BRCA2 c.2164A>T, p.(Lys722Ter)

Reasoning

NM_000059.4:c.2164A>T creates a premature termination codon, NP_000050.3:p.(Lys722Ter)/p.(K722*), in BRCA2 exon 11. The ENIGMA BRCA1/BRCA2 VCEP specification (v1.2) lists PVS1 as applicable for BRCA2 with default strength Pathogenic Very Strong for null variants (nonsense, frameshift, canonical splice site, initiation codon, single/multi-exon deletion) once germline loss of function is an established disease mechanism, which is confirmed for BRCA2 by the CSPEC itself. Exon 11 is a large, constitutively included coding exon well upstream of the final exon (BRCA2 has 27 coding exons), so the transcript is predicted to be a substrate for nonsense-mediated decay; exon 11 is not among the VCEP's PM5_N/A exon list (E12, E27, E6) and the VCEP exon-level PVS1 decision table (Table 4) shows exon-11 truncating events assigned PVS1 with no exon-specific downgrade instructions for mid-exon nonsense variants. No exon skipping, alternative-transcript rescue, or escape from NMD is indicated in the case materials. Applying the ClinGen SVI generic PVS1 decision framework (used here to interpret the VCEP default because the case-provided pvs1_variant_assessment explicitly frames this as a nonsense variant evaluated under the generic PVS1 flowchart once gene-level LOF eligibility is confirmed) supports full-strength PVS1.

Rule

ENIGMA BRCA1/BRCA2 VCEP Specification v1.2, PVS1 (defaultStrength Pathogenic Very Strong) applied via ClinGen SVI generic PVS1 decision framework for nonsense/NMD-predicted PTC variants.

Evidence considered

• cspec (ENIGMA BRCA1/BRCA2 Specification v1.2): PVS1 defaultStrength 'Pathogenic Very Strong' for null variants (nonsense, frameshift, canonical splice site +/-1,2, initiation codon, single/multi-exon deletion) in BRCA2, applied per PVS1 flowchart considering clinically important functional domains.
• pvs1_gene_context: germline_disease_context_found=true, lof_mechanism_supported=true, pvs1_gene_gate='eligible' because official CSPEC/VCEP criteria for BRCA2 include PVS1 guidance, establishing LOF as a recognized disease mechanism.
• pvs1_variant_assessment: consequence_class='nonsense', variant_bucket='nonsense', suggested_default_strength='PVS1', framework_source PMC6185798 (ClinGen SVI PVS1 recommendations) applied since gene-level LOF eligibility is established.
• vcep_specifications_table4_v1_2_2024_11_18 (Specifications_Table4_V1.2_2024-11-18.xlsx): exon-level PVS1 decision table returned 'BRCA2 Exon 11 rows' for this variant lookup; exon 11 is not in the PM5_N/A exclusion list (E12, E27, E6), and exon-11 truncating/PTC events in this table are assigned PVS1 strength with no downgrade flag for this variant.

Still missing

• The bundle's pvs1_variant_assessment left 'exon' as null; exon 11 assignment is inferred from the VCEP materials pull explicitly returning 'BRCA2 Exon 11 rows' from Specifications_Table4 for this variant and from the c.2164 coding position falling within the known large exon 11 coding range, but an explicit exon-number confirmation from the transcript model was not present in the bundle.
• No BRCA2-specific NMD-escape exception (e.g., proximity to final exon, exon skip-and-frame-preservation) was documented in the retrieved VCEP materials for this exact variant; general LOF biology (loss of C-terminal RAD51-binding/NLS/DNA-binding domains) is described only in gene-level literature (PMIDs 10570174, 11239455, 20878484, 22193408, 24312913) that does not mention this specific variant, so it is used only as general mechanistic background, not as variant-specific grounded evidence.

