BRCA1 c.5266dup, p.(Gln1756ProfsTer74)

Reasoning

NM_007294.4 is the ENIGMA-preferred BRCA1 transcript. c.5266dup is an exonic frameshift in BRCA1 exon E19(20), predicting p.(Gln1756ProfsTer74). The resulting premature termination lies in the distal transcript and may escape canonical NMD, but the applicable ENIGMA BRCA1/2 v1.2 exon-level Table 4 explicitly assigns PVS1 to protein-truncating variants in E19(20), rather than a downgraded or N/A weight. This gene-specific rule governs the assessment. Variant-specific studies additionally show loss of BRCT-dependent nuclear localization/foci and failure to restore homologous-recombination activity, consistent with a damaging loss-of-function consequence; these studies are recorded as corroboration and are not separately assigned as another criterion here.

Rule

ENIGMA BRCA1/2 VCEP v1.2: null variants are assigned PVS1 at the exon-specific weight in Specifications Table 4; BRCA1 E19(20) protein-truncating variants are assigned PVS1.

Evidence considered

• The case normalization on the ENIGMA-preferred transcript NM_007294.4 predicts BRCA1 p.(Gln1756ProfsTer74); the variant is in exon 19 of this transcript, corresponding to ENIGMA E19(20).
• ENIGMA BRCA1/2 VCEP v1.2 Table 4 assigns PVS1 to protein-truncating variants in BRCA1 E19(20); c.5266dup is within that exon (c.5194-c.5277).
• The predicted stop is distal and within the terminal coding exon, so canonical NMD escape is a consideration; the gene-specific E19(20) PTC assignment nevertheless specifies full PVS1 for this exon.
• PMID:14729053 directly studied the corresponding BRCA1 5382insC/c.5266dup mutant and reported increased cytoplasmic localization and failure to form DNA-damage-inducible foci when targeted to the nucleus by BARD1.
• PMID:23867111 directly studied 5382insC/c.5266dup and reported that it did not restore homologous-recombination activity in BRCA1-deficient embryonic stem cells.

Sources

Reasoning

Variant-specific protein-level functional evidence supports a damaging effect: BRCA1 5382insC/c.5266dup caused abnormal cytoplasmic localization, impaired nuclear damage-focus formation, and failed to restore homologous-recombination activity in BRCA1-deficient cells. The cisplatin complementation result was not fully classifiable because one of three transfection series scored neutral, so that assay is not treated as independently decisive. ENIGMA BRCA1 v1.2 directs PS3 to well-established assays measuring protein function and reserves calibrated strength assignments to Table 9; the exact c.5266dup frameshift is not assigned in the available Table 9 extract. Therefore the convergent protein-level findings support PS3 at Supporting strength, but not the VCEP Strong level without expert confirmation of assay calibration and applicability to this frameshift.

Rule

ENIGMA BRCA1 v1.2 PS3: apply for well-established in vitro or in vivo functional studies showing a damaging effect when the assay measures protein function; use Table 9 calibrated published-assay recommendations for strength. Because the exact frameshift lacks a Table 9 assignment and one assay is technically ambiguous, apply a conservative Supporting strength pending human review rather than Strong.

Evidence considered

• ENIGMA BRCA1/2 Specification v1.2 states that PS3 is for well-established in vitro or in vivo functional studies supportive of a damaging effect when the assay measures protein function, and refers to Table 9 for calibrated published-assay code recommendations.
• ENIGMA Table 9 contains calibrated assignments for other c.5266 substitutions, including PS3 Strong for c.5266C>T (p.Gln1756Ter), but the available extract contains no exact c.5266dup entry; the missense/nonsense entries cannot be transferred to this frameshift.
• PMID:14729053 directly studied BRCA1 5382insC, corresponding to c.5266dup, and reported increased cytoplasmic staining and failure to form DNA-damage-inducible foci when targeted to the nucleus by BARD1. The paper used wild-type and endogenous-cell comparisons, localization microscopy, fractionation/Western blotting, and a DNA-damage focus assay.
• Direct quote from PMID:14729053: "Furthermore, we observed increased cytoplasmic staining of both the ectopic and endogenous forms of the BRCA1-5382insC mutant (deleted BRCT domain) in HCC1937 breast cancer cells. Unlike wild-type BRCA1, the BRCA1-5382insC mutant failed to form DNA damage-inducible foci when targeted to the nucleus by BARD1."
• PMID:23867111 included c.5266dupC/5382insC as a deleterious control. It did not restore homologous-recombination activity in the DR-GFP assay in BRCA1-deficient mouse embryonic stem cells; the cisplatin assay was labeled Not clear because one of three transfection series scored neutral, with the authors attributing this to technical reasons.
• Direct quote from PMID:23867111: "The pathogenic 5382insC truncation mutation could not be classified because it scored as neutral in one of three transfection series. This was probably due to technical reasons, as the 5382insC mutation did not restore HR activity in Brca1-deficient embryonic stem cells."

