NTHL1 c.31C>T, p.(Arg11Trp)

Reasoning

PM2 is met at supporting strength. The allele is rare in large population datasets: gnomAD v4.1 AF is 0.00528% overall with a maximum ancestry-specific AF of 0.01472% (African/African American), and gnomAD v2.1 AF is 0.00376% overall with a maximum ancestry-specific AF of 0.01919% (African/African American). Both are below the generic 0.1% PM2 rarity threshold. The absence of homozygotes is consistent with, but does not independently establish, rarity evidence.

Rule

Generic ACMG/AMP fallback: rare population frequency below the generic 0.1% PM2 threshold supports PM2 at supporting strength when no gene-specific specification is available.

Evidence considered

• gnomAD v4.1 reports 84/1,591,356 alleles (AF 0.00528%), African/African American 11/74,734 (AF 0.01472%), grpmax filtering AF 0.008173%, and 0 homozygotes.
• gnomAD v2.1 reports 9/239,502 alleles (AF 0.00376%), African/African American 4/20,842 (AF 0.01919%), grpmax filtering AF 0.006724%, and 0 homozygotes.
• The v4.1 and v2.1 maximum AFs are both below 0.1%; sparse ancestry counts do not create a threshold artifact that would negate PM2.

Still missing

• No NTHL1-specific PM2 frequency threshold or disease-specific maximum credible allele-frequency calculation was retrieved.

Sources

Reasoning

NM_002528.7:c.31C>T (p.Arg11Trp) is a missense substitution with no NTHL1-specific VCEP PP3/BP4 lookup available (cspec.found=false; framework_mode=generic_acmg). Applying this lab's generic ACMG/AMP PP3/BP4 in-silico calibration for missense variants (REVEL-only path), the REVEL score of 0.196 is below the BP4 supporting threshold of <0.250, indicating the variant is computationally predicted as likely benign at the protein level. BP4 is met at supporting strength. SpliceAI (max delta score = 0.00) was not used to determine BP4 here because this variant is missense and the calibration explicitly reserves SpliceAI for intronic/synonymous/non-canonical-splice-position variants, not missense substitutions with their own REVEL-assessable protein effect; the low SpliceAI delta is consistent with (not independently used to support) the absence of a competing splice-disruption signal.

Rule

generic_acmg_missense_revel

Evidence considered

• REVEL score = 0.196 (source_registry 'revel', local REVEL v1.3 lookup) for NM_002528.7:c.31C>T (p.Arg11Trp), below the generic ACMG/AMP fallback BP4 threshold of <0.250 for missense variants.
• generic_acmg_classification_rules.md PP3/BP4 in-silico calibration section specifies REVEL-only assessment for missense variants and instructs not to combine or substitute a splice-path score for a missense variant.
• SpliceAI max delta score = 0.00 noted in prefetch data as supplementary context only; not independently applied for BP4 because the variant type is missense, per calibration rule restricting SpliceAI use to non-missense variant types.
• No NTHL1 VCEP-specific PP3/BP4 lookup spreadsheet was retrieved (vcep_materials.json: found=false, no cspec url available), so generic ACMG/AMP fallback governs per generic_acmg_classification_rules.md guardrail.

Sources

Reasoning

NM_002528.7:c.31C>T (p.Arg11Trp) is a missense substitution. No NTHL1 ClinGen CSPEC / VCEP-specific PP3/BP4 in-silico lookup was retrieved for this case (cspec.found=false; vcep_materials note='no cspec url available'; final_classification_framework.found=false, framework falls back to generic_acmg). Per this lab's generic ACMG/AMP PP3/BP4 in-silico calibration for a missense variant, REVEL is the sole predictor to use (SpliceAI is reserved for intronic/synonymous/non-canonical-splice-position variants and must not be substituted for a missense variant's own protein-level effect). REVEL score = 0.196, which falls below the BP4 threshold of <0.250, not above the PP3 threshold of >0.750. Therefore PP3 is not met. BayesDel score (-0.229484) exists but has no verified published calibration threshold available to this pipeline and is not usable for PP3/BP4 per lab policy.

