MUTYH c.505-4A>G, p.?

Reasoning

BS1 (strong benign) applies when the allele frequency in a control or general population is greater than expected for the disorder; the generic SVI default threshold is a filtering allele frequency of >= 0.01 (supplied calibration block, citing Whiffin et al. 2017, PMID:28518168), applied here because the governing InSiGHT MUTYH v1.0 specification was retrieved with an empty rule payload and supplied no gene-specific threshold or mandated population source. The variant exceeds 0.01 in every dataset view, on both the grpmax FAF and the popmax AF convention. gnomAD v2.1 all-comers: grpmax FAF 0.0164467 with African/African American AF 0.0177913 (444/24956 alleles). gnomAD v4.1 all-comers: grpmax FAF 0.0166596 with African/African American AF 0.0174459 (1309/75032 alleles). The non-cancer subsets are equally decisive: gnomAD v2.1 non-cancer exomes-only exome grpmax FAF 0.0162815 with African exome-only 269/14900 (1.805%), and gnomAD v3.1 non-cancer genomes-only grpmax FAF 0.0162335 with African 0.0172951 (711/41110). gnomAD-Canada v1.0 adds African 0.0107843 (11/1020). Whichever view is used, the highest-frequency ancestry sits at ~1.6-1.8%, i.e. 1.6-fold or more above the 1% threshold, and the conclusion does not depend on choosing between the all-comers totals and the non-cancer/exome-only sources. Corroborating biology: MUTYH-associated polyposis is an autosomal recessive, adult-onset, incompletely penetrant condition (PMID:25452455), so its expected pathogenic-allele frequency is genuinely higher than that of a fully penetrant dominant disorder; even so, a single allele at ~1.7% in one ancestry exceeds the range compatible with the observed rarity of MAP, and gnomAD v4.1 reports 8 homozygotes in that ancestry (5 XX, 3 XY) consistent with Hardy-Weinberg expectation for a neutral polymorphism, not for a highly penetrant biallelic disease allele. BS1 is met at strong strength. Flagged for human review because the threshold used is the generic default rather than a gene-specific recessive-derived value, and this variant sits only ~1.6-fold above it.

Rule

ACMG/AMP 2015 BS1 (strong benign): allele frequency greater than expected for the disorder; generic SVI default filtering allele frequency >= 0.01 (threshold block citing Whiffin et al. 2017, PMID:28518168; criterion PMID:25741868)

Evidence considered

• BS1 threshold applied verbatim from the supplied generic calibration block: filtering allele frequency >= 0.01, described there as the generic SVI default with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168); the criterion itself is defined in ACMG/AMP 2015 as an allele frequency greater than expected for the disorder (PMID:25741868).
• gnomAD v2.1 all-comers: grpmax FAF 0.0164467; highest ancestry African/African American AF 0.0177913 (444/24956 alleles, 0 homozygotes); total AF 0.00167588 (474/282836).
• gnomAD v4.1 all-comers: grpmax FAF 0.0166596 (exome grpmax FAF 0.0165617); highest ancestry African/African American AF 0.0174459 (1309/75032 alleles, 8 homozygotes); total AF 0.00090076 (1454/1614192).
• gnomAD v2.1 non-cancer subset (exomes-only figures): exome grpmax FAF 0.0162815; African/African American exome-only 269/14900 (1.805%); exome AF 0.00125357 (297/236924, 0 homozygotes).
• gnomAD v3.1 non-cancer subset (genomes only): grpmax FAF 0.0162335; African/African American 0.0172951 (711/41110 alleles, 4 homozygotes); total AF 0.00504293 (746/147930).
• gnomAD-Canada v1.0 (HostSeq genomes): African/African American 0.0107843 (11/1020 alleles); total AF 0.00070591 (13/18416, 0 homozygotes).
• Both frequency conventions exceed 0.01 in every view: grpmax filtering allele frequency is 0.0162-0.0167 (gnomAD v2.1, v4.1, v2.1 non-cancer exomes, v3.1 non-cancer), and the highest ancestry allele frequency is 0.0108-0.0178; the smallest of these figures (gnomAD-Canada African ancestry, 1.078%, 11/1020) is still above the threshold.
• PMID:25452455 (ASCO endorsement of the ESMO familial-risk colorectal cancer guidelines) states that MUTYH-associated polyposis is autosomal recessive with an average age of onset in approximately the mid-50s; this context explains why the frequency expected for this disorder is higher than for a dominant condition, and it was taken into account rather than applying a dominant-disorder expectation uncritically.
• Homozygote structure in gnomAD v4.1 (8 homozygotes in 75032 African alleles, 5 XX and 3 XY) is close to the number expected under Hardy-Weinberg equilibrium for a neutral allele at that frequency (~11), which argues that the ~1.7% African frequency reflects a common neutral polymorphism rather than a pathogenic MAP allele.
• Governing framework consulted first: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis specification for MUTYH v1.0 (cspec doc 1742141534) returned an empty rule payload, so the generic threshold was applied and the all-comers sources were used as the default while the non-cancer subsets listed above were also evaluated.

Still missing

• The governing InSiGHT MUTYH v1.0 specification (cspec doc 1742141534) was retrieved with an empty criteria/rule payload (framework_complete=false, framework_mode=unstructured_ruleset), so no gene-specific BS1 threshold and no mandated population source (all-comers vs non-cancer or exome-only) could be read; the generic SVI default of 0.01 was applied.
• The 0.01 default is a generic value; a threshold derived for an autosomal recessive disorder with a high pathogenic-allele burden would in principle be higher, so the ~1.6-fold margin by which the observed frequencies exceed 0.01 is the remaining uncertainty in this call. No published MUTYH-specific threshold is available in this case to substitute.

Sources

Reasoning

Scope: NM_001128425.2:c.505-4A>G is intronic (intron 6, 4 nt from the exon boundary), so BP4 is assessed on the SpliceAI path only. The governing InSiGHT MUTYH v1.0 specification (CSPEC doc 1742141534) provides no gene-specific BP4 rule in the retrieved framework, so the generic ClinGen-SVI SpliceAI cutoff applies: BP4 supporting when the maximum delta score is <= 0.1 (Jaganathan et al. 2019, Cell, PMID:30661751). SpliceAI maximum delta for this variant is 0.024 (MANE Plus Clinical transcript ENST00000710952.2 = NM_001128425.2, consistent across all transcripts and in both hg37 and hg38), which is well below 0.1 and satisfies BP4 at supporting strength. This is a single calibrated predictor's result on the applicable path for an intronic variant; the Pangolin scores present in the case data (Pangolin SG 0.014, SL -0.006) were not used because no published ACMG-strength calibration for Pangolin was available to this group, and no second in-silico path was consulted. BP4 is being applied at supporting, not moderate/strong, because only one calibrated predictor was evaluable.

Evidence considered

• SpliceAI max delta score 0.024 for NM_001128425.2:c.505-4A>G (hg37 1-45798510-T-C and hg38 chr1-45332838-T-C), retrieved directly from the Broad SpliceAI Lookup scoring service; identical value for the MANE Plus Clinical transcript ENST00000710952.2 and for all other annotated transcripts.
• Applied generic ClinGen-SVI SpliceAI cutoff: BP4 supporting when max delta <= 0.1 (Jaganathan et al. 2019, Cell, PMID:30661751). 0.024 <= 0.1, so BP4 supporting is met.
• BP4 taken from the SpliceAI path only, because the variant is intronic; no REVEL score exists or applies, and a clean/absent splice signal was not mixed with any protein-level predictor.
• The ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MUTYH v1.0 (CSPEC doc 1742141534) is the governing framework but its retrieved criteria payload is empty, so no gene-specific BP4 rule or threshold was available and generic calibration was applied.
• Pangolin scores in the case data (Pangolin SG 0.014, SL -0.006) were deliberately not used: no published ACMG-strength calibration for Pangolin was available, so per the threshold rules it is treated as not_available rather than as supporting evidence.
• The case's own SpliceAI web scrape had timed out (max_delta_score null in evidence.json); the 0.024 value used here is a fresh, successful query of the same Broad SpliceAI source, not an interpretation of the missing value.

Sources

Reasoning

Not met, and no strength tier is assigned. NM_001128425.2:c.505-4A>G (NC_000001.10:g.45798510T>C / NC_000001.11:g.45332838T>C; ClinVar variation 138311 carries the same genomic change on the equivalent transcript NM_001048174.2:c.421-4A>G) is a single-nucleotide substitution in intron 6, four nucleotides upstream of the adjacent exon boundary (VariantValidator variant_exonic_positions = '6i' on both GRCh37 and GRCh38; MANE_Plus_Clinical / RefSeq Select transcript NM_001128425.2, protein NP_001121897.1). Position -4 lies in the acceptor pyrimidine-tract region, not at a canonical +/-1 or +/-2 splice consensus dinucleotide, and the variant-level consequence scaffold records canonical_splice_consensus = false, consequence_class 'unknown', variant_bucket 'other' and apply_generic_pvs1_framework = false. The predicted protein consequence is NP_001121897.1:p.? - no nonsense, frameshift, initiation-codon loss, stop-loss or single/multi-exon deletion is created, and no amino-acid change is predicted. Under the ClinGen SVI PVS1 recommendations (PMC6185798), PVS1 is restricted to variant types predicted to be null (nonsense, frameshift, canonical +/-1,2 splice, initiation codon, single/multi-exon deletion); for a non-canonical splice-region change such as this one, an aberrant transcript must be demonstrated rather than assumed, and ACMG/AMP 2015 (PMID:25741868) states plainly that although splice-site variants are predicted to have a null effect, 'confirmation of impact requires functional analysis by either RNA or protein analysis'. Neither route is satisfied for this variant: SpliceAI returned no scores (timeout), so no SpliceAI delta exists at all - this is missing data and must not be read as a delta of 0 - and no RNA or protein assay of this variant exists in the case or in the reviewed literature (no paper in this case names c.505-4A>G or c.421-4A>G). The only computational signal present is Pangolin SG 0.014 / SL -0.006; it is low, but no calibrated threshold applies to Pangolin in this framework and it is not relied upon either way. Exon-importance and NMD reasoning were also considered and do not help: the only route to a truncating transcript would be loss of the downstream acceptor with skipping of the following exon, in which case NMD-sensitivity and the functional importance of that exon would have to be established, and no such product is predicted or demonstrated. MUTYH loss of function is the established mechanism of MUTYH-associated polyposis, so a genuine null allele would be PVS1-eligible, but this variant is not a null variant on the available evidence. No SVI/VCEP downgrade rule was applied because no null effect was established in the first place.

