The BRCA2 NM_000059.3:c.10043del (NP_000050.2:p.(Asn3348IlefsTer35)) frameshift variant has not previously been reported in somatic cancers (COSMIC). Based on available guidelines, this variant is considered to be a Variant of Uncertain Significance (VUS).
Five-step scale from benign to pathogenic. This variant is uncertain. Select a step to read what it means.
This variantScenario
BenignPathogenic
Uncertain.The recorded classification is uncertain significance.
In plain language
Gene-level background from the recorded interpretation.
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About BRCA2
BRCA2 encodes a DNA repair protein that maintains genome stability by repairing double-strand breaks through homologous recombination and by protecting DNA replication forks. It acts as a tumor suppressor, and inherited loss-of-function changes cause hereditary breast and ovarian cancer syndrome, with elevated lifetime risks of breast, ovarian, prostate, and pancreatic cancers; biallelic changes cause Fanconi anemia complementation group D1. Reduced or altered BRCA2 activity is implicated in multiple tumor types, and PARP inhibitors are an approved treatment for BRCA2-associated ovarian and breast cancers.
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01The reasoning
How it was classified
The recorded framework is ClinGen BRCA2 VCEP v1.2. The criteria and strengths shown below come from the saved assessment; this page does not recalculate the classification.
Recorded applied criteria
The applied criteria and their strengths are shown as assessed under the stated framework.
No pathogenic criterion marked as applied in the recorded assessment.
Benign criteria applied · 0
No benign criterion marked as applied in the recorded assessment.
Recorded classification: Uncertain significance
VUS: no criterion is met - ENIGMA BRCA2 v1.2 pre-assigns PVS1 and PM5 (PTC) as not applicable distal to p.E3309 and excludes PM2 for deletions, so no Table 3 rule is satisfied.
02The sources
Evidence at a glance
Evidence summaries and captured source records, where available. Missing entries should not be interpreted as negative findings.
gnomAD v4.1
gnomAD v2.1
Population frequency
Not recorded
gnomAD v4.1 allele counts were not recorded for this variant.
ACMG/AMP criteria under ClinGen BRCA2 VCEP v1.2, with the data and sources behind each one.
28 criteria with recorded statuses: 0 met · 6 not met · 5 not assessed · 17 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
PVS1 is not applicable, not met: Specifications Table 4 assigns PVS1_N/A to BRCA2 exon 27 termination codons downstream of p.E3309, and this frameshift begins at codon 3348 with a new stop near codon 3382.
Because PVS1_N/A is a pre-assigned not-applicable code, no loss-of-function points accrue at any strength from this group; any pathogenic classification must rest on the other evidence groups (PS4, PM5 (PTC), PP1, PP4 etc.).
PM4 and BP3 are both marked 'Do not use' / 'Not applicable' by the ENIGMA BRCA2 v1.2 specification, and the variant lies outside their in-frame-indel variant classes in any case, so neither contributes evidence.
No strength value is recorded for any of the three criteria because none is met; PVS1 strength is deliberately null rather than a strength word, since PVS1_N/A denotes absence of the code rather than a downgraded PVS1.
All five codon/residue/domain criteria are inapplicable to this variant rather than refuted: c.10043del is a frameshift in BRCA2 exon 27 (p.Asn3348IlefsTer35, termination codon p.Ter3382).
The VCEP itself disables PM1 and PP2 ('Do not use' in Table 1); PS1 is limited to missense substitutions or splice-equivalent variants and no classified comparator exists at codon 3348 (variant absent from ClinVar); BP1_Strong is limited to silent/missense/in-frame variants with SpliceAI <=0.1.
PM5 is now PM5 (PTC) in this specification, and Table 4 pre-assigns PM5_N/A for BRCA2 exon 27 PTCs terminating after p.E3309 - confirmed by Supplementary Table 1's exon 27 escape-NMD boundary of p.3309 - so the p.Ter3382 termination codon receives no PM5 weight.
Net contribution of this group to the final classification: zero pathogenic and zero benign points.
Neither PS3 nor BS3 contributes: c.10043del is absent from the VCEP's curated functional assay table (Specifications Table 9) and from ST4 of the Supplementary Tables, and no retrieved publication reports a functional assay of this variant.