Sources

Reasoning

NM_000059.4:c.2164A>T is NP_000050.3:p.(Lys722Ter), a nonsense variant, so the ENIGMA BRCA1/BRCA2 CSpec PP3 missense/silent bioinformatic path (BayesDel no-AF and SpliceAI thresholds for missense/silent/intronic variant types) does not apply to this variant type. The only PP3 sub-path available to a nonsense substitution under this framework is the predicted-splicing path, which is evaluated on the variant's SpliceAI score irrespective of coding consequence class. SpliceAI predicts no significant splice impact for c.2164A>T (max delta score = 0.01: DS_AG=0.0, DS_AL=0.001, DS_DG=0.0, DS_DL=0.005), far below the ENIGMA PP3 predicted-splicing threshold of SpliceAI >=0.2. Therefore PP3 is not met via either the missense or splicing sub-path.

Rule

ENIGMA BRCA1/BRCA2 CSpec v1.2 PP3 (Specifications Figure 1A, Appendix J): predicted-splicing sub-path requires SpliceAI >=0.2; not met here (max delta 0.01).

Evidence considered

• ENIGMA BRCA1/BRCA2 CSpec v1.2 PP3 rule: apply PP3(missense path) only for missense or in-frame indel variants inside a clinically important functional domain with BayesDel no-AF >=0.30; apply PP3(splicing path) for predicted splicing (SpliceAI >=0.2) for silent, missense/in-frame variants and intronic variants outside donor/acceptor +/-1,2 sites.
• Variant is NP_000050.3:p.(Lys722Ter) (nonsense), not missense, silent, or intronic, so the CSpec's missense-domain sub-path (BayesDel) is not applicable to this variant type; only the splicing sub-path (SpliceAI) is relevant regardless of coding consequence.
• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01).' Scores: DS_AG=0.0, DS_AL=0.001, DS_DG=0.0, DS_DL=0.005 -- all well below the 0.2 PP3 threshold.

Sources

Reasoning

BA1 is not met. The variant was absent from gnomAD v2.1 and gnomAD v4.1; therefore no filter allele frequency above the ENIGMA threshold of 0.1% (FAF > 0.001) was observed.

Rule

Filter allele frequency (FAF) is above 0.1% (FAF > 0.001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s). Do not apply if read depth <20. Do not apply to well-established pathogenic founder variants.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies BA1 as FAF > 0.001 in a gnomAD non-cancer, non-founder population, with exome and genome data considered separately and no application when read depth is <20.
• The variant NM_000059.4:c.2164A>T (NP_000050.3:p.(Lys722Ter)) was reported absent from gnomAD v2.1 and gnomAD v4.1, so the BA1 frequency threshold is not reached.

Still missing

• The case bundle does not provide numeric FAF or read-depth values, although the variant-level searches returned absent in the available gnomAD datasets.

Sources

Reasoning

BS1 is not met. The variant was absent from gnomAD v2.1 and gnomAD v4.1, so no FAF above the ENIGMA BS1 thresholds was observed: >0.0001 for BS1_Strong or >0.00002 and <=0.0001 for BS1_Supporting.

Rule

Filter allele frequency (FAF) is above 0.01% (FAF > 0.0001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s) for BS1_Strong; FAF is above 0.002% (FAF > 0.00002) and <= 0.01% (FAF <= 0.0001) for BS1_Supporting. Do not apply if read depth <20 or for well-established pathogenic founder variants.

Evidence considered

• ENIGMA BRCA2 v1.2 applies BS1 using maximum FAF in a gnomAD non-cancer, non-founder population, with BS1_Strong at FAF > 0.0001 and BS1_Supporting at FAF > 0.00002 through <= 0.0001; read depth must not be <20.
• The variant NM_000059.4:c.2164A>T (NP_000050.3:p.(Lys722Ter)) was reported absent from gnomAD v2.1 and gnomAD v4.1, so neither BS1 frequency range is met.

Still missing

• The case bundle does not provide numeric FAF or read-depth values, although the variant-level searches returned absent in the available gnomAD datasets.