Still missing

• The exact c.5266dup frameshift is not present among the available ENIGMA Table 9 variant-level calibrated PS3 assignments.
• The cisplatin-sensitivity assay in PMID:23867111 was technically ambiguous for 5382insC/c.5266dup because one of three transfection series scored neutral.
• No quantitative assay-specific likelihood ratio or formal calibration sufficient to justify PS3 Strong is provided in the case bundle for these variant-specific results.

Sources

Reasoning

The exact BRCA1 c.5266dupC row in the ENIGMA clinical-history LR table reports 183 probands and LR=8.080955291458534e+19. This exceeds the ENIGMA PP4_Very Strong threshold of LR>=350.

Rule

ENIGMA BRCA1/2 PP4: use multifactorial clinical-history LR only; Very Strong at LR>=350.

Evidence considered

• The exact table row is BRCA1 c.5266dupC (5382insC), with LOG(LR)=45.83862686157227, N_Probands=183, and LR=8.080955291458534e+19.
• ENIGMA PP4 is restricted to multifactorial clinical-history likelihood evidence and assigns Very Strong at LR>=350.

Sources

Reasoning

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

Rule

Exact-variant ClinVar Pathogenic/Likely pathogenic expert-panel assertion supports PP5 at Supporting strength.

Evidence considered

• The exact-variant ClinVar record VCV000017677 lists ENIGMA as an expert-panel submitter with a Pathogenic assertion and expert-panel review status.
• ClinVar expert panel classification

Sources

Reasoning

BA1 is not met under the ENIGMA BRCA1 specification. The relevant maximum non-founder population FAF is 0.00012672 in gnomAD v2.1 and 0.00004346 in gnomAD v4.1, both below the BA1 threshold of FAF >0.001 (0.1%). The higher Ashkenazi Jewish frequencies are founder-population observations and do not satisfy the specified non-founder BA1 rule.

Rule

ENIGMA BRCA1 v1.2 BA1: FAF >0.001 in gnomAD v2.1 non-cancer exome-only and/or gnomAD v3.1 non-cancer, non-founder population(s).

Evidence considered

• ENIGMA BRCA1 v1.2 defines BA1 as FAF >0.001 in gnomAD v2.1 and/or v3.1 non-cancer, non-founder populations.
• gnomAD v2.1 reports total AF 0.000180281, grpmax FAF 0.00012672, and Ashkenazi Jewish AF 0.00231437; the maximum non-founder FAF remains below 0.001.
• gnomAD v4.1 reports total AF 0.0000675378 and grpmax FAF 0.00004346; Ashkenazi Jewish AF is 0.00118275, but this is a founder population and is not the applicable non-founder BA1 measure.

Sources

Reasoning

No variant-specific, well-established functional study in the case bundle demonstrates no damaging effect on BRCA1 protein function. The available studies instead report loss-of-function or abnormal protein behavior for c.5266dup/5382insC, and the only partially neutral result was explicitly considered technically unreliable and was not a no-damage conclusion. Therefore BS3 is not met.

Rule

ENIGMA BRCA1 v1.2 BS3: require well-established functional studies showing no damaging effect on protein function, with calibrated assay recommendations from Table 9. No such evidence is present for this exact frameshift.

Evidence considered

• ENIGMA BRCA1/2 Specification v1.2 defines BS3 as well-established in vitro or in vivo functional evidence showing no damaging effect on protein function and directs reviewers to Table 9 for calibrated assay recommendations.
• The available ENIGMA Table 9 extract has no exact c.5266dup frameshift BS3 assignment; BS3 Strong assignments for c.5266C>A and c.5266C>G are different missense substitutions and cannot be extrapolated.
• PMID:14729053 reported abnormal localization and loss of DNA-damage-induced foci for the exact 5382insC/c.5266dup mutant, which is inconsistent with benign protein function.
• PMID:23867111 reported failure of 5382insC/c.5266dup to restore homologous-recombination activity; its isolated neutral cisplatin result was classified as Not clear and attributed to technical reasons, not interpreted as evidence of normal function.