Adjacent-variant assignments in exon 27 are not transferable: Table 9 gives PS3 Strong to c.9672dup, c.9699_9702del and c.9924C>G but BS3 Strong to c.9945del, c.9976A>T and c.10184del, so the region cannot be used as a proxy for this untested truncation.
For a frameshift variant this VCEP applies the loss-of-function evidence through PVS1 (Specifications Table 4, exon-level weighting) rather than through PS3.
This group contributes no pathogenic or benign weight: PS4, PP4 and BP5 are unevaluable for BRCA2 c.10043del because no case-control OR/CI and no combined multifactorial likelihood LR exists for the variant in any declared ENIGMA source, and PP5/BP6 are removed from the code set by the specification itself.
The absence of entries in the ENIGMA multifactorial tables is expected rather than exculpatory: those tables cover missense, in-frame and splice variants, while ENIGMA routes clinical/prevalence evidence for protein-truncating variants through the exon-specific PM5 (PTC) weight, determined for this frameshift by exon 27.
Final classification therefore rests on the other criteria groups; no positive or negative phenotype/prevalence evidence should be counted from this group.
Allelic group contributes no pathogenic or benign points: PM3 is not met because the VCEP restricts it to Fanconi Anaemia probands with a co-occurrent P/LP variant in BRCA2 and no such proband or co-occurrent variant is documented for c.10043del.
BP2 cannot contribute benign weight because the ENIGMA BRCA2 v1.2 specification marks it 'Do not use / Applied only in the context of BS2'; allelic-phase evidence in BRCA2 is scored through BS2 (Table 8) instead.
Population-frequency evidence is uniformly uninformative for BRCA2 c.10043del: the variant is absent from gnomAD v2.1 exomes (13-32972690-TA-T), gnomAD v4.1 (chr13-32398553-TA-T) and both non-cancer subsets, so no benign frequency code (BA1 FAF >0.001, BS1 FAF >0.0001 or BS1_Supporting FAF >0.00002) can be met, and BS2 fails for want of any proband-level co-occurrence or homozygous observation (0 of >=4 points).
PM2 is not applicable rather than met: ENIGMA BRCA2 v1.2 excludes insertion/deletion/delins variants from PM2_Supporting because indel recall in gnomAD is poor (Appendix G, PMID: 33600021), and this is a 1-bp frameshift deletion.
All four criteria therefore contribute no population evidence in either direction; classification rests entirely on the other evidence groups.
No criteria match.
Not met · 6 · evaluated, threshold not reached
PS3VCEPNot met: no calibrated functional assay reports this variant, and Specifications Table 9 carries no row for c.10043del or p.Asn3348IlefsTer35.
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Reasoning
The governing ENIGMA BRCA2 v1.2 PS3 rule requires well-established in vitro or in vivo functional studies supportive of a damaging effect on protein function, with Specifications Table 9 supplying the VCEP's pre-assigned code strength for calibrated published assays. Specifications_Table9_V1.2_2024-11-18.xlsx was searched for c.10043del, p.(Asn3348Ilefs*35), p.N3348Ifs*35, N3348 and 3348 and contains no row for this variant; Supplementary Table 4 (ST4 Fxn assay results) in SupplementaryTables_V1.2_2024-11-18.xlsx likewise has no BRCA2 row at c.10043, and the Parsons 2019 multifactorial supplementary table (HUMU-40-1557-s001.xlsx) has no entry either. None of the five retrieved full texts reports this variant or any assay of it. Table 9 does contain pre-assigned functional codes for other exon 27 truncating variants, but those assignments are variant-specific and are not transferable: c.9672dup p.(Tyr3225Ilefs*30), c.9699_9702del p.(Cys3233Trpfs*15) and c.9924C>G p.(Tyr3308*) carry PS3 Strong, while c.9945del p.(Glu3316Asnfs*2), c.9976A>T p.(Lys3326Ter) and c.10184del p.(E3395Gfs*32) carry BS3 Strong. Because no calibrated assay reports c.10043del, there is no functional evidence of a damaging effect and PS3 is not met; the rule's mRNA-only branch is likewise unaddressed because no mRNA transcript assay of this variant exists (SpliceAI delta 0.205 is a prediction, not an assay). A truncating variant is in any case handled by this VCEP through PVS1 (Specifications Table 4) rather than PS3.
