BRCA2 c.8537_8538del, p.(Glu2846GlyfsTer22)

Reasoning

NM_000059.4:c.8537_8538del is a two-base deletion predicted to cause the frameshift p.(Glu2846GlyfsTer22). The applicable ENIGMA BRCA2 v1.2 specification recognizes BRCA2 loss of function as a disease mechanism and directs use of its exon-level PVS1 decision table for frameshift variants. The affected transcript is the framework's preferred transcript, and the variant is in BRCA2 exon 20, for which the table assigns PVS1 rather than PVS1_N/A. The premature stop is upstream of the terminal exon and is expected to undergo NMD; if translated, it would truncate the final approximately 552 amino acids of the 3,418-residue protein, including distal C-terminal sequence. PVS1 is therefore met at Very Strong strength.

Rule

ENIGMA BRCA1/BRCA2 Expert Panel Specifications v1.2: apply PVS1 at the table-directed strength for a null variant in BRCA2, a gene with established loss-of-function disease mechanism; use the exon-level Table 4 decision table.

Evidence considered

• The normalized consequence on the ENIGMA preferred transcript NM_000059.4 is NP_000050.3:p.(Glu2846GlyfsTer22), a frameshift with a premature termination codon 22 codons downstream.
• ENIGMA BRCA1/BRCA2 v1.2 specifies PVS1 for null variants in BRCA2, with strength determined by its PVS1 flowchart and Table 4 exon-level decision table.
• The ENIGMA BRCA2 Table 4 summary identifies exon 20 as PVS1-applicable; only exons 6, 12, and 27 are designated PVS1_N/A in the supplied table summary.

Sources

Reasoning

PM5 is met as the ENIGMA-repurposed PM5 (PTC) code, not as classic same-residue missense PM5. NM_000059.4:c.8537_8538del is a genomic frameshift in BRCA2 exon 20 and is predicted to create a premature termination codon at p.(Glu2846GlyfsTer22). ENIGMA Table 4 assigns PM5_Strong (PTC) to genomic PTC variants in exon 20. The variant is not a splice-site variant, and the exon is not one of the PM5_N/A exons.

Rule

ENIGMA BRCA2 VCEP v1.2 PM5 (PTC): apply the exon-specific PM5 weight to a genomic PTC variant in an exon where a different proven pathogenic PTC has been observed; exon 20 is assigned Strong.

Evidence considered

• ENIGMA BRCA2 VCEP v1.2 repurposes PM5 for an additional weight for a genomic protein-termination-codon variant when a different proven pathogenic PTC has been observed in the same exon; the weight is determined by exon.
• ENIGMA Specifications Table 4 places c.8537_8538del within exon 20 because exon 19 ends at c.8487 and exon 20 spans c.8488-c.8632; the exon 20 PTC row assigns PM5_Strong (PTC).
• The normalized protein consequence is NP_000050.3:p.(E2846Gfs*22), confirming a truncating PTC consequence; the variant is not at a canonical donor/acceptor +/-1,2 position.

Sources

Reasoning

Expert panel Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) classified as Pathogenic.

Rule

Apply PP5 at Supporting only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion.

Evidence considered

• ClinVar identifies the exact record as NM_000059.4(BRCA2):c.8537_8538del (p.Glu2846fs) and reports a Pathogenic expert-panel submission by ENIGMA.
• ClinVar expert panel classification

Sources

Reasoning

The BRCA2 ENIGMA VCEP PM2 rule requires the variant to be absent from controls in gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer data, with adequate regional coverage. This variant is observed in gnomAD v2.1 (3/251142 alleles; AF 1.19454e-05; 0 homozygotes) and gnomAD v4.1 (10/1614188 alleles; AF 6.19507e-06; 0 homozygotes). Although rare and absent from gnomAD-Canada, it is not absent from the required general population datasets, so PM2 is not met.

Rule

Absent from controls in gnomAD v2.1 non-cancer exome data and gnomAD v3.1 non-cancer data, with regional average read depth >=25; apply at Supporting strength.

Evidence considered

• ENIGMA BRCA2 specification v1.2 defines PM2 as absence from controls in gnomAD v2.1 non-cancer exome data and gnomAD v3.1 non-cancer data; the surrounding region must have average read depth >=25.
• gnomAD v2.1 reports the variant present at AF 1.19454e-05 (3/251142 alleles) with 0 homozygotes.
• gnomAD v4.1 reports the variant present at AF 6.19507e-06 (10/1614188 alleles) with 0 homozygotes.
• gnomAD-Canada v1.0 reports the variant absent, but absence in that additional dataset does not overcome its presence in the required general gnomAD datasets.
• The case bundle does not provide a separate regional read-depth value; regardless, the explicit presence in gnomAD v2.1 and the available newer gnomAD dataset fails the VCEP absence requirement.