Rule

Generic ACMG/AMP 2015 PVS1 (PMID:25741868) with the ClinGen SVI PVS1 recommendations (source_registry key 'pvs1_generic_framework', PMC6185798), applied because the InSiGHT MUTYH specification available for this case contained no criterion-level rules: PVS1 requires a variant predicted to be null (nonsense, frameshift, canonical +/-1,2 splice, initiation codon, single/multi-exon deletion) or, for a non-canonical splice-region variant, a demonstrated aberrant transcript. NM_001128425.2:c.505-4A>G is an intronic -4 substitution that is not a canonical splice-consensus change and has no RNA/protein confirmation, so PVS1 is not met and no strength tier is assigned.

Evidence considered

• Variant normalization (prefetch / variant_validator): NM_001128425.2:c.505-4A>G, genomic NC_000001.10:g.45798510T>C and NC_000001.11:g.45332838T>C; variant_exonic_positions = '6i' on both assemblies (intron 6, four nucleotides from the adjacent exon boundary); MANE_Plus_Clinical / RefSeq Select transcript NM_001128425.2, gene MUTYH (HGNC:7527, OMIM 604933); predicted protein consequence NP_001121897.1:p.? with no amino-acid change; primary_assembly_loci confirm a single T>C substitution.
• Variant-level PVS1 scaffold in the case data (source_registry key 'pvs1_variant_assessment'): canonical_splice_consensus = false, consequence_class 'unknown', variant_bucket 'other', apply_generic_pvs1_framework = false, with the rationale that the variant does not fall into the generic null-variant buckets of nonsense, frameshift or canonical +/-1,2 splice consensus variants.
• ClinGen SVI PVS1 recommendations (source_registry key 'pvs1_generic_framework', PMC6185798): PVS1 is restricted to variants predicted to be null (nonsense, frameshift, canonical +/-1/2 splice, initiation codon, single- or multi-exon deletion); for non-canonical splice-region variants an aberrant transcript must be demonstrated, not assumed, before PVS1 can be applied, and null-effect strength tiers (very strong down to supporting) are only reached once that null effect is established.
• ACMG/AMP 2015 full text (PMID:25741868, PVS1 null-variants section): 'For splice site variants, the variant may lead to exon skipping, shortening or inclusion of intronic material, due to alternative donor/acceptor site usage or creation of new sites. Although splice site variants are predicted to lead to a null effect, confirmation of impact requires functional analysis by either RNA or protein analysis.' The same section warns that truncating variants in the last exon or the last 50 bp of the penultimate exon escape NMD and that alternatively spliced exons can make an assumed null benign - none of which can be evaluated here because no aberrant transcript is predicted or demonstrated.
• SpliceAI Lookup (source_registry key 'spliceai'): available = false, max_delta_score = null, error 'timeout: The read operation timed out'. The case data explicitly flags that a null SpliceAI result is missing data rather than a delta of 0 and must not be used to argue for or against a splice effect. Accompanying Pangolin scores SG 0.014 / SL -0.006 are low but have no calibration applicable to this decision.
• Governing framework check: the InSiGHT Hereditary Colorectal Cancer/Polyposis specification v1.0 (source_registry key 'cspec', doc 1742141534) is present for MUTYH but its retrieved content contains no criterion-level rules (empty criteria/rule payload), so there is no applicable gene-specific PVS1 decision tree; generic ACMG/AMP 2015 plus the ClinGen SVI PVS1 recommendations govern (source_registry key 'final_classification_framework').
• Gene-level LoF gate audit: the case's gene-level scaffold (source_registry key 'pvs1_gene_context') marks pvs1_gene_gate = 'eligible' for MUTYH but justifies it with PMIDs whose content concerns unrelated genes and conditions (placental chorangioma/EPAS1, a hereditary breast cancer cohort, an MSH2 Lynch syndrome case series, PIGA duodenal tumorigenesis, and a long-read polyposis study) and with a mis-assigned disease name; none of those papers reports a MUTYH loss-of-function mechanism for this variant, so the gate was not used as PVS1 evidence. MUTYH-associated polyposis is nonetheless a well-established loss-of-function recessive disease, so the PVS1 verdict rests on the variant consequence rather than on disease mechanism.
• Literature check for a splice-null mechanism: none of the reviewed papers (the 8 PMIDs in the literature packet, plus the papers supporting the gene-level gate) names c.505-4A>G or c.421-4A>G or reports RNA studies of this position; the ClinVar record for variation 138311 is on the equivalent transcript NM_001048174.2:c.421-4A>G, and its submitted classifications (majority benign) were not used to infer this criterion.

Still missing

• SpliceAI returned no scores for this variant (the lookup timed out in this run), so no computational splice prediction is available; this is missing data and was not read as a splice delta of 0.
• No RNA or protein-based splice assay of c.505-4A>G / c.421-4A>G exists in this case or in the reviewed literature; a demonstrated aberrant (e.g. out-of-frame, NMD-triggering) transcript would be required to revisit PVS1.
• No gene-specific MUTYH PVS1 decision tree could be applied: the InSiGHT Hereditary Colorectal Cancer/Polyposis specification v1.0 retrieved for this case carried no parseable criterion-level rules, so PVS1 was assessed under generic ACMG/AMP 2015 plus the ClinGen SVI PVS1 recommendations.

Sources

Reasoning

PS3 requires well-established in vitro or in vivo functional evidence that the variant has a deleterious effect on the gene or gene product, with assay validity, controls and biological relevance to MUTYH-associated polyposis established. NM_001128425.2:c.505-4A>G is an intronic substitution 4 bp upstream of the exon containing c.505 (the -4 position of the acceptor region, outside the canonical -1/-2 dinucleotide; ClinVar lists the same genomic change on the shorter transcript as NM_001048174.2:c.421-4A>G). For this variant class the governing generic framework requires direct RNA or protein analysis to confirm impact: 'Although splice site variants are predicted to lead to a null effect, confirmation of impact requires functional analysis by either RNA or protein analysis' (PMID:25741868). No such study exists in the evidence retrieved for this case. No RT-PCR/transcript analysis, minigene splicing assay, or MUTYH 8-oxoguanine glycosylase activity assay for c.505-4A>G is reported in any retrieved source: the seven available full texts are clinical guidelines, secondary-findings policy statements and general variant-classification methodology (PMID:21325953, PMID:25645574, PMID:25452455, PMID:23788249, PMID:27854360, PMID:26467025, PMID:25741868), none of which reports assay data for this variant. The gene-level MUTYH literature compiled for the PVS1 assessment (pvs1_gene_context) supports loss of function as the disease mechanism but assays no MUTYH variant, and the single paper naming one (PMID:41634840) reports a different change (somatic c.650G>A, p.R217H) with expression not confirmable by RNA-Seq. ClinVar holds 17 submissions for this variant, 13 usable for classification, and none provides a functional criterion lead (usable_for_criterion_leads = 0; criterion_leads empty), so no submitter-reported functional evidence is available either. Computational splice predictions recorded for this case (Pangolin splice gain 0.014, splice loss -0.006; SpliceAI returned no scores because the lookup timed out) are in-silico evidence feeding PP3/PVS1, not assay evidence, and are not used as PS3 support. The benign ClinVar consensus and the gnomAD population frequency are population/assertion evidence belonging to other criteria and are not converted into functional evidence here. PS3 is therefore not met; because this rests on the absence of any identified functional study rather than on a negative assay result, confidence is medium and the criterion is flagged for human review of the primary functional literature.

Evidence considered

• Governing framework check: the InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for MUTYH v1.0 (CSpec doc 1742141534, https://cspec.genome.network/cspec/ui/svi/doc/1742141534) is registered with transcripts NM_001128425.2 / NM_001048174.2 for familial adenomatous polyposis 2 (MONDO:0012041, autosomal recessive), but its classification rules are still listed as 'Classification Rules In Prep' on the registry page and no criterion payload was retrievable, so no VCEP-specific PS3 rule exists to apply and generic ACMG/AMP 2015 PS3 is used.
• ClinVar variation 138311 (17 submissions; 13 usable for classification; 0 usable criterion leads; 0 expert-panel submissions; criterion_leads empty): searched for submitter-provided functional evidence of a damaging effect, including any PS3/RNA-assay citation. No functional criterion lead is present, so no PS3 support exists from ClinVar. The single-submitter Benign/Likely benign classifications were not used as functional evidence.
• Seven full-text papers were screened for functional data on this variant: PMID:21325953 (clinical review of MYH-associated polyposis), PMID:25645574 (ACG hereditary GI cancer guideline), PMID:25452455 (ASCO/ESMO endorsement), PMID:23788249 and PMID:27854360 (ACMG incidental/secondary findings policy statements), PMID:26467025 (laboratory variant-scoring methodology) and PMID:25741868 (ACMG/AMP 2015 framework). None names c.505-4A>G, none reports an assay of any MUTYH splice variant, and none provides functional evidence for PS3.
• PMID:25741868 (ACMG/AMP 2015 standards): for splice-region variants 'confirmation of impact requires functional analysis by either RNA or protein analysis'; this is the evidentiary requirement PS3 would have to satisfy here, and it is unmet.
• PMID:26467025 (retrospective laboratory scoring system, 11,894 variants across 338 genes): reports that intronic variants outside the consensus splice sites are enriched among variants scored pathogenic on predicted splice effects, that such variants cluster at +3 to +5 at the intron start and -3 to -10 at the intron end (the region containing this -4 acceptor change), and that published transcript-splicing assays were generally not accepted as functional evidence of pathogenicity unless the aberrant splicing truncated the mRNA. Used as calibration context for how a splicing assay would have to behave to support PS3; no assay of this variant is reported.
• Gene-level MUTYH loss-of-function literature compiled for the PVS1 assessment (pvs1_gene_context: PMID:41634840, PMID:42135811, PMID:42286372, PMID:38546397, PMID:40571398) was reviewed for variant-level functional results: it supports MUTYH loss of function as the germline disease mechanism but contains no assay of c.505-4A>G. The only one of these naming a MUTYH variant reports a different change (somatic NM_001048174.2:c.650G>A, p.R217H) whose expression could not be confirmed in RNA-Seq data.
• Computational splice predictions recorded in this case: Pangolin splice gain 0.014 and splice loss -0.006; SpliceAI returned no scores (lookup timeout), which is missing data and not evidence of absent splice impact. These in-silico values inform PP3/PVS1 only and were explicitly not used as functional evidence for PS3.
• Search terms used when screening the retrieved sources for variant-specific functional evidence: 'c.505-4A>G', 'MUTYH c.505-4A>G', 'NM_001128425.2:c.505-4A>G', 'NM_001048174.2:c.421-4A>G', 'minigene', 'RT-PCR', 'transcript analysis', 'glycosylase', '8-oxoG', 'functional assay'.
• The benign-direction material in this case - ClinVar Benign/Likely benign consensus (16 of 17 submissions) and gnomAD v4.1 frequency (AF 0.000900, 1454/1614192 alleles, 8 homozygotes, FAF 1.67% in African/African American ancestry) - is population and assertion evidence for other criteria and was not treated as functional (PS3/BS3) evidence.

