FANCD2 c.2255T>C, p.(Ile752Thr)

Reasoning

PM2 is met at moderate strength under the generic ACMG/AMP framework. Although present in gnomAD v4.1, the allele is extremely rare (AF 4.35e-06 overall; maximum observed ancestry-specific AF 2.23e-05), with no homozygotes; it is absent from gnomAD v2.1 and gnomAD-Canada v1.0.

Rule

Generic ACMG/AMP PM2: absent from controls or present at an extremely low frequency in population databases.

Evidence considered

• No FANCD2-specific VCEP/CSPEC was available; the generic ACMG/AMP framework applies.
• gnomAD v4.1 reports 7/1,608,286 alleles (AF 4.35246e-06), no homozygotes, a maximum observed East Asian AF of 2.22896e-05 (1/44,864 alleles), and grpmax FAF 8e-07.
• The matched variant is absent from gnomAD v2.1 and gnomAD-Canada v1.0.

Sources

Reasoning

No FANCD2-specific VCEP/CSPEC PP3/BP4 framework was retrieved (case_summary.cspec.found=false; vcep_materials.final_classification_framework.found=false; framework_mode=generic_acmg), so the lab's generic ACMG/AMP in-silico calibration rules apply. NM_033084.4:c.2255T>C is a missense substitution (NP_149075.2:p.(Ile752Thr)), so per the generic rule set the missense path (REVEL only) governs and the variant's own SpliceAI score must not be substituted for it. REVEL score = 0.578, which falls in the gray zone [0.250, 0.750] defined by this lab's rules, so PP3 is not triggered.

Rule

generic_acmg_fallback: missense variant -> REVEL only; REVEL 0.578 in [0.250,0.750] gray zone -> PP3 not met

Evidence considered

• REVEL score = 0.578 for NM_033084.4:c.2255T>C / NP_149075.2:p.(Ile752Thr), which lies within the generic ACMG/AMP fallback gray zone [0.250, 0.750] where neither PP3 nor BP4 is met (per this lab's local generic_acmg_classification_rules.md, PP3/BP4 in-silico calibration section, itself grounded in Richards et al. 2015 ACMG/AMP framework, PMID:25741868).
• Variant is a missense substitution, so the generic rules require using the REVEL (missense) path only, not the SpliceAI (splice) path, even though SpliceAI data is available for this variant.

Still missing

• No gene-specific VCEP CSPEC PP3/BP4 lookup table available for FANCD2 to override the generic REVEL gray-zone call.
• BayesDel score (0.207275) exists but has no verified published calibration threshold available to this pipeline and cannot be used for PP3/BP4.

Sources

Reasoning

The exact ClinVar record has one Labcorp clinical-testing submission classified as uncertain significance and no expert-panel submission. Therefore, there is no exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion.

Rule

Apply PP5 at supporting strength only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification.

Evidence considered

• ClinVar variation 2913870 is an exact genomic match for FANCD2 c.2255T>C (p.Ile752Thr); its submission audit records zero expert-panel submissions and one single-submitter uncertain-significance assertion.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification would be required.

Sources

Reasoning

BA1 is not met. The highest observed population AF is 4.35e-05 overall in gnomAD v4.1 (East Asian AF 2.23e-05), far below a stand-alone benign frequency threshold.

Rule

Generic ACMG/AMP BA1: stand-alone benign evidence requires an allele frequency too high for the disorder.

Evidence considered

• No FANCD2-specific VCEP/CSPEC was available; the generic ACMG/AMP framework applies.
• gnomAD v4.1 reports 7/1,608,286 alleles (AF 4.35246e-06), zero homozygotes, and a maximum observed ancestry-specific AF of 2.22896e-05 in East Asian individuals; the grpmax filtering allele frequency is 8e-07.

Sources

Reasoning

BS1 is not met. The gnomAD v4.1 overall and ancestry-specific frequencies are very low and do not establish a frequency greater than expected for a pathogenic FANCD2 allele.

Rule

Generic ACMG/AMP BS1: allele frequency must be greater than expected for the disorder.

Evidence considered

• No FANCD2-specific VCEP/CSPEC was available; the generic ACMG/AMP framework applies.
• gnomAD v4.1 reports AF 4.35246e-06 overall (7/1,608,286 alleles); the highest reported subpopulation AF is 2.22896e-05 in East Asian individuals (1/44,864 alleles), with grpmax FAF 8e-07.

