BRCA2 c.6748A>G, p.(Thr2250Ala)

Reasoning

BS1 Strong is met under the ENIGMA BRCA2 specification. The gnomAD v2.1 non-cancer exome grpmax FAF is 0.00010864, which is above 0.0001 (0.01%) and therefore satisfies the specified BS1 Strong threshold.

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). See Appendix G for details.

Evidence considered

• The ENIGMA BRCA1/2 specification, version 1.2, defines BS1 Strong as FAF >0.0001 (0.01%) in gnomAD v2.1 non-cancer exome and/or gnomAD v3.1 non-cancer non-founder populations; BS1 Supporting applies only when FAF is >0.00002 and <=0.0001.
• gnomAD v2.1 reports grpmax FAF 0.00010864 (0.010864%), exceeding the BS1 Strong cutoff; the highest observed subpopulation AF is 0.000173481 in Admixed American individuals, with zero homozygotes.

Sources

Reasoning

The variant is a missense substitution at residue 2250. Under the ENIGMA BRCA2 v1.2 BP1 rule, BP1_Strong applies to missense variants outside the clinically important functional domains when splicing is not predicted. The specified domains are aa 10-40 and aa 2481-3186; residue 2250 is outside both. SpliceAI maximum delta is 0.016, which is at or below the required 0.1 threshold. BP1_Strong is therefore met.

Rule

ENIGMA BRCA1/2 v1.2: BP1_Strong for silent, missense, or in-frame insertion/deletion/delins variants outside a clinically important functional domain and with no predicted splicing impact (SpliceAI <=0.1).

Evidence considered

• The ENIGMA BRCA2 v1.2 BP1_Strong rule applies to missense variants outside the clinically important functional domains when SpliceAI is <=0.1.
• The case variant is NP_000050.3:p.(Thr2250Ala), so the altered residue is 2250; the specified domains are aa 10-40 and aa 2481-3186, placing residue 2250 outside the domains.
• SpliceAI reports a maximum delta score of 0.016, below the ENIGMA threshold of 0.1.

Sources

Reasoning

The reported exact-variant multifactorial odds are >100:1 in favor of neutrality, corresponding to LR<0.01 and meeting the ENIGMA BP5_Strong threshold of LR<=0.05.

Rule

ENIGMA BRCA2 v1.2 BP5: combined clinical-data LR against pathogenicity; Strong requires LR<=0.05.

Evidence considered

• ENIGMA BRCA2 v1.2 directs BP5 to capture only combined clinical-data LR against pathogenicity; Strong requires LR<=0.05.
• PMID:23683081 explicitly reports BRCA2 c.6748A>G among variants with odds >100:1 in favor of neutrality according to a multifactorial model.

Sources

Reasoning

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

Rule

Apply BP6_Supporting only for an exact-variant ClinVar expert-panel Benign or Likely benign assertion.

Evidence considered

• ClinVar variation 38064 exactly matches NM_000059.4:c.6748A>G (p.Thr2250Ala) and records a Benign ENIGMA assertion with review status 'reviewed by expert panel'.
• No ordinary laboratory submission or aggregate ClinVar label was used to trigger BP6.
• ClinVar expert panel classification

Sources

Reasoning

NM_000059.4:c.6748A>G is the missense substitution p.(Thr2250Ala), not a null variant or canonical splice-site variant. No mRNA assay demonstrating an abnormal transcript is provided. Therefore it does not meet the BRCA2 ENIGMA PVS1 rule.

Rule

ENIGMA BRCA2 v1.2 PVS1: apply to specified null variants in a gene with established loss of function, with strength determined by the PVS1 flowchart; mRNA-only functional evidence may also support PVS1 at the appropriate strength.

