COSV60693777, n=1). Domain context: E595 is a non-cysteine residue in the extracellular cysteine-rich domain of RET (positioned between C585 and C609; transmembrane helix starts at residue 638 and the kinase domain at ~730 in NP_066124.1), 14 residues N-terminal of the first established MEN2A hotspot cysteine C609; the well-established germline MEN2 hotspot residues are the cysteine codons C609/C611/C618/C620 (exon 10) and C630/C634 (exon 11) plus kinase-domain codons - E595 is not among them. The broad cysteine-rich domain does not satisfy the generic ACMG PM1 domain bar ('all missense variants identified to date in these domains have been shown to be pathogenic... must also lack benign variants'), and no RET-specific VCEP rule defining a PM1 domain was available in this case. PM1 therefore not met under the governing generic framework.

Rule

PM1 (generic ACMG/AMP 2015, PMID:25741868): Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation.

Evidence considered

• PMID:25741868
• oncokb
• clinvar

Still missing

• A RET-specific VCEP (ClinGen CSpec) PM1 rule was not retrieved for this case; if one existed, domain-boundary definitions could differ from generic ACMG.

Sources

Reasoning

Missense variant NM_020975.6:c.1783G>A (p.Glu595Lys, exon 10; not in canonical splice consensus). Generic ACMG/AMP 2015 PP3 requires MULTIPLE lines of computational evidence supporting a deleterious effect; the retrieved predictors do not supply them. (1) SpliceAI max delta = 0.00 (DS_AG/DS_AL/DS_DG/DS_DL all 0.0): no splice impact predicted - provides no PP3 support via the splice path. (2) REVEL = 0.47: lies in the calibrated indeterminate zone (between the BP4-supporting boundary ~0.207 and the PP3-supporting boundary 0.644 per Pejaver et al. 2022, PMID:36413997, the REVEL calibration verified for this pipeline) and below the commonly applied damaging range (~0.5-0.75) - no PP3 support. (3) BayesDel noAF = 0.197098 is the only nominally pathogenic-leaning score, but its threshold is NOT verified in this pipeline (REVEL calibration is verified; BayesDel is not) and it falls between proposed noAF cutoffs (>=0.16 in some conventions, >=0.28 in ENIGMA v1.2), so it cannot serve as a calibrated line. Even granting BayesDel full credit, only a single line would support a deleterious effect while REVEL (indeterminate) and SpliceAI (no impact) fail to corroborate, so the PP3 'multiple lines' requirement is not met. External ClinVar single-submitter assertions ('predicted to be deleterious by in silico analysis', Ambry SCV002714960) were not adopted: they carry no validated PMID trail and per policy a criterion is never inferred from another submitter's statement, especially when the retrieved deterministic scores contradict it.

Rule

PP3 (Richards et al. 2015, PMID:25741868; generic ACMG fallback via generic_acmg_combination_rules): Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.); supporting strength by default.

Evidence considered

• SpliceAI max delta 0.00 (all DS scores 0.0): no predicted splice impact - no PP3 support via splice path
• REVEL 0.47: indeterminate zone (0.207-0.644 calibrated; Pejaver et al. 2022, PMID:36413997) - no PP3 support
• BayesDel noAF 0.197098: single unverified, weakly pathogenic-leaning predictor - insufficient for the multiple-lines requirement
• Variant is a deep-exonic missense (exon 10, c.1783, p.Glu595Lys), canonical_splice_consensus=false per pvs1_variant_assessment

Still missing

• Only one predictor (BayesDel noAF 0.197098) leans pathogenic and its calibrated threshold is not verified in this pipeline; no additional calibrated missense predictors (e.g., AlphaMissense, EVE, CADD, MAPP) were retrieved to reach the PP3 multiple-lines requirement.
• REVEL 0.47 falls in the calibrated indeterminate zone (0.207-0.644); a score in the damaging range (>=0.644) or a concordant second calibrated line would be needed to meet PP3.
• No RNA/minigene splice assay data exist; not required for this call since SpliceAI is negative, but it is the only route to splice-based PP3.

Sources

Reasoning

Under the project PP5/BP6 rule, PP5 is triggered ONLY by an exact-variant ClinVar expert-panel classification of Pathogenic/Likely pathogenic (3-star expert panel); ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger PP5. ClinVar VCV000966506 has an empty expert_panels field (0 expert-panel submissions), a review status of 'criteria provided, multiple submitters, no conflicts' (2 stars, not 3-star expert panel), and 5 submissions from ordinary clinical-testing laboratories, all classified 'Uncertain significance'. No expert-panel classification exists for this exact variant, so the PP5 trigger is absent and the criterion is not met.

