BRCA2 c.7617+16C>T, p.?

Reasoning

The variant is intronic and outside the native donor/acceptor +/-1,2 positions (+16). Its maximum SpliceAI delta score is 0.018, below the ENIGMA BRCA2 v1.2 BP4 threshold of 0.1; BP4_Supporting is met.

Rule

ENIGMA BRCA2 v1.2 BP4: intronic variant outside +/-1,2 and SpliceAI <=0.1.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies BP4_Supporting for intronic variants outside native donor and acceptor +/-1,2 positions with SpliceAI <=0.1.
• SpliceAI for NM_000059.4:c.7617+16C>T reports a maximum delta score of 0.018.

Sources

Reasoning

ENIGMA BRCA2 v1.2 permits BP7_Supporting for an intronic variant at or beyond +7/-21 when BP4 is met. This variant is at +16 and meets BP4 based on SpliceAI 0.018. BP7 is applied as the specification's positional criterion conditional on BP4, not as an independent second splice-prediction result.

Rule

ENIGMA BRCA2 v1.2 BP7: intronic variant at or beyond +7/-21 if BP4 is met.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies BP7_Supporting for intronic variants at or beyond positions +7/-21 if BP4 is met.
• NM_000059.4:c.7617+16C>T is 16 nucleotides into the intron and SpliceAI maximum delta is 0.018, which meets the linked BP4 requirement.

Sources

Reasoning

NM_000059.4:c.7617+16C>T is a non-canonical intronic substitution (+16), not a null variant or a canonical donor/acceptor ±1,2 splice-site variant. The ENIGMA BRCA2 PVS1 specification therefore does not assign PVS1 from sequence consequence. SpliceAI predicts no significant splice impact (maximum delta 0.018), providing no evidence that this variant creates a loss-of-function transcript.

Rule

ENIGMA BRCA2 v1.2 PVS1: apply to null variants (including splice donor/acceptor ±1,2) or to well-established mRNA-only studies demonstrating a damaging transcript effect; apply the PVS1 flowchart and exon-level table.

Evidence considered

• The ENIGMA BRCA2 specification limits PVS1 to null variants, including canonical donor/acceptor ±1,2 splice variants, with strength determined by its PVS1 framework; it also permits RNA-based PVS1 only for well-established mRNA studies showing a damaging transcript effect.
• The submitted transcript consequence is c.7617+16C>T, which is 16 bases into the intron and outside the canonical ±1,2 splice positions.
• SpliceAI maximum delta score is 0.018 (DS_AG 0.018; DS_DG 0.005), reported as no significant predicted splice impact.

Still missing

• No variant-specific RNA study is available; such evidence would be required to reconsider PVS1 if a splice defect were demonstrated.

Sources

Reasoning

The variant is intronic (NM_000059.4:c.7617+16C>T) with no protein change assigned (p.?), and SpliceAI predicts no significant splice impact (maximum delta score 0.018). ENIGMA BRCA2 PS1 requires either the same protein-level missense substitution as a previously pathogenic variant or a precisely matching predicted/proven splice event to a previously (likely) pathogenic variant. No such same amino-acid change or matching splice event is documented for this variant; the nearby pathogenic c.7617+1G>T and c.7617+2T>G observations are different canonical donor variants with reported exon-skipping effects and do not establish the required equivalent impact for c.7617+16C>T.

Rule

ENIGMA BRCA2 v1.2 PS1: same pathogenic protein-level missense change, or precisely matching splice impact with comparable or stronger prediction; avoid applying when the reference or target effect is not established at the required level.

Evidence considered

• The governing ENIGMA BRCA2 specification v1.2 permits PS1 for a predicted missense substitution when the same protein change was previously classified as pathogenic, or for variants with a precisely matching predicted splice impact; it also requires consideration that the effect is not instead due to splicing.
• This case has no assigned protein consequence (NP_000050.3:p.?), is intronic at c.7617+16C>T, and SpliceAI reports a maximum delta score of 0.018, below the ENIGMA no-significant-splice-impact threshold of 0.1.
• The VCEP-extracted BRCA2 evidence lists pathogenic c.7617+1G>T and c.7617+2T>G splice-site variants with exon 15 skipping, but these are not the same predicted event established for c.7617+16C>T and cannot by themselves satisfy PS1.

