BRCA2 c.3073A>G, p.(Lys1025Glu)

Reasoning

BS1 Supporting is met. Under ENIGMA BRCA2 v1.2, FAF >0.00002 and <=0.0001 in a gnomAD non-founder population qualifies for Supporting. The variant has maximum FAF 5.405e-05 in gnomAD v2.1 and 5.795e-05 in gnomAD v4.1, within this interval.

Rule

Filter allele frequency (FAF) is above 0.002% (FAF > 0.00002) and less than or equal to 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). See Appendix G for details.

Evidence considered

• The applicable ClinGen ENIGMA BRCA1/2 specification v1.2 defines BS1 Supporting as maximum non-founder gnomAD FAF above 0.002% and at or below 0.01% (0.00002 < FAF <= 0.0001).
• gnomAD v2.1 reports maximum group FAF 5.405e-05 (0.005405%), with 12/250060 total alleles and zero homozygotes; gnomAD v4.1 reports maximum group FAF 5.795e-05 (0.005795%), with 86/1613120 total alleles and zero homozygotes.
• The gnomAD v2.1 and v4.1 FAF values are above the Supporting lower threshold and below the Strong threshold of FAF >0.0001; the small-subgroup raw AF values are not substituted for FAF.

Sources

Reasoning

BS4 Supporting is met because the exact-variant ENIGMA multifactorial dataset reports a segregation likelihood ratio of 0.445, which is <=0.48 and therefore meets the ENIGMA Supporting threshold for lack of segregation. The evidence is quantitative, although the bundle does not provide the underlying pedigree or individual meiosis-level observations.

Rule

ClinGen ENIGMA BRCA2 v1.2 Appendix I / quantitative Bayes-score approach: BS4 Supporting requires LR <=0.48; BS4 Moderate requires LR <=0.23; BS4 Strong requires LR <=0.05.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies BS4 Supporting for quantitative lack-of-segregation LR <=0.48, Moderate for LR <=0.23, Strong for LR <=0.05, and Very Strong for LR <=0.00285.
• The exact-variant ENIGMA multifactorial row for BRCA2 c.3073A>G (p.Lys1025Glu) reports Segregation LR = 0.445, meeting the Supporting threshold.
• The variant-specific paper extraction for PMID:32599251 reports one observation of rs80358550 (p.K1025E) but no segregation analysis; it therefore does not independently establish BS4.

Still missing

• The underlying pedigree, affected-relative genotypes, informative meioses, and calculation details for the reported segregation LR are not included in the case bundle.

Sources

Reasoning

The variant is a missense substitution at Lys1025, outside both ENIGMA-defined clinically important domains (aa 10-40 and aa 2481-3186), and SpliceAI predicts no significant splice impact (maximum delta score 0.00). The governing ENIGMA rule therefore supports BP1_Strong for a missense variant outside a clinically important domain without predicted splicing impact.

Rule

Apply BP1_Strong for a silent, missense, or in-frame insertion/deletion/delins variant outside a clinically important functional domain with no predicted splicing impact (SpliceAI <=0.1).

Evidence considered

• ENIGMA BRCA2 v1.2 specifies BP1_Strong for silent, missense, or in-frame insertion/deletion/delins variants outside a clinically important domain when SpliceAI is <=0.1.
• The variant is NP_000050.3:p.(Lys1025Glu), and Lys1025 is outside the ENIGMA domains aa 10-40 and aa 2481-3186.
• SpliceAI maximum delta score is 0.00, below the ENIGMA threshold of 0.1.

Sources

Reasoning

NM_000059.4:c.3073A>G is a MANE Select exonic missense variant, NP_000050.3:p.(Lys1025Glu), rather than a nonsense, frameshift, initiation-codon, canonical splice-site, or exon-deletion variant. The ENIGMA BRCA2 specification permits PVS1 for null variants or for well-established mRNA-only assays showing a damaging transcript effect. No such RNA evidence is present, and SpliceAI reports a maximum delta score of 0.00. PVS1 is therefore not met.

Rule

ENIGMA BRCA2 VCEP v1.2 PVS1: apply to eligible null variants at flowchart-determined strength, or as PVS1(RNA) only for well-established mRNA-only studies showing a damaging transcript profile.

Evidence considered

• Variant normalization identifies NM_000059.4:c.3073A>G as the exonic missense substitution NP_000050.3:p.(Lys1025Glu) on the MANE Select transcript; it is not a default PVS1 null-variant class.
• ENIGMA BRCA2 VCEP v1.2 specifies PVS1 for null variants and permits PVS1(RNA) only when well-established mRNA-only functional studies support a damaging transcript effect.
• SpliceAI reports no significant splice impact, with maximum delta score 0.00.
• PMID:32599251 explicitly identifies the exact variant as BRCA2 p.K1025E in exon 11 and reports only in-silico analyses; its extraction reports no experimental functional assay or segregation analysis for this variant.