Sources

Reasoning

BS2 is not met. gnomAD v4.1 reports no homozygotes, so there are no observations in unaffected individuals that could support BS2.

Rule

Generic ACMG/AMP BS2: observe the variant in healthy adults for a disorder expected to be fully penetrant early in life.

Evidence considered

• No FANCD2-specific VCEP/CSPEC was available; the generic ACMG/AMP framework applies.
• gnomAD v4.1 reports 7 alleles among 1,608,286 alleles and zero homozygotes; zero homozygotes are also reported in the East Asian group with the highest observed allele frequency.

Still missing

• No curated evidence of this variant in well-phenotyped unaffected adult homozygotes was provided.

Sources

Reasoning

Same generic ACMG/AMP fallback calibration as PP3 applies (no FANCD2 VCEP-specific PP3/BP4 framework retrieved). As a missense variant, only REVEL is used per the local rule set. REVEL score = 0.578 falls in the gray zone [0.250, 0.750], which is below the BP4 threshold (REVEL < 0.250) required to trigger BP4, so BP4 is not met.

Rule

generic_acmg_fallback: missense variant -> REVEL only; REVEL 0.578 in [0.250,0.750] gray zone -> BP4 not met

Evidence considered

• REVEL score = 0.578 for NM_033084.4:c.2255T>C / NP_149075.2:p.(Ile752Thr) is above the BP4 threshold of REVEL < 0.250, so it does not qualify as benign-supporting in-silico evidence under the generic ACMG/AMP fallback rules (generic_acmg_classification_rules.md, PP3/BP4 in-silico calibration section, grounded in Richards et al. 2015, PMID:25741868).

Still missing

• No gene-specific VCEP CSPEC PP3/BP4 lookup table available for FANCD2 to override the generic REVEL gray-zone call.
• BayesDel score (0.207275) exists but has no verified published calibration threshold available to this pipeline and cannot be used for PP3/BP4.

Sources

Reasoning

The exact ClinVar record has no expert-panel submission and its sole single-submitter assertion is uncertain significance. Therefore, there is no exact-variant ClinVar expert-panel Benign or Likely benign assertion.

Rule

Apply BP6 at supporting strength only for an exact-variant ClinVar expert-panel Benign or Likely benign classification.

Evidence considered

• ClinVar variation 2913870 is an exact genomic match for FANCD2 c.2255T>C (p.Ile752Thr); its submission audit records zero expert-panel submissions and one single-submitter uncertain-significance assertion.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign classification would be required.

Sources

Reasoning

PS1 requires an established pathogenic variant that produces the identical amino-acid change (p.Ile752Thr) via a different nucleotide substitution. No CSPEC/VCEP framework exists for FANCD2 (framework_mode=generic_acmg, cspec.found=false), and the case's dedicated same-residue comparator search (pm5_candidates.json) explicitly declined to run, flagging uncertainty in classic same-residue semantics and returning zero candidates. No other source in this bundle reports an alternate codon producing Ile752Thr with an established classification, so PS1 cannot be evaluated from the retrieved evidence.

Evidence considered

• pm5_candidates.json: eligible_for_classic_pm5_search=false, candidate_summary.total_candidates_seen=0, reason='Unable to confirm classic same-residue PM5 semantics safely; skipping automatic candidate harvesting.' This same artifact is the only source in the bundle that would surface a same-amino-acid-change (PS1) comparator, and it returned no candidates.

Still missing

• No ClinVar/literature search was performed or retrieved for other nucleotide changes at codon 752 that also encode Thr (would need direct lookup, not present in this bundle)
• pm5_candidates.json explicitly skipped automated candidate harvesting for this variant

Sources

Reasoning

No proband-level parental testing or confirmed de novo observation is available for NM_033084.4:c.2255T>C (p.Ile752Thr).

Rule

Generic ACMG/AMP PS2: apply only for a de novo variant in a patient with the disease and no family history, with maternity and paternity confirmed.

Evidence considered

• No FANCD2 gene-specific VCEP/CSPEC or local framework was available; generic ACMG/AMP criteria were used.
• The consolidated case-evidence bundle contains no proband phenotype, parental test result, or de novo confirmation for this variant.