Still missing

• No validated benign functional assay result for the exact c.5266dup frameshift is available.

Sources

Reasoning

The exact variant's ENIGMA clinical-history LR is 8.080955291458534e+19, which is in the pathogenic direction and does not meet any BP5 threshold (LR<=0.48 for Supporting or lower).

Rule

ENIGMA BRCA1/2 BP5: use multifactorial clinical-history LR only; Supporting requires LR<=0.48.

Evidence considered

• The exact BRCA1 c.5266dupC table row has LR=8.080955291458534e+19 from 183 probands.
• ENIGMA BP5 is limited to combined multifactorial LR against pathogenicity and requires LR<=0.48 for Supporting, <=0.23 for Moderate, <=0.05 for Strong, or <=0.00285 for Very Strong.

Sources

Reasoning

The exact-variant ClinVar expert-panel assertion is Pathogenic, not Benign or Likely benign; no exact-variant benign expert-panel assertion is present.

Rule

BP6 requires an exact-variant ClinVar Benign/Likely benign expert-panel assertion at Supporting strength.

Evidence considered

• The exact-variant ClinVar record VCV000017677 lists the ENIGMA expert-panel assertion as Pathogenic.

Sources

Reasoning

The ENIGMA BRCA1/2 specification requires a case-control result with p<=0.05, OR>=4, and a lower confidence bound excluding 2.0. No such exact-variant affected-versus-control analysis is available in the consolidated evidence.

Rule

ENIGMA BRCA1/2 PS4: affected-versus-control enrichment; p<=0.05 and OR>=4 with lower CI excluding 2.0.

Evidence considered

• ENIGMA BRCA1/2 PS4 requires case-control enrichment with p<=0.05, OR>=4, and a lower confidence interval excluding 2.0.
• PMID:20104584 identified c.5266dupC (5382insC) in 10 women in the WECARE cohort, but the extracted result concerns contralateral versus unilateral breast cancer and supplies no affected-versus-control prevalence, OR, p value, or confidence interval for PS4.

Still missing

• An exact-variant case-control study reporting affected and control counts, OR, p value, and confidence interval is required.

Sources

Reasoning

The applicable ENIGMA BRCA1/2 VCEP rule restricts PM3 to a patient with a phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia and co-occurring variants in the same gene, with strength assigned from the PM3 points accumulated per proband. The case bundle provides no affected-proband Fanconi anemia phenotype, chromosome-breakage result, second BRCA1 pathogenic/likely pathogenic variant, or parental/offspring testing establishing phase. Therefore PM3 cannot be assessed from the available case evidence.

Rule

Apply PM3 for a patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi anemia and co-occurring variants in the same gene. Use ENIGMA Specifications Table 6 to assign points per proband; PM3 Supporting = 1 point, PM3 = 2 points, and PM3 Strong = at least 4 points. Phase requires parental or offspring testing.

Evidence considered

• The ENIGMA BRCA1/2 VCEP v1.2 rule states that PM3 requires a Fanconi anemia-consistent phenotype and co-occurring variants in the same gene; it assigns PM3 strength using Table 6 points per proband.
• The bundle identifies BRCA1-related cancer predisposition as autosomal dominant, but it does not document a biallelic BRCA1 observation, an affected Fanconi anemia proband, or phase information for this case.
• The available paper extracts concern variant-specific functional or cancer-cohort observations and do not report a second BRCA1 variant in trans or cis with c.5266dup in an affected Fanconi anemia proband.

Still missing

• Affected-proband Fanconi anemia phenotype and relevant clinical features are not provided.
• Chromosome-breakage testing result is not provided.
• No co-occurring BRCA1 pathogenic/likely pathogenic variant is documented.
• No parental or offspring testing establishing trans/cis phase is provided.
• PM3 point total per proband cannot be calculated.

Sources

Reasoning

The ENIGMA BRCA1 specification allows PM5_PTC as additional weight for a PVS1-annotated protein-termination-codon variant when a different proven pathogenic PTC variant has been observed in the same exon. NM_007294.4:c.5266dup is a frameshift producing p.(Gln1756ProfsTer74), but the consolidated bundle does not contain the PM5 candidate collection or an exact same-exon proven-pathogenic PTC comparator for this case. PM5 cannot be assigned without that comparator and the exon-specific PM5_PTC strength.

Rule

Protein termination codon variant in an exon where a different proven pathogenic PTC variant has been seen before; use as additional weight for PTC variants annotated as PVS1, with strength determined by ENIGMA Table 4/ST1.