Rule

generic_acmg_missense_revel

Evidence considered

• REVEL score = 0.196 (source_registry 'revel', local REVEL v1.3 lookup) for NM_002528.7:c.31C>T (p.Arg11Trp), a missense variant.
• generic_acmg_classification_rules.md PP3/BP4 in-silico calibration section: missense variants use REVEL only, REVEL >0.750 -> PP3 supporting, REVEL <0.250 -> BP4 supporting, REVEL in [0.250,0.750] -> neither met.
• No NTHL1-specific ClinGen VCEP CSPEC PP3/BP4 lookup table was found in vcep_materials.json (found=false, no cspec url available), so the generic ACMG/AMP fallback threshold set governs.
• BayesDel score -0.229484 present in prefetch data but excluded from consideration because no named, citable publication establishes a verified BayesDel threshold for PP3/BP4 in this pipeline.

Sources

Reasoning

ClinVar has no exact-variant expert-panel submission for a Pathogenic or Likely pathogenic classification; the recorded submissions are non-expert Uncertain significance assertions.

Rule

Apply PP5 only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion.

Evidence considered

• The exact-variant ClinVar submission audit reports zero expert-panel submissions; all reported clinical classifications are Uncertain significance.

Sources

Reasoning

BA1 is not met. The highest observed population AF is 0.01472% in gnomAD v4.1 African/African American (11/74,734 alleles); this is well below the generic 1% BA1 threshold. gnomAD v2.1 similarly reports a highest AF of 0.01919% (4/20,842 alleles).

Rule

Generic ACMG/AMP fallback: BA1 requires an allele frequency above the stand-alone benign threshold; the highest observed ancestry-specific frequency is below 1%.

Evidence considered

• No NTHL1 CSPEC/VCEP or local gene-specific framework was retrieved; generic ACMG/AMP population-frequency assessment applies.
• gnomAD v4.1: 84/1,591,356 alleles overall (AF 0.00528%); maximum ancestry-specific AF is African/African American 11/74,734 (0.01472%), with zero homozygotes.
• gnomAD v2.1: 9/239,502 alleles overall (AF 0.00376%); maximum ancestry-specific AF is African/African American 4/20,842 (0.01919%), with zero homozygotes.

Sources

Reasoning

BS1 is not met. The highest observed population AF, 0.01472% in gnomAD v4.1 African/African American, is below the generic 0.3% BS1 threshold; the v2.1 African/African American estimate is 0.01919%, also below this threshold.

Rule

Generic ACMG/AMP fallback: BS1 requires a frequency greater than expected for the disorder; neither observed ancestry-specific AF reaches the generic 0.3% threshold.

Evidence considered

• gnomAD v4.1 African/African American AF is 11/74,734 alleles (0.01472%); overall AF is 84/1,591,356 (0.00528%).
• gnomAD v2.1 African/African American AF is 4/20,842 alleles (0.01919%); overall AF is 9/239,502 (0.00376%).
• The modest difference between v2.1 and v4.1 ancestry estimates reflects sparse allele counts and does not approach a generic BS1 threshold.

Still missing

• No NTHL1 disease-specific prevalence, penetrance, genetic-heterogeneity, or maximum credible allele-frequency calculation was retrieved; no gene-specific BS1 threshold can be applied.

Sources

Reasoning

BS2 is not met. gnomAD v4.1 reports zero homozygotes among 84 alternate alleles and gnomAD v2.1 reports zero homozygotes among 9 alternate alleles. These population database counts do not document the variant in healthy adult individuals, and there are no homozygotes to support a benign observation in the recessive NTHL1 disease context.

Rule

Generic ACMG/AMP fallback: BS2 requires observation in healthy adult individual(s) for a disorder expected to be fully penetrant at an early age; unphenotyped population data with no homozygotes do not satisfy this requirement.

Evidence considered

• gnomAD v4.1: 0 homozygotes, including 0 in the African/African American population where the highest AF is observed.
• gnomAD v2.1: 0 homozygotes, including 0 in the African/African American population where the highest AF is observed.

Still missing

• No curated observations of unaffected adult homozygotes or other well-phenotyped healthy individuals were retrieved.

Sources

Reasoning

ClinVar has no exact-variant expert-panel submission for a Benign or Likely benign classification; the recorded submissions are non-expert Uncertain significance assertions.