Still missing

• No variant-specific functional assay (RNA/transcript analysis such as RT-PCR or minigene, or a MUTYH 8-oxoguanine glycosylase activity assay) for c.505-4A>G was identified in any source retrieved for this case; a targeted search of the primary functional-splicing literature is required before PS3 can be excluded with high confidence.
• The InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for MUTYH v1.0 (CSpec doc 1742141534) exists but its classification rules are not released, so no VCEP-specific PS3 assay-acceptance criteria or strength calibration could be applied; generic ACMG/AMP 2015 PS3 was used.
• SpliceAI returned no scores (lookup timeout), so no SpliceAI-based splice prediction is available to complement an RNA assay for this -4 acceptor change.

Sources

Reasoning

Governing framework: the case is flagged framework_mode=vcep against the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MUTYH v1.0 (SVI doc 1742141534), but that specification's criterion-level text could not be retrieved: its machine-readable payload is empty in the case materials and in vcep_db/MUTYH, and the captured cspec page holds only the registry landing page (title, version 1.0, AR inheritance, transcripts NM_001128425.2/NM_001048174.2), not the rules. Generic ACMG/AMP 2015 definitions (PMID:25741868) were therefore applied. Generic PS4 requires that the prevalence of the variant in affected individuals be significantly increased relative to controls; the operator as literally written is 'Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0' - it strengthens as the value goes UP, i.e. met requires value > 5.0. Substituted explicitly: 'OR > 5.0?' - no OR was reported or estimable from any retrieved source, because no case-control study or affected-individual series for MUTYH c.505-4A>G exists; the inequality therefore cannot be satisfied and PS4 is not met. No retrieved publication names the variant (all seven full-text extractions record variant_mentioned=false), and the exact-variant ClinVar record (variation 138311) contains no case reports, no expert-panel assertion and no criterion leads (submission audit: 0 usable criterion leads, 0 validated PMIDs). The only quantitative population data available run against enrichment: gnomAD v4.1 grpmax filtering AF 0.01666 (AFR 1.7446%, 1309/75032 alleles, 8 homozygotes) and gnomAD v2.1 grpmax FAF 0.01645 (AFR 1.779%), allele frequencies far above what a variant enriched in biallelic polyposis cases would show.

Rule

Generic ACMG/AMP 2015 PS4 (PMID:25741868): prevalence of the variant in affected individuals significantly increased vs controls; RR or OR > 5.0 with CI excluding 1.0 (strengthens as the value increases). No MUTYH VCEP-specific PS4 rule was retrievable.

Evidence considered

• ClinVar variation 138311 (exact match to NM_001048174.2:c.421-4A>G = NM_001128425.2:c.505-4A>G): 16 submissions, all clinical laboratories, 0 expert-panel submissions, consensus Benign (12 labs)/Likely benign (4 labs)/Uncertain significance (1 lab), 2-star review status 'criteria provided, multiple submitters, no conflicts'. No case reports or case-control data attached.
• ClinVar submission audit: usable_for_criterion_leads = 0, validated_pmids = 0, evidence_signals criteria_found = [] for every submission - no submitter provided affected-individual counts.
• gnomAD v4.1: total AF 0.00090076 (1454/1614192 alleles), grpmax FAF 0.0166596, AFR AF 1.74459% (1309/75032), 8 homozygotes.
• gnomAD v2.1: total AF 0.00167588 (474/282836 alleles), grpmax FAF 0.0164467, AFR AF 1.77913% (444/24956), 0 homozygotes; gnomAD v2.1 non-cancer exomes AF 0.00125357.
• Full-text search of all seven retrieved publications for 'c.505-4A>G', '421-4A>G', '45798510' and '45332838' returned no hits; no paper reports affected carriers of this variant.
• ACMG/AMP 2015 (PMID:25741868) PS4 definition: RR or OR >5.0 from case-control studies with the confidence interval excluding 1.0.

Still missing

• No case-control or affected-individual dataset for MUTYH c.505-4A>G was identified in any retrieved source, so no OR/RR could be computed.
• The MUTYH v1.0 VCEP specification text (including any gene-specific PS4 rule such as a phenotype-point or OR-based scheme) could not be retrieved; generic ACMG/AMP 2015 PS4 was used instead.

Sources

Reasoning

PM1 requires the variant to lie in a mutational hotspot and/or a critical, well-established functional domain without benign variation (ACMG/AMP 2015). Three checks were made and all fail. (1) vcep_materials.json contains an empty domain_tables object for MUTYH, so no VCEP-approved critical functional domain list exists for this gene to test the residue against; there is therefore no per-gene domain entry that could place this variant inside an approved domain. (2) The governing specification was retrieved but is empty at the rule level: vcep_materials/cspec for MUTYH InSiGHT Hereditary Colorectal Cancer/Polyposis Specification v1.0 (doc 1742141534) returns "criteria": [] with final_classification_framework.rule_payload {} and framework_complete=false, so no gene-specific PM1 definition or hotspot rule is available and generic ACMG PM1 is the only applicable rule. (3) The variant itself has no protein consequence (NP_001121897.1:p.?): c.505-4A>G sits 4 bp inside the intron in the acceptor/polypyrimidine region, so it is not located in any protein functional domain (MUTYH's catalytic NUDIX hydrolase domain and helix-hairpin-helix motif are coding), and the variant is explicitly not a canonical +/-1,2 splice consensus change. No hotspot annotation is available either: the hotspot lookup returned residue_significant=false with exact_variant_listed='no'. Additionally, the premise of PM1 ('without benign variation') is contradicted for this position - gnomAD v4.1 reports 1454/1,614,192 alleles with 8 homozygotes and population-max AF 1.74% (African/African American) - and the variant is a 2-star ClinVar Benign/Likely benign allele, so this region is demonstrably tolerant of variation.

Evidence considered

• ACMG/AMP 2015 PM1 definition (PMID:25741868): located in a mutational hotspot and/or critical and well-established functional domain without benign variation.
• vcep_materials.json for MUTYH: "domain_tables": {} - no gene-specific approved domain table was supplied for this case.
• InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MUTYH v1.0 (doc 1742141534) was retrieved (found=true) but its payload is empty: "criteria": [] and final_classification_framework "framework_complete": false, rule_payload {} - so no gene-specific PM1 rule could be applied.
• Normalized consequence NP_001121897.1:p.? with c.505-4A>G located 4 bp from the exon boundary (acceptor region); pvs1_variant_assessment.json records canonical_splice_consensus=false and consequence_class=unknown - the change is not in a coding/domain region.
• Hotspot lookup returned residue_significant=false and exact_variant_listed="no" - no mutational hotspot evidence supports PM1.
• gnomAD v4.1 frequency for this position (AF 0.00090076; 1454/1614192 alleles; 8 homozygotes; popmax AF 1.74459% in African/African American) and the 2-star ClinVar Benign/Likely benign record (variation 138311) show this region tolerates variation, contrary to PM1's 'without benign variation' premise.

Still missing

• vcep_materials.json has no domain_tables entry for MUTYH (empty object), so PM1 could not be evaluated against a VCEP-approved critical-domain list; the InSiGHT MUTYH v1.0 CSPEC in this case also carries an empty criteria/rule payload.
• The conclusion is nevertheless robust because the variant is intronic with no amino-acid change and is not a canonical splice-consensus change, so no protein functional domain can contain it.

Sources

Reasoning

PM2 (supporting) requires the variant to be absent from, or at extremely low frequency in, a large general population or control cohort; the SVI supporting threshold used here, taken verbatim from the supplied calibration block, is a filtering allele frequency of <= 0.0001 (PMID:25741868). NM_001128425.2:c.505-4A>G fails decisively in every source view. gnomAD v2.1 all-comers: total AF 0.00167588 (474/282836 alleles, 0 homozygotes) - about 17-fold above the threshold - with grpmax FAF 0.0164467 and African/African American 0.0177913 (444/24956). gnomAD v4.1 all-comers: total AF 0.00090076 (1454/1614192 alleles, 8 homozygotes) with grpmax FAF 0.0166596 and African/African American 0.0174459 (1309/75032). The non-cancer subsets, evaluated in case the governing specification mandates them, are also far above threshold: gnomAD v2.1 non-cancer exomes-only exome AF 0.00125357 (297/236924 alleles, 0 homozygotes), i.e. about 12-fold above 0.0001, with exome grpmax FAF 0.0162815 and African exome-only 269/14900 (1.805%); gnomAD v3.1 non-cancer genomes-only AF 0.00504293 (746/147930 alleles, 4 homozygotes) with grpmax FAF 0.0162335. gnomAD-Canada v1.0 adds 13/18416 alleles (AF 0.00070591, 0 homozygotes; African 11/1020). The lowest allele-frequency figure anywhere in the case is gnomAD-Canada's total of 0.00070591, still about 7-fold above the supporting threshold, and the lowest per-dataset total (gnomAD v4.1, 0.00090076) is about 9-fold above it; applying the ancestry-specific comparison that ACMG/AMP 2015 recommends for a race-matched control population (PMID:25741868) widens the gap further, since the variant reaches 1.6-1.8% in African ancestry. With 474 and 1454 carrier alleles in gnomAD v2.1 and v4.1 respectively, the variant is plainly not absent from controls. PM2 is not met.

Rule

ClinGen SVI / ACMG/AMP 2015 PM2 (supporting): absent or extremely low frequency in population databases, filtering allele frequency <= 0.0001 (PMID:25741868)

Evidence considered

• PM2 threshold applied verbatim from the supplied generic calibration block: PM2 (supporting) at filtering allele frequency <= 0.0001 (SVI recommendation to downgrade PM2 to supporting; criterion defined in ACMG/AMP 2015, PMID:25741868).
• gnomAD v4.1 all-comers (default source): total AF 0.00090076 (1454/1614192 alleles, 8 homozygotes); grpmax FAF 0.0166596; highest ancestry African/African American 0.0174459 (1309/75032 alleles).
• gnomAD v2.1 all-comers (default source): total AF 0.00167588 (474/282836 alleles, 0 homozygotes); grpmax FAF 0.0164467; highest ancestry African/African American 0.0177913 (444/24956 alleles, 0 homozygotes).
• gnomAD v2.1 non-cancer subset (exomes-only figures): exome AF 0.00125357 (297/236924 alleles, 0 homozygotes); African/African American exome-only 269/14900 (1.805%); exome grpmax FAF 0.0162815.
• gnomAD v3.1 non-cancer subset (genomes only, since v3 has no exome cohort): total AF 0.00504293 (746/147930 alleles, 4 homozygotes); African/African American 0.0172951 (711/41110); grpmax FAF 0.0162335.
• gnomAD-Canada v1.0 (HostSeq genomes): total AF 0.00070591 (13/18416 alleles, 0 homozygotes); African/African American 0.0107843 (11/1020); European (non-Finnish) 0/11740.
• ACMG/AMP 2015 frames PM2 for recessive disease against the expected carrier frequency and a race-matched control population (PMID:25741868); the race-matched (African ancestry) frequencies here are 1.1-1.8% across gnomAD v2.1, v4.1, v3.1 non-cancer and gnomAD-Canada, which is incompatible with an absent or vanishingly rare control-population observation.
• No source view places this variant anywhere near the threshold: the smallest total allele frequency in any dataset or subset is gnomAD-Canada 0.00070591 (about 7-fold above 0.0001) and the smallest grpmax FAF is 0.0162335 (about 160-fold above it), so the call does not depend on the choice between all-comers and non-cancer/exome-only sources.
• Governing framework consulted first: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis specification for MUTYH v1.0 (cspec doc 1742141534) contains no frequency criteria or mandated population source in its retrieved payload (autosomal recessive, MONDO:0012041, NM_001128425.2 only), so the generic ACMG/SVI default was applied.

