Variant classification summary

NM_000059.4:c.750G>A

BRCA2 · NP_000050.3:p.(Val250=) · NM_000059.4

GRCh37: chr13:32905124 G>A · GRCh38: chr13:32330987 G>A

Gene: BRCA2 Transcript: NM_000059.4

Final call

Likely Benign

BP1 strong benign BS1 supporting benign BP6 supporting benign

Variant details

Gene

BRCA2

Transcript

NM_000059.4

Protein

NP_000050.3:p.(Val250=)

gnomAD AF

8.305411037271461e-05 (v4.1)

ClinVar

Likely benign

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

BP1_Strong is met: c.750G>A is a silent (synonymous) substitution (p.Val250=) located outside both BRCA2 clinically important functional domains (PALB2 binding domain aa10-40 and DNA binding domain aa2481-3186), and SpliceAI predicts no splicing impact (max delta = 0.00, ≤ 0.1 threshold).

2

BS1_Supporting is met: the gnomAD v2.1 non-cancer exome filter allele frequency (FAF) is 4.75e-05 (0.00475%), falling in the ENIGMA BS1_Supporting range (FAF > 0.002% and ≤ 0.01%). The highest population sub-frequency is in European (non-Finnish) at 7.77e-05 (10/128,766 alleles).

3

PM2_Supporting is not met because the variant is present in gnomAD population databases (11 alleles in v2.1, 134 alleles in v4.1), failing the ENIGMA requirement of absence from outbred population controls.

4

PP3 is not met: SpliceAI max delta is 0.00 (< 0.2) and the variant is outside clinically important functional domains, failing both PP3 application pathways in the ENIGMA specification.

5

BA1 (Stand-Alone Benign) is not met: gnomAD grpmax FAF of 4.75e-05 (v2.1) and 9.28e-05 (v4.1) are both well below the 0.1% (FAF > 0.001) threshold.

6

BS3, BS4, PP1, PP4, BP5, and BP7 are not assessed due to absence of variant-specific functional, segregation, or clinical-history LR data in the ENIGMA VCEP-curated datasets (Table 9, clinical_history_LR.xlsx, HUMU-40-1557-s001, ST7).

7

PVS1, PS1, PS2, PS3, PS4, PS5, PM1, PM5, PM6, PP2, PP5, BP2, BP3, BP6 are not applicable for this synonymous variant under the ENIGMA VCEP framework.

8

Under the ENIGMA Table 3 combination rules, 1 Strong Benign criterion (BP1_Strong) plus 1 Supporting Benign criterion (BS1_Supporting) reaches the threshold for Likely Benign. Under the ENIGMA point system: BP1_Strong = -4 points, BS1_Supporting = -1 point, total = -5 points (Likely Benign range: -6 to -2).

9

ClinVar lists this variant as Likely Benign with expert panel review status by ENIGMA (ClinVar ID 135814), with 10 clinical laboratories reporting Likely Benign and 5 reporting Benign. This clinical consensus is concordant with the ENIGMA framework-based assessment.