Sources

Reasoning

No qualifying case-control enrichment was identified for BRCA2 c.3073A>G (p.(Lys1025Glu)). The ENIGMA multifactorial record has no case-control LR for this exact variant, so the ENIGMA PS4 requirement is not satisfied.

Rule

ENIGMA BRCA2 v1.2 PS4: case-control p<=0.05 and OR>=4 with lower confidence interval excluding 2.0.

Evidence considered

• ENIGMA BRCA2 PS4 requires significant affected-versus-control enrichment with p<=0.05, OR>=4, and a lower confidence bound excluding 2.0.
• The exact ENIGMA SuppT1 record for c.3073A>G lists Case-Control LR as blank; it does not supply the required case-control enrichment result.

Still missing

• A variant-specific case-control study reporting an odds ratio, confidence interval, and p-value would be required to reassess PS4.

Sources

Reasoning

PM2 Supporting is not met because the variant is observed in gnomAD non-cancer controls. ENIGMA requires absence from controls in an outbred population, with adequate local read depth; this variant is present at 12/250060 alleles in gnomAD v2.1 and 86/1613120 alleles in gnomAD v4.1, with zero homozygotes.

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

• The applicable ClinGen ENIGMA BRCA1/2 specification v1.2 defines PM2 Supporting as absence from controls in an outbred population in gnomAD v2.1 and v3.1, with average local read depth at least 25; the criterion does not apply when the variant is observed.
• gnomAD v2.1 reports the variant at AF 4.79885e-05 (12/250060 alleles) and gnomAD v4.1 reports AF 5.33128e-05 (86/1613120 alleles); both report zero homozygotes. Presence in controls prevents PM2, regardless of the absence of homozygotes.

Sources

Reasoning

PP1 is not supported. The ENIGMA multifactorial dataset reports a segregation likelihood ratio of 0.445 for BRCA2 c.3073A>G, which is below 1 and therefore favors lack of cosegregation rather than cosegregation. It does not meet the ENIGMA PP1 threshold of LR >=2.08 for supporting evidence, and no additional affected relatives, meioses, or positive segregation series are documented.

Rule

ClinGen ENIGMA BRCA2 v1.2 Appendix I / quantitative Bayes-score approach: PP1 Supporting requires LR >=2.08; apply only for cosegregation with disease.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies PP1 Supporting for quantitative cosegregation LR >=2.08, Moderate for LR >=4.3, Strong for LR >=18.7, and Very Strong for LR >=350.
• The exact-variant ENIGMA multifactorial row for BRCA2 c.3073A>G (p.Lys1025Glu) reports Segregation LR = 0.445, which is not positive cosegregation evidence.

Still missing

• No detailed pedigree, number of informative meioses, affected-relative genotypes, or independent positive cosegregation analysis is provided.

Sources

Reasoning

ENIGMA BRCA2 v1.2 PP3 Supporting requires either a missense/in-frame variant within the PALB2-binding (aa 10-40) or DNA-binding (aa 2481-3186) domain with BayesDel no-AF >=0.30, or predicted splice impact with SpliceAI >=0.20. p.(Lys1025Glu) is outside both defined domains and SpliceAI maximum delta is 0.00. The available BayesDel value was not used because no verified published calibration threshold is available for this pipeline. The exact-variant paper reports uncalibrated/discordant prediction-tool outputs only and no functional or RNA assay.

Rule

ENIGMA BRCA2 v1.2 PP3: missense/in-frame in aa 10-40 or aa 2481-3186 with BayesDel no-AF >=0.30, or predicted splicing with SpliceAI >=0.20.

Evidence considered

• The governing ENIGMA BRCA2 v1.2 specification defines PP3 Supporting for missense/in-frame variants only in the PALB2-binding domain (aa 10-40) or DNA-binding domain (aa 2481-3186) when BayesDel no-AF is >=0.30, or for predicted splicing with SpliceAI >=0.20.
• The case is p.(Lys1025Glu), outside the two ENIGMA-defined domains; SpliceAI maximum delta is 0.00, below the PP3 splice threshold.
• BayesDel -0.185286 is available but is not used for PP3 because this pipeline has no verified published BayesDel calibration threshold.
• PMID:32599251 directly mentions rs80358550/p.K1025E but reports only discordant in-silico predictions and no variant-specific functional or RNA assay.