Sources

Reasoning

Scope: NM_001128425.2:c.505-4A>G is an intronic variant in intron 6, 4 nt upstream of the next exon boundary (VariantValidator exonic position '6i', predicted protein consequence NP_001121897.1:p.?, genomic NC_000001.10:g.45798510T>C / NC_000001.11:g.45332838T>C). PP3/BP4 are therefore evaluated on the SpliceAI path only; no REVEL or BayesDel score applies to an intronic variant and none was used. Governing specification: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MUTYH v1.0 (CSPEC doc 1742141534) is present as the governing framework but its criteria payload is empty in this case, so it supplies no gene-specific PP3/BP4 rule or SpliceAI cutoff; the generic ClinGen-SVI SpliceAI cutoffs apply (PP3 supporting >= 0.2, PP3 moderate >= 0.5; Jaganathan et al. 2019, Cell, PMID:30661751). SpliceAI maximum delta score for this variant is 0.024 (DS_AG 0.024, DS_AL 0.001, DS_DG 0.003, DS_DL 0.002; DP_AL -4, i.e. the altered acceptor signal is the one 4 nt away, consistent with the c.505-4 position). The same 0.024 maximum holds for the MANE Plus Clinical transcript ENST00000710952.2 = NM_001128425.2 and for every other annotated transcript, and was reproduced in both genome builds (hg37 1-45798510-T-C and hg38 chr1-45332838-T-C); the SpliceAI 10k aberration model returned no aberration calls. 0.024 is below the 0.2 supporting threshold, so no splicing-based PP3 is warranted at any strength. The score reported here was retrieved directly from the Broad SpliceAI Lookup scoring API for this exact variant/transcript; the case's own scrape of the Lookup web page had timed out and stored max_delta_score as null, which is why the in-case SpliceAI record reads 'unavailable' - that is a retrieval artefact, not a second, conflicting measurement.

Evidence considered

• Variant consequence used to select the predictor path: intronic, 4 nt from the exon boundary (c.505-4A>G; exonic position 6i; protein consequence p.?), so PP3/BP4 were evaluated via SpliceAI only. REVEL and BayesDel were not used and are not applicable; the case prefetch records REVEL 'found: false' and BayesDel 'found: false'.
• SpliceAI max delta score 0.024 (DS_AG 0.024, DS_AL 0.001, DS_DG 0.003, DS_DL 0.002), identical for the MANE Plus Clinical transcript ENST00000710952.2 (RefSeq NM_001128425.2) and across all annotated MUTYH transcripts; retrieved from the Broad SpliceAI Lookup scoring service for hg37 1-45798510-T-C and independently for hg38 chr1-45332838-T-C (both returned source 'spliceai:model', variant echoed back correctly, raw input NM_001128425.2:c.505-4A>G).
• Applied generic ClinGen-SVI SpliceAI cutoffs: PP3 supporting >= 0.2 and PP3 moderate >= 0.5 (Jaganathan et al. 2019, Cell, PMID:30661751); 0.024 falls below both.
• The ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MUTYH v1.0 (CSPEC doc 1742141534) is the governing framework for this gene, but the retrieved criteria payload is empty, so it specifies no PP3/BP4 rule and no SpliceAI or REVEL cutoff; generic calibration was therefore used as the documented fallback.
• The case's own SpliceAI scrape (screenshots/spliceai.png, evidence.json 'spliceai') failed with a read timeout and stored spliceai_available=false with max_delta_score=null; that null was not treated as a score, and the 0.024 value above comes from a direct query of the same Broad SpliceAI source, not from the null record.
• SpliceAI 10k aberration predictions returned an empty aberration list for this variant, providing no independent splice-aberration signal.

Sources

Reasoning

PP5 (reputable source recently reports the variant as pathogenic) is applied in this group only for an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. The exact-variant record exists - ClinVar variation 138311, whose title NM_001048174.2(MUTYH):c.421-4A>G is the same variant as NM_001128425.2:c.505-4A>G, confirmed by the submission audit (exact_match = true, matched_by = transcript_hgvs, match_score 16) - but it contains no pathogenic reputable-source assertion of any kind. All 16 submissions come from clinical laboratories with is_expert_panel = false, expert-panel submissions = 0, and the consensus is the opposite direction: Benign (12 clinical laboratories), Likely benign (4), Uncertain significance (1), with 2-star review status 'criteria provided, multiple submitters, no conflicts' (aggregate API label 'Benign/Likely benign'). There is no count of 3-star expert-panel review and no other reputable external source in the registry asserting pathogenicity (the consulted guideline and policy documents - PMID:23788249, PMID:25452455, PMID:27854360 - make gene-level or reporting-policy statements only and never name the variant). PP5 is therefore not met.

Rule

Group rule for PP5 as adjudicated here: exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic is required; ordinary laboratory submissions, aggregate labels and non-expert assertions never trigger it. Also assessed against generic ACMG/AMP 2015 PP5 (PMID:25741868) - 'reputable source recently reports variant as pathogenic' - which likewise requires an actual pathogenic external assertion that is absent.

Evidence considered

• ClinVar variation 138311 exact-variant match (NM_001048174.2:c.421-4A>G = NM_001128425.2:c.505-4A>G), variation name reproduced in the submission audit with exact_match = true.
• ClinVar review status 'criteria provided, multiple submitters, no conflicts' (2 stars, api review_stars = 2) - not 'reviewed by expert panel' (3 stars).
• ClinVar submission audit: 16 submissions, expert_panel_submissions = 0, every submission flagged is_expert_panel = false, most recent evaluation 2026-03-03.
• ClinVar consensus: Benign 12 clinical laboratories, Likely benign 4, Uncertain significance 1 (aggregate API classification 'Benign/Likely benign').
• ACMG incidental/secondary findings policy statements (PMID:23788249, PMID:27854360) were screened as potential 'reputable source' claims and contain only gene-list/reporting recommendations; neither names the variant.
• ASCO endorsement guideline (PMID:25452455) was screened for a variant-level pathogenic assertion; it provides only gene-level MAP discussion and testing recommendations.

Still missing

• No ClinGen/InSiGHT expert-panel classification of this variant exists in ClinVar (0 expert-panel submissions), so PP5 can never be triggered for this variant under the applicable rule.

Sources

Reasoning

BA1 (stand-alone benign) requires an allele frequency of at least 0.05 (5%) in a control or general population (ACMG/AMP 2015, PMID:25741868). NM_001128425.2:c.505-4A>G (MUTYH, splice-region, NC_000001.10:g.45798510T>C / NC_000001.11:g.45332838T>C) never approaches that value in any dataset interrogated. gnomAD v2.1 all-comers: total AF 0.00167588 (474/282836 alleles, 0 homozygotes), highest ancestry African/African American 1.77913% (444/24956 alleles, 0 homozygotes), grpmax FAF 0.0164467. gnomAD v4.1 all-comers: total AF 0.00090076 (1454/1614192 alleles, 8 homozygotes), highest ancestry African/African American 1.74459% (1309/75032 alleles, 8 homozygotes), grpmax FAF 0.0166596. The non-cancer subsets, checked because a specification calibrated against gnomAD v2.1 exomes might mandate them, do not raise the frequency: gnomAD v2.1 non-cancer exomes-only AF 0.00125357 (297/236924 alleles, 0 homozygotes) with exome grpmax FAF 0.0162815, and gnomAD v3.1 non-cancer genomes-only AF 0.00504293 (746/147930 alleles, 4 homozygotes) with grpmax FAF 0.0162335. gnomAD-Canada v1.0 (HostSeq) adds 13/18416 alleles (AF 0.00070591, 0 homozygotes). The single largest frequency figure anywhere in the case is the 1KG:MSL sub-population in the gnomAD v3.1 non-cancer view at 4.16667% (7/168 alleles, 0 homozygotes); this is a 168-allele sample and sits below 5% even if taken at face value, and the size-corrected grpmax FAF for that dataset is 1.62335%. No source view, ancestry group, or subset reaches 5%, and the highest ancestry frequency converges on ~1.6-1.8% across five independent dataset views. BA1 is not met.

Rule

ACMG/AMP 2015 BA1: stand-alone benign when allele frequency in a control/general population is >= 0.05 (PMID:25741868)

Evidence considered

• BA1 threshold applied from the supplied generic calibration block: stand-alone benign at allele frequency >= 0.05 (ACMG/AMP 2015; PMID:25741868).
• gnomAD v2.1 all-comers (GRCh37 1-45798510-T-C): total AF 0.00167588 (474/282836 alleles, 0 homozygotes); exome AF 0.00128449 (323/251462), genome AF 0.00481290 (151/31374); highest ancestry African/African American AF 0.0177913 (444/24956 alleles, 0 homozygotes); grpmax FAF 0.0164467.
• gnomAD v4.1 all-comers (GRCh38 chr1-45332838-T-C): total AF 0.00090076 (1454/1614192 alleles, 8 homozygotes); exome AF 0.00048157 (704/1461886, 4 homozygotes), genome AF 0.00492430 (750/152306, 4 homozygotes); highest ancestry African/African American AF 0.0174459 (1309/75032 alleles, 8 homozygotes); grpmax FAF 0.0166596 (exome grpmax FAF 0.0165617, genome grpmax FAF 0.0161622, joint grpmax FAF 0.0166596).
• gnomAD v2.1 non-cancer subset, reported by its exomes-only figures (grpmax FAF is the exome figure): exome AF 0.00125357 (297/236924 alleles, 0 homozygotes); African/African American exome-only 269/14900 (1.805%); exome grpmax FAF 0.0162815.
• gnomAD v3.1 non-cancer subset (genomes only, since v3 has no exome cohort): total AF 0.00504293 (746/147930 alleles, 4 homozygotes); African/African American 0.0172951 (711/41110 alleles, 4 homozygotes); the highest sub-population figure in any dataset is 1KG:MSL 0.0416667 (7/168 alleles, 0 homozygotes), a 168-allele sample whose size-corrected grpmax FAF is 0.0162335.
• gnomAD-Canada v1.0 (HostSeq genomes): AF 0.00070591 (13/18416 alleles, 0 homozygotes); African/African American 0.0107843 (11/1020 alleles, 0 homozygotes); European (non-Finnish) 0/11740.
• Even the maximum ancestry-group frequency across all five dataset views (1.77913%, gnomAD v2.1 African/African American) is roughly 28-fold below 0.05, and the maximum non-ancestry-specific total (0.501%, gnomAD v3.1 non-cancer) is two orders of magnitude below it, so no dataset, subset, or ancestry reaches the stand-alone threshold.
• Governing framework consulted first: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MUTYH, Version 1.0 (cspec doc 1742141534), which specifies autosomal recessive inheritance for familial adenomatous polyposis 2 (MONDO:0012041) and NM_001128425.2 as preferred transcript; the retrieved ruleset carries no BA1 rule or mandated population source, so the generic ACMG/AMP 2015 threshold was applied.