Sources

Reasoning

As with PP3, the ENIGMA CSpec BP4 rule's missense/silent computational sub-path (BayesDel no-AF <=0.18 AND SpliceAI <=0.1, for missense/in-frame variants in a clinically important domain; or SpliceAI <=0.1 alone for silent/intronic variants) is not directly applicable because c.2164A>T is a nonsense variant (p.(Lys722Ter)), not missense, silent, or intronic. However, the underlying SpliceAI evidence needed to evaluate any splicing-based benign computational signal is available: SpliceAI max delta = 0.01, which is below the <=0.1 no-splice-impact threshold used throughout the CSpec bioinformatic flowchart. Despite the low SpliceAI score being consistent with 'no predicted splice impact', BP4 as specified is scoped to missense/silent/intronic variant classes under this VCEP framework and there is no CSpec provision to apply BP4 to a nonsense/PTC-generating variant (such variants are instead evaluated under PVS1/PM5_PTC pathways, which are outside this group's remit). BP4 is therefore not met/not applicable via the missense path (wrong variant class) and the splicing evidence alone does not constitute grounds for BP4 given the CSpec does not extend this code to nonsense substitutions.

Rule

ENIGMA BRCA1/BRCA2 CSpec v1.2 BP4 (Specifications Figure 1A, Appendix J): scoped to missense/in-frame, silent, and intronic variant classes; not applicable to nonsense variant p.(Lys722Ter).

Evidence considered

• ENIGMA BRCA1/BRCA2 CSpec v1.2 BP4 rule: missense/in-frame indel in a clinically important domain requires BayesDel no-AF <=0.18 AND SpliceAI <=0.1; silent variant in a clinically important domain requires SpliceAI <=0.1; intronic variants outside +/-1,2 splice sites require SpliceAI <=0.1.
• Variant is nonsense (p.(Lys722Ter)), not missense/silent/intronic, so none of the enumerated BP4 variant-class conditions in the CSpec apply.
• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01).'

Still missing

• CSpec BP4 rule text (Specifications Figure 1A / Appendix J) enumerates missense/in-frame, silent, and intronic variant classes explicitly; it does not describe an application of BP4 to nonsense/PTC variants, so no code is applied here for that reason.

Sources

Reasoning

NM_000059.4:c.2164A>T (NP_000050.3:p.(Lys722Ter)) is a premature termination codon (nonsense) variant in BRCA2 exon 11. The ENIGMA BRCA1/BRCA2 VCEP specification v1.2 allows PS3 for well-established in vitro/in vivo functional studies (protein-only, or mRNA+protein combined, or mRNA-only supporting PVS1(RNA)) with strength assigned per the curated ENIGMA Table 9 calibrated-assay list. Table 9 (Specifications_Table9_V1.2_2024-11-18.xlsx) was checked and contains no entry for c.2164A>T / p.(Lys722Ter) - the only nearby-codon entries are for the unrelated missense cluster at codon 2722 (c.8165C>G/A, c.8164A>G). The multifactorial likelihood dataset (HUMU-40-1557-s001.xlsx, SuppT1/ST7) and the clinical-history LR file (PMID:31853058) were also checked; neither contains a row for this variant. The five literature-search hits retrieved for this case (PMID:10570174, PMID:11239455, PMID:20878484, PMID:22193408, PMID:24312913) are general BRCA2 biology/mutation-spectrum papers describing homology-directed-repair function of BRCA2 and nuclear-localization loss for other, unrelated truncating variants (6174delT, 7525_7526insT); paper_extracts.json confirms none of them mention c.2164A>T or p.(K722*) specifically, so per strict citation-grounding rules they cannot be used to support a variant-specific PS3 call. No calibrated, variant-specific functional assay (e.g. saturation genome editing, HDR reporter, mRNA splicing assay) for c.2164A>T/p.(Lys722Ter) was located in the case bundle.