VCEP 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. See Specifications Table 9 for code recommendations from calibrated published assays. Also see Figure1C and Appendix E for details.
Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details.
Evidence considered
Specifications_Table9_V1.2_2024-11-18.xlsx, sheet Table9_BRCA12VCEP_specs: searched by c.10043del, p.(Asn3348Ilefs*35), p.N3348Ifs*35, '3348' and 'N3348'; no matching row. The nearest coding rows are c.10045A>G p.(Thr3349Ala) (BS3, Strong) and c.10184del p.(Glu3395Glyfs*32) (BS3, Strong).
Specifications_Table9_V1.2_2024-11-18.xlsx, header note: 'This Table is provided as a separate file in excel format to facilitate searches and look-ups by variant c. and p. nomenclature... This is not an exhaustive list'.
SupplementaryTables_V1.2_2024-11-18.xlsx, sheet ST4 Fxn assay results, BRCA2 exon-27-region rows: c.9925G>T p.(Glu3309Ter), c.9945del p.(Glu3316Asn-fs*2), c.9976A>T p.(Lys3326Ter), c.10045A>G p.(Thr3349Ala) and c.10184del p.(Glu3395Gly-fs*32) are all recorded as 'No functional impact'; no c.10043del row is present.
SupplementaryTables_V1.2_2024-11-18.xlsx, sheet ST5: the BRCA2 protein-function sources used by the VCEP are Hart et al. 2019 (PMID:29884841) and Mesman et al. 2019 (PMID:29988080); neither paper was retrieved for this case and neither reports c.10043del in any available extract.
Appendices_V1.2_2024-11-18.docx, Appendix E: 'PS3 and BS3 codes are reserved for assigning variant impact on protein function, but are required to take into consideration functional assay design, variant location, and predicted/observed impact on splicing as well as predicted impact on the encoded protein sequence.'
Specifications_V1.2_2024-11-18.docx, Table 9 preamble: additional published assay results not listed in Table 9 should be reviewed following Figure 1C before application of PS3 and BS3 codes - no such additional assay exists for c.10043del.
HUMU-40-1557-s001.xlsx (Parsons et al. 2019 multifactorial table): searched by c.10043del and by residue 3348; no row for this variant.
Still missing
Specifications_Table9_V1.2_2024-11-18.xlsx was searched for c.10043del, p.(Asn3348Ilefs*35), p.N3348Ifs*35, N3348 and 3348: no entry for this variant exists anywhere in the file.
SupplementaryTables_V1.2_2024-11-18.xlsx sheet ST4 (Fxn assay results) was searched; BRCA2 rows in the c.96xx-c.101xx range include c.9945del, c.9976A>T, c.10045A>G and c.10184del, but there is no c.10043del row.
HUMU-40-1557-s001.xlsx (Parsons et al. 2019) was searched by coding change and by residue 3348: no entry for this variant.
None of the five retrieved publications reports this variant, so no published assay of c.10043del was available to assess.
PM3VCEPNot met: no Fanconi Anaemia proband and no co-occurrent BRCA2 P/LP variant is reported, so zero of the >=1 PM3 point threshold can be assigned.
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Reasoning
Under ENIGMA BRCA2 v1.2, PM3 is repurposed exclusively for recessive Fanconi Anaemia: it is applied only for a patient whose phenotype is consistent with BRCA1/BRCA2-related FA (increased chromosome breakage by DEB/MMC or spontaneous, plus at least one clinical FA feature; or, if breakage testing is unknown, at least two FA features spanning at least two of the three categories) who also carries a co-occurrent P/LP variant in the same gene, with points assigned per proband (2 points for P/LP in trans or homozygote with FA phenotype, 1 point for phase-unknown, summing to PM3_Supporting = 1, PM3 = 2, PM3_Strong >= 4). None of those elements is documented here: BRCA2 c.10043del (p.Asn3348IlefsTer35) is absent from ClinVar, so no proband, pedigree, phase, or co-occurrent BRCA2 variant has been reported; the retrieved publications (10570174, 11239455, 20878484, 22193408, 24312913) do not name the variant; and nothing in the case evidence describes a Fanconi Anaemia phenotype. With no FA-affected proband and no co-occurrent P/LP variant, the PM3 trigger condition is not satisfied and no points can be assigned, so PM3 is not met. Note this is a dominant cancer-predisposition context (BRCA2-related cancer predisposition, autosomal dominant), where the VCEP does not use the classic 'in trans with pathogenic variant' formulation of PM3 at all.