Sources

Reasoning

The ENIGMA BRCA2 v1.2 PP3 splice-prediction rule requires SpliceAI >=0.2 for intronic variants outside donor/acceptor +/-1,2 positions. This intronic +16 variant has a maximum SpliceAI delta score of 0.018 and therefore does not meet PP3.

Rule

ENIGMA BRCA2 v1.2 PP3: intronic variants outside donor/acceptor +/-1,2 require SpliceAI >=0.2.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies PP3_Supporting for predicted splicing in eligible intronic variants when SpliceAI is at least 0.2.
• SpliceAI for NM_000059.4:c.7617+16C>T reports DS_AG 0.018, DS_DG 0.005, DS_AL 0, and DS_DL 0 (maximum delta 0.018).

Sources

Reasoning

ClinVar Variation ID 531528 is an exact match to c.7617+16C>T but has one Likely benign submission from a clinical laboratory, with no expert-panel submission. It cannot support PP5.

Rule

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

Evidence considered

• ClinVar reports one exact-variant Likely benign submission from Labcorp Genetics with review status 'criteria provided, single submitter'; the submission audit records zero expert-panel submissions.
• The ENIGMA BRCA2 specification lists PP5 as not applicable; independently, the required exact-variant expert-panel condition is not present.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion would be required for PP5.

Sources

Reasoning

BA1 is not met. The BRCA2 ENIGMA v1.2 rule requires FAF >0.001 (0.1%) in gnomAD v2.1 non-cancer exomes and/or gnomAD v3.1 non-cancer non-founder populations. The variant is absent from gnomAD v2.1 and the available gnomAD v4.1 data show total AF 2.48e-6, grpmax FAF 3.65e-6, and highest subpopulation AF 3.38e-5 in Ashkenazi Jewish individuals, all far below 0.001. gnomAD v4.1 is not used as a substitute for the specified v3.1 dataset.

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). See Appendix G for details.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies BA1 when FAF is above 0.1% (FAF >0.001) in gnomAD v2.1 non-cancer exome-only and/or gnomAD v3.1 non-cancer non-founder populations.
• gnomAD v2.1 reports the variant as absent.
• gnomAD v4.1 reports total AF 2.48041e-06 (4/1,612,638 alleles), grpmax FAF 3.65e-06, and highest observed subpopulation AF 3.37861e-05 (1/29,598 alleles) in Ashkenazi Jewish individuals; all are below the BA1 threshold.

Sources

Reasoning

BS1 is not met. The ENIGMA BRCA2 v1.2 thresholds are FAF >0.0001 for benign strong or FAF >0.00002 and <=0.0001 for benign supporting, assessed in gnomAD v2.1 and/or gnomAD v3.1 non-cancer non-founder populations. The variant is absent from gnomAD v2.1; gnomAD v4.1 shows grpmax FAF 3.65e-6 and highest subpopulation AF 3.38e-5, below the BS1 supporting lower threshold of 2.0e-5 for grpmax FAF, although the single Ashkenazi Jewish subpopulation estimate is above that lower threshold. Because the framework specifies FAF and the qualifying datasets are v2.1/v3.1, the gnomAD v4.1 observation cannot independently establish BS1; no gnomAD v3.1 result is provided.

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); supporting strength applies when FAF is above 0.002% (FAF >0.00002) and <=0.01% (FAF <=0.0001). See Appendix G for details.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies BS1 strong for FAF >0.0001 and BS1 supporting for FAF >0.00002 and <=0.0001 in gnomAD v2.1 and/or gnomAD v3.1 non-cancer non-founder populations.
• gnomAD v2.1 reports the variant as absent.
• gnomAD v4.1 reports grpmax FAF 3.65e-6, below the BS1 supporting threshold, while the highest single subpopulation AF is 3.37861e-5 in Ashkenazi Jewish individuals. The required framework dataset gnomAD v3.1 is not present, so the v4.1 subpopulation observation is not independently sufficient to apply BS1.

Sources

Reasoning

ClinVar Variation ID 531528 is an exact match to c.7617+16C>T but its only Likely benign assertion is from a single clinical laboratory, not an expert panel. It cannot support BP6.

Rule

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

Evidence considered

• ClinVar reports one exact-variant Likely benign submission from Labcorp Genetics with review status 'criteria provided, single submitter'; the submission audit records zero expert-panel submissions.
• The ENIGMA BRCA2 specification lists BP6 as not applicable; independently, the required exact-variant expert-panel condition is not present.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign assertion would be required for BP6.