Still missing

• Obtain parental genotypes for the proband and confirm biological parentage if the variant is absent from both parents.
• Document whether the proband phenotype is consistent with the relevant FANCD2-associated condition.

Reasoning

No functional/experimental studies of NM_033084.4:c.2255T>C (p.Ile752Thr) were located in this case's evidence bundle. There is no FANCD2 ClinGen VCEP specification in this case (cspec.found=false, framework_mode=generic_acmg), so generic ACMG/AMP guidance for PS3 applies. Searches of the compiled evidence.json, literature_pass.json, and prefetch.json content for functional-assay terms (complementation, mitomycin C/MMC sensitivity, crosslinker sensitivity, FANCD2 monoubiquitination/ubiquitination) returned no hits. OncoKB and ClinVar sources both confirm no variant-specific functional data is available (OncoKB explicitly notes 'did not identify variant-specific reviewed functional evidence'; ClinVar's single submitter cites only PM2 via Sherloc methodology, not a functional assay). The one full-text paper available in this case (PMID:19888064, ACOG carrier-screening opinion) is a clinical guideline document with no experimental content and does not mention this variant.

Evidence considered

• OncoKB gene/variant page (oncokb source) explicitly states no variant-specific reviewed functional evidence was found for FANCD2 I752T; only gene-level Fanconi Anemia context is provided.
• ClinVar record 2913870 (single submitter, Labcorp/Invitae) cites only PMID 28492532 (Sherloc methodology paper) and applies PM2 (population frequency), not a PS3/functional criterion.
• Keyword search of the full consolidated evidence bundle for functional-assay terminology (complementation, mitomycin C, MMC, crosslinker, monoubiquitination) returned zero matches.

Still missing

• No cell-based or biochemical functional assay data (e.g., patient-derived fibroblast MMC/DEB sensitivity, FANCD2 monoubiquitination status, complementation assay) identified for this specific variant in the reviewed sources.
• No FANCD2 ClinGen VCEP-approved functional assay framework was available in this case to calibrate assay validity or PS3 strength.

Reasoning

No case-control study, affected-case count, or control comparison for FANCD2 c.2255T>C (p.Ile752Thr) was provided. The reviewed carrier-screening publication concerns Fanconi anemia group C, not FANCD2, and does not mention this variant.

Rule

Generic ACMG/AMP PS4: apply only when the variant is significantly enriched in affected individuals compared with controls. No FANCD2-specific VCEP/CSPEC was retrieved.

Evidence considered

• PMID:19888064 was reviewed for possible case/prevalence evidence. Its Table 1 and text discuss Fanconi anemia group C and do not report FANCD2 c.2255T>C or p.Ile752Thr; it supplies no variant-specific case-control evidence.

Still missing

• Variant-specific, independently ascertained affected-case data and an appropriate control comparison are required to evaluate enrichment.

Sources

Reasoning

PM1 requires the variant to fall in a well-established mutational hotspot or critical/well-characterized functional domain without benign variation. No FANCD2 CSPEC/VCEP domain framework was retrieved (gene_vcep_dir=null, vcep_materials.json shows no cspec url). Cancer somatic mutational-hotspot screening (cancerhotspots.org) returned no result for FANCD2 I752, but this is a somatic-cancer-mutation database, not a germline domain/functional-region annotation, and its absence of a hit is only weakly informative for germline PM1. OncoKB gene-level review found no variant-specific reviewed functional evidence and classified FANCD2 as tumor suppressor via LOF, with no domain-level detail for residue 752. No UniProt/protein-domain boundary data for the FANCD2 protein around residue 752 was retrieved in this bundle.

Evidence considered

• oncokb gene-level page found no variant-specific reviewed functional evidence for FANCD2 I752T and classified it 'Unknown Oncogenic Effect'; only gene-level tumor-suppressor/LOF context was available, with no domain boundary information for residue 752.
• cancerhotspots.org query 'FANCD2 I752' returned no_result ('This variant does not lie in a statistically significant hotspot'), but this database captures somatic cancer mutation hotspots and is not a direct substitute for a germline critical-domain annotation.

Still missing

• No UniProt or structural domain annotation for FANCD2 residue 752 was retrieved
• No germline mutational cluster/hotspot data (as opposed to somatic cancerhotspots.org) was retrieved for this region
• OncoKB review was gene-level only, not variant- or domain-specific

Sources

Reasoning

No affected-proband observation of NM_033084.4(FANCD2):c.2255T>C / p.(Ile752Thr) with a pathogenic or likely pathogenic FANCD2 variant was provided, and no phase information is available. PM3 cannot be assessed.