VCEP rule
Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene. ... Assign final PM3 code based on sum of PM3-related points from observations across multiple unrelated individuals, as follows: PM3_Strong = 4 (or more) points; PM3 = 2 points; PM3_Supporting = 1 point.
Evidence considered
ENIGMA BRCA2 v1.2 Table 1, PM3 row: PM3_VariableWeight applies only to a patient with a phenotype consistent with BRCA1- or BRCA2-related Fanconi Anaemia and co-occurrent variants in the same gene; the phenotype criteria are (i) increased chromosome breakage (DEB, MMC, or spontaneous) plus at least one FA clinical feature, or (ii) chromosome breakage result unknown plus at least two FA clinical features in at least two of the three categories (physical features, pathology and laboratory findings, cancer diagnosis <=5yr).
ENIGMA BRCA2 v1.2 Table 6, rows 'P or LP in trans or homozygote' = 2 points and 'Phase unknown' = 1 point for a proband with FA-consistent phenotype; final code assigned from the sum across unrelated individuals: PM3_Strong = 4 (or more) points; PM3 = 2 points; PM3_Supporting = 1 point.
ClinVar: search for BRCA2 c.10043del returned no record ('This variant is absent from ClinVar'), so there is no submitted proband, phase, or co-occurrent variant observation to score.
gnomAD v2.1, gnomAD v4.1 and gnomAD non-cancer subsets: variant absent; no homozygote or biallelic observation available that could bear on a recessive/FA scenario.
Publications retrieved for this case (PMID:10570174, PMID:11239455, PMID:20878484, PMID:22193408, PMID:24312913) were each searched for c.10043del / p.N3348Ifs*35 and none names the variant; the only family data in the set concerns a different BRCA2 frameshift (7525_7526insT, p.Ser2509fs) in PMID:20878484 and is not transferable to this variant.
Still missing
No proband-level data (pedigree, phase, co-occurrent BRCA2 variant) exists for c.10043del: the variant is absent from ClinVar and is not named in any of the five publications retrieved, so the FA-phenotype precondition of PM3 could not be observed in any individual.
Searched the full converted text of Specifications_V1.2_2024-11-18.docx, Specifications_Table4/Table9, SupplementaryTables_V1.2, HUMU-40-1557-s001, the Li 2020 clinical-history LR table and the three VCEP reference PDFs for 'c.10043del', 'p.Asn3348IlefsTer35' and 'p.N3348Ifs*35': no entry for this variant in any of them (PM3 has no per-variant table, so no pre-assigned code exists to defer to).
BA1VCEPNot met: filter allele frequency is 0 in gnomAD v2.1 non-cancer exomes and v4.1, versus the >0.001 BA1 threshold.
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Reasoning
BRCA2 c.10043del (p.Asn3348IlefsTer35), a 1-bp deletion in exon 27 (NM_000059.3:c.10043del; GRCh38 chr13-32398553-TA-T), is absent from gnomAD v2.1 exomes (13-32972690-TA-T) and from gnomAD v4.1 (chr13-32398553-TA-T); the case gnomAD records include non-cancer subsets (GNOMAD_V2_1_NON_CANCER exomes-only, GNOMAD_V3_1_NON_CANCER genomes) and all report absent/no variant record, so there is no observed allele count and no filter allele frequency of any magnitude. The ENIGMA BRCA2 v1.2 BA1 rule requires a filter allele frequency above 0.1% (FAF > 0.001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder populations; an observed FAF of 0 is three orders of magnitude below that stand-alone threshold, so BA1 cannot be met. The VCEP's applicability conditions for BA1 (do not apply if read depth <20; do not apply to well-established pathogenic founder variants) do not rescue the criterion here, and the ACMG/AMP 2015 BA1 threshold (>5% in ESP/1000G/ExAC) is superseded by this VCEP-specific 0.001 FAF rule and would also not be met. ClinVar also holds no record for this variant, so no population-frequency-based benign assertion exists anywhere in the evidence set.
VCEP 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
gnomAD v2.1 (13-32972690-TA-T, GRCh37): search_status 'absent', evidence sentence 'Absent from gnomAD v2.1'; no allele count, allele number or filter allele frequency available.