Rule

Project PP5 rule (per pp5-bp6-rule-engine-gap): PP5 triggered ONLY by an exact-variant ClinVar expert-panel (3-star) Pathogenic/Likely pathogenic classification; ordinary laboratory submissions, aggregate labels, and non-expert assertions never trigger PP5

Evidence considered

• ClinVar VCV000966506 expert_panels field empty (0 expert-panel submissions); API review stars = 2
• All 5 submissions from ordinary clinical-testing laboratories (Baylor, All of Us, Fulgent, Ambry, Labcorp), all 'Uncertain significance' - aggregate label is VUS, not an expert-panel Pathogenic/LP classification

Sources

Reasoning

No population dataset approaches a stand-alone benign allele frequency. Maximum observed allele frequency anywhere is 0.00336% (Admixed American, gnomAD v4.1, 2/59,550 alleles); total AF is 0.00019% (3/1,610,212 alleles) in gnomAD v4.1 and 0.00041% (1/241,470) in gnomAD v2.1; variant is absent from gnomAD-Canada v1.0; 0 homozygotes in all datasets. This is >300-fold below the generic ACMG/AMP 2015 stand-alone BA1 threshold (>5%) and far below the operational non-VCEP threshold used here (>1%).

Rule

Generic ACMG/AMP 2015 (PMID:25741868), BA1: allele frequency >5% in ESP/1000 Genomes/ExAC/gnomAD (operational non-VCEP threshold: >1%). Max observed AF 0.00336% -> not met.

Evidence considered

• gnomad_v2
• gnomad_v4
• gnomad_canada

Sources

Reasoning

The variant is ultra-rare, not over-represented. gnomAD v4.1 total AF 0.00019% (3/1,610,212 alleles; 0 homozygotes), gnomAD v2.1 total AF 0.00041% (1/241,470; 0 homozygotes), maximum subpopulation AF 0.00336% (AMR v4.1), grpmax FAF 5.57e-06, absent from gnomAD-Canada. For RET (MEN2/FMTC, autosomal dominant, high penetrance), the expected disease-contributing allele frequency is far lower than what would be needed for BS1; the observed AF is also ~90-fold below the operational non-VCEP BS1 threshold of >0.3%. Frequency is therefore NOT greater than expected for the disorder.

Rule

Generic ACMG/AMP 2015 (PMID:25741868), BS1: allele frequency greater than expected for the disorder (operational non-VCEP threshold: >0.3% in gnomAD). Max observed AF 0.00336% -> not met.

Evidence considered

• gnomad_v2
• gnomad_v4

Sources

Reasoning

BS2 requires observation of the variant in a healthy adult with full penetrance expected at an early age. gnomAD provides no phenotype, age, or unaffected-adult confirmation for the 1 (v2.1) or 3 (v4.1) heterozygous carriers, all with 0 homozygotes; RET MEN2/FMTC has age-dependent and incomplete penetrance, so population-database carrier status cannot establish 'healthy adult with full penetrance'. The tiny carrier count and absence of homozygotes actually argue against benignity, not for it. No phenotyped unaffected-adult cohort data are available to support BS2.

Rule

Generic ACMG/AMP 2015 (PMID:25741868), BS2: observed in a healthy adult individual for a recessive (homozygous), autosomal dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance expected at an early age. No healthy-adult phenotype confirmation available -> not met.

Evidence considered

• gnomad_v2
• gnomad_v4

Still missing

• Phenotyped unaffected-adult carrier data (age/phenotype of gnomAD or cohort carriers) are unavailable; without such data BS2 cannot be applied.

Sources

Reasoning

Generic ACMG/AMP 2015 (PMID:25741868) applies. BS3 requires well-established functional studies showing no damaging effect on protein function or splicing. No functional studies exist for p.Glu595Lys (see PS3 rationale), so none demonstrate a lack of damaging effect. The only potentially mitigating datum is SpliceAI (max delta score 0.00), but SpliceAI is an in-silico splice prediction, not a well-established functional study (no RNA/minigene assay), and under ACMG 2015 in-silico predictions do not satisfy BS3; absence of predicted splice impact is at most contextual. No RNA-based or enzymatic assay evidence was retrieved. Therefore BS3 is not met.