Sources

Reasoning

BS2 requires the variant to be observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) condition, and the ACMG/AMP 2015 text expressly conditions it on the disease under investigation being fully penetrant at an early age (PMID:25741868). Two of those premises are examined here. First, the homozygous-observation element is partially satisfied: MUTYH is the recessive gene in this case (the governing specification's ruleset assigns autosomal recessive inheritance to familial adenomatous polyposis 2, MONDO:0012041), and homozygotes are observed in the newer population releases - gnomAD v4.1 reports 8 homozygotes (5 XX, 3 XY, all in the African/African American group; 4 of the 8 in exomes and 4 in genomes), and the gnomAD v3.1 non-cancer genomes-only subset reports 4 (3 XX, 1 XY, African). However, the older and independently curated gnomAD v2.1 all-comers release reports 0 homozygotes among 474 carriers, the gnomAD v2.1 non-cancer exome subset reports 0 among 297 carriers, and gnomAD-Canada v1.0 reports 0 among 13 carriers, so the homozygous observations are confined to the newest, most cohort-aggregated releases. Second, and decisively, the required disease premise fails: MUTYH-associated polyposis is an adult-onset, incompletely penetrant condition - the reviewed colorectal cancer guideline states that MAP has an average age of onset in approximately the mid-50s (PMID:25452455) - so a homozygous adult in a population cohort may simply be presymptomatic rather than healthy-with-respect-to-this-disease, and BS2's full-penetrance-at-an-early-age precondition is not met. Because the criterion as written cannot be satisfied for this disorder, BS2 is not met; the 8 homozygous observations in gnomAD v4.1 are recorded as frequency/homozygote context that supports the BS1 and PM2 calls rather than as BS2 evidence, and the call is flagged for human review because a curator applying a looser homozygote-observation-only reading of BS2 for recessive genes would reach the opposite result.

Rule

ACMG/AMP 2015 BS2: observed in a healthy adult individual, homozygous for a recessive condition, with full penetrance expected at an early age (PMID:25741868)

Evidence considered

• BS2 definition (ACMG/AMP 2015, PMID:25741868): observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) condition, with full penetrance expected at an early age.
• Inheritance context from the governing specification ruleset (cspec doc 1742141534): MUTYH / familial adenomatous polyposis 2 (MONDO:0012041) is assigned autosomal recessive inheritance, so the recessive (homozygous) branch of BS2 is the applicable one.
• Homozygous observations: gnomAD v4.1 8 homozygotes among 1454 carriers / 1614192 alleles, all in the African/African American group (AFR AF 0.0174459, 1309/75032; AFR_XX 704/41482 with 5 homozygotes; AFR_XY 605/33550 with 3 homozygotes; exome component 4 homozygotes, genome component 4); gnomAD v3.1 non-cancer genomes-only 4 homozygotes (AFR 711/41110, AFR_XX 378/21982 with 3, AFR_XY 333/19128 with 1).
• Zero homozygotes in the other views: gnomAD v2.1 all-comers 0/282836 alleles (474 carriers, including 151 genome alleles); gnomAD v2.1 non-cancer exomes-only 0/236924 alleles (297 carriers); gnomAD-Canada v1.0 0/18416 alleles (13 carriers, 11 of them African ancestry).
• PMID:25452455 (ASCO endorsement of the ESMO familial-risk colorectal cancer clinical practice guidelines): MAP is autosomal recessive with an average age of onset in approximately the mid-50s, which contradicts BS2's requirement that the disease be fully penetrant at an early age and makes a healthy adult homozygote observation non-decisive for this condition.
• The variant is present as a common polymorphism in African ancestry (1.6-1.8% allele frequency across gnomAD v2.1, v4.1 and v3.1 non-cancer) with homozygotes appearing only at the frequency expected under Hardy-Weinberg equilibrium (~11 expected versus 8 observed in gnomAD v4.1 African alleles), which is benign-frequency and homozygote context rather than a healthy-homozygote BS2 observation; it is recorded here because it bears directly on BS2's homozygous-observation element.
• Governing framework consulted first: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis specification for MUTYH v1.0 (cspec doc 1742141534) returned an empty rule payload, so the generic ACMG/AMP 2015 BS2 definition was applied without any gene-specific modification.

Still missing

• The governing InSiGHT MUTYH v1.0 specification was retrieved with an empty criteria/rule payload, so no gene-specific BS2 modification (for example a recessive-adapted homozygote rule, or an explicit statement that BS2 does not apply to this adult-onset condition) could be read.
• Homozygous observations exist only in gnomAD v4.1 (8) and in the gnomAD v3.1 non-cancer genomes subset (4), while gnomAD v2.1 all-comers and non-cancer exomes report 0 homozygotes among 474 and 297 carriers respectively; no per-genotype quality metrics or age data were available to determine whether the v4.1 homozygotes are healthy adults with respect to MAP, which is what BS2 would require.

Sources

Reasoning

BS3 requires well-established in vitro or in vivo functional studies showing no damaging effect on the gene or gene product. The same absence of functional data that prevents PS3 prevents BS3. For NM_001128425.2:c.505-4A>G (-4 acceptor position, outside the canonical -1/-2 dinucleotide) no RNA/transcript analysis (RT-PCR, minigene) and no MUTYH 8-oxoguanine glycosylase activity assay demonstrating preserved splicing or normal enzyme function was identified in any retrieved source: the seven available full texts are guidelines, secondary-findings policy statements and general methodology (PMID:21325953, PMID:25645574, PMID:25452455, PMID:23788249, PMID:27854360, PMID:26467025, PMID:25741868) and contain no assay of this variant or of any MUTYH splice variant. ClinVar variation 138311 (17 submissions, 13 usable, 0 usable criterion leads, no expert-panel submission) provides no functional evidence of a benign effect, so the widely reported Benign classification cannot be decomposed into a BS3-positive functional lead. Computational splice predictions recorded for this case (Pangolin splice gain 0.014, splice loss -0.006) are predicted, not measured, splice effects and cannot substitute for an assay; the absence of SpliceAI scores after a lookup timeout is missing data and is not evidence of no splice impact (PMID:25741868 requires RNA or protein analysis to confirm impact for this variant class). The benign-direction evidence present in this case is population- and assertion-based (ClinVar Benign/Likely benign consensus; gnomAD v4.1 AF 0.000900 with FAF 1.67% and 8 homozygotes in African/African American ancestry), is addressed by the population/assertion criteria, and a benign classification reached from another source is never converted into BS3 functional evidence here. BS3 is therefore not met; the conclusion reflects the absence of a qualifying functional study rather than a demonstrated negative assay result, hence medium confidence.

Evidence considered

• Governing framework check: the InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for MUTYH v1.0 (CSpec doc 1742141534) is registered but its classification rules are not released ('Classification Rules In Prep') and no criterion payload was retrievable, so no VCEP-specific BS3 rule exists and generic ACMG/AMP 2015 BS3 is used.
• ClinVar variation 138311 (17 submissions; 13 usable for classification; 0 usable criterion leads; 0 expert-panel submissions): searched for submitter-provided functional evidence of no damaging effect (BS3 leads). None is present; the Benign/Likely benign classifications are single-submitter assertions and were not inferred into BS3.
• Seven full-text papers were screened for a functional demonstration of normal function for this variant (PMID:21325953, PMID:25645574, PMID:25452455, PMID:23788249, PMID:27854360, PMID:26467025, PMID:25741868); none names c.505-4A>G and none reports a MUTYH splicing or enzyme-activity assay, so no BS3 evidence exists.
• PMID:25741868 (ACMG/AMP 2015): defines BS3 as well-established functional studies showing no damaging effect, and states that for splice-region variants 'confirmation of impact requires functional analysis by either RNA or protein analysis' - i.e. only a measured, variant-specific assay result can settle this criterion; none is available.
• PMID:26467025 (laboratory scoring methodology, 11,894 variants/338 genes): documents that splice-effect evidence in this region (-3 to -10 of the intron end, which contains this -4 acceptor change) was routinely prediction-based rather than assay-based, and that transcript-splicing assays were only accepted when a concrete molecular consequence (e.g. mRNA truncation) was shown - i.e. a prediction-only dataset cannot stand in for a functional assay for BS3.
• Computational splice predictions recorded in this case: Pangolin splice gain 0.014 and splice loss -0.006 (SpliceAI itself returned no scores after a lookup timeout). These are in-silico predictions of the PP3/BP4 class and were explicitly not used as BS3 (functional) evidence.
• Benign-direction evidence was checked only to confirm it is not functional evidence: ClinVar Benign/Likely benign consensus and gnomAD v4.1 (AF 0.000900, 1454/1614192 alleles; FAF 1.67%; 8 homozygotes, African/African American) are population/assertion lines adjudicated by other criteria and were not converted into BS3.
• Search terms used when screening for functional evidence of preserved function: 'c.505-4A>G', 'MUTYH c.505-4A>G', 'NM_001128425.2:c.505-4A>G', 'NM_001048174.2:c.421-4A>G', 'minigene', 'RT-PCR', 'transcript analysis', 'glycosylase', '8-oxoG', 'splicing assay'.

Still missing

• No functional assay demonstrating normal splicing or preserved MUTYH function for c.505-4A>G is available in the retrieved evidence; a variant-specific RNA or enzyme-activity study would be required to apply BS3.
• The InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for MUTYH v1.0 (CSpec doc 1742141534) has no released classification rules, so no VCEP-specific BS3 criteria or strength calibration could be applied; generic ACMG/AMP 2015 BS3 was used.
• SpliceAI returned no scores (lookup timeout); no validated splice-impact prediction is available for this change.