ENIGMA BRCA2 specification v1.2, Table 1 BA1 row: FAF >0.1% (0.001) in gnomAD v2.1 non-cancer exomes and/or gnomAD v3.1 non-cancer genomes, applied to the maximum filter allele frequency in a non-founder population, exome and genome data considered separately; do not apply if read depth <20; do not apply to well-established pathogenic founder variants.
ENIGMA v1.2 Appendix G (BA1 calibration): Whiffin-calculator minimal credible allele frequency for BRCA2 was 0.000906 (prevalence 0.125, genetic heterogeneity 0.01, allelic heterogeneity 1.00, penetrance 0.69, PMID:28632866), rounded up to 0.001, with the LR-based approach giving LR 0.0009 (Benign Stand-alone) for MAF >0.001 to <=0.01; no BRCA2 variant reaches that frequency here.
ClinVar: no record for BRCA2 c.10043del, therefore no population-frequency-based benign assertion is available.
Still missing
The GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER records in this case were resolved against the same all-comers gnomAD URLs (gnomad_r2_1 and gnomad_r4) rather than separate non-cancer dataset pages, so the non-cancer subset figures were not independently retrieved; the V3_1_NON_CANCER record also resolves to a v4 URL rather than a gnomAD v3.1 genomes query. Absence is concordant across all four records, so this does not change the BA1 verdict.
No gnomAD regional read-depth/coverage figures were captured for this position, so the VCEP's 'do not apply if read depth <20' guard cannot be formally checked; this is immaterial because absence cannot satisfy a frequency-above-threshold rule.
ENIGMA BRCA2 v1.2 SupplementaryTables_V1.2_2024-11-18.xlsx, Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx and HUMU-40-1557-s001.xlsx were searched for 'c.10043del', 'p.Asn3348IlefsTer35' and 'p.N3348Ifs*35'; no row for this variant exists in any of them (all '3348' hits are unrelated variants or numeric scores), so no pre-assigned population code was available to apply.
BS1VCEPNot met: filter allele frequency is 0 in gnomAD v2.1 non-cancer exomes and v4.1, versus the >0.00002 BS1_Supporting floor.
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Reasoning
BS1 in ENIGMA BRCA2 v1.2 has two tiers: Strong for a filter allele frequency above 0.01% (FAF > 0.0001) and Supporting for FAF above 0.002% and up to 0.01% (FAF > 0.00002 and <= 0.0001), assessed on the maximum FAF in a gnomAD non-founder population using gnomAD v2.1 non-cancer exomes and/or gnomAD v3.1 non-cancer genomes, exome and genome data considered separately. This variant is absent from gnomAD v2.1 exomes (13-32972690-TA-T), gnomAD v4.1 (chr13-32398553-TA-T), the gnomAD v2.1 non-cancer exome subset and the gnomAD v3.1 non-cancer genome subset, so its observed FAF is 0 - below the Supporting floor of 0.00002 by more than four orders of magnitude and therefore also below the Strong threshold. A variant absent from controls cannot be 'more common than expected for the disorder', so neither BS1 strength tier is met. The VCEP guards (read depth >20, exclusion of quality-control-failed data, no application to well-established founder variants) are not engaged because there is no observed frequency to evaluate, and the ACMG/AMP 2015 BS1 wording ('greater than expected for disorder') is operationalised by this VCEP threshold rather than any generic cutoff.
VCEP 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. ... 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
gnomAD v2.1 (13-32972690-TA-T): absent, no allele count or filter allele frequency reported.
gnomAD v4.1 (chr13-32398553-TA-T): absent.
gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer genomes: reported absent in both records.
ENIGMA BRCA2 specification v1.2, Table 1 BS1/BS1_Supporting row: BS1 - above 0.0001 (0.01%); BS1_Supporting - >0.00002 (0.002%) to <=0.0001 (0.01%); preferred datasets gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer genomes; apply on maximum filter allele frequency in a non-founder population; do not apply if read depth <20; exclude data failing quality-control filters.
ENIGMA v1.2 Appendix G (BS1 calibration): minimal credible allele frequency 0.0000906 for BRCA2 (prevalence 0.125, genetic heterogeneity 0.01, allelic heterogeneity 0.10, penetrance 0.69), rounded to 0.0001 for Benign Strong, with BS1_Supporting permitted for MAF >0.00002 to <=0.0001; none of these bands is approached by an absent variant.