Sources

Reasoning

The exact ENIGMA multifactorial record gives a combined LR of 2.035204629841311, below the ENIGMA PP4_Supporting threshold of 2.08. No exact-variant clinical-history LR is present in the ENIGMA clinical-history LR table. The one-person report in PMID:32599251 does not provide a phenotype-specific likelihood ratio.

Rule

ENIGMA BRCA2 v1.2 PP4: apply only to combined multifactorial clinical LR; Supporting LR>=2.08, Moderate>=4.3, Strong>=18.7, Very Strong>=350.

Evidence considered

• ENIGMA BRCA2 permits PP4 only for combined multifactorial clinical data; supporting strength requires LR>=2.08.
• The exact ENIGMA SuppT1 record for c.3073A>G has combined LR 2.035204629841311, below 2.08.
• The exact c.3073A>G entry was not present in the BRCA2 clinical-history LR table used for PP4/BP5 assessment.
• PMID:32599251 reports p.K1025E in one 125-person BRCA-related-cancer referral cohort but reports no breast, ovarian, stomach, jejunal, thyroid, or family-history cancer count for that variant and provides no clinical LR.

Still missing

• A validated, independent multifactorial clinical LR of at least 2.08 for this exact variant would be required to reassess PP4.

Sources

Reasoning

No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion is present. ClinVar reports no expert-panel submissions for this variant; laboratory and aggregate assertions were not used.

Rule

Requested PP5 gate: exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion only.

Evidence considered

• ClinVar variation 51393 has no expert-panel submissions for NM_000059.4:c.3073A>G (p.Lys1025Glu); available submissions are laboratory submissions with conflicting classifications.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion would be required to apply PP5 under the requested gate.

Sources

Reasoning

BA1 is not met. The ENIGMA BRCA2 v1.2 threshold is FAF >0.001 (0.1%) in a gnomAD non-founder population. The maximum reported FAF is 5.405e-05 in gnomAD v2.1 and 5.795e-05 in gnomAD v4.1, both below 0.001.

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

• The applicable ClinGen ENIGMA BRCA1/2 specification v1.2 defines BA1 as maximum gnomAD non-founder filter allele frequency (FAF) above 0.1% (FAF >0.001), with read-depth and founder-variant caveats.
• gnomAD v2.1 reports total AF 4.79885e-05, zero homozygotes, and maximum group FAF 5.405e-05; gnomAD v4.1 reports total AF 5.33128e-05, zero homozygotes, and maximum group FAF 5.795e-05. These are below the BA1 threshold.
• The higher raw AF in the small gnomAD v2.1 NFE_BGR subgroup is not used as the criterion value because the ENIGMA rule specifies filter allele frequency (FAF), and the reported maximum FAF remains below the BA1 cutoff.

Sources

Reasoning

ENIGMA BRCA2 v1.2 BP4 Supporting for missense variants requires location inside a defined clinically important domain plus BayesDel no-AF <=0.18 and SpliceAI <=0.10. Although SpliceAI is 0.00, p.(Lys1025Glu) is outside the PALB2-binding and DNA-binding domains. BayesDel is additionally not used because no verified published calibration threshold is available for this pipeline. Therefore the gene-specific BP4 rule is not satisfied.

Rule

ENIGMA BRCA2 v1.2 BP4: missense/in-frame in aa 10-40 or aa 2481-3186 with BayesDel no-AF <=0.18 and SpliceAI <=0.10.

Evidence considered

• The governing ENIGMA BRCA2 v1.2 BP4 rule requires a missense/in-frame variant in the PALB2-binding domain (aa 10-40) or DNA-binding domain (aa 2481-3186), BayesDel no-AF <=0.18, and SpliceAI <=0.10.
• p.(Lys1025Glu) is outside both ENIGMA-defined domains; SpliceAI maximum delta is 0.00.
• BayesDel -0.185286 is not used because no verified published BayesDel calibration threshold is available for this pipeline.

Sources

Reasoning

The exact ENIGMA multifactorial record gives a combined LR of 2.035204629841311, which is above the ENIGMA BP5_Supporting threshold of 0.48. No exact-variant clinical-history LR is present in the ENIGMA clinical-history LR table. Co-observation in another breast/ovarian cancer-risk gene was not used.

Rule

ENIGMA BRCA2 v1.2 BP5: apply only to combined multifactorial clinical LR; Supporting LR<=0.48, Moderate<=0.23, Strong<=0.05, Very Strong<=0.00285; do not use co-observation in other breast/ovarian cancer-risk genes.