Still missing

• No ENIGMA Table 9 calibrated-assay entry for c.2164A>T / p.(Lys722Ter)
• No variant-specific functional (protein/mRNA) assay data located; retrieved literature discusses BRCA2 function generally or other truncating variants, not this exact variant
• This is a nonsense (null) variant already covered mechanistically by PVS1/loss-of-function; a dedicated PS3 functional study specific to this allele was not found

Sources

Reasoning

No variant-specific case-control enrichment data are available for BRCA2 NM_000059.4:c.2164A>T (p.Lys722Ter).

Rule

ENIGMA BRCA2 PS4: significant case-control enrichment (p<=0.05, OR>=4, lower confidence interval excludes 2.0).

Evidence considered

• The ENIGMA BRCA2 specification requires affected-versus-control enrichment with p<=0.05, OR>=4, and lower confidence interval excluding 2.0 for PS4.
• The reviewed ENIGMA multifactorial dataset contains no exact c.2164A>T or p.Lys722Ter entry; it therefore supplies no case-control estimate for this variant.

Still missing

• A variant-specific case-control study reporting odds ratio, confidence interval, and p-value is needed.

Sources

Reasoning

PM2 is not assessable at the specified BRCA2 strength because the variant is absent from gnomAD v2.1, but the required average read depth of at least 25 around the variant is not reported in the case bundle. The available gnomAD v4.1 and gnomAD-Canada absence results are supplementary but do not replace the ENIGMA v2.1/v3.1 and depth requirements.

Rule

Absent from controls in an outbred population, from gnomAD v2.1 (non-cancer, exome only subset) and gnomAD v3.1 (non-cancer). Region around the variant must have an average read depth >=25. Observation of a variant only once in a gnomAD outbred population is not informative; do not apply for insertion, deletion or delins variants or if read depth <25.

Evidence considered

• ENIGMA BRCA2 v1.2 permits PM2 only at Supporting strength when the variant is absent from controls in an outbred population in gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer, and requires average read depth >=25 around the variant.
• The variant was reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, but no average read-depth value is supplied and gnomAD v3.1 is not represented in the bundle; therefore PM2 cannot be confirmed under the governing rule.

Still missing

• Average read depth around the variant is not reported for the required gnomAD population dataset(s).
• The case bundle reports gnomAD v4.1 and gnomAD-Canada results but does not provide a gnomAD v3.1 result.

Sources

Reasoning

The ENIGMA BRCA2 VCEP specification makes PM3 applicable only when the variant under assessment co-occurs with a pathogenic or likely pathogenic variant in the same gene in a patient with a phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia, with strength assigned from the Table 6 point system. The case bundle contains no affected-proband Fanconi-anemia phenotype, chromosome-breakage result, second BRCA2 pathogenic/likely pathogenic variant, phase information, or trans/cis determination. PM3 therefore cannot be assessed.

Rule

Apply PM3 for a patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia and co-occurring variants in the same gene. The phenotype requires either increased chromosome breakage plus at least one qualifying clinical feature, or unknown chromosome-breakage status plus at least two qualifying clinical features from at least two categories. Assign PM3 strength from the Specifications Table 6 point system: PM3_Supporting = 1 point, PM3 = 2 points, PM3_Strong = at least 4 points.

Evidence considered

• The ENIGMA BRCA2 specification states that the co-occurring variant should be classified using VCEP specifications, the assessed variant must be sufficiently rare, and PM3 strength is assigned from the Table 6 point system.
• The ENIGMA BRCA2 PM3 rule requires a patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia and co-occurring variants in the same gene; the available case evidence does not provide the required phenotype, second variant, or phase data.