Sources

Reasoning

The BRCA2 ENIGMA VCEP BA1 threshold is FAF >0.001 (0.1%) in gnomAD v2.1 or v3.1 non-cancer, non-founder populations. The observed gnomAD v2.1 grpmax FAF is 0.00000703 (0.000703%), and the observed gnomAD v4.1 grpmax FAF is 0.00000429 (0.000429%); neither approaches the BA1 threshold.

Rule

Filter allele frequency (FAF) > 0.001 in gnomAD v2.1 or v3.1 non-cancer, non-founder populations.

Evidence considered

• ENIGMA BRCA2 specification v1.2 defines BA1 as FAF >0.001 (0.1%) in gnomAD v2.1 or v3.1 non-cancer, non-founder populations.
• gnomAD v2.1 reports grpmax FAF 7.03e-06 (0.000703%), with 3/251142 alleles and 0 homozygotes.
• gnomAD v4.1 reports grpmax FAF 4.29e-06 (0.000429%), with 10/1614188 total alleles and 0 homozygotes.

Sources

Reasoning

The BRCA2 ENIGMA VCEP BS1 thresholds are FAF >0.0001 for BS1_Strong and FAF >0.00002 and <=0.0001 for BS1_Supporting. The highest observed group FAF is 7.03e-06 in gnomAD v2.1 and 4.29e-06 in gnomAD v4.1, both below 0.00002; therefore BS1 is not met.

Rule

FAF >0.0001 = BS1_Strong; FAF >0.00002 and <=0.0001 = BS1_Supporting, using gnomAD non-cancer, non-founder populations.

Evidence considered

• ENIGMA BRCA2 specification v1.2 defines BS1_Strong as FAF >0.0001 and BS1_Supporting as FAF >0.00002 and <=0.0001 in the specified gnomAD non-cancer populations.
• gnomAD v2.1 reports grpmax FAF 7.03e-06; the highest subpopulation AF is 2.64387e-05 in European non-Finnish individuals, but the VCEP rule uses FAF and the group FAF remains below the supporting threshold.
• gnomAD v4.1 reports grpmax FAF 4.29e-06; the highest subpopulation AF is 8.47443e-06 in European non-Finnish individuals, also below the supporting threshold.

Sources

Reasoning

The exact ClinVar record has a Pathogenic ENIGMA expert-panel assertion, not a Benign or Likely benign expert-panel assertion. Therefore the required trigger for BP6 is absent.

Rule

Apply BP6 at Supporting only for an exact-variant ClinVar expert-panel Benign or Likely benign assertion.

Evidence considered

• ClinVar identifies the exact record as NM_000059.4(BRCA2):c.8537_8538del (p.Glu2846fs) and reports a Pathogenic expert-panel submission by ENIGMA; no benign or likely benign expert-panel assertion is reported.

Sources

Reasoning

The applicable ClinGen ENIGMA BRCA2 v1.2 specification permits PS3 for well-established functional studies measuring protein function, or mRNA and protein together; mRNA-only evidence is directed to PVS1 (RNA), not PS3. The curated ENIGMA Table 9 contains no entry for NM_000059.4:c.8537_8538del / p.(Glu2846GlyfsTer22), and the reviewed functional publications either study different BRCA2 variants or provide gene-level biological context without testing this variant. Therefore, no variant-specific validated protein-function assay supports PS3.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect. Apply PS3 for assays measuring effect via protein only OR mRNA and protein combined. Well-established studies measuring mRNA transcript profile only are assigned as PVS1 (RNA) at the appropriate strength.

Evidence considered

• ENIGMA BRCA1/2 specification v1.2 states that PS3 is applied to well-established in vitro or in vivo studies showing a damaging effect when the assay measures protein function alone or mRNA and protein together; mRNA-only functional evidence is assigned through PVS1 (RNA).
• ENIGMA Table 9 is the curated calibrated-assay table for PS3/BS3 and contains no row for c.8537_8538del, p.(Glu2846GlyfsTer22), or the equivalent variant notation.
• PMID:11239455 reports homology-directed-repair defects for different BRCA2 mutations (including 6174delT and mouse C-terminal deletions), not this variant; this cannot be transferred as variant-specific PS3 evidence.
• PMID:10570174 reports cytoplasmic localization for BRCA2 deletion constructs and endogenous 6174delT, not this variant; this provides biological context only and cannot establish PS3.
• PMID:20878484 reports a different BRCA2 frameshift, 7525_7526insT, in an Italian family and does not test c.8537_8538del.
• PMID:22193408 and PMID:24312913 provide gene-level discussion of BRCA2 homologous-recombination function and truncating variants but do not test or mention c.8537_8538del.