Rule

ACMG/AMP 2015 (PMID:25741868) BS3: Well-established functional studies show no damaging effect on protein function or splicing (Strong, Benign). Applied under generic ACMG fallback because no RET-specific CSPEC/VCEP framework was available in the workspace.

Evidence considered

• ClinVar variation 966506: no submission cites any functional study showing no damaging effect
• OncoKB RET E595K: no variant-specific reviewed functional evidence
• SpliceAI: max delta score 0.00 (in-silico only; does not qualify as a functional study for BS3)
• PMID:25741868: defines BS3 evidence requirements (well-established functional studies showing no damaging effect on protein function or splicing)

Still missing

• No functional assay demonstrating normal RET protein function for p.Glu595Lys (e.g., kinase activity, signaling, transformation assays)
• No RNA-level or splicing assay for c.1783G>A; SpliceAI (in-silico, max delta 0.00) is insufficient alone to meet BS3 under generic ACMG 2015

Sources

Reasoning

BP1 applies to missense variants in genes for which primarily truncating variants are known to cause disease. RET does not meet this profile: germline missense gain-of-function variants are a primary disease mechanism (MEN2A, MEN2B, familial medullary thyroid carcinoma), and RET is a receptor tyrosine kinase proto-oncogene altered by point mutation in medullary thyroid cancers (OncoKB gene-level context; ClinVar conditions for this record include MEN2A/MEN2B/FMTC). Truncating loss-of-function RET variants cause Hirschsprung disease, but the gene is not 'primarily truncating' for its cancer phenotypes. BP1 is therefore not met.

Rule

BP1 (generic ACMG/AMP 2015, PMID:25741868): Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• PMID:25741868
• oncokb
• clinvar

Sources

Reasoning

BP2 (Richards et al. 2015) requires observing the variant in cis with a pathogenic variant in any inheritance pattern, or in trans with a pathogenic variant for a fully dominant disorder ('finding the second variant in cis would be supporting evidence, though not definitive, for a benign role (BP2)... In the context of dominant disorders, the detection of a variant in trans with a pathogenic variant can be considered supporting evidence for a benign impact'). No collected source contains a second RET variant or any phase determination for this variant: the 5 ClinVar submissions carry no co-occurrence/phase information, no parental/family testing data exists in the case bundle, and the only ClinVar-validated literature PMID (35534704) never mentions the exact variant (RET cited at gene level only). With no cis- or trans-phase observation reported, BP2 is not met. Note: the absence of any reported co-occurring variant is not itself benign evidence; it reflects absence of allelic data.

Rule

BP2 (Richards et al. 2015, PMID:25741868): Observed in trans with a pathogenic variant for a fully dominant disease/disorder OR observed in cis with a pathogenic variant in any inheritance pattern (supporting evidence for benign impact).

Evidence considered

• Rule definition of BP2 (cis with pathogenic variant, or trans with pathogenic variant in fully dominant disorder) from Richards et al. 2015 (PMID:25741868) full text, 'PM3 BP2 Cis/trans testing' section.
• ClinVar record VCV000966506 and submission audit (SCV001414200, SCV002714960, SCV004206723, SCV005430350, SCV005674412): no co-occurrence or phase information present in any submission.
• Full-text check of PMID:35534704: exact variant absent; no family/phase data relevant to this variant.
• Case bundle contains no proband family-history or parental-testing section (review_manifest, case_summary, prefetch, evidence.json all lack allelic/phase fields).

Still missing

• No parental/family phase-testing data for this variant in any collected source.
• No co-occurring second RET variant reported in any ClinVar submission or publication; no evidence of cis/trans configuration to evaluate.

Sources

Reasoning

Generic ACMG/AMP 2015 BP4 requires MULTIPLE lines of computational evidence suggesting no impact on the gene/gene product. Only one of the three retrieved predictors supports no impact. (1) SpliceAI max delta = 0.00 (all DS scores 0.0): a single line supporting absence of splice impact, and this covers only the splicing mechanism - for a missense variant it does not clear the protein-level effect. (2) REVEL = 0.47: lies above the calibrated BP4-supporting boundary (~0.207; Pejaver et al. 2022, PMID:36413997) and is therefore indeterminate, not benign-supporting. (3) BayesDel noAF = 0.197098: pathogenic-leaning, actively opposes a no-impact interpretation. With one line for and one line against (plus one indeterminate), the BP4 'multiple lines of no-impact evidence' requirement is not met. External ClinVar single-submitter assertion ('computational prediction suggests that this variant may not impact protein structure and function', All of Us SCV005430350) was not adopted: no validated PMID trail, and the retrieved deterministic missense predictors (REVEL gray zone, BayesDel pathogenic-leaning) do not corroborate a no-impact interpretation.