Evidence considered

• The BRCA2 ENIGMA VCEP specification uses NM_000059.4 as the preferred transcript and reserves PVS1 for null variants (including nonsense, frameshift, canonical ±1/2 splice, initiation-codon, and exon-deletion variants) or an established mRNA-only functional assay showing a damaging transcript effect.
• The case normalization identifies c.6748A>G as NP_000050.3:p.(Thr2250Ala), a missense consequence; the PVS1 variant assessment identifies it as noncanonical for splicing and outside the null-variant buckets.
• SpliceAI maximum delta is 0.016, which does not indicate a predicted splice effect; this is computational information only and not an mRNA assay.

Sources

Reasoning

The variant is a missense substitution, and the ENIGMA BRCA1/2 specification permits PS1 when a previously classified pathogenic or likely pathogenic variant produces the same amino-acid change (with no relevant splicing effect). The reviewed case evidence identifies c.6748A>G/p.Thr2250Ala as benign, likely benign, or IARC Class 1, but provides no qualifying pathogenic or likely pathogenic same-amino-acid comparator at residue 2250. Therefore PS1 is not met.

Rule

ENIGMA BRCA1/2 v1.2: apply PS1 for a predicted missense substitution when a previously classified pathogenic variant is considered to act through protein change and has no confirmed or predicted splicing effect; apply PS1_Moderate for a previously classified likely pathogenic comparator.

Evidence considered

• The ENIGMA BRCA2 v1.2 rule requires a previously classified pathogenic variant for PS1, or a likely pathogenic variant for PS1_Moderate, with the relevant protein-change or splicing condition.
• PMID:16683254 reports p.Thr2250Ala as an unclassified variant with unknown effect, not as a pathogenic comparator.
• PMID:23683081 reports c.6748A>G/p.Thr2250Ala as probably non-pathogenic under a multifactorial model, with odds greater than 100:1 in favor of neutrality.
• PMID:24323938 lists p.Thr2250Ala as IARC Class 1, defined in that paper as not pathogenic or of no clinical significance; it does not provide a pathogenic same-residue comparator.

Still missing

• No verified pathogenic or likely pathogenic BRCA2 variant producing p.Thr2250Ala was identified in the provided evidence bundle.

Sources

Reasoning

PM2 is not met because the variant is observed in population controls. It is present in gnomAD v2.1 at 28/251,358 alleles and in gnomAD v4.1 at 175/1,613,990 alleles; both datasets report zero homozygotes. Presence in gnomAD fails the ENIGMA requirement that the variant be absent from controls, regardless of the zero homozygote count.

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 ENIGMA BRCA1/2 specification, version 1.2, defines PM2 Supporting as absence from controls in an outbred population in gnomAD v2.1 non-cancer exome and gnomAD v3.1 non-cancer, with average regional read depth >=25.
• gnomAD v2.1 reports the variant in 28/251,358 alleles (AF 0.000111395; zero homozygotes), so it is not absent from controls.
• gnomAD v4.1 independently reports the variant in 175/1,613,990 alleles (AF 0.000108427; zero homozygotes), confirming population presence; gnomAD-Canada v1.0 is unavailable due to an HTTP 500 response.

Sources

Reasoning

ENIGMA BRCA2 v1.2 PP3 is not met. p.(Thr2250Ala) is outside the specified PALB2-binding (aa 10–40) and DNA-binding (aa 2481–3186) domains, so it is ineligible for the missense/protein-prediction PP3 path. The SpliceAI maximum delta score (0.016) is below the ENIGMA PP3 splicing threshold (≥0.20).

Rule

ENIGMA BRCA2 v1.2 PP3: missense/in-frame variants require location in aa 10–40 or aa 2481–3186 plus BayesDel no-AF ≥0.30; splice-path PP3 requires SpliceAI ≥0.20.

Evidence considered

• The governing ENIGMA BRCA2 v1.2 specification limits missense PP3 to variants in the PALB2-binding domain (aa 10–40) or DNA-binding domain (aa 2481–3186) with BayesDel no-AF score ≥0.30, and permits PP3 for predicted splicing at SpliceAI ≥0.20.
• The case is BRCA2 NM_000059.4:c.6748A>G, NP_000050.3:p.(Thr2250Ala); residue 2250 is outside both ENIGMA-defined domains.
• SpliceAI reports maximum delta 0.016 (DS_AG 0.016; DS_DG 0.010), below 0.20.