Still missing

• No variant-specific calibrated assay measuring BRCA2 protein function, or mRNA and protein together, was identified.
• Any future RNA-only result should be evaluated under the ENIGMA PVS1 (RNA) framework rather than PS3.

Sources

Reasoning

The BRCA2-specific ENIGMA rule requires a case-control study with p<=0.05, OR>=4, and a lower confidence bound excluding 2.0. No such exact-variant analysis for NM_000059.4:c.8537_8538del was supplied.

Rule

ENIGMA BRCA2 PS4: case-control enrichment requires p<=0.05, OR>=4, and lower CI excludes 2.0.

Evidence considered

• The applicable ENIGMA BRCA2 specification sets PS4 at p<=0.05 and OR>=4 with the lower confidence interval excluding 2.0.
• The selectively extracted full-text study PMID:17640379 concerns a different BRCA2 variant, 8765delAG, not c.8537_8538del; it cannot supply PS4 evidence for this variant.

Still missing

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

Sources

Reasoning

The applicable ENIGMA BRCA2 v1.2 rule requires a co-occurring pathogenic or likely pathogenic BRCA2 variant in the same patient, phase/inheritance information, and a phenotype consistent with BRCA1/2-related Fanconi anemia. The bundle contains no patient-level affected-proband observations, second BRCA2 variant, parental/offspring testing, or cis/trans phase data; therefore PM3 cannot be assigned.

Rule

ENIGMA BRCA2 v1.2 PM3: co-occurring pathogenic or likely pathogenic variant in the same gene, with a phenotype consistent with BRCA1/2-related Fanconi anemia; assign PM3 strength from the Table 6 point total (Supporting = 1 point, Moderate = 2 points, Strong = 4 or more points).

Evidence considered

• ENIGMA BRCA2 specification v1.2 states that PM3 is applicable to a patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia and co-occurring variants in the same gene; strength is assigned from PM3-related points in Specifications Table 6.
• The case bundle identifies the assessed variant as NM_000059.4:c.8537_8538del in BRCA2 but provides no case-level phase, co-occurring BRCA2 variant, parental/offspring testing, or Fanconi-anemia phenotype information.

Still missing

• Patient-level phenotype and affected-proband data, including chromosome-breakage results and Fanconi-anemia features
• A documented co-occurring pathogenic or likely pathogenic BRCA2 variant
• Parental, offspring, or other informative testing establishing cis/trans phase

Sources

Reasoning

PP1 is applicable under the ENIGMA BRCA2 framework only when co-segregation with disease in multiple affected family members is supported by a quantitative co-segregation analysis. The case bundle contains no pedigree, affected-relative genotype data, phase information, meioses, segregation likelihood ratio, or other quantitative co-segregation result for NM_000059.4:c.8537_8538del.

Rule

Co-segregation with disease in multiple affected family members, measured by a quantitative co-segregation analysis method; PP1 Supporting LR >=2.08:1, PP1 Moderate LR >=4.3:1, PP1 Strong LR >=18.7:1, and PP1 Very Strong LR >=350:1.

Evidence considered

• ENIGMA BRCA2 Version 1.2 requires quantitative co-segregation analysis and assigns PP1 Supporting at LR >=2.08:1, PP1 Moderate at LR >=4.3:1, PP1 Strong at LR >=18.7:1, and PP1 Very Strong at LR >=350:1; no such result is present in the case bundle.

Still missing

• Pedigree and affected-relative phenotypes are not provided.
• Genotypes of affected and unaffected relatives are not provided.
• Number of informative meioses and a quantitative segregation likelihood ratio are not provided.

Sources

Reasoning

For BRCA2, PP4 may be used only for a combined multifactorial clinical-data likelihood ratio toward pathogenicity. No exact-variant clinical-history or combined likelihood ratio was supplied for NM_000059.4:c.8537_8538del.

Rule

ENIGMA BRCA2 PP4: use only a combined multifactorial clinical-data LR; Supporting LR>=2.08, Moderate>=4.3, Strong>=18.7, Very Strong>=350.

Evidence considered

• The ENIGMA BRCA2 specification restricts PP4 to combined multifactorial likelihood clinical data and sets Supporting at LR>=2.08.
• The supplied BRCA2 clinical-history LR resource requires an exact Gene and HGVS_Nucleotide match; no matching c.8537_8538del record was provided in the case evidence.

Still missing

• An exact BRCA2 c.8537_8538del multifactorial or clinical-history likelihood ratio is needed; PP4 Supporting requires LR>=2.08.

Sources

Reasoning

The BRCA2 ENIGMA VCEP applies BS2 in the absence of features of the recessive Fanconi Anemia phenotype and assigns strength from BS2-related points per proband. The case bundle provides population observations but does not provide the affected individuals' Fanconi Anemia phenotype assessment, chromosome-breakage results, or the proband-level data needed to calculate BS2 points. Population homozygote absence alone does not establish the VCEP BS2 requirement.