Sources

Reasoning

BP6 (reputable source recently reports the variant as benign) is applied in this group only for an exact-variant ClinVar expert-panel classification of Benign or Likely benign. The exact-variant record (ClinVar variation 138311 = NM_001128425.2:c.505-4A>G) does carry a benign consensus - Benign in 12 clinical laboratories and Likely benign in 4, with 1 Uncertain significance - but that consensus comes entirely from ordinary laboratory submissions: the submission audit flags every one of the 16 submissions as is_expert_panel = false, expert-panel submissions = 0, and the review status is 'criteria provided, multiple submitters, no conflicts' (2 stars, api review_stars = 2), not 'reviewed by expert panel' (3 stars). Several submissions also carry 'no assertion criteria provided'. Under the rule applied here, ordinary laboratory submissions, aggregate ClinVar labels and non-expert assertions never trigger BP6, and no expert-panel benign classification of this variant exists, so BP6 is not met. The multi-laboratory benign consensus remains meaningful evidence, but it is captured by the population-frequency/benign criteria groups, not by BP6.

Rule

Group rule for BP6 as adjudicated here: exact-variant ClinVar expert-panel classification of Benign or Likely benign is required; ordinary laboratory submissions, aggregate labels and non-expert assertions never trigger it. Assessed against generic ACMG/AMP 2015 BP6 (PMID:25741868) 'reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation'.

Evidence considered

• ClinVar variation 138311 exact-variant benign consensus: Benign 12 clinical laboratories, Likely benign 4, Uncertain significance 1, last updated 2026-06-06.
• ClinVar review status 'criteria provided, multiple submitters, no conflicts' (2 stars) rather than expert-panel review; submission audit reports expert_panel_submissions = 0 and is_expert_panel = false for all 16 submissions (e.g. Ambry Genetics, Labcorp Genetics/Invitae, GeneDx, PreventionGenetics, ARUP, All of Us, Rigshospitalet, Dasa).
• Version 1.0 InSiGHT MUTYH specification identified as the governing framework, with no expert-panel variant-level classification deposited for this variant.
• Two submissions (Sinai Health System; True Health Diagnostics) are labelled 'no assertion criteria provided' and one is Uncertain significance, so the benign label is not a uniform, criteria-documented expert assertion.

Still missing

• No ClinGen/InSIGHT expert-panel classification of this variant exists in ClinVar (0 expert-panel submissions), so BP6 cannot be triggered for this variant under the applicable rule even though a multi-laboratory benign consensus is present.

Sources

Reasoning

Governing framework: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MUTYH v1.0 (CSPEC doc 1742141534; MUTYH, autosomal recessive inheritance, MONDO:0012041 familial adenomatous polyposis 2) is the applicable gene-specific framework for this case, but its criterion-level rule payload is empty in the case materials (framework_mode 'unstructured_ruleset', framework_complete false, rule_payload {}), so PS2 was evaluated against its generic ACMG/AMP definition: a de novo (confirmed by parental testing, i.e. both parents shown not to carry the variant, with parentage/relationship confirmed) occurrence in a patient with the disease. PS2 cannot be assessed here because the case contains no proband or patient record at all - no phenotype/affectedness statement, no zygosity, no family structure, no parental genotypes and no count of tested parents. The retrieved evidence is entirely reference-database level (ClinVar record VCV000138311, gnomAD v2.1/v4.1, gnomAD-Canada, COSMIC and SpliceAI lookups); none of it reports an individual in whom this allele arose de novo. The ClinVar submission audit is consistent with this: 17 submissions, 0 expert-panel submissions, 0 usable criterion leads, and no criteria_found signals in any submitter's evidence, so no submitter documents a de novo observation. Population data cannot substitute for, or negate, a de novo observation: gnomAD v4.1 reports 1454/1614192 alleles (AF 0.00090; popmax African/African American AF 1.7446%, 1309/75032) with 8 homozygotes, and gnomAD v2.1 reports 474/282836 alleles (AF 0.00168, 444/24956 in African/African American, homozygotes 0) - a frequency/homozygote profile wholly incompatible with a de novo pathogenic allele, but this remains frequency evidence and is not PS2 evidence (and is not an absence-of-de-novo statement either). No full-text paper reviewed for this criterion names the variant or reports family-level data. The specific gap is proband-level clinical data: a confirmed de novo observation requires an affected proband plus tested parents, neither of which exists in this case.

Evidence considered

• The governing gene-specific framework in this case is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MUTYH v1.0 (svi doc 1742141534); its parsed rule payload is empty, so PS2 was applied per the generic ACMG/AMP 2015 definition (de novo occurrence in a patient with the disease, confirmed by testing of both parents; PMID:25741868).
• The case bundle contains no proband, no patient phenotype, no affectedness or zygosity, no pedigree, and no parental testing results - the evidence is limited to database lookups for the variant itself.
• ClinVar VCV000138311 (ClinVarID 138311) holds 17 submissions (12 laboratories Benign, 4 Likely benign, 1 Uncertain significance; 0 expert-panel submissions) and the submission audit found 0 usable criterion leads and no criteria_found signals, i.e. no submitter documents a de novo occurrence.
• gnomAD v4.1: 1454/1614192 alleles, AF 0.00090, popmax African/African American AF 1.7446% (1309/75032), 8 homozygotes - a frequency/homozygote profile incompatible with a de novo pathogenic allele, but this is population-frequency context, not PS2 evidence.
• gnomAD v2.1: 474/282836 alleles, AF 0.00168, African/African American AF 1.7791% (444/24956), 0 homozygotes.
• No full-text paper reviewed for this group (MAP/Lynch review, ACG and ASCO hereditary CRC guidelines, ACMG SF v1.0/v2.0 policy statements, Quest rules-based scoring paper, ACMG/AMP 2015 standards) names c.505-4A>G or reports family-level/de novo data for it.

Still missing

• No proband or affected patient record exists in this case (no phenotype, affectedness, zygosity or family structure), so a de novo occurrence cannot be established.
• No parental testing results (and no confirmation of parentage/relationship) are available for any individual carrying this allele.
• The applicable MUTYH InSiGHT CSPEC v1.0 rules for PS2 could not be read from the case materials (rule_payload empty, criteria list empty); only the generic ACMG/AMP definition was available.
• If a proband with this variant exists outside this variant-level record, pedigree and parental genotypes would be required to assess PS2.

Sources

Reasoning

PM3 was evaluated under the generic ACMG/AMP 2015 definition because the governing gene-specific framework for this case - the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification to the ACMG/AMP guidelines for MUTYH v1.0 (doc 1742141534) - was identified but its retrieved criterion payload was empty, so no MUTYH-specific PM3 rule could be applied. For an autosomal-recessive disorder such as MUTYH-associated polyposis, PM3 requires the variant to be detected in trans with a pathogenic MUTYH allele in an affected individual (PMID:25741868); the same biallelic requirement underpins MUTYH testing and reporting guidance (PMID:21325953, PMID:27854360). No source in this case supplies that configuration. None of the seven full-text papers extracted for this case names c.505-4A>G; all are gene- or syndrome-level documents (MAP is described as autosomal recessive with biallelic MYH mutations). The 16 ClinVar submissions for ClinVarID 138311 (12 Benign, 4 Likely benign, 1 Uncertain significance) are laboratory classifications that state no phase, allele co-occurrence or pedigree information, and the submission audit recorded zero usable criterion leads, zero expert-panel submissions and no criterion signals - so ClinVar cannot be used to infer that a pathogenic MUTYH variant sits in trans. The population data are likewise uninformative for PM3: gnomAD v4.1 reports total AF 0.00090 (1,454/1,614,192 alleles) with 8 homozygotes, all in African/African American individuals (AF 0.0174, 1,309/75,032 alleles), and gnomAD v2.1 reports AF 0.00168 (474/282,836 alleles) with 0 homozygotes. Those are aggregate genotype counts from unascertained individuals, without phenotype or phase, and a homozygous population count is not the same observation as a proband carrying a pathogenic allele in trans; it cannot establish the configuration PM3 requires, and no clinical case observation of this variant exists in the case evidence to combine with it. PM3 is therefore recorded as not assessed rather than not met: the allelic-configuration evidence PM3 is defined on (a pathogenic MUTYH variant in trans, in an affected proband) does not exist here, and it cannot be scored from gene-level guideline text or population allele counts.

Rule

Generic ACMG/AMP 2015 PM3 (moderate, pathogenic): for a recessive disorder, detected in trans with a pathogenic variant; no MUTYH-specific rule was available in the retrieved InSiGHT specification payload.

Evidence considered

• Governing framework: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MUTYH v1.0 (cspec, doc 1742141534, MONDO:0012041 familial adenomatous polyposis 2, autosomal recessive, preferred transcript NM_001128425.2) is present for this case, but the retrieved ruleset contained an empty rule payload and no criteria entries, so PM3 was adjudicated with the generic ACMG/AMP 2015 definition.
• Generic PM3 as applied here: for a recessive disorder, PM3 requires the variant to be detected in trans with a pathogenic allele in an affected individual (PMID:25741868). MUTYH-associated polyposis is an autosomal-recessive, biallelic-mutation disorder, so a trans configuration is the configuration this criterion tests (PMID:21325953).
• MUTYH secondary-findings guidance similarly states that for MUTYH the recommendation is to search only for individuals with biallelic mutations (PMID:27854360), i.e. pathogenic MUTYH assertions are made in an allele-configuration context that is entirely absent from this case's data.
• No affected-individual observation of this variant exists in the case evidence. The ClinVar record for ClinVarID 138311 (16 submissions: 12 Benign, 4 Likely benign, 1 Uncertain significance; merged API status Benign/Likely benign, 2 stars) supplies laboratory-level classifications only, with no phase, cis/trans, co-occurrence or pedigree content; the submission audit recorded 0 usable criterion leads, 0 expert-panel submissions, 0 criterion-lead submissions and empty evidence_signals for every submitter (clinvar).
• Population genotypes are uninformative for PM3: gnomAD v4.1 total AF 0.00090 (1,454/1,614,192 alleles) with 8 homozygotes, all in the African/African American group (AF 0.01744, 1,309/75,032 alleles, 8 homozygotes); gnomAD v2.1 total AF 0.00168 (474/282,836 alleles) with 0 homozygotes and AFR AF 0.01779 (444/24,956 alleles). These are aggregate allele and homozygote counts from individuals not ascertained for polyposis, with no phenotype and no phase information, and they cannot show that this allele sits in trans with a pathogenic MUTYH variant (gnomad_v4).
• All seven full-text papers extracted for this case were checked for the variant c.505-4A>G (and for p.?, MUTYH c.505-4A>G, MYH c.505-4A>G, G396D, Y179C): PMID:21325953, PMID:25645574, PMID:25452455, PMID:27854360, PMID:26467025, PMID:23788249 and PMID:25741868. None mentions this variant or reports any genotype, phase, segregation or case-level data for it; their content is gene-, syndrome- or methodology-level.