Sources

Reasoning

The exact-variant multifactorial clinical evidence is in the benign direction, not the pathogenic direction required for PP4.

Rule

ENIGMA BRCA2 v1.2 PP4: use only a combined clinical-data LR; Supporting requires LR>=2.08.

Evidence considered

• ENIGMA BRCA2 v1.2 permits PP4 only for a combined clinical-data likelihood ratio toward pathogenicity; Supporting requires LR>=2.08.
• PMID:23683081 explicitly states that BRCA2 c.6748A>G is among variants with odds >100:1 in favor of neutrality under a multifactorial model.

Sources

Reasoning

The exact-variant ClinVar expert-panel assertion is Benign, not Pathogenic or Likely pathogenic; ordinary laboratory assertions were not used.

Rule

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

Evidence considered

• ClinVar variation 38064 exactly matches NM_000059.4:c.6748A>G (p.Thr2250Ala) and has an ENIGMA expert-panel Benign assertion reviewed by expert panel.

Sources

Reasoning

BA1 is not met. The ENIGMA BRCA2 specification requires gnomAD v2.1 and/or v3.1 non-cancer, non-founder population FAF >0.001 (0.1%). The observed gnomAD v2.1 grpmax FAF is 0.00010864 (0.010864%), below the BA1 threshold.

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 ENIGMA BRCA1/2 specification, version 1.2, defines BA1 as a filter allele frequency above 0.1% (FAF >0.001) in gnomAD v2.1 non-cancer exome and/or gnomAD v3.1 non-cancer non-founder populations.
• gnomAD v2.1 reports 28/251,358 alleles, total AF 0.000111395, zero homozygotes, and grpmax FAF 0.00010864 (0.010864%); this is below 0.001.

Sources

Reasoning

The applicable ClinGen ENIGMA BRCA2 Version 1.2 framework reserves PS3 for well-established functional assays measuring protein function, or mRNA and protein together; mRNA-only splicing evidence is handled under the alternative RNA framework. The variant is not listed in the ENIGMA calibrated functional-assay Table 9. The exact-variant literature reviewed here reports clinical/multifactorial or validation-panel information but no qualifying protein-function or combined mRNA-and-protein assay result for BRCA2 c.6748A>G (p.Thr2250Ala). Therefore PS3 cannot be assessed from the available evidence.

Rule

ENIGMA BRCA2 V1.2: PS3 is for well-established in vitro or in vivo studies showing a damaging effect when the assay measures protein function alone or mRNA and protein combined; mRNA-only evidence is handled as PVS1 (RNA) at the applicable strength.

Evidence considered

• ENIGMA BRCA1/2 VCEP Version 1.2 specifies that PS3 applies to well-established functional studies measuring protein function, or mRNA and protein combined, and directs curated assay lookups to Table 9; mRNA-only assay evidence is assigned under the alternative RNA framework rather than PS3.
• The exact variant c.6748A>G (p.Thr2250Ala) is absent from the BRCA2 calibrated functional-assay Table 9 lookup; Table 9 contains other BRCA2 variants with assigned PS3/BS3 codes but no row for this variant.
• PMID:24323938 explicitly lists c.6748A>G/p.Thr2250Ala in a validation panel and assigns IARC Class 1, but its extracted variant-level record states that no functional-assay result for this exact variant is reported in Table 2. This classification/validation-panel result was not treated as functional evidence.

Still missing

• No qualifying validated protein-function or combined mRNA-and-protein assay result for c.6748A>G/p.Thr2250Ala was identified.
• No ENIGMA Table 9 calibrated PS3 assignment exists for this exact variant.