Rule

Generic ACMG/AMP fallback: PM3 requires the variant to be observed in trans with a pathogenic variant in an affected individual for a recessive disorder.

Evidence considered

• No FANCD2 allelic, affected-proband, co-occurrence, or phase evidence was present in the consolidated case-evidence bundle.
• No FANCD2 VCEP/ClinGen gene-specific specification was retrieved; the case framework identifies generic ACMG/AMP 2015 fallback.

Still missing

• Affected-proband genotype(s) containing this variant and a second FANCD2 variant.
• Classification evidence for any second FANCD2 variant.
• Parental or molecular phasing evidence demonstrating the variants are in trans.

Sources

Reasoning

PM5 requires a different missense change at the same residue (position 752) previously established as pathogenic/likely pathogenic. The case's dedicated pm5_candidates.json artifact explicitly marked this variant ineligible for classic same-residue PM5 search ('Unable to confirm classic same-residue PM5 semantics safely; skipping automatic candidate harvesting'), returning zero same-residue candidates and a recommendation level of not_applicable pending manual review, so no PM5 comparator evidence is available in this bundle.

Evidence considered

• pm5_candidates.json: pm5_mode='unknown', eligible_for_classic_pm5_search=false, candidates=[] (empty), recommendation.level='not_applicable', recommendation.summary='Unable to confirm classic same-residue PM5 semantics safely; skipping automatic candidate harvesting.'

Still missing

• pm5_candidates.json harvesting was skipped entirely; no alternate-residue-752 missense variant comparator data was collected
• Reviewer should manually query ClinVar for other FANCD2 codon-752 missense variants before applying PM5

Sources

Reasoning

No reported de novo occurrence without confirmed parentage is available for NM_033084.4:c.2255T>C (p.Ile752Thr).

Rule

Generic ACMG/AMP PM6: apply only for an assumed de novo variant in a patient with the disease when parental confirmation is unavailable.

Evidence considered

• No FANCD2 gene-specific VCEP/CSPEC or local framework was available; generic ACMG/AMP criteria were used.
• The consolidated case-evidence bundle contains no proband phenotype, parental test result, or de novo report for this variant.

Still missing

• Obtain parental genotypes for the proband.
• If both parents are negative but parentage is not confirmed, document the proband phenotype and testing context for potential PM6 assessment.

Reasoning

No informative family segregation data, affected carrier observations, unaffected non-carrier observations, or meiosis count is available for NM_033084.4:c.2255T>C (p.Ile752Thr).

Rule

Generic ACMG/AMP PP1: apply only when the variant cosegregates with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• No FANCD2 gene-specific VCEP/CSPEC or local framework was available; generic ACMG/AMP criteria were used.
• The consolidated case-evidence bundle contains no family-level genotype-phenotype segregation data for this variant.

Still missing

• Provide pedigree data with genotypes and clinically adjudicated phenotypes for informative relatives.
• Report the number of informative segregations/meioses and the inheritance model before applying segregation evidence.

Reasoning

PP2 requires the gene to have a low rate of benign missense variation and missense to be a common mechanism of disease. No FANCD2-specific VCEP framework or missense-constraint metric (e.g., gnomAD missense Z-score/o:e ratio, ClinVar missense pathogenic density) was retrieved in this bundle. pvs1_gene_context.json establishes that FANCD2 loss-of-function (truncating) variants are an accepted germline disease mechanism (Fanconi anemia and cancer predisposition), but this documents LOF mechanism only; it does not quantify the tolerance/intolerance of the gene to missense variation, which is required to assess PP2 specifically.

Evidence considered

• pvs1_gene_context.json documents that FANCD2 loss-of-function/truncating variants are an established germline Fanconi anemia and cancer-predisposition disease mechanism (lof_mechanism_supported=true), but provides no missense-variant-tolerance statistic needed to assess PP2.

Still missing

• No gnomAD missense constraint (Z-score / observed:expected) statistic for FANCD2 was retrieved
• No published statement on the proportion of FANCD2 disease-causing variants that are missense vs. truncating was retrieved

Sources

Reasoning

No phenotype, age of onset, laboratory findings, or diagnostic work-up for a carrier of this exact variant was provided. The ClinVar condition label alone cannot establish that an individual has a phenotype highly specific for a FANCD2-related disorder.