Evidence considered

• The ENIGMA rule defines PM5 for a PTC variant in an exon where a different proven pathogenic PTC variant has previously been seen and limits it to PTC variants annotated as PVS1.
• The VCEP index states that Table 4 provides exon-level PM5_PTC applicability and strength, while Supplementary Table ST1 lists PM5_PTC weights by exon.
• The case bundle notes that the PM5 candidate file is not present, so no exact same-exon comparator can be verified from the supplied evidence.

Still missing

• An exact different proven pathogenic PTC variant in the same BRCA1 exon is required.
• The exon-specific PM5_PTC strength from ENIGMA Table 4/ST1 must be confirmed.

Sources

Reasoning

The ENIGMA BRCA1/2 v1.2 framework requires quantitative co-segregation analysis in multiple affected family members. The bundle contains no variant-specific segregation likelihood ratio, Bayes score, pedigree-level transmission data, or documented affected relatives who tested positive for this variant. PMID:20104584 reports c.5266dupC/5382insC in women in a population-based cohort, but does not provide variant-specific familial co-segregation evidence for this case; it cannot establish PP1.

Rule

ENIGMA BRCA1/2 v1.2 PP1: quantitative co-segregation with disease in multiple affected family members; Supporting LR >=2.08:1, Moderate LR >=4.3:1, Strong LR >=18.7:1, Very Strong LR >=350:1.

Evidence considered

• ENIGMA requires quantitative co-segregation analysis and assigns PP1 Supporting at LR >=2.08:1, Moderate at LR >=4.3:1, Strong at LR >=18.7:1, and Very Strong at LR >=350:1.
• The paper-extraction record for PMID:20104584 states that c.5266dupC (BIC: 5382insC) was carried by 10 women in the WECARE cohort, but reports cohort mutation carriage and cancer outcomes rather than segregation through affected families; no usable PP1 likelihood ratio is provided.
• No case-specific affected-relative genotype, phenotype, meiosis count, or co-segregation calculation appears in the consolidated evidence bundle.

Still missing

• Variant-specific pedigree and phenotype data for affected relatives
• Parental and relative genotypes with informative meioses
• Quantitative co-segregation likelihood ratio or Bayes score

Sources

Reasoning

BS2 cannot be adjudicated from this case bundle. The ENIGMA rule requires documented absence of Fanconi Anemia features and assigns strength from BS2 point totals, but no eligible proband-level phenotype, chromosome-breakage, genotype, or point-count data are provided.

Rule

ENIGMA BRCA1 v1.2 BS2: apply in the absence of Fanconi Anemia features; Supporting = 1 point, Moderate = 2 points, Strong = >=4 points, subject to Table 8 stipulations.

Evidence considered

• ENIGMA BRCA1 v1.2 defines BS2 as absence of features of recessive disease, specifically Fanconi Anemia phenotype, with Supporting, Moderate, or Strong strength determined by Table 8 point totals.
• The consolidated case-evidence bundle contains no proband-level Fanconi Anemia phenotype assessment or BS2 point calculation.

Still missing

• Proband-level evidence that the evaluated individuals lack a BRCA1-related Fanconi Anemia phenotype.
• The ENIGMA Table 8 BS2 point assignments and aggregate point total for eligible probands.

Sources

Reasoning

The ENIGMA BRCA1/2 v1.2 framework requires quantitative evidence for lack of segregation in affected family members. The bundle contains no variant-specific non-segregating affected relatives, informative pedigree, segregation likelihood ratio, or Bayes score. The absence of such data is insufficient to call BS4, and no paper-extracted source documents lack of segregation for c.5266dup.

Rule

ENIGMA BRCA1/2 v1.2 BS4: quantitative lack of segregation in affected family members; Supporting LR <=0.48:1, Moderate LR <=0.23:1, Strong LR <=0.05:1, Very Strong LR <=0.00285:1.

Evidence considered

• ENIGMA requires quantitative analysis of lack of segregation and assigns BS4 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 consolidated case-evidence bundle contains no documented affected relative who lacks the variant, no pedigree-level non-segregation result, and no quantitative BS4 likelihood ratio.
• The ENIGMA framework cautions that phenocopies and more than one pathogenic variant in a family can mimic lack of segregation; neither issue can be evaluated from the supplied case data.

Still missing

• Variant-specific affected-relative non-segregation observations
• Informative pedigree and relative genotypes
• Quantitative co-segregation likelihood ratio or Bayes score, including assessment of phenocopies or another causative pathogenic variant

Sources