Sources

Reasoning

No ethnicity- and country-matched case-control analysis reporting the ENIGMA-required significance and effect-size thresholds was identified for c.6748A>G.

Rule

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

Evidence considered

• The ENIGMA BRCA2 v1.2 PS4 rule requires a case-control study with p<=0.05, OR>=4, and lower confidence interval excluding 2.0.
• The reviewed exact-variant literature records report occurrence/classification context, not a qualifying matched case-control estimate.

Still missing

• A matched case-control study for c.6748A>G with p-value, odds ratio, and confidence interval is needed.

Sources

Reasoning

The applicable ENIGMA BRCA2 v1.2 specification 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 determined by PM3 points. The case bundle contains no documented affected-proband Fanconi-anemia phenotype, second BRCA2 pathogenic/likely pathogenic variant, phase, or inheritance information. 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; assign Supporting for 1 point, Moderate for 2 points, or Strong for at least 4 points.

Evidence considered

• The ENIGMA BRCA2 v1.2 rule requires a Fanconi-anemia-consistent phenotype plus co-occurring variants in the same gene; PM3 strength is assigned from the specified PM3 point total.
• The case-evidence bundle documents the index variant as BRCA2 c.6748A>G (p.Thr2250Ala) but provides no affected-proband allelic observation, second BRCA2 variant, phase, or inheritance evidence.
• The exact-variant literature findings available in the bundle report occurrence or multifactorial classification, not a qualifying Fanconi-anemia compound-genotype observation with phase.

Still missing

• Documented Fanconi-anemia phenotype and chromosome-breakage result for the proband
• Identity and clinical classification of any co-occurring BRCA2 variant
• Phase of the variants and parental/inheritance data

Sources

Reasoning

The ENIGMA BRCA2 specification requires co-segregation with disease in multiple affected family members measured by a quantitative co-segregation method, with strength assigned from the resulting likelihood ratio. The case bundle provides no quantitative co-segregation likelihood ratio, meiosis count, affected-relative genotype table, or sufficiently detailed family segregation data for NM_000059.4:c.6748A>G (p.Thr2250Ala). The literature reports the variant in one family and discusses a multifactorial neutrality assessment, but does not provide variant-specific segregation data adequate for PP1.

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 PP1 requires quantitative co-segregation evidence and assigns 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.
• Blay et al. explicitly report BRCA2 c.6748A>G (p.Thr2250Ala) as listed in one high-risk family, but the extracted report does not provide the relative-level segregation genotypes, informative meioses, or a PP1-specific LR.
• The paper's statement that the variant had odds greater than 100:1 in favor of neutrality is a multifactorial-model result and is not treated as a PP1 segregation LR.

Still missing

• Variant-specific genotypes and phenotypes for affected and unaffected relatives
• Number of informative meioses and verified familial relationships
• Quantitative co-segregation likelihood ratio or Bayes score

Sources

Reasoning

BS2 cannot be assessed from the available case bundle. The BRCA2-specific rule requires evaluation of individuals lacking features of recessive BRCA2-related Fanconi anemia and assignment of points according to the specification's Table 8. No adequately documented proband-level Fanconi anemia phenotype assessment or Table 8 point data are provided.

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. BS2 = >=4 points for Strong, BS2_Moderate = 2 points, and BS2_Supporting = 1 point.

Evidence considered

• The ENIGMA BRCA1/2 specification, version 1.2, states that BS2 is applied in the absence of features of recessive disease, specifically Fanconi anemia, using additional stipulations and point assignment from Specification Table 8; BS2 Strong requires at least 4 points, Moderate 2 points, and Supporting 1 point.
• The case bundle provides population allele counts and homozygote counts but does not provide the required individual-level Fanconi anemia phenotype evaluation or BS2 point calculation.