Final determination: ENIGMA Table 3 Likely Benign rule: 1 Strong Benign criterion (BP1_Strong) plus 1 Supporting Benign criterion (BS1_Supporting) satisfies the 'all_of: 1 Strong (Benign) + 1 Supporting (Benign)' combination, and 2 Supporting Benign criteria (BS1_Supporting + BP6_Supporting) independently satisfy the 'all_of: 2 Supporting (Benign)' combination; both rules yield Likely Benign.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	N/A	PVS1 is not applicable for synonymous (silent) variants. c.750G>A (p.Val250=) does not create a null variant (nonsense, frameshift, or canonical ±1,2 splice site) and no mRNA splicing disruption is predicted (SpliceAI max delta = 0.00). The variant is not in the ENIGMA PVS1/PM5_PTC exon decision table (Specifications Table 4).	spliceai cspec pvs1_variant_assessment
PS1	N/A	PS1 requires a previously classified pathogenic variant with the same amino acid change or same predicted splicing impact. c.750G>A is synonymous (p.Val250=) and does not change the amino acid. No pathogenic comparator variant at this residue was identified.	cspec
PS2	N/A	PS2 is not applicable per ENIGMA VCEP. BRCA1/2-related cancers occur relatively commonly and de novo occurrences cannot be calibrated for predictive capacity.	cspec
PS3	Not assessed	No well-established functional studies demonstrating a damaging effect were identified. The ENIGMA Specifications Table 9 (curated functional assay results) does not list c.750G>A. The variant is synonymous and SpliceAI predicts no splicing impact (max delta 0.00), making a pathogenic functional effect unlikely. None of the ClinVar-associated PMIDs (25741868, 26467025, 27495310, 28492532, 31843900) mention this specific variant in their full text.	cspec spliceai
PS4	Not assessed	No case-control studies demonstrating enrichment of c.750G>A in affected individuals were identified. The variant is present in gnomAD population databases at low frequency, suggesting it does not exhibit strong case enrichment. The ENIGMA Supplementary Tables (ST7) do not list c.750G>A. None of the reviewed publications contained variant-specific case-control data.	gnomad_v2 cspec
PS5	N/A	PS5 criterion (reputable source reports variant as pathogenic) is not present in the ENIGMA BRCA2 VCEP criteria set. The VCEP does not define a PS5 criterion.	cspec
PM1	N/A	PM1 is not applicable per ENIGMA VCEP. It is considered as a component of the bioinformatic analysis captured under PP3/BP4.	cspec
PM2	Not met	PM2_Supporting per ENIGMA requires the variant to be absent from gnomAD v2.1 (non-cancer, exome only) AND gnomAD v3.1 (non-cancer). c.750G>A is present in gnomAD v2.1 at AF=3.9e-05 (11/282,244 alleles) and in gnomAD v4.1 at AF=8.3e-05 (134/1,613,406 alleles). The variant is not absent from population controls, so PM2 is not met.	gnomad_v2 gnomad_v4 cspec
PM5	N/A	PM5 per ENIGMA is repurposed exclusively for protein termination codon (PTC) variants in exons where proven pathogenic PTCs exist (PM5_PTC). c.750G>A is a synonymous variant that does not create a termination codon. Additionally, PM5 candidate assessment confirmed no classic same-residue missense comparator pathway applies.	cspec pm5_candidates
PM6	N/A	PM6 is not applicable per ENIGMA VCEP. BRCA1/2-related cancers occur relatively commonly and de novo occurrences cannot be calibrated for predictive capacity.	cspec
PP1	Not assessed	No co-segregation data were identified for c.750G>A. The ENIGMA VCEP requires quantitative co-segregation analysis with LR ≥ 2.08 for PP1. No segregation studies mentioning this specific variant were found in the VCEP materials or reviewed publications.	cspec
PP2	N/A	PP2 is not applicable per ENIGMA VCEP. The VCEP does not define PP2 as an applicable criterion for BRCA2.	cspec
PP3	Not met	PP3 per ENIGMA requires either (a) missense/in-frame variant inside a clinically important functional domain with BayesDel no-AF ≥ 0.30, or (b) SpliceAI ≥ 0.2 for any variant type regardless of location. c.750G>A is synonymous and located at Val250, which is outside both clinically important functional domains (PALB2 binding domain aa10-40 and DNA binding domain aa2481-3186). SpliceAI max delta score is 0.00, well below the 0.2 threshold. BayesDel score was not available. PP3 is not met under either pathway.	spliceai cspec
PP4	Not assessed	PP4 per ENIGMA requires a multifactorial likelihood ratio (LR ≥ 2.08) from calibrated clinical data. c.750G>A was not found in the Li et al. 2020 clinical-history LR table (PMID_31853058_BRCA2_clinical_history_LR.xlsx) or in the Parsons et al. 2019 multifactorial dataset (HUMU-40-1557-s001.xlsx). The variant is also absent from the ENIGMA ST7 Reference Set. Without calibrated LR data, PP4 cannot be applied.	cspec vcep_pmid_31853058_brca2_clinical_history_lr vcep_humu_40_1557_s001