Still missing

• Documented Fanconi-anemia phenotype in an affected proband, including chromosome-breakage testing and qualifying clinical features
• A co-occurring BRCA2 pathogenic or likely pathogenic variant classified under the VCEP specification
• Parent/offspring or equivalent testing establishing phase, specifically whether the two BRCA2 variants are in trans
• Table 6 PM3 point assignment for each informative proband

Sources

Reasoning

The ENIGMA CSPEC v1.2 repurposes PM5 for BRCA2 as a PTC (protein termination codon)-specific rule: 'Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before,' with per-exon applicability/strength defined in Specifications Table 4. NM_000059.4:c.2164A>T produces NP_000050.3:p.(Lys722Ter), a PTC in exon 11 (BRCA2 coding range c.1910-c.6841 / p.637-p.2281 per Table 4), and Table 4's exon-11 PTC row is annotated 'PM5_Strong (PTC)', confirming this exon is eligible for PM5 stacking at Strong strength if a comparator pathogenic PTC has previously been observed in exon 11. However, the case's own pm5_candidates.json artifact reports pm5_mode='ptc_gene_specific', eligible_for_classic_pm5_search=false, and zero candidates collected (total_candidates_seen=0, same_residue_candidates=0), explicitly instructing not to apply PM5 from that artifact alone. No specific previously-classified pathogenic PTC variant in BRCA2 exon 11 was identified or reviewed in this evidence bundle to serve as the required comparator, so PM5 cannot be confirmed met or not_met.

Rule

ENIGMA BRCA1/BRCA2 CSPEC v1.2 Table 1, PM5 (PTC repurposing): apply per-exon PM5_PTC strength from Specifications Table 4 when a different proven pathogenic PTC variant has previously been observed in the same exon; BRCA2 exon 11 is annotated PM5_Strong (PTC)-eligible.

Evidence considered

• Specifications Table 4 (ENIGMA BRCA2 exon-level PVS1/PM5 decision table) annotates BRCA2 exon 11 (c.1910-c.6841, p.637-p.2281, containing c.2164) with 'PVS1 / PTC / PM5_Strong (PTC)', confirming exon 11 is PM5_PTC-applicable (not PM5_N/A) at Strong strength when a comparator proven pathogenic PTC exists in the same exon.
• pm5_candidates.json for this case reports pm5_mode='ptc_gene_specific', eligible_for_classic_pm5_search=false, 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,' indicating no comparator PTC variant was actually identified in this workup.
• ENIGMA CSPEC v1.2 PM5 rule text: 'Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before. Use to justify additional weight for PTC variants annotated as PVS1. See Specifications Table 4 for PM5_PTC code strengths applicable per exon.'

Still missing

• No comparator search was performed to identify a specific previously-classified pathogenic PTC variant elsewhere in BRCA2 exon 11 (c.1910-c.6841) that would satisfy the CSPEC PM5_PTC rule; pm5_candidates.json returned zero candidates and flagged this variant type as excluded from the classic same-residue PM5 search path.
• A ClinVar or literature lookup for other exon-11 PTC pathogenic variants (e.g. against Specifications Table 4 / Supplementary Table 1 PM5_PTC weighting) is needed before PM5 can be scored.

Sources

Reasoning

PP1 is applicable under the ENIGMA BRCA2 specification only when quantitative co-segregation evidence supports an LR of at least 2.08:1. The consolidated case-evidence bundle contains no proband-level parental testing, affected-relative genotypes and phenotypes, meioses, family structure, or quantitative co-segregation LR for NM_000059.4:c.2164A>T; therefore PP1 cannot be assessed.

Rule

Quantitative co-segregation with disease in multiple affected family members; PP1 Supporting LR >=2.08:1, PP1 Moderate LR >=4.3:1, PP1 Strong LR >=18.7:1.

Evidence considered

• ENIGMA BRCA1/2 Specification v1.2 recommends quantitative co-segregation analysis and states that LR >0.48 and <2.08 provides no supporting evidence in either direction; PP1 Supporting requires LR >=2.08:1, Moderate requires LR >=4.3:1, and Strong requires LR >=18.7:1.
• The case-evidence bundle provides no case-specific segregation, parental, proband, affected-relative, meiosis, or quantitative LR evidence for this variant.

Still missing

• Obtain family-level segregation data identifying affected and unaffected relatives, their variant testing results, phenotype status, number of informative meioses, and a quantitative co-segregation LR.