Rule

BP4 (Richards et al. 2015, PMID:25741868; generic ACMG fallback via generic_acmg_combination_rules): Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.); supporting (benign) strength by default.

Evidence considered

• SpliceAI max delta 0.00: one line supporting no splice impact (splice path only)
• REVEL 0.47: above the calibrated BP4-supporting boundary (~0.207; Pejaver et al. 2022, PMID:36413997) - indeterminate, does not support no-impact
• BayesDel noAF 0.197098: pathogenic-leaning, opposes a no-impact interpretation
• Only one of three predictors supports no impact, so the BP4 multiple-lines requirement is unmet

Still missing

• REVEL 0.47 sits in the calibrated indeterminate zone (0.207-0.644): the missense path can neither meet BP4 (requires <=~0.207) nor PP3 (requires >=0.644).
• No RNA/minigene splice assay is available to confirm the SpliceAI no-impact prediction; a functional splice readout would be the only additional no-impact line available for this variant.
• No additional calibrated missense predictors were retrieved that might supply a second benign-leaning line.

Sources

Reasoning

Under the project PP5/BP6 rule, BP6 is triggered ONLY by an exact-variant ClinVar expert-panel classification of Benign/Likely benign (3-star expert panel); ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger BP6. ClinVar VCV000966506 has an empty expert_panels field (0 expert-panel submissions), 2-star review status, and an aggregate classification of 'Uncertain significance' from 5 ordinary clinical-testing laboratories. No expert-panel benign classification exists for this exact variant, so the BP6 trigger is absent and the criterion is not met.

Rule

Project BP6 rule (per pp5-bp6-rule-engine-gap): BP6 triggered ONLY by an exact-variant ClinVar expert-panel (3-star) Benign/Likely benign classification; ordinary laboratory submissions, aggregate labels, and non-expert assertions never trigger BP6

Evidence considered

• ClinVar VCV000966506 expert_panels field empty (0 expert-panel submissions); aggregate classification 'Uncertain significance' (2 stars); no exact-variant expert-panel Benign/Likely benign classification exists

Sources

Reasoning

PS2 (Richards et al. 2015, PMID:25741868) requires a documented de novo occurrence of the variant in a proband with the disease and no family history, with maternity and paternity confirmed. No proband-level data, parental testing results, or published de novo report for NM_020975.6:c.1783G>A (RET p.Glu595Lys) exists anywhere in this case bundle: case_summary.json contains no proband/phenotype section, no publication among the five full texts retrieved mentions the variant (searches for c.1783G>A, p.Glu595Lys, p.E595K, 1783, 595, RET returned zero hits, including in the ClinVar-validated PMID:35534704), and none of the five ClinVar submissions (all VUS, single submitter) carries a de novo signal. Evidence insufficient to apply PS2.

Rule

PS2: De novo (maternity and paternity confirmed) in a patient with the disease and no family history (Strong, pathogenic; Richards et al. 2015).

Still missing

• No proband clinical/phenotype information provided for this case
• No parental genotyping or maternity/paternity confirmation data
• No published or submitted de novo occurrence of c.1783G>A in the literature or ClinVar submissions reviewed

Sources

Reasoning

PS4 requires case-control or cohort enrichment showing a significantly increased prevalence of the variant in affected individuals versus controls. No such dataset exists for this exact variant in the evidence bundle. ClinVar VCV000966506 carries 5 submissions, all 'Uncertain significance' from ordinary clinical-testing laboratories (Baylor Genetics, All of Us, Fulgent, Ambry, Labcorp) with no case counts, no controls, and no enrichment statistics; the aggregate 2-star VUS label is not usable as case evidence. COSMIC COSV60693777 is a single somatic occurrence (n=1) in a cancer database, which is not germline case-control evidence (and 'cosmic' is not a source_registry key, so it is noted descriptively only). Population data (gnomAD v2.1 AF 4.14e-06, 1/241,470; gnomAD v4.1 AF 1.86e-06, 3/1,610,212) show extreme rarity in controls, which is the opposite of an enrichment signal. The only validated ClinVar PMID for this variant, PMID:35534704 (Brazilian hereditary cancer cohort of 1682 patients), was read in full text and mentions RET only at gene level - the exact variant c.1783G>A / p.Glu595Lys is absent from the entire paper (0 matches for '1783', 'E595', 'Glu595'), so it cannot contribute PS4 case counts. Evidence for this criterion is insufficient; PS4 is not assessed pending case-control or cohort data for the exact variant.