Evidence considered

• ENIGMA BRCA2 permits BP5 only for combined multifactorial clinical evidence against pathogenicity; supporting strength requires LR<=0.48.
• The exact ENIGMA SuppT1 record for c.3073A>G has combined LR 2.035204629841311, above 0.48.
• The exact c.3073A>G entry was not present in the BRCA2 clinical-history LR table used for PP4/BP5 assessment.

Still missing

• A validated, independent multifactorial clinical LR of 0.48 or lower for this exact variant would be required to reassess BP5.

Sources

Reasoning

No exact-variant ClinVar expert-panel Benign or Likely benign assertion is present. ClinVar reports no expert-panel submissions for this variant; laboratory and aggregate assertions were not used.

Rule

Requested BP6 gate: exact-variant ClinVar expert-panel Benign or Likely benign assertion only.

Evidence considered

• ClinVar variation 51393 has no expert-panel submissions for NM_000059.4:c.3073A>G (p.Lys1025Glu); available submissions are laboratory submissions with conflicting classifications.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign assertion would be required to apply BP6 under the requested gate.

Sources

Reasoning

The variant is a missense substitution, and SpliceAI predicts no significant splice impact (maximum delta score 0.00), satisfying the ENIGMA prerequisite for protein-change PS1 consideration. However, the case bundle does not document a previously classified pathogenic or likely pathogenic BRCA2 variant producing the same amino-acid change or a qualifying pathogenic alternate substitution at Lys1025. The exact variant's conflicting ClinVar submissions cannot be used as a substitute for comparator evidence.

Rule

Apply PS1 for a predicted missense substitution when a previously classified pathogenic variant is considered to act via protein change and there is no confirmed or predicted splice effect; apply PS1_Moderate for a previously classified likely pathogenic comparator.

Evidence considered

• ENIGMA BRCA2 v1.2 specifies PS1 for a missense substitution when a previously classified pathogenic or likely pathogenic variant has the relevant protein-change relationship and no confirmed or predicted splice effect.
• The case variant is NM_000059.4:c.3073A>G, NP_000050.3:p.(Lys1025Glu), and SpliceAI maximum delta is 0.00.
• The bundle contains no documented qualifying pathogenic/likely pathogenic same-change or alternate-at-Lys1025 comparator; the exact variant has conflicting single-submitter ClinVar classifications and those classifications were not treated as criterion evidence.

Still missing

• No qualifying previously classified pathogenic or likely pathogenic same-amino-acid or same-residue alternate variant is documented in the case bundle.

Sources

Reasoning

The applicable ENIGMA BRCA1/2 VCEP framework requires well-established, calibrated functional evidence showing a damaging effect and directs reviewers to Table 9. Table 9 contains calibrated entries for other BRCA2 variants but no entry for NM_000059.4:c.3073A>G (p.Lys1025Glu/K1025E). The only fetched paper that explicitly mentions the variant reports in-silico predictions and one cohort observation, not a validated functional assay. Therefore PS3 cannot be assessed.

Rule

ENIGMA BRCA2 VCEP v1.2: PS3 requires well-established in vitro or in vivo functional studies supportive of a damaging effect; use protein-only or combined mRNA/protein assays and consult calibrated published assay recommendations in Table 9.

Evidence considered

• The ENIGMA BRCA2 Version 1.2 rule states that PS3 is applied for well-established in vitro or in vivo studies supportive of a damaging effect, with protein-only or combined mRNA/protein assays, and recommends calibrated published assay results from Table 9.
• The extracted Table 9 material lists calibrated PS3 assignments for BRCA2 c.9218A>G (p.Asp3073Gly) and c.9217G>T (p.Asp3073Tyr), but does not list c.3073A>G or p.Lys1025Glu.
• PMID:32599251 explicitly identifies rs80358550 as BRCA2 p.K1025E and reports SIFT, MutationTaster, PolyPhen-2, and AWESOME predictions in one individual; the extraction states that no experimental functional assay or segregation analysis for this variant was reported.

Still missing

• No variant-specific calibrated protein-function assay result was identified.
• No variant-specific assay controls, replicate data, or quantitative functional result were identified.

Sources

Reasoning

The applicable ClinGen ENIGMA BRCA2 v1.2 specification restricts PM3 to a patient with a phenotype consistent with BRCA1/2-related Fanconi anemia and co-occurring variants in the same gene, with strength assigned from phenotype/variant observations. The bundle contains no documented Fanconi-anemia phenotype, chromosome-breakage result, second BRCA2 pathogenic or likely pathogenic variant, phase, or inheritance observation for this case. Therefore PM3 cannot be assessed.