Sources

Reasoning

No exact-variant BRCA2 clinical-history likelihood ratio is available for c.2164A>T, so phenotype specificity cannot be quantified under the ENIGMA framework.

Rule

ENIGMA BRCA2 PP4: use only a combined multifactorial likelihood ratio toward pathogenicity; minimum Supporting threshold LR>=2.08.

Evidence considered

• ENIGMA limits PP4 to combined multifactorial clinical data and assigns Supporting at LR>=2.08, Moderate at LR>=4.3, Strong at LR>=18.7, and Very Strong at LR>=350.
• The BRCA2 clinical-history likelihood-ratio table was checked for c.2164A>T and has no exact entry.

Still missing

• An exact-variant BRCA2 personal/family-history likelihood ratio or a qualifying combined multifactorial likelihood ratio is needed.

Sources

Reasoning

BS2 could not be assessed. The ENIGMA rule requires evaluation of individuals carrying the variant in the absence of Fanconi Anemia features, with point assignment per proband; the case bundle contains no homozygote observations, carrier phenotype data, or proband-level BS2 pointable evidence.

Rule

Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See Specifications Table 8 for additional stipulations, approach to assign points per proband, and final BS2 code assignment based on the sum of BS2-related points. BS2 = >=4 points for Strong, 2 points for Moderate, and 1 point for Supporting.

Evidence considered

• ENIGMA BRCA2 v1.2 defines BS2 around absence of features of recessive disease, specifically Fanconi Anemia phenotype, and requires ENIGMA Table 8 point assignment per proband; those data are absent from this case bundle.

Still missing

• No homozygous or other carrier observations with clinical phenotype are provided.
• No Fanconi Anemia phenotype assessment or ENIGMA Table 8 BS2 point calculation is provided.

Sources

Reasoning

BS4 is applicable under the ENIGMA BRCA2 specification only when quantitative co-segregation evidence demonstrates lack of segregation in affected family members with an LR of 0.48:1 or lower. The consolidated case-evidence bundle contains no affected-relative genotypes and phenotypes, family structure, non-segregation observations, meioses, or quantitative co-segregation LR for NM_000059.4:c.2164A>T; therefore BS4 cannot be assessed.

Rule

Quantitative lack of segregation in affected family members; BS4 Supporting LR <=0.48:1, BS4 Moderate LR <=0.23:1, BS4 Strong LR <=0.05:1, BS4 Very Strong LR <=0.00285:1.

Evidence considered

• ENIGMA BRCA1/2 Specification v1.2 recommends quantitative co-segregation analysis and states that LR >0.48 and <2.08 provides no supporting evidence in either direction; BS4 Supporting requires LR <=0.48:1, Moderate requires LR <=0.23:1, and Strong requires LR <=0.05:1.
• The case-evidence bundle provides no case-specific non-segregation, affected-relative, family, meiosis, or quantitative LR evidence for this variant.

Still missing

• Obtain family-level non-segregation data identifying affected relatives who lack the variant, confirmation of phenotype and testing quality, number of informative meioses, and a quantitative co-segregation LR.

Sources

Reasoning

No exact-variant BRCA2 clinical-history likelihood ratio is available for c.2164A>T, so benign-direction multifactorial phenotype evidence cannot be assessed.

Rule

ENIGMA BRCA2 BP5: use only a combined multifactorial likelihood ratio against pathogenicity; minimum Supporting threshold LR<=0.48.

Evidence considered

• ENIGMA limits BP5 to combined multifactorial clinical data and assigns Supporting at LR<=0.48, Moderate at LR<=0.23, Strong at LR<=0.05, and Very Strong at LR<=0.00285.
• The BRCA2 clinical-history likelihood-ratio table was checked for c.2164A>T and has no exact entry.

Still missing

• An exact-variant BRCA2 personal/family-history likelihood ratio or a qualifying combined multifactorial likelihood ratio is needed.

Sources