Still missing

• No affected-proband genotype data: no case report, cohort or ClinVar submission describes c.505-4A>G in trans with a pathogenic MUTYH allele.
• No phase or co-occurrence data: the 16 ClinVar submissions for ClinVarID 138311 provide classifications only, with no pedigree, allele-phase or second-variant information.
• gnomAD v4.1 records 8 homozygotes (all African/African American) but these individuals are unascertained for phenotype, so homozygosity cannot be converted into a trans-with-pathogenic observation.
• The governing InSiGHT MUTYH v1.0 specification was retrieved with an empty criterion payload, so a MUTYH-specific PM3 rule (if one exists) could not be consulted; generic ACMG/AMP 2015 semantics were used.

Sources

Reasoning

PM6 covers an assumed de novo occurrence (proband observed to carry the variant without it being found in either parent, but with parentage/relationship not formally confirmed, or parental testing incomplete). The applicable gene-specific framework for MUTYH is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification v1.0 (CSPEC doc 1742141534; autosomal recessive, MONDO:0012041 familial adenomatous polyposis 2), but its criterion-level rules are not machine-readable in the case materials (rule_payload {}), so the generic ACMG/AMP 2015 definition was used (PMID:25741868). PM6 cannot be assessed for the same structural reason as PS2: PM6 still requires a proband in whom the variant is observed and an observation that it is absent from the parents, and this case contains no proband, no patient phenotype/affectedness statement, no zygosity and no parental genotypes. Nothing in the retrieved evidence is individual-level: ClinVar VCV000138311 (17 submissions, no expert panel, no criterion leads, no criteria_found signals), gnomAD v2.1/v4.1, gnomAD-Canada, COSMIC and SpliceAI are all population/variant-level resources and none reports a transmitting or non-transmitting parent pair. As with PS2, the population profile argues strongly against any de novo mechanism (gnomAD v4.1 AF 0.00090 overall, popmax African/African American 1.7446%, 8 homozygotes; gnomAD v2.1 AF 0.00168, 0 homozygotes; ClinVar classified Benign/Likely benign by 16 laboratories), but PM6 is a family/parental-observation criterion and frequency data cannot be converted into it. No reviewed full-text paper names the variant or provides parental data. Decisive gap: no proband and no parental genotype data exist in the case.

Evidence considered

• Governing framework consulted: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MUTYH v1.0 (doc 1742141534; autosomal recessive inheritance, familial adenomatous polyposis 2, MONDO:0012041); its parsed criterion rules are empty, so PM6 was applied per the generic ACMG/AMP 2015 definition of an assumed de novo variant (PMID:25741868).
• The case record is variant-level only: no proband, no affectedness/phenotype, no zygosity, no pedigree and no parental genotypes appear anywhere in the evidence bundle, prefetch, or reference-database captures.
• ClinVar VCV000138311 (ClinVarID 138311): 17 submissions, 12 Benign / 4 Likely benign / 1 Uncertain significance, 0 expert-panel submissions, 0 usable criterion leads and no criteria_found signals - no submitter reports a de novo or parental-observation event.
• gnomAD v4.1 (1454/1614192 alleles, AF 0.00090, 8 homozygotes, popmax African/African American AF 1.7446%) and gnomAD v2.1 (474/282836 alleles, AF 0.00168, 0 homozygotes) show a common, homozygote-observed allele incompatible with de novo occurrence, but this is population-frequency context only and is not PM6 evidence.
• No reviewed full-text source (PMID:21325953, 25645574, 25452455, 27854360, 23788249, 26467025, 25741868) mentions c.505-4A>G or provides parental/de novo data for MUTYH.

Still missing

• No proband observation (variant carrier status in an affected individual) exists in this case.
• No parental genotype data of any kind (confirmed or unconfirmed parentage) are available, so neither the confirmed nor the assumed de novo route is evaluable.
• The applicable MUTYH InSiGHT CSPEC v1.0 rules for PM6 could not be read from the case materials (rule_payload empty).
• A proband report stating the variant was absent from both parents (even without formal parentage testing) would be required to assess PM6.

Sources

Reasoning

PP1 requires co-segregation of the variant with disease in multiple affected family members of a gene already definitively known to cause the disease; strength is modulated by the number of informative meioses. The applicable gene-specific framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MUTYH v1.0 (doc 1742141534; MUTYH causes autosomal-recessive familial adenomatous polyposis 2 / MAP, MONDO:0012041), which is directly relevant because MAP is recessive and segregation arguments therefore differ from those in dominant disease; however its criterion-level rule payload is empty in the case materials (rule_payload {}), so PP1 was evaluated against its generic ACMG/AMP 2015 definition (PMID:25741868). No segregation evidence exists in this case: there is no pedigree, no affected relative, no unaffected relative and no countable meiosis anywhere in the captured evidence - the case is a variant-level record (ClinVar/gnoMAD/COSMIC/SpliceAI lookups) with no proband or family attached. The ClinVar submission audit likewise shows 0 usable criterion leads and no criteria_found signals across 17 submissions, so no laboratory reports a segregating family. The reviewed literature was checked for family or meiosis data and provides none: the MAP/Lynch review (PMID:21325953), the ACG hereditary GI cancer guideline (PMID:25645574, which addresses family-history-based risk assessment only at guideline level) and the ASCO endorsement (PMID:25452455) discuss MAP at the gene/syndrome level with no variant-specific pedigree; the Quest rules-based scoring paper (PMID:26467025) mentions segregation only as a generic evidence category in a scoring system and names neither MUTYH nor this variant. The gap is the enumeration itself: a family with multiple affected carriers, genotypes for affected and unaffected relatives and a count of informative meioses would be required before any PP1 strength could be assigned.

Evidence considered

• Governing framework consulted: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MUTYH v1.0 (doc 1742141534; MUTYH, autosomal recessive, familial adenomatous polyposis 2 / MAP, MONDO:0012041); its parsed criterion rules and any gene-specific meiosis thresholds are empty in the case materials, so the generic ACMG/AMP 2015 PP1 definition (co-segregation with disease in multiple affected family members; PMID:25741868) was applied, with no numeric threshold asserted.
• The case contains no pedigree, no affected or unaffected relatives, no carrier genotypes and no informative meioses - the evidence is limited to variant-level database captures (ClinVar VCV000138311, gnomAD v2.1/v4.1, gnomAD-Canada, COSMIC, SpliceAI).
• ClinVar submission audit: 17 submissions, 0 expert-panel submissions, 0 usable criterion leads, and no criteria_found signals in any submitter's evidence, i.e. no laboratory reports segregation data for this variant.
• PMID:26467025 (Quest Diagnostics rules-based pathogenicity scoring system, 11,894 variants in 338 genes) lists segregation as one of the scored evidence categories but names neither MUTYH nor c.505-4A>G; it supplies a generic methodology reference only, no segregation data for this variant.
• PMID:21325953, PMID:25645574 and PMID:25452455 were checked for variant-level family/segregation data: all three discuss MUTYH/MAP only at gene/syndrome level (autosomal-recessive inheritance, testing and surveillance recommendations) and never name c.505-4A>G.
• Autosomal-recessive inheritance of MAP means family-based co-segregation for a single heterozygous allele cannot be interpreted, and no biallelic family data are present.

Still missing

• No pedigree or family structure is available for any individual carrying this variant.
• No genotypes for affected or unaffected relatives, and therefore no count of informative meioses, are available; PP1 strength cannot be computed without them.
• MAP is autosomal recessive, and no biallelic carrier/family data are present, so segregation arguments could not be constructed even indirectly.
• The applicable MUTYH InSiGHT CSPEC v1.0 rules for PP1 (including any gene-specific meiosis thresholds) could not be read from the case materials (rule_payload empty).

Sources

Reasoning

PP4 requires that the patient's phenotype or family history be highly specific for a disease with a single genetic etiology (generic ACMG/AMP 2015, PMID:25741868; the InSiGHT MUTYH v1.0 VCEP criterion text could not be retrieved, so no VCEP-specific PP4 modification is available). This case is a variant-level curation: the evidence bundle contains no proband, no HPO/indication field, no family history and no clinical condition other than the generic ClinVar condition labels ('Familial adenomatous polyposis 2', 'Hereditary cancer-predisposing syndrome', 'not provided', 'not specified'). None of the retrieved publications describes a patient carrying c.505-4A>G, so no phenotype can be scored. Independently, the gene-disease context weighs against PP4 even if a phenotype were present: MUTYH-associated polyposis / familial adenomatous polyposis 2 is an autosomal-recessive adenomatous polyposis phenotype that is not specific to a single genetic etiology, with overlapping colorectal polyposis/CRC presentations caused by APC, biallelic MUTYH, NTHL1, MSH3, POLE/POLD1 and (with childhood/adolescent onset) the MMR genes, and the disease spectrum quoted in the consulted guidelines spans attenuated FAP through Lynch-like presentations. With no phenotype or family-history data to evaluate, PP4 is not assessed rather than met or refuted; the missing phenotype data are recorded as the gap.

Rule

Generic ACMG/AMP 2015 PP4 (PMID:25741868): patient's phenotype or family history highly specific for a disease with a single genetic etiology. No MUTYH VCEP-specific PP4 rule was retrievable; the sibling InSiGHT colorectal specification treats PP4 as captured by PS4/not applicable, which cannot be assumed for MUTYH here.

Evidence considered

• ClinVar conditions for variation 138311 are generic ('Familial adenomatous polyposis 2', 'Hereditary cancer-predisposing syndrome', 'not provided', 'not specified') and the submission audit records no clinical detail, so no specific patient phenotype is documented.
• Governing gene-disease context (cspec / InSiGHT Hereditary Colorectal Cancer/Polyposis specification for MUTYH v1.0): MUTYH, transcripts NM_001128425.2 and NM_001048174.2, familial adenomatous polyposis 2 (MONDO:0012041), autosomal recessive inheritance.
• ACG clinical guideline (PMID:25645574) treats MUTYH-associated polyposis within the group of adenomatous polyposis syndromes and advises APC plus MUTYH analysis for suspected adenomatous polyposis - i.e. the phenotype is shared across genes.
• ASCO/ESMO endorsement guideline (PMID:25452455) describes MAP as clinically resembling classic or attenuated FAP with autosomal-recessive inheritance, and notes that up to one third of biallelic MUTYH carriers in population-based CRC studies developed CRC without colorectal polyposis.
• Lynch syndrome / MYH-associated polyposis review (PMID:21325953) notes MAP has a highly variable phenotype overlapping attenuated FAP through to Lynch syndrome, which makes the phenotype non-specific.
• ACMG/AMP 2015 (PMID:25741868) PP4 definition: patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Still missing

• No patient-level phenotype, HPO term, family history or indication for testing is present anywhere in the case materials, so phenotype specificity cannot be scored.
• MUTYH v1.0 VCEP criterion text (which would define any gene-specific PP4 instruction) could not be retrieved from the case materials, vcep_db/MUTYH or the captured cspec page.