Rule

Generic ACMG/AMP 2015 (PMID:25741868) PS4: prevalence of variant in affected individuals significantly increased vs controls (case-control/cohort enrichment)

Evidence considered

• ClinVar VCV000966506: 5 submissions, all 'Uncertain significance' from ordinary clinical-testing laboratories; no case counts, controls, or enrichment data; expert_panels empty
• COSMIC COSV60693777: single somatic occurrence (n=1) - somatic observation, not germline case-control evidence; noted descriptively (cosmic is not a source_registry key)
• gnomAD v2.1/v4.1: ultra-rare in population controls (1/241,470 and 3/1,610,212 alleles; 0 homozygotes) - no enrichment signal
• PMID:35534704 full text: RET reported only at gene level; exact variant c.1783G>A absent (0 matches for '1783'/'E595'/'Glu595')

Still missing

• No case-control or cohort study reporting the exact variant (c.1783G>A / p.Glu595Lys) was retrieved; enrichment (prevalence in affected individuals vs controls) cannot be computed without such a study. A broader literature search beyond the retrieved bundle would be required.

Sources

Reasoning

PM5 requires a different missense change at the same residue (E595) that has been determined pathogenic. The pipeline's pm5_candidates.json found no same-residue comparator candidates: 'found': false, pm5_mode 'unknown', eligible_for_classic_pm5_search=false, total_candidates_seen=0, candidates=[], recommendation level 'not_applicable' with instruction 'do not apply PM5 from this artifact alone'. No alternate missense change at Glu595 (e.g., E595Q/E595A/E595G/E595V/E595D) is mentioned anywhere in the evidence bundle, and no literature or ClinVar record of a pathogenic alternate change at this residue was retrieved. Because the automatic same-residue comparator search was skipped rather than performed, the evidence is insufficient to apply PM5, and it is also not yet safe to conclude PM5 is definitively not met.

Rule

PM5 (generic ACMG/AMP 2015, PMID:25741868): Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• pm5_candidates
• PMID:25741868
• clinvar

Still missing

• Manual ClinVar and literature search for alternate missense changes at RET p.Glu595 (E595Q/E595A/E595G/E595V/E595D) and their classifications is required; pm5 candidate harvesting was skipped by the pipeline (pm5_candidates.json: eligible_for_classic_pm5_search=false).

Sources

Reasoning

PM6 (Richards et al. 2015) requires an assumed de novo occurrence without confirmation of paternity and maternity. The case contains no evidence of a de novo event: no proband data, no parental testing, and no publication or ClinVar submission reporting the variant as de novo. Since PS2-level (confirmed) de novo evidence is absent, PM6-level (assumed de novo) evidence is likewise absent. Evidence insufficient to apply PM6.

Rule

PM6: Assumed de novo, but without confirmation of paternity and maternity (Moderate, pathogenic; Richards et al. 2015).

Still missing

• No documentation of de novo occurrence (with or without parental identity confirmation)
• No parental testing data at all for any proband carrying c.1783G>A
• No literature report of de novo origin for this variant

Sources

Reasoning

PP1 (Richards et al. 2015) requires co-segregation of the variant with disease in multiple affected family members in a gene definitively known to cause the disease. RET is definitively associated with MEN2 (autosomal dominant), so PP1 would be applicable in principle, but no segregation data exists for this variant: the case bundle contains no pedigree, no affected-relative genotyping, and no literature or ClinVar segregation evidence (literature_pass.json lists no segregation-themed paper; criteria_index contains no PP1 entries; full-text searches for 'segregat' returned zero hits). Evidence insufficient to apply PP1.

Rule

PP1: Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease (Supporting, pathogenic; Richards et al. 2015).

Still missing

• No pedigree or family history data in the case
• No co-segregation study of c.1783G>A in affected family members published or submitted
• No meiosis/segregation counts available

Sources

Reasoning

PP2 requires a gene with (a) a low rate of benign missense variation AND (b) missense variants as a common mechanism of disease. For RET, part (b) is supported by gene-level context: RET is a proto-oncogene activated by point mutations and germline missense gain-of-function variants cause MEN2A/MEN2B/FMTC (OncoKB gene-level context; ClinVar condition list for this record includes MEN2A, MEN2B and familial medullary thyroid carcinoma). However, no gene-level missense constraint evidence (e.g., gnomAD missense Z-score or observed/expected ratio) was retrieved in the evidence bundle, so part (a) - 'low rate of benign missense variation' - cannot be evaluated. Under the governing generic ACMG rules, PP2 cannot be applied without this constraint data.