Rule

Apply BP6 only for an exact-variant ClinVar expert-panel Benign or Likely benign assertion.

Evidence considered

• The exact-variant ClinVar submission audit reports zero expert-panel submissions; all reported clinical classifications are Uncertain significance.

Sources

Reasoning

PS1 requires identification of a different nucleotide change producing the identical amino acid substitution p.(Arg11Trp) that has established pathogenic classification. No CSPEC/VCEP framework exists for NTHL1 (case_summary.compact_evidence.cspec.found=false; vcep_materials.json note='no cspec url available'), so generic ACMG PS1 semantics would apply, but no source in this case bundle performed or returned a search for alternate-codon same-amino-acid-change comparator variants. ClinVar record for this exact variant (VariationID 664916) shows only entries for this transcript/codon change itself, not for an independently-arising p.Arg11Trp allele via a different codon.

Evidence considered

• ClinVar VariationID 664916 for NM_002528.7:c.31C>T / NP_002519.2:p.(Arg11Trp) shows only this variant's own submissions (Uncertain significance, 7 labs); no independent same-amino-acid-change comparator record was surfaced in this case bundle.

Still missing

• No dedicated search was performed in this case for other nucleotide substitutions at codon 11 that also yield p.(Arg11Trp) with an established pathogenic/likely pathogenic ClinVar classification.
• pm5_candidates.json (the only same-residue comparator artifact retrieved) explicitly did not attempt same-amino-acid PS1-style harvesting.

Sources

Reasoning

No proband-level parental genotype results, parentage confirmation, phenotype details, or de novo observations for NM_002528.7:c.31C>T (p.Arg11Trp) were provided. PS2 cannot be assessed.

Rule

Generic ACMG/AMP PS2 requires a confirmed de novo occurrence in an affected individual with a consistent phenotype and no family history.

Evidence considered

• No NTHL1 VCEP/ClinGen gene-specific specification was retrieved; the case is governed by the generic ACMG/AMP fallback.
• The consolidated case-evidence bundle contains no proband-level parental testing, confirmed de novo result, parentage confirmation, or phenotype information for this variant.

Still missing

• Obtain the proband phenotype and indication for testing.
• Obtain maternal and paternal test results for the exact variant and confirm biological parentage when de novo evidence is claimed.
• Document whether any reported de novo observation has a phenotype consistent with biallelic NTHL1-associated disease.

Reasoning

No functional/biochemical assay data (e.g., glycosylase activity, base excision repair complementation, cell-based repair assays) for NM_002528.7:c.31C>T (p.Arg11Trp) is present anywhere in the case evidence bundle. No ClinGen VCEP specification exists for NTHL1 (framework_mode is generic_acmg, cspec.found=false, vcep_materials.json empty). OncoKB was queried but explicitly returned no variant-specific reviewed functional evidence (is_variant_specific=false, not_reviewed=true), only gene-level narrative about NTHL1's role in DNA base excision repair. No PMID in the literature pass or ClinVar citation trail is tagged with a functional-assay candidate_criteria entry for PS3.

Still missing

• No validated functional assay (activity, complementation, or clinical validation study) identified for this variant in the current source set.
• No ClinGen NTHL1 VCEP specification available to check for a gene-specific PS3 assay list or strength calibration.

Reasoning

No exact-variant case-control counts, odds ratio, or independently documented affected-case series were provided for NM_002528.7:c.31C>T (p.Arg11Trp).

Rule

Generic ACMG/AMP PS4: apply only with statistically significant enrichment of the exact variant in affected individuals versus controls.

Evidence considered

• No NTHL1 CSPEC/VCEP or local gene-specific framework is available for this case; generic ACMG/AMP fallback applies.
• The selectively reviewed NTHL1 breast-cancer case-control paper did not mention c.31C>T or p.Arg11Trp, so its gene-level missense association cannot be used as PS4 evidence for this variant.

Still missing

• An exact-variant, phenotype-defined case-control analysis or sufficiently documented affected-case series with appropriate controls is needed.