Rule

ENIGMA BRCA2 v1.2 PM3: apply for a patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia and co-occurring variants in the same gene; PM3 Supporting = 1 point, PM3 Moderate = 2 points, PM3 Strong = at least 4 points, using the specified phenotype and chromosome-breakage criteria.

Evidence considered

• The ENIGMA BRCA2 v1.2 rule 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 bundle provides no qualifying phenotype or chromosome-breakage result.
• PMID:32599251 reports rs80358550/p.K1025E (the queried variant) in one person from a 125-person cohort, but reports no second BRCA2 variant, phase, trans configuration, or segregation result for that individual.

Still missing

• Documented Fanconi-anemia phenotype and chromosome-breakage testing result for the proband
• A second BRCA2 pathogenic or likely pathogenic variant in the same proband
• Phase or inheritance evidence establishing the two BRCA2 variants in trans

Sources

Reasoning

BS2 cannot be assessed from the available population data. gnomAD reports zero homozygotes but does not provide the adult health status, age, penetrance-relevant follow-up, or Fanconi Anemia phenotype assessment required for the ENIGMA BRCA2 BS2 point-based framework. Heterozygote observations in an ostensibly general-population database are not sufficient by themselves for a fully penetrant early-onset dominant criterion, particularly for BRCA2 cancer susceptibility.

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, and final BS2 code assignment based on the sum of BS2-related points. See Appendix H for additional details.

Evidence considered

• The applicable ClinGen ENIGMA BRCA1/2 specification v1.2 applies BS2 using point-based evaluation of observations in the absence of recessive Fanconi Anemia features; BS2 strength depends on the accumulated points.
• gnomAD v2.1 and v4.1 report zero homozygotes for the variant, but the case bundle supplies no individual-level age, health, penetrance, or Fanconi Anemia phenotype data.

Still missing

• No curated healthy-adult carrier observations with age and phenotype information were provided.
• No ENIGMA Table 8 BS2 point assignments are available for this variant or identified carriers.
• No information is available to establish absence of Fanconi Anemia features in the relevant individuals.

Sources

Reasoning

The applicable ENIGMA BRCA1/2 VCEP framework requires well-established, calibrated functional evidence showing no damaging effect on protein function and directs reviewers to Table 9. Table 9 contains calibrated entries for other BRCA2 variants but no entry for NM_000059.4:c.3073A>G (p.Lys1025Glu/K1025E). The only fetched paper that explicitly mentions the variant reports in-silico predictions and one cohort observation, not a validated benign functional assay. Therefore BS3 cannot be assessed.

Rule

ENIGMA BRCA2 VCEP v1.2: BS3 requires well-established in vitro or in vivo functional studies showing no damaging effect on protein function; use protein-only or combined mRNA/protein assays and consult calibrated published assay recommendations in Table 9.

Evidence considered

• The ENIGMA BRCA2 Version 1.2 rule states that BS3 is applied for well-established in vitro or in vivo functional studies showing no damaging effect on protein function, with protein-only or combined mRNA/protein assays, and recommends calibrated published assay results from Table 9.
• The extracted Table 9 material lists calibrated functional classifications for other BRCA2 variants, but does not list c.3073A>G or p.Lys1025Glu.
• PMID:32599251 explicitly identifies rs80358550 as BRCA2 p.K1025E but reports only computational predictions and a single cohort observation; the extraction states that no experimental functional assay was reported for this variant.

Still missing

• No variant-specific calibrated assay demonstrating preserved protein function was identified.
• No variant-specific assay controls, replicate data, or quantitative benign functional result were identified.

Sources

Reasoning

This is a missense variant outside an ENIGMA-defined clinically important domain. ENIGMA permits BP7_Strong (RNA) for such a missense variant only when a well-established mRNA-only assay shows no damaging effect. No variant-specific RNA splicing assay result is available. BP7 Supporting is specified only for silent variants in a defined domain or qualifying intronic variants, so it does not apply to this missense variant.

Rule

ENIGMA BRCA2 v1.2 BP7: BP7_Strong (RNA) requires a well-established mRNA-only assay showing no damaging effect; BP7 Supporting is limited to specified silent and intronic variants.

Evidence considered

• The governing ENIGMA BRCA2 v1.2 BP7 rule permits BP7_Strong (RNA) for a missense variant outside a clinically important domain only when a well-established mRNA-only assay shows no damaging effect; BP7 Supporting applies to specified silent or intronic variants.
• PMID:32599251 directly mentions p.K1025E but reports in-silico analysis only and no variant-specific RNA assay.

Still missing

• A well-established variant-specific mRNA-only assay demonstrating no damaging effect is required to assess BP7_Strong (RNA).

Sources