Sources

Reasoning

No validated variant-specific functional assay evidence was identified for BRCA2 NM_000059.4:c.7617+16C>T. The ENIGMA BRCA1/2 VCEP framework reserves PS3 for well-established assays measuring protein function, or combined mRNA and protein effects, and directs curated calibrated assay results to Table 9. The variant has no matching entry in the curated Table 9 functional-assay dataset, and the case bundle contains no variant-specific protein or combined mRNA/protein assay result. Therefore, a damaging functional effect cannot be assigned under PS3.

Rule

ENIGMA BRCA2 VCEP v1.2 PS3: apply only for well-established functional studies supportive of a damaging effect when the assay measures protein function alone or mRNA and protein combined; use calibrated published assay recommendations from Table 9.

Evidence considered

• The ENIGMA BRCA1/2 VCEP specification states that PS3 is for well-established in vitro or in vivo functional studies measuring protein function, or mRNA and protein together, and refers to Table 9 for calibrated published assay recommendations.
• The variant NM_000059.4:c.7617+16C>T was searched in the ENIGMA Table 9 workbook; no matching variant-specific functional-assay row was found.
• The case evidence bundle reports SpliceAI max delta 0.018 and no variant-specific functional assay finding; computational splice prediction alone is not validated functional assay evidence for PS3.

Still missing

• No variant-specific validated protein-function or combined mRNA/protein assay result with appropriate controls and calibrated interpretation was available.

Sources

Reasoning

The ENIGMA BRCA2 specification requires an ethnicity- and country-matched case-control study with p<=0.05, OR>=4, and lower confidence interval excluding 2.0. No such exact-variant dataset or case-control result was provided for BRCA2 c.7617+16C>T.

Rule

ENIGMA BRCA2 PS4: case-control p<=0.05 and OR>=4 with lower confidence interval excluding 2.0; do not use proband counting.

Evidence considered

• The ENIGMA BRCA2 specification applies PS4 only to significantly enriched case-control evidence (p<=0.05, OR>=4, lower confidence interval excluding 2.0) and does not permit proband counting.
• The literature bundle contains no exact-variant case-control publication or quantitative enrichment result for c.7617+16C>T.

Still missing

• An exact-variant, ancestry-matched case-control study reporting case and control counts, odds ratio, confidence interval, and p-value is needed.

Sources

Reasoning

PM2 was not assessed because the required BRCA2 ENIGMA v1.2 population datasets are incomplete. The rule requires absence from controls in both gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer, with local read depth >=25. The variant is absent from gnomAD v2.1, but no gnomAD v3.1 result or regional read-depth value is provided. gnomAD v4.1 and gnomAD-Canada are not specified substitutes; gnomAD v4.1 does contain the variant at AF 2.48e-6 with zero homozygotes, so the variant is not globally absent from available population data.

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. See Appendix G for details.

Evidence considered

• ENIGMA BRCA2 v1.2 PM2 Supporting requires absence from controls in gnomAD v2.1 non-cancer exome-only and gnomAD v3.1 non-cancer, with average read depth around the variant >=25.
• gnomAD v2.1 reports the variant as absent.
• gnomAD v4.1 reports the variant present at total AF 2.48041e-06 (4/1,612,638 alleles), grpmax FAF 3.65e-6, and zero homozygotes; gnomAD-Canada v1.0 reports it as absent.
• The case bundle does not provide gnomAD v3.1 data or the required regional average read-depth metric.

Still missing

• No gnomAD v3.1 non-cancer result is provided.
• No average read-depth value for the region is provided.
• The available gnomAD v4.1 presence cannot be used to satisfy the v3.1-specific ENIGMA rule, but it prevents an uncomplicated claim of absence from all available population datasets.

Sources

Reasoning

The applicable ENIGMA BRCA2 specification limits PM3 to a patient with a phenotype consistent with BRCA1/2-related Fanconi anemia and a co-occurring variant in the same gene, with strength assigned from PM3 points per proband. The case bundle provides no affected-proband Fanconi anemia phenotype, no second BRCA2 pathogenic/likely pathogenic variant, and no phase information establishing trans or cis. Therefore PM3 cannot be assessed.