Reasoning

PM1 requires the variant to fall in a well-established mutational hotspot or critical/well-characterized functional domain without benign variation. No NTHL1-specific VCEP/CSPEC framework is available (cspec.found=false) to define domain boundaries or hotspot residues. A CancerHotspots.org query for NTHL1 R11 returned no result ('This variant does not lie in a statistically significant hotspot'), but CancerHotspots is a somatic-cancer hotspot resource and not informative for this gene's germline base-excision-repair mechanism. No UniProt/Pfam/InterPro domain annotation for the NTHL1 HhH-GPD glycosylase domain boundaries, nor any structural/conservation data localizing residue 11 to a catalytic or structurally critical region, was retrieved anywhere in this case bundle. Residue 11 is very close to the N-terminus of the 312-residue NTHL1 protein, a region not established in the retrieved literature as part of the catalytic glycosylase domain, but this cannot be confirmed without domain-boundary data that was not collected in this case.

Evidence considered

• CancerHotspots.org query for 'NTHL1 R11' returned search_status='no_result' / found=false, with no source_data populated.
• OncoKB page for NTHL1 R11W provided only gene-level context (NTHL1 as a DNA damage repair protein mutated in germline hereditary cancer families) with no variant-specific or domain-specific functional annotation.

Still missing

• No UniProt/Pfam/InterPro domain-boundary annotation for NTHL1 was retrieved to confirm or exclude residue 11 from a critical functional domain.
• CancerHotspots.org search returned no result and is a somatic-hotspot resource, not directly applicable to this germline recessive DNA-repair gene mechanism.
• No local/gene-specific PM1 framework or domain map was available in this case for NTHL1.

Reasoning

Not assessed: NTHL1-related tumor predisposition is a biallelic loss-of-function condition, but the consolidated evidence contains no affected individual carrying NM_002528.7:c.31C>T (p.Arg11Trp) with a pathogenic/likely pathogenic NTHL1 variant in trans, no phase data, and no qualifying affected-proband observation for this variant.

Rule

Generic ACMG/AMP PM3: for a recessive disorder, the variant is observed in trans with a pathogenic variant.

Evidence considered

• No NTHL1 CSPEC/VCEP or local gene-specific framework was retrieved; the generic ACMG/AMP framework was used.
• Richards et al. defines PM3 as detection, for a recessive disorder, in trans with a pathogenic variant. The required affected-proband, pathogenic-partner, and trans-phase evidence is absent from the bundle.
• Li et al. describes NTHL1 disease predisposition in carriers of bi-allelic loss-of-function variants, establishing that a recessive allelic context is relevant; it does not report c.31C>T (p.Arg11Trp).

Still missing

• Affected-proband data for NM_002528.7:c.31C>T with a second NTHL1 variant classified as pathogenic or likely pathogenic.
• Phase evidence demonstrating that c.31C>T and the second variant are in trans.

Sources

Reasoning

PM5 requires a different missense change at the same residue (Arg11) with established pathogenic/likely pathogenic classification. The dedicated pm5_candidates.json artifact for this case explicitly declined to perform classic same-residue PM5 candidate harvesting ('Unable to confirm classic same-residue PM5 semantics safely; skipping automatic candidate harvesting'), returning zero candidates and recommendation level 'not_applicable' for the harvesting step itself (not a clinical determination). No other source in the case bundle lists alternate amino-acid substitutions at NTHL1 codon 11.

Evidence considered

• pm5_candidates.json reports pm5_mode='unknown', eligible_for_classic_pm5_search=false, candidate_summary.total_candidates_seen=0, and explicitly instructs: 'Review whether any collected same-residue comparator variants satisfy the governing PM5 rule; do not apply PM5 from this artifact alone.'

Still missing

• pm5_candidates.json harvesting was explicitly skipped for this variant; no same-residue alternate-missense comparator search was completed.
• A manual/targeted ClinVar search for other NTHL1 p.Arg11X missense variants with Pathogenic/Likely Pathogenic classification is needed before PM5 can be adjudicated.

Sources

Reasoning

No reported de novo occurrence with unavailable or unconfirmed parental testing was provided for NM_002528.7:c.31C>T (p.Arg11Trp). PM6 cannot be assessed.