Still missing
The GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER records in this case resolve to the all-comers gnomAD URLs (gnomad_r2_1, gnomad_r4) rather than dedicated non-cancer dataset pages, and the V3_1_NON_CANCER record does not query gnomAD v3.1 genomes specifically; non-cancer-subset frequencies were therefore not independently retrieved. All records agree on absence, so the BS1 verdict is unaffected.
Regional read depth (>20 required by the VCEP for benign frequency codes) was not retrieved for this position; immaterial here because absence cannot meet a frequency-above-threshold rule.
SupplementaryTables_V1.2_2024-11-18.xlsx, Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx and HUMU-40-1557-s001.xlsx were searched for 'c.10043del', 'p.Asn3348IlefsTer35' and 'p.N3348Ifs*35' and contain no row for this variant, so no pre-assigned BS1 code or published frequency comparison for this variant exists in the VCEP tables.
BS2VCEPNot met: no proband-level in-trans or homozygous observation yields any of the >=4 BS2 points required.
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Reasoning
In ENIGMA BRCA2 v1.2 BS2 is not a general 'seen in a healthy adult' criterion driven by population databases: it is a variable-weight, proband-counting code applied when the variant is seen in trans with a pathogenic/likely pathogenic variant in the same gene, or as a homozygote, in a presumed Fanconi-anemia-unaffected individual, with points assigned per proband by age at first cancer diagnosis or last follow-up (4 points for in trans with first cancer >50y or cancer-unaffected >50y; 2 points for homozygote in a phenotyped individual at that age; 1 point for phase-unknown co-occurrence), and BS2 requires a sum of >= 4 points. The evidence set contains no proband-level co-occurrence or phase data for this variant, no homozygous observation in any phenotyped individual, and gnomAD returns no homozygote or heterozygote observations at all, so the accumulated BS2-related point total is 0 against a threshold of >= 4. The Appendix H stipulation that the homozygote column applies only to phenotyped individuals from clinical or research cohorts and must NOT be derived from gnomAD homozygote counts independently blocks any inference from population data. BS2 is therefore not met rather than not assessed: the rule can be fully evaluated and no qualifying observation exists.
VCEP rule
Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. ... Assign final BS2 code based on sum of BS2-related points: BS2 = 4 (or more) points; BS2_moderate = 2 points; BS2_Supporting = 1 point.
Evidence considered
ENIGMA BRCA2 specification v1.2, Table 1 BS2_VariableWeight row: 'Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype', with points assigned per proband via Table 8 and the final code from the summed BS2-related points.
ENIGMA v1.2 Table 8 / Appendix H Table 13, BS2-related points per proband: VUA in trans with P or LP in same gene = 4 points (first cancer onset >50y or cancer-unaffected at >50y), homozygote = 2 points, phase-unknown co-occurrence with P or LP = 1 point; halved values for first cancer onset/last follow-up 40-50y; final code 'BS2 = 4 (or more) points; BS2_moderate = 2 points; BS2_Supporting = 1 point'.
ENIGMA v1.2 Table 8 footnote **: homozygote points apply only for phenotyped individuals from clinical or research cohorts and 'NOT to be applied for data used to assign frequency-based codes'.
gnomAD v2.1 and gnomAD v4.1: variant absent, therefore no homozygote observations are available from population data.
ClinVar: no record for this variant, so no reported healthy-adult or co-occurrence observation is available.
Still missing
No proband-level data (co-occurrence with a P/LP BRCA2 variant, phase, age at diagnosis or last follow-up) are available in this case for a BS2 points calculation; a BS2 determination would require such case-level records.
BS3VCEPNot met: no assay reports c.10043del, although Table 9 assigns BS3 Strong to neighbouring exon 27 truncations c.9945del and c.10184del.