Rule

PP2 (generic ACMG/AMP 2015, PMID:25741868): Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.

Evidence considered

• PMID:25741868
• oncokb

Still missing

• RET missense constraint metrics (gnomAD missense Z-score / o/e ratio or equivalent benign-missense-rate evidence) not retrieved in this case; needed to satisfy the 'low rate of benign missense variation' component of PP2.

Sources

Reasoning

PP4 requires the variant to be observed in a phenotype or family history highly specific for a disease with a single genetic etiology (for RET: medullary thyroid carcinoma / MEN2 spectrum). No case-level phenotype documentation exists for this exact variant. The ClinVar record lists record-level conditions (familial medullary thyroid carcinoma, MEN2A, MEN2B, MEN2, pheochromocytoma, Hirschsprung susceptibility 1, hereditary cancer-predisposing syndrome), but these are asserted record labels - every individual SCV has an empty condition list and no submission documents the observed phenotype of a carrier. No retrieved publication mentions the exact variant; gene-level sources (PMID:15604628 NSGC recommendations, PMID:24493721 ASCO statement, PMID:34012068 ACMG SF v3.0) establish only that RET is associated with MEN2/MTC, which cannot link this specific variant to a phenotype. Evidence for this criterion is insufficient; PP4 is not assessed pending case-level phenotype data for the exact variant.

Rule

Generic ACMG/AMP 2015 (PMID:25741868) PP4: phenotype or family history highly specific for a disease with a single genetic etiology

Evidence considered

• ClinVar VCV000966506: record-level conditions (FMTC, MEN2A/B, MEN2, pheochromocytoma, Hirschsprung, hereditary cancer) are asserted labels; all 5 SCV condition lists empty; no observed carrier phenotype documented
• No retrieved publication (full texts of PMID:25741868, 15604628, 24493721, 34012068, 35534704) mentions the exact variant; gene-level RET-MEN2/MTC associations only

Still missing

• No case reports or submissions documenting the exact variant (c.1783G>A / p.Glu595Lys) in individuals with a RET-specific phenotype (MTC, MEN2A/B, FMTC, pheochromocytoma); case-level phenotype data are needed.

Sources

Reasoning

BS4 (Richards et al. 2015) requires non-segregation of the variant with disease in affected family members. No non-segregation evidence exists in this case: there are no family studies of c.1783G>A at all, and no unaffected-carrier or affected-non-carrier observations in the case bundle. Note that BS4 must not be inferred from population frequency data (the variant is present in gnomAD v4.1 at AF ~1.86e-6, 3 alleles, 0 homozygotes); such data are handled by BS1/BA1 in the population group and do not constitute family non-segregation. Evidence insufficient to apply BS4.

Rule

BS4: Non-segregation with disease in affected family members (Strong, benign; Richards et al. 2015).

Still missing

• No family segregation studies (affected/non-carrier or unaffected/carrier observations) for c.1783G>A
• No pedigree data permitting non-segregation assessment
• Population frequency data (gnomAD) cannot substitute for family non-segregation evidence

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease (e.g., a second, explanatory variant in the same or another gene). No case-level data exist in the bundle: no retrieved publication mentions the exact variant, and no ClinVar submission reports a carrier of c.1783G>A with an identified alternate molecular cause (all SCV condition lists are empty and submissions provide no case narratives). Evidence for this criterion is insufficient; BP5 is not assessed pending case-level data documenting an alternate molecular explanation in a carrier of the exact variant.

Rule

Generic ACMG/AMP 2015 (PMID:25741868) BP5: variant found in a case with an alternate molecular basis for disease

Evidence considered

• No publication or ClinVar submission in the bundle documents an alternate molecular basis in a carrier of c.1783G>A; ClinVar SCVs contain no case narratives
• PMID:35534704 reviewed in full text: RET at gene level only, no variant-level case detail

Still missing

• No case-level data reporting an alternate molecular basis (second variant or non-genetic cause) in a carrier of the exact variant c.1783G>A / p.Glu595Lys.

Sources