Rule

Generic ACMG/AMP PM6 applies to an assumed de novo occurrence in an affected individual when parental confirmation is unavailable.

Evidence considered

• No NTHL1 VCEP/ClinGen gene-specific specification was retrieved; the case is governed by the generic ACMG/AMP fallback.
• The consolidated case-evidence bundle contains no proband-level de novo observation or parental testing information for this variant.

Still missing

• Obtain any reported de novo case observation for the exact variant.
• For each claimed occurrence, obtain the proband phenotype and the status and result of maternal and paternal testing.

Reasoning

No informative pedigree, affected-relative genotypes, phenotype data, or meiosis count was provided for NM_002528.7:c.31C>T (p.Arg11Trp). Co-segregation cannot be assessed.

Rule

Generic ACMG/AMP PP1 requires co-segregation of the variant with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• No NTHL1 VCEP/ClinGen gene-specific specification was retrieved; the case is governed by the generic ACMG/AMP fallback.
• The consolidated case-evidence bundle contains no family segregation data or informative meioses for this variant.

Still missing

• Obtain a pedigree with diagnoses and ages of onset.
• Obtain genotype results for affected and informative unaffected relatives for the exact variant.
• Establish phase and identify the disease-relevant genotype before counting informative segregations in this recessive NTHL1 context.

Reasoning

PP2 requires that missense variants be a common mechanism of disease in this gene and that the gene has a low rate of benign missense variation. No NTHL1-specific VCEP/CSPEC framework was found to define this. The germline disease literature retrieved for NTHL1 (pvs1_gene_context.json) establishes NTHL1 tumor syndrome as an autosomal-recessive condition caused by biallelic loss-of-function (predominantly nonsense/frameshift) variants -- the founder mutation described (Weren et al., PMID not in this group's registry) is a nonsense mutation, and the case's own PVS1 gene-context assessment centers on a LoF disease mechanism, not missense. No gnomAD/ExAC missense constraint metric (missense Z-score or O/E ratio, e.g. via gnomAD constraint tab) was retrieved for NTHL1 in this case bundle to quantify the rate of benign missense variation gene-wide.

Evidence considered

• pvs1_gene_context.json: NTHL1 germline disease mechanism established in the literature review is loss-of-function ('lof_mechanism_supported': true; disease description centers on 'A germline homozygous mutation in the base-excision repair gene NTHL1 causes adenomatous polyposis and colorectal cancer', describing a homozygous nonsense mutation as the founder pathogenic allele), with no missense-mechanism literature identified.

Still missing

• No gnomAD missense constraint statistic (missense Z-score / O/E) for NTHL1 was retrieved in this case.
• No VCEP/CSPEC-defined statement on whether missense variation is an established common disease mechanism for NTHL1 was available; the retrieved germline literature centers on LoF (nonsense/frameshift) as the disease mechanism, which argues against but does not conclusively establish/exclude a common missense mechanism.

Sources

Reasoning

No individual-level phenotype, tumor phenotype, or genotype context was provided for the carrier; phenotype specificity for NTHL1-associated disease cannot be evaluated.

Rule

Generic ACMG/AMP PP4: apply only when the individual's phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• No NTHL1 CSPEC/VCEP or local gene-specific framework is available for this case; generic ACMG/AMP fallback applies.

Still missing

• Provide the proband phenotype, relevant tumor or polyposis findings, age at onset, and whether a second pathogenic NTHL1 allele is present.

Reasoning

No functional/biochemical assay data showing normal (wild-type-like) activity for NM_002528.7:c.31C>T (p.Arg11Trp) is present anywhere in the case evidence bundle. No ClinGen VCEP specification exists for NTHL1 (framework_mode is generic_acmg, cspec.found=false, vcep_materials.json empty). OncoKB returned no variant-specific reviewed functional evidence (is_variant_specific=false, not_reviewed=true). No PMID in the literature pass or ClinVar citation trail is tagged with a functional-assay candidate_criteria entry for BS3.

Still missing

• No validated functional assay demonstrating normal/wild-type activity identified for this variant in the current source set.
• No ClinGen NTHL1 VCEP specification available to check for a gene-specific BS3 assay list or strength calibration.