Rule

Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene. PM3_Supporting = 1 point, PM3 = 2 points, and PM3_Strong = >=4 points, using the Specifications Table 6 point system per proband.

Evidence considered

• ENIGMA BRCA1/2 Specification version 1.2 states that PM3 requires a patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia and co-occurring variants in the same gene; the PM3 strength is based on points assigned per proband.
• The case bundle identifies the assessed variant as NM_000059.4:c.7617+16C>T in BRCA2 and reports no co-occurring BRCA2 variant, affected-proband observation, or phase result.
• The assessed variant is rare in the available population data and does not meet a benign population evidence code, but rarity alone does not establish PM3 without the required biallelic Fanconi anemia observation and phase information.

Still missing

• Document whether the proband has a phenotype consistent with BRCA1/2-related Fanconi anemia, including chromosome-breakage testing and qualifying clinical features.
• Identify any co-occurring BRCA2 pathogenic or likely pathogenic variant in the same proband.
• Establish phase of the two BRCA2 variants by parental, offspring, or equivalent phasing studies.
• Apply the ENIGMA PM3 point system per qualifying proband once the above data are available.

Sources

Reasoning

PP1 is applicable under the ENIGMA BRCA2 specification, but the case-evidence bundle provides no affected-family-member genotypes, phenotype-linked segregation observations, meioses, quantitative co-segregation analysis, or likelihood ratio. Therefore PP1 cannot be assigned.

Rule

Apply PP1 only for co-segregation with disease in multiple affected family members using a quantitative co-segregation method; LR >= 2.08:1 Supporting, >= 4.3:1 Moderate, >= 18.7:1 Strong, and >= 350:1 Very Strong. LR > 0.48 and < 2.08 provides no supporting evidence in either direction.

Evidence considered

• ENIGMA BRCA2 specification v1.2 requires quantitative co-segregation evidence and assigns PP1 strength by likelihood ratio: Supporting at LR >= 2.08:1, Moderate at LR >= 4.3:1, and Strong at LR >= 18.7:1; Very Strong is described at LR >= 350:1.
• The bundle contains no family segregation dataset or quantitative co-segregation result. The single-submitter ClinVar classification is not segregation evidence and was not used to infer PP1.

Still missing

• Affected and unaffected relatives with confirmed BRCA2 c.7617+16C>T genotypes
• Phenotype and age information for informative relatives
• Number of informative meioses
• Quantitative co-segregation analysis or likelihood ratio from COOL or CAL-Leiden
• Assessment of possible phenocopies or an alternative causative pathogenic variant

Sources

Reasoning

For BRCA2, ENIGMA permits PP4 only for a combined multifactorial clinical-data LR toward pathogenicity. No combined LR was supplied for c.7617+16C>T, and the variant is absent from the Li et al. BRCA2 clinical-history LR table; therefore no PP4 strength can be assigned.

Rule

ENIGMA BRCA2 PP4: combined clinical-data LR >=2.08 (Supporting), >=4.3 (Moderate), >=18.7 (Strong), or >=350 (Very Strong).

Evidence considered

• The ENIGMA BRCA2 specification restricts PP4 for the genetically heterogeneous breast-cancer phenotype to combined multifactorial clinical-data LR evidence.
• The Li et al. BRCA2 clinical-history LR workbook was queried for c.7617+16C>T and contained no matching row. This resource is a clinical-history component only, not a combined LR.

Still missing

• A variant-specific, independently reviewable combined multifactorial clinical LR is needed; PP4 Supporting requires LR>=2.08, Moderate >=4.3, Strong >=18.7, and Very Strong >=350.

Sources

Reasoning

BS2 was not assessed. The BRCA2 ENIGMA v1.2 rule applies BS2 in the absence of Fanconi anemia features and requires point assignment per proband from the specified clinical evidence. The case bundle provides no proband phenotype, age, clinical findings, chromosome-breakage results, or BS2 point calculation; population allele counts alone cannot establish BS2.

Rule

Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See Specifications Table 8 for additional stipulations and approach to assign points per proband; BS2 Strong = >=4 points, Moderate = 2 points, Supporting = 1 point.

Evidence considered

• ENIGMA BRCA2 v1.2 defines BS2 as applied in the absence of features of recessive disease, specifically Fanconi anemia phenotype, with strength based on the summed BS2-related points from the specified clinical assessment.
• The case bundle contains population-frequency data but no proband-level Fanconi anemia assessment or BS2 point total.