Still missing

• Proband-level assessment for absence of BRCA2-related Fanconi anemia features
• The ENIGMA Table 8 BS2 point assignments and eligible individuals

Sources

Reasoning

The applicable ClinGen ENIGMA BRCA2 Version 1.2 framework reserves BS3 for well-established functional assays showing no damaging effect on protein function, using protein-only or combined mRNA-and-protein assays; mRNA-only splicing evidence is handled under the alternative RNA framework. The variant is not listed in the ENIGMA calibrated functional-assay Table 9, and the exact-variant literature reviewed provides no qualifying benign functional assay result. The available benign or likely-benign classifications were not used to infer BS3. Therefore BS3 cannot be assessed from the available evidence.

Rule

ENIGMA BRCA2 V1.2: BS3 is for well-established in vitro or in vivo studies showing no damaging effect on protein function when the assay measures protein function alone or mRNA and protein combined; mRNA-only evidence is handled as BP7 (RNA) at the applicable strength.

Evidence considered

• ENIGMA BRCA1/2 VCEP Version 1.2 specifies that BS3 applies to well-established in vitro or in vivo studies showing no damaging effect on protein function when the assay measures protein function alone or mRNA and protein combined; mRNA-only evidence is handled as BP7 (RNA) at the applicable strength.
• The exact variant c.6748A>G (p.Thr2250Ala) is absent from the BRCA2 calibrated functional-assay Table 9 lookup; Table 9 contains other BRCA2 variants with assigned PS3/BS3 codes but no row for this variant.
• PMID:24323938 explicitly lists c.6748A>G/p.Thr2250Ala in a validation panel and assigns IARC Class 1, but its extracted variant-level record states that no functional-assay result for this exact variant is reported in Table 2. The benign classification was not used as a substitute for direct functional evidence.

Still missing

• No qualifying validated protein-function or combined mRNA-and-protein assay result showing preserved function for c.6748A>G/p.Thr2250Ala was identified.
• No ENIGMA Table 9 calibrated BS3 assignment exists for this exact variant.

Sources

Reasoning

The ENIGMA BRCA2 specification requires lack of segregation in affected family members measured by a quantitative co-segregation analysis method, with strength assigned from a likelihood ratio against pathogenicity. The case bundle contains no verified affected non-carriers, family-level genotype/phenotype data, informative meiosis count, or quantitative BS4 LR for this variant. The report of one family and a multifactorial neutrality assessment does 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 BS4 requires quantitative lack-of-segregation evidence and assigns 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.
• Blay et al. report the variant in one family and cite a multifactorial model favoring neutrality, but the extracted source does not identify affected non-carriers or provide a BS4-specific quantitative LR.
• A multifactorial odds result is not substituted for direct affected-relative non-segregation evidence.

Still missing

• Documented affected relatives who do not carry the variant
• Variant testing methods and genotypes for those affected relatives
• Informative meioses and quantitative non-segregation likelihood ratio or Bayes score

Sources

Reasoning

The variant is missense, not silent or intronic, so the BP7_Supporting sequence-based paths do not apply. ENIGMA permits BP7_Strong (RNA) for a missense variant outside a clinically important domain only when a well-established mRNA-only assay shows no damaging transcript effect. No such variant-specific RNA assay result is supplied.

Rule

ENIGMA BRCA2 v1.2 BP7: BP7_Strong (RNA) requires a well-established mRNA-only no-effect result; BP7_Supporting is limited to specified silent or intronic contexts.

Evidence considered

• The governing ENIGMA BRCA2 v1.2 specification restricts BP7_Supporting to silent variants in a clinically important domain with BP4 met, or qualifying deep intronic variants with BP4 met.
• The ENIGMA BP7_Strong (RNA) path allows missense variants outside a clinically important domain only with a well-established mRNA-only assay showing no damaging effect; no such result is present for c.6748A>G (p.Thr2250Ala).

Still missing

• A well-established, variant-specific mRNA-only assay demonstrating no damaging transcript effect would be required to assess ENIGMA BP7_Strong (RNA).

Sources