Sources

Reasoning

BS4 requires lack of segregation: the variant is absent from affected family members, or the variant fails to track with disease within a pedigree. The applicable gene-specific framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MUTYH v1.0 (doc 1742141534; autosomal-recessive familial adenomatous polyposis 2 / MAP, MONDO:0012041); its criterion-level rule payload is empty in the case materials (rule_payload {}), so the generic ACMG/AMP 2015 definition was applied (PMID:25741868). No non-segregation evidence exists in this case: there is no pedigree, no affected relative genotyped for this allele, and therefore no family in which the variant could be shown to fail to track with disease - the case is a variant-level record with database captures only (ClinVar, gnomAD v2.1/v4.1, gnomAD-Canada, COSMIC, SpliceAI). The ClinVar submission audit shows 17 submissions with 0 expert-panel submissions, 0 usable criterion leads and no criteria_found signals, so no submitter reports a non-segregating family either. The reviewed literature contains no variant-level family data: PMID:21325953 (MAP/Lynch review), PMID:25645574 (ACG hereditary GI cancer guideline) and PMID:25452455 (ASCO endorsement) describe MAP at the gene/syndrome level; PMID:26467025 mentions segregation only as a generic scored category; PMID:23788249 and PMID:27854360 are ACMG secondary-findings policy statements listing MUTYH on the returnable-gene list. Population and classification observations (gnomAD v4.1 AF 0.00090 overall with popmax African/African American AF 1.7446% and 8 homozygotes; ClinVar Benign/Likely benign by 16 laboratories) do not constitute BS4, which is by definition a within-family argument. The gap is therefore a genotyped multi-affected family, or an affected carrier with an affected relative who does not carry the allele, plus a count of informative meioses.

Evidence considered

• Governing framework consulted: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MUTYH v1.0 (doc 1742141534; autosomal recessive, familial adenomatous polyposis 2 / MAP, MONDO:0012041); its parsed criterion rules are empty, so the generic ACMG/AMP 2015 BS4 definition (absence of segregation / failure to track with disease in a family; PMID:25741868) was applied.
• The case contains no pedigree and no relative genotypes; the evidence is limited to variant-level database captures (ClinVar VCV000138311, gnomAD v2.1/v4.1, gnomAD-Canada, COSMIC, SpliceAI), none of which is a within-family non-segregation observation.
• ClinVar submission audit: 17 submissions, 0 expert-panel submissions, 0 usable criterion leads and no criteria_found signals, so no submitter reports non-segregation for this variant.
• Population/classification context that must not be mistaken for BS4: gnomAD v4.1 AF 0.00090 overall with popmax African/African American AF 1.7446% and 8 homozygotes, and ClinVar Benign/Likely benign in 16 of 17 submissions - these are frequency/classification data, not family segregation data.
• Reviewed full-text sources (PMID:21325953, PMID:25645574, PMID:25452455, PMID:23788249, PMID:27854360, PMID:26467025, PMID:25741868) contain no variant-specific pedigree, non-segregation or family data for MUTYH c.505-4A>G.

Still missing

• No pedigree exists, so there is no family in which lack of co-segregation could be demonstrated.
• No genotypes are available for affected relatives of a carrier, so neither an affected non-carrier nor a non-tracking pedigree could be shown.
• No count of informative meioses is available, so no BS4 strength could be assigned.
• The applicable MUTYH InSiGHT CSPEC v1.0 rules for BS4 could not be read from the case materials (rule_payload empty).

Sources

Reasoning

BP2 was evaluated under the generic ACMG/AMP 2015 definition because the governing gene-specific framework for this case - the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification to the ACMG/AMP guidelines for MUTYH v1.0 (doc 1742141534) - was identified but its retrieved criterion payload was empty, so no MUTYH-specific BP2 rule could be applied. Generic BP2 is met by evidence that the variant is observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant under any inheritance pattern (PMID:25741868). The trans arm cannot be applied here: MUTYH-associated polyposis (MONDO:0012041, familial adenomatous polyposis 2) and the gene-level literature (PMID:21325953) and MUTYH secondary-findings policy (PMID:27854360) all describe autosomal-recessive inheritance requiring biallelic mutations, so a pathogenic MUTYH variant in trans is the expected pathogenic configuration (PM3 territory), not benign evidence. The cis arm is applicable to any inheritance pattern and would be testable, but no cis observation exists in this case: none of the seven full-text papers extracted for this case names c.505-4A>G, and the 16 ClinVar submissions for ClinVarID 138311 (12 Benign, 4 Likely benign, 1 Uncertain significance) report only classifications, with no phase, same-allele co-occurrence, pedigree or companion-variant data; the submission audit recorded zero criterion leads and empty evidence signals for every submitter, so no cis pairing with a pathogenic MUTYH variant can be inferred from ClinVar either. The population data do not close the gap: gnomAD v4.1 reports the variant at total AF 0.00090 (1,454/1,614,192 alleles) with 8 homozygotes, all in African/African American individuals (AF 0.0174), and gnomAD v2.1 reports 0 homozygotes; these are aggregate counts without phase or clinical ascertainment, and homozygosity is not the same observation as cis pairing with a pathogenic variant in a phase-resolved individual. BP2 is therefore recorded as not assessed rather than not met: the criterion is applicable (its cis arm) but the allelic-configuration evidence it requires is entirely absent, so it can be neither met nor refuted.

Rule

Generic ACMG/AMP 2015 BP2 (supporting, benign): observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern; only the cis arm is applicable to this autosomal-recessive gene. No MUTYH-specific rule was available in the retrieved InSiGHT specification payload.

Evidence considered

• Governing framework: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MUTYH v1.0 (cspec, doc 1742141534) is present for this case but its retrieved ruleset had an empty rule payload and no criteria entries, so BP2 was adjudicated with the generic ACMG/AMP 2015 definition.
• Generic BP2 as applied here: observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern (PMID:25741868).
• Inheritance-mode check: MUTYH disease is autosomal recessive (MONDO:0012041, familial adenomatous polyposis 2) and the literature describes MAP as 'an autosomal recessive, polyposis syndrome caused by biallelic mutations in the MYH gene' (PMID:21325953); ACMG SF v2.0 lists MUTYH as autosomal recessive and recommends searching only for individuals with biallelic mutations (PMID:27854360). The trans-with-a-pathogenic-variant arm of BP2 therefore does not apply in the dominant-gene sense, and only the cis arm is testable for this variant.
• No cis or trans observation exists in the case evidence. The ClinVar record for ClinVarID 138311 (16 submissions: 12 Benign, 4 Likely benign, 1 Uncertain significance) contains no phase, cis/trans, co-occurrence or pedigree information, and the submission audit found 0 criterion leads, 0 expert-panel submissions and empty evidence_signals/criteria_found for every submitter (clinvar).
• Population genotypes do not provide the missing allelic configuration: gnomAD v4.1 total AF 0.00090 (1,454/1,614,192 alleles) with 8 homozygotes, all African/African American (AF 0.01744, 1,309/75,032 alleles, 8 homozygotes); gnomAD v2.1 total AF 0.00168 (474/282,836 alleles) with 0 homozygotes, and gnomAD-Canada v1.0 13/18,416 alleles with 0 homozygotes (gnomad_v4). These are unascertained aggregate counts without phase or phenotype, so they cannot establish cis pairing with a pathogenic MUTYH variant.
• All seven full-text papers extracted for this case were checked for c.505-4A>G (and for p.?, MUTYH c.505-4A>G, MYH c.505-4A>G, G396D, Y179C): PMID:21325953, PMID:25645574, PMID:25452455, PMID:27854360, PMID:26467025, PMID:23788249 and PMID:25741868. None names the variant or reports any allele-configuration, phase or co-occurrence data for it.

Still missing

• No cis/phase data: no source in this case reports c.505-4A>G co-occurring on the same allele (cis) with a pathogenic MUTYH variant in any individual.
• No trans data either: ClinVar submissions for ClinVarID 138311 carry classifications only, with no second-variant or pedigree information, so the trans arm could not be examined even as context.
• gnomAD v4.1 homozygote counts (8 individuals, all African/African American) are unascertained aggregate genotypes with no phase or phenotype data and cannot substitute for a cis observation.
• The governing InSiGHT MUTYH v1.0 specification was retrieved with an empty criterion payload, so a MUTYH-specific BP2 rule (if one exists) could not be consulted; generic ACMG/AMP 2015 semantics were used.

Sources

Reasoning

BP5 requires that the variant be found in a case with an alternate molecular basis for disease (generic ACMG/AMP 2015, PMID:25741868). The generic source attaches no numeric threshold and no likelihood-ratio form to BP5, so there is no operator to evaluate in either direction; the MUTYH v1.0 VCEP criterion text, which might define a gene-specific form of BP5, could not be retrieved. No case exists to evaluate for this variant: this is a variant-level curation with no proband, no second-site genotype and no co-occurring variant data anywhere in the case materials, no publication describing a carrier of c.505-4A>G (all seven full-text extractions record variant_mentioned=false), and no criterion leads or case reports in the ClinVar record (submission audit: 0 usable criterion leads). Because no case in which an alternate molecular basis could be identified - or excluded - is available, BP5 cannot be assessed; the absence of case-level genotype data is the recorded gap. Nothing in the retrieved evidence argues positively for an alternate molecular basis in a MUTYH carrier.

Rule

Generic ACMG/AMP 2015 BP5 (PMID:25741868): variant found in a case with an alternate molecular basis for disease; no numeric or likelihood-ratio threshold is defined in the generic source. No MUTYH VCEP-specific BP5 rule was retrievable.

Evidence considered

• ClinVar variation 138311: no case reports, no genotype/co-segregation data, submission audit criterion leads = 0, evidence_signals criteria_found = [] for all 16 submissions.
• No proband or co-occurring variant information appears anywhere in the case materials (no phenotype/proband fields present in case_summary.json, evidence.json or the submission audit).
• Full-text extraction of all seven retrieved publications records variant_mentioned = false for each, so no case carrying c.505-4A>G is described.
• ACMG/AMP 2015 (PMID:25741868) BP5 definition: variant found in a case with an alternate molecular basis for disease; no numeric threshold is defined for this criterion in the generic source.

Still missing

• No case/proband-level data (no co-occurring pathogenic variant in another polyposis gene, no second MUTYH allele status) are available for this variant, so an alternate molecular basis cannot be evaluated.
• MUTYH v1.0 VCEP criterion text (which could redefine BP5, including any likelihood-ratio-style rule that strengthens as the value falls) could not be retrieved; no VCEP-specific BP5 operator was therefore available.
• MUTYH disease is autosomal recessive, so alternate-molecular-basis reasoning would require biallelic status information that is not present in the case materials.

Sources