Rule

Generic ACMG/AMP PP4: apply only for a phenotype or family history highly specific for a disease with a single genetic etiology. No FANCD2-specific VCEP/CSPEC was retrieved.

Evidence considered

• The exact ClinVar record lists Fanconi anemia as a condition but provides no individual-level phenotype or diagnostic work-up in the supplied evidence.

Still missing

• Variant-carrier phenotype and diagnostic testing details are required to assess phenotype specificity and alternative etiologies.

Sources

Reasoning

No functional/experimental studies of NM_033084.4:c.2255T>C (p.Ile752Thr) were located in this case's evidence bundle, so there is no basis to assess whether a validated assay showed no damaging effect. No FANCD2 ClinGen VCEP specification is available (cspec.found=false), so generic ACMG/AMP BS3 guidance applies but cannot be invoked absent functional data.

Evidence considered

• Keyword search of the full consolidated evidence bundle for functional-assay terminology returned zero matches; OncoKB and ClinVar sources confirm absence of variant-specific functional evidence.

Still missing

• No functional assay data of any kind (damaging or benign-consistent) identified for this variant in the reviewed sources.

Reasoning

No informative non-segregation evidence is available for NM_033084.4:c.2255T>C (p.Ile752Thr).

Rule

Generic ACMG/AMP BS4: apply only when the variant demonstrably does not segregate with disease in affected family members, after considering penetrance and phenocopies.

Evidence considered

• No FANCD2 gene-specific VCEP/CSPEC or local framework was available; generic ACMG/AMP criteria were used.
• The consolidated case-evidence bundle contains no family-level genotype-phenotype non-segregation data for this variant.

Still missing

• Provide genotypes and clinically adjudicated phenotypes for informative relatives showing that the variant does not segregate with the relevant condition.
• Establish the relevant disease model and account for age-dependent onset, reduced penetrance, and phenocopies before using non-segregation evidence.

Reasoning

BP1 requires evidence that the gene's disease mechanism is primarily truncating, such that missense variants are generally not pathogenic. pvs1_gene_context.json documents FANCD2 loss-of-function/truncating variants as an accepted germline disease mechanism, which is consistent with -- but not sufficient on its own to establish -- a BP1-qualifying gene-level pattern, since FANCD2 missense variants are also reported in the literature and disease databases (e.g., ClinVar) without a quantified benign-missense rate being available. No missense constraint metric or systematic missense-vs-truncating pathogenicity comparison for FANCD2 was retrieved in this bundle.

Evidence considered

• pvs1_gene_context.json documents FANCD2 loss-of-function/truncating variants as the established germline disease mechanism (lof_mechanism_supported=true), which is relevant context for BP1 but insufficient alone to confirm a low background rate of pathogenic missense variation in this gene.

Still missing

• No gnomAD missense constraint (Z-score / observed:expected) statistic for FANCD2 was retrieved
• No systematic comparison of missense vs. truncating variant pathogenicity rates for FANCD2 was retrieved

Sources

Reasoning

No case observation documents this variant in cis with a pathogenic or likely pathogenic FANCD2 variant, nor is there a documented trans observation in a setting where biallelic FANCD2 variants would be non-causative. BP2 cannot be assessed.

Rule

Generic ACMG/AMP fallback: BP2 requires observed co-occurrence with a pathogenic variant in cis, or a trans observation with a pathogenic variant in a disorder for which the observed biallelic state is not expected to be causative.

Evidence considered

• No FANCD2 allelic, co-occurrence, or phase evidence was present in the consolidated case-evidence bundle.
• No FANCD2 VCEP/ClinGen gene-specific specification was retrieved; the case framework identifies generic ACMG/AMP 2015 fallback.

Still missing

• A documented co-occurring pathogenic or likely pathogenic FANCD2 variant in the same individual.
• Validated cis/trans phase evidence and clinical context establishing that the co-occurrence is non-causative.

Sources

Reasoning

No affected individual carrying this variant, phenotype, or independent molecular diagnosis was provided. BP5 cannot be assessed without documentation that the phenotype is explained by an alternative molecular cause.

Rule

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

Still missing

• An affected individual's phenotype and independently established alternative molecular diagnosis are required.