Still missing

• Proband-level phenotype and clinical findings relevant to Fanconi anemia are not provided.
• No chromosome-breakage result or ENIGMA Table 8 BS2 point calculation is available.
• The number and eligibility of informative probands for BS2 are unknown.

Sources

Reasoning

No validated variant-specific functional assay evidence was identified for BRCA2 NM_000059.4:c.7617+16C>T. The ENIGMA BRCA1/2 VCEP framework reserves BS3 for well-established assays showing no damaging effect on protein function, or combined mRNA and protein effects, and directs curated calibrated assay results to Table 9. The variant has no matching entry in the curated Table 9 functional-assay dataset, and the case bundle contains no variant-specific protein or combined mRNA/protein assay result. Therefore, a benign functional effect cannot be assigned under BS3.

Rule

ENIGMA BRCA2 VCEP v1.2 BS3: apply only for well-established functional studies showing no damaging effect on protein function when the assay measures protein function alone or mRNA and protein combined; use calibrated published assay recommendations from Table 9.

Evidence considered

• The ENIGMA BRCA1/2 VCEP specification states that BS3 is for well-established in vitro or in vivo functional studies showing no damaging effect on protein function, with the assay measuring protein function alone or mRNA and protein together, and refers to Table 9 for calibrated published assay recommendations.
• The variant NM_000059.4:c.7617+16C>T was searched in the ENIGMA Table 9 workbook; no matching variant-specific functional-assay row was found.
• The case evidence bundle reports SpliceAI max delta 0.018 and no variant-specific functional assay finding; absence of a significant computational splice prediction does not establish a benign protein-function result for BS3.

Still missing

• No variant-specific validated protein-function or combined mRNA/protein assay result with appropriate controls and calibrated interpretation was available.

Sources

Reasoning

BS4 is applicable under the ENIGMA BRCA2 specification, but the case-evidence bundle provides no affected-family-member genotypes, phenotype-linked non-segregation observations, meioses, quantitative co-segregation analysis, or likelihood ratio. Therefore BS4 cannot be assigned.

Rule

Apply BS4 only for lack of segregation in affected family members using a quantitative co-segregation method; LR <= 0.48:1 Supporting, <= 0.23:1 Moderate, <= 0.05:1 Strong, and <= 0.00285:1 Very Strong. LR > 0.48 and < 2.08 provides no supporting evidence in either direction.

Evidence considered

• ENIGMA BRCA2 specification v1.2 requires quantitative evidence of lack of segregation and assigns BS4 strength by likelihood ratio: Supporting at LR <= 0.48:1, Moderate at LR <= 0.23:1, Strong at LR <= 0.05:1, and Very Strong at LR <= 0.00285:1.
• The bundle contains no family segregation or non-segregation dataset and no quantitative likelihood ratio. The ClinVar classification and population observations do not establish BS4 and were not used to infer it.

Still missing

• Affected relatives who lack the variant despite a phenotype consistent with BRCA2-related cancer predisposition
• Confirmed genotypes and phenotype/age information for informative relatives
• Number of informative meioses
• Quantitative co-segregation analysis or likelihood ratio from COOL or CAL-Leiden
• Assessment of phenocopies and the possibility of more than one pathogenic variant in the family

Sources

Reasoning

For BRCA2, ENIGMA restricts BP5 to a combined multifactorial clinical-data LR against pathogenicity and excludes use for co-observation with another breast-ovarian cancer risk gene. No combined LR was supplied for c.7617+16C>T, and the Li et al. clinical-history LR table has no matching row; BP5 cannot be assigned.

Rule

ENIGMA BRCA2 BP5: combined clinical-data LR <=0.48 (Supporting), <=0.23 (Moderate), <=0.05 (Strong), or <=0.00285 (Very Strong).

Evidence considered

• The ENIGMA BRCA2 specification permits BP5 only for combined multifactorial clinical-data evidence against pathogenicity, not for alternate-gene co-observation.
• The Li et al. BRCA2 clinical-history LR workbook was queried for c.7617+16C>T and contained no matching row. This resource is a clinical-history component only, not a combined LR.

Still missing

• A variant-specific, independently reviewable combined multifactorial clinical LR is needed; BP5 Supporting requires LR<=0.48, Moderate <=0.23, Strong <=0.05, and Very Strong <=0.00285.

Sources