Reasoning

No informative non-segregation evidence was provided: there are no genotype-phenotype data showing that the exact variant fails to track with the disease-relevant genotype or phenotype in a family. BS4 cannot be assessed.

Rule

Generic ACMG/AMP BS4 requires lack of segregation of the variant with disease in affected family members; it should not be applied from an uninformative pedigree or without accounting for the disease inheritance model.

Evidence considered

• No NTHL1 VCEP/ClinGen gene-specific specification was retrieved; the case is governed by the generic ACMG/AMP fallback.
• The consolidated case-evidence bundle contains no family data demonstrating lack of segregation for this variant.

Still missing

• Obtain pedigree, phenotype, and genotype data for affected and informative unaffected relatives.
• Confirm that any apparent non-segregation is informative for the recessive NTHL1 disease model, including assessment of the second allele and phase.

Reasoning

BP1 requires that the gene's disease mechanism is established as predominantly truncating/LoF, such that missense variants are generally not expected to be pathogenic. The retrieved germline literature (pvs1_gene_context.json) does support NTHL1 disease as LoF-driven (biallelic nonsense/frameshift causing NTHL1 tumor syndrome), which would be directionally supportive of BP1 applicability for missense variants generally in this gene. However, no formal gene-specific missense pathogenicity-rate statistic, no VCEP/CSPEC BP1 threshold, and no statement confirming that missense variants as a class are established as benign/non-pathogenic in NTHL1 was retrieved in this case bundle, so the criterion cannot be confidently scored either met or not_met without that quantitative or VCEP-defined basis.

Evidence considered

• pvs1_gene_context.json indicates NTHL1 disease mechanism is loss-of-function (biallelic nonsense/frameshift), directionally consistent with a gene where missense variants are not the primary disease mechanism, but no quantitative missense pathogenicity-rate data or VCEP BP1 rule was available to confirm applicability.

Still missing

• No VCEP/CSPEC-defined BP1 rule or missense-pathogenicity-rate statistic for NTHL1 was retrieved.
• No gene-level statement was found confirming that missense variants in NTHL1 are generally considered non-pathogenic as a class (as opposed to simply under-studied).

Sources

Reasoning

Not assessed: the consolidated evidence provides no individual-level co-occurrence or phase data showing NM_002528.7:c.31C>T (p.Arg11Trp) in cis with a pathogenic/likely pathogenic variant, and no homozygous or biallelic affected/unaffected observations that would support benignity. The 84 gnomAD v4.1 alleles have zero reported homozygotes and do not establish BP2.

Rule

Generic ACMG/AMP BP2: observed in trans with a pathogenic variant for a fully penetrant dominant disorder, or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• No NTHL1 CSPEC/VCEP or local gene-specific framework was retrieved; the generic ACMG/AMP framework was used.
• Richards et al. defines BP2 as observation in trans with a pathogenic variant for a fully penetrant dominant disorder, or observation in cis with a pathogenic variant in any inheritance pattern. Neither form of evidence is present for c.31C>T.
• Li et al. reports that NTHL1-associated predisposition is associated with bi-allelic loss-of-function variants; it does not report c.31C>T (p.Arg11Trp) or allele-level co-occurrence/phase evidence.
• gnomAD v4.1 reports 84 alleles and zero homozygotes for c.31C>T; aggregate population data cannot establish the requisite pathogenic-variant co-occurrence or an unaffected homozygous observation.

Still missing

• Individual-level co-occurrence data identifying a pathogenic/likely pathogenic NTHL1 variant and cis/trans phase for c.31C>T.
• If considering a homozygous observation, phenotype and age data demonstrating that the individual is unaffected for the relevant NTHL1-related condition.

Sources

Reasoning

No affected individual, phenotype, or alternate molecular diagnosis was supplied; an alternate cause for the observed phenotype cannot be assessed.

Rule

Generic ACMG/AMP BP5: apply only when the variant is found in an affected individual whose phenotype is explained by an alternate molecular diagnosis.

Evidence considered

• No NTHL1 CSPEC/VCEP or local gene-specific framework is available for this case; generic ACMG/AMP fallback applies.

Still missing

• Provide phenotype and diagnostic testing results sufficient to determine whether another molecular etiology fully explains the presentation.