PP5	N/A	PP5 is not applicable per ENIGMA VCEP. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BA1	Not met	BA1 (Stand-Alone Benign) per ENIGMA requires filter allele frequency (FAF) > 0.001 (0.1%) in gnomAD non-cancer, non-founder populations. The gnomAD v2.1 grpmax FAF is 4.75e-05 (0.00475%) and gnomAD v4.1 grpmax FAF is 9.28e-05 (0.00928%). Both are well below the 0.1% BA1 threshold.	gnomad_v2 gnomad_v4 cspec
BS1	Met	BS1_Supporting per ENIGMA is met. The filter allele frequency (FAF) in gnomAD v2.1 (non-cancer, exome only) is 4.75e-05 (0.00475%), which falls in the range FAF > 0.00002 (0.002%) and ≤ 0.0001 (0.01%). The highest population sub-frequency is in European (non-Finnish) at AF=7.77e-05 (10/128,766 alleles, 0 homozygotes). The variant is also present in gnomAD v4.1 with grpmax FAF 9.28e-05, consistent with the BS1_Supporting range. This frequency is higher than expected for a highly penetrant pathogenic BRCA2 variant.	gnomad_v2 gnomad_v4 cspec
BS2	Not assessed	BS2 per ENIGMA requires observation in the absence of Fanconi Anemia (FA) phenotype features, with a point system per proband (Specifications Table 8). No proband-level data with documented absence of FA features were identified for c.750G>A. While the variant is observed in gnomAD (suggesting healthy adult carriers), the ENIGMA BS2 pathway requires structured proband assessment that was not available.	cspec
BS3	Not assessed	BS3 per ENIGMA requires well-established functional studies showing no damaging effect, with code recommendations from calibrated published assays in Specifications Table 9. c.750G>A is not listed in ENIGMA Table 9. The exploratory evidence pass noted that Whiley et al. 2014 (PMID:24323938) reported normal splicing for c.750G>A, but this assay was not calibrated under the ENIGMA framework and the full text was not available for verification. Without VCEP-endorsed functional data, BS3 cannot be applied.	cspec
BS4	Not assessed	BS4 per ENIGMA requires quantitative co-segregation analysis showing lack of segregation (LR ≤ 0.48). No locus-specific segregation data for c.750G>A were found in the VCEP materials, ENIGMA ST7 Reference Set, or the Parsons et al. 2019 multifactorial dataset (HUMU-40-1557-s001).	cspec vcep_humu_40_1557_s001
BP1	Met	BP1_Strong per ENIGMA is met. c.750G>A is a silent (synonymous) substitution located at Val250, which is outside both BRCA2 clinically important functional domains (PALB2 binding domain aa10-40 and DNA binding domain aa2481-3186). SpliceAI predicts no splicing impact (max delta score = 0.00, well below the 0.1 threshold). The ENIGMA specification states: 'Apply BP1_Strong for silent substitution, missense or in-frame insertion, deletion or delins variants outside a (potentially) clinically important functional domain AND no splicing predicted (SpliceAI ≤ 0.1).' All conditions are satisfied.	spliceai cspec
BP2	N/A	BP2 is not applicable per ENIGMA VCEP. The VCEP does not define BP2 as an applicable criterion for BRCA2.	cspec
BP3	N/A	BP3 (in-frame deletions/insertions in repetitive regions) is not applicable to this substitution variant, as specified in the skip list.
BP4	Not met	BP4 per ENIGMA for silent variants applies only when the variant is inside a clinically important functional domain AND no splicing impact is predicted (SpliceAI ≤ 0.1). Val250 is outside both domains (PALB2: aa10-40, DNA binding: aa2481-3186). The BP4 rule for silent variants inside the domain is therefore not triggered. For missense variants outside the domain, BP4 is not applicable per the ENIGMA bioinformatic flowchart (Figure 1A). The synonymous variant at this position is better captured by BP1_Strong.	spliceai cspec
BP5	Not assessed	BP5 per ENIGMA requires a multifactorial likelihood ratio (LR ≤ 0.48) from calibrated clinical data. c.750G>A was not found in the Li et al. 2020 clinical-history LR table or the Parsons et al. 2019 multifactorial dataset. Without calibrated LR data in the benign direction, BP5 cannot be applied.	cspec vcep_pmid_31853058_brca2_clinical_history_lr vcep_humu_40_1557_s001
BP6	Met	Expert panel Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) classified as Likely benign.	cspec clinvar
BP7	Not assessed	BP7 per ENIGMA can be applied at Strong strength for silent variants with well-established mRNA assay data showing no damaging effect, or at Supporting strength for silent variants inside a clinically important domain if BP4 is met. c.750G>A is outside the domain, so the BP7_Supporting pathway does not apply. For BP7_Strong (RNA), a well-established mRNA functional assay is needed, but no VCEP-endorsed BP7 functional data are listed in Specifications Table 9 for this variant. While the exploratory evidence pass noted that Whiley et al. 2014 (PMID:24323938) reported normal splicing, this assay has not been calibrated under the ENIGMA framework and the full text was unavailable for independent verification.	cspec spliceai

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