Rule

Apply BS2 for absence of Fanconi Anemia features; BS2_Supporting = 1 point, BS2_Moderate = 2 points, and BS2_Strong >=4 points under the VCEP point-based approach.

Evidence considered

• ENIGMA BRCA2 specification v1.2 states that BS2 is applied in the absence of features of recessive disease, namely Fanconi Anemia phenotype, with BS2 strength determined from the sum of BS2-related points per proband.
• The available gnomAD data show zero homozygotes in gnomAD v2.1 and v4.1, but this is population evidence and does not substitute for the VCEP's required Fanconi Anemia phenotype and point-based assessment.

Still missing

• Fanconi Anemia phenotype and chromosome-breakage status for eligible probands are not provided.
• The VCEP Table 8 BS2 point calculation cannot be performed from the available case evidence.

Sources

Reasoning

The applicable ClinGen ENIGMA BRCA2 v1.2 specification requires well-established functional evidence showing no damaging effect on protein function, using protein-only or combined mRNA-and-protein assays. The curated ENIGMA Table 9 contains no entry for NM_000059.4:c.8537_8538del / p.(Glu2846GlyfsTer22), and no reviewed publication reports a benign calibrated functional result for this variant. Gene-level or alternate-variant functional findings cannot establish BS3.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function. Apply BS3 for assays measuring effect via protein only OR mRNA and protein combined. mRNA-only evidence of no damaging effect is assigned as BP7 (RNA) at the appropriate strength.

Evidence considered

• ENIGMA BRCA1/2 specification v1.2 defines BS3 as well-established in vitro or in vivo evidence of no damaging effect on protein function, with protein-only or combined mRNA-and-protein measurement; mRNA-only benign evidence is handled under BP7 (RNA).
• ENIGMA Table 9 is the curated calibrated-assay table for PS3/BS3 and contains no row for c.8537_8538del, p.(Glu2846GlyfsTer22), or the equivalent variant notation.
• The reviewed functional papers do not provide a benign functional result for this exact variant: PMID:11239455 and PMID:10570174 study other BRCA2 mutations or constructs; PMID:20878484 studies a different frameshift; and PMID:22193408 and PMID:24312913 are gene-level reviews without variant-specific assays.

Still missing

• No variant-specific calibrated assay demonstrating preserved BRCA2 protein function, or preserved mRNA and protein function together, was identified.
• No benign-control-equivalent functional result for this exact frameshift variant was identified.

Sources

Reasoning

BS4 is applicable under the ENIGMA BRCA2 framework only when lack of segregation in affected family members is supported by a quantitative co-segregation analysis. The case bundle contains no non-segregating affected relatives, pedigree, informative meioses, or quantitative likelihood ratio for this variant. Population frequency, ClinVar classification, and reports of different BRCA2 variants do not establish BS4.

Rule

Lack of segregation in affected family members, measured by a quantitative co-segregation analysis method; BS4 Supporting LR <=0.48:1, BS4 Moderate LR <=0.23:1, BS4 Strong LR <=0.05:1, and BS4 Very Strong LR <=0.00285:1.

Evidence considered

• ENIGMA BRCA2 Version 1.2 requires quantitative analysis of lack of segregation in affected family members and assigns BS4 Supporting at LR <=0.48:1, BS4 Moderate at LR <=0.23:1, BS4 Strong at LR <=0.05:1, and BS4 Very Strong at LR <=0.00285:1; no such result is present in the case bundle.

Still missing

• No affected family members with confirmed absence of the variant are documented.
• No pedigree, family phenotypes, or informative meioses are provided.
• No quantitative non-segregation likelihood ratio is provided.

Sources

Reasoning

For BRCA2, BP5 may be used only for a combined multifactorial clinical-data likelihood ratio against pathogenicity and is not applicable to co-observation. No exact-variant likelihood ratio was supplied.

Rule

ENIGMA BRCA2 BP5: use only a combined multifactorial clinical-data LR; Supporting LR<=0.48, Moderate<=0.23, Strong<=0.05, Very Strong<=0.00285; not for co-observation.

Evidence considered

• The ENIGMA BRCA2 specification restricts BP5 to a combined multifactorial clinical-data LR against pathogenicity and excludes co-observation as a basis for BP5.
• The supplied BRCA2 clinical-history LR resource requires an exact Gene and HGVS_Nucleotide match; no matching c.8537_8538del record was provided in the case evidence.

Still missing

• An exact BRCA2 c.8537_8538del multifactorial or clinical-history likelihood ratio is needed; BP5 Supporting requires LR<=0.48.

Sources