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Reasoning
The governing ENIGMA BRCA2 v1.2 BS3 rule requires well-established in vitro or in vivo functional studies showing no damaging effect on protein function, again with Specifications Table 9 supplying the VCEP's pre-assigned strength for calibrated published assays. No assay of c.10043del p.(Asn3348Ilefs*35) exists: Specifications Table 9 has no row for the variant, Supplementary Table 4 (Fxn assay results) has no BRCA2 row at c.10043, the Parsons 2019 multifactorial table has no entry, and none of the five retrieved full texts names the variant. BS3 therefore cannot be assigned on variant-specific functional evidence. The nearest Table 9 assignments are nevertheless notable and warrant curator review rather than silent omission: this frameshift begins at codon 3348 and introduces a stop about 35 codons downstream, placing it in the same exon 27 C-terminal segment where the VCEP assigned BS3 Strong to c.9945del p.(Glu3316Asnfs*2) and c.10184del p.(E3395Glyfs*32) on Mesman et al. 2019 (PMID:29988080) mESC complementation/HDR data, and where c.9976A>T p.(Lys3326Ter) is also BS3 Strong; conversely c.9672dup, c.9699_9702del and c.9924C>G in the same exon carry PS3 Strong. Those codes belong to those variants only and cannot be extrapolated to c.10043del under this framework, so BS3 is recorded as not met with a flag that the VCEP's own exon 27 assignments are internally discordant in this region.
VCEP rule
Well-established in vitro or in vivo functional studies shows no damaging effect on protein function. Assay measures effect via protein only OR mRNA and protein combined. See Specifications Table 9 for code recommendations from calibrated published assays. Also see Figure1C and Appendix E for details.
Well-established _in vitro_ or _in vivo_ functional studies supportive of no damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as BP7 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details.
Evidence considered
Specifications_Table9_V1.2_2024-11-18.xlsx, sheet Table9_BRCA12VCEP_specs: no row for c.10043del p.(Asn3348Ilefs*35); searched by coding change, three-letter and one-letter protein notation, 'N3348' and '3348'.
Specifications_Table9_V1.2_2024-11-18.xlsx, rows c.9945del p.(Glu3316Asnfs*2) and c.10184del p.(Glu3395Glyfs*32): Assigned Code BS3, Code Weight Strong, both supported by 'Mesman 2019 (PMID:29988080) - Complementation Yes; HDR 63' and 'Complementation Yes; HDR 67' respectively.
SupplementaryTables_V1.2_2024-11-18.xlsx, sheet ST4, BRCA2 exon-27 rows: c.9945del p.(Glu3316Asn-fs*2) 'No functional impact', c.10184del p.(Glu3395Gly-fs*32) 'No functional impact', c.10045A>G p.(Thr3349Ala) 'No functional impact'; the BRCA2 c.10043 position is not assayed.
SupplementaryTables_V1.2_2024-11-18.xlsx, sheet ST5: Mesman et al. 2019 (PMID:29988080) is a mouse embryonic stem cell complementation assay of full-length genomic human BRCA2 with follow-on homologous recombination assays, reported to capture impact at the mRNA level.
Appendices_V1.2_2024-11-18.docx, Appendix E: 'PS3 and BS3 codes are reserved for assigning variant impact on protein function' - splicing-assay data are not assigned PS3/BS3, so the SpliceAI delta of 0.205 is not usable as BS3 evidence here.
HUMU-40-1557-s001.xlsx (Parsons et al. 2019): no row for c.10043del, so no multifactorial functional cross-reference exists.
ClinVar: variant absent (no submission, no classification), so no curator-applied PS3/BS3 exists in any public record.
Still missing
Specifications_Table9_V1.2_2024-11-18.xlsx was searched for c.10043del, p.(Asn3348Ilefs*35), p.N3348Ifs*35, N3348 and 3348: no entry for this variant exists in the file.
SupplementaryTables_V1.2_2024-11-18.xlsx ST4 (Fxn assay results) was searched; no c.10043del row exists, so the Mesman et al. 2019 mESC complementation/HDR dataset that underpins the BS3 Strong codes for c.9945del and c.10184del contains no result for this variant.
The primary assay publication cited by Table 9 in this region (Mesman et al. 2019, PMID:29988080) was not retrieved for this case, so its supplementary variant list could not be checked for an alternative notation of c.10043del.
No published functional assay of c.10043del p.(Asn3348Ilefs*35) was identified in any of the five retrieved papers or in ClinVar (variant absent from ClinVar).
Curator decision needed: whether the exon 27 truncation BS3 Strong pattern in Table 9 (c.9945del, p.Lys3326Ter, c.10184del) should inform a BS3 assignment for this untested truncation, given that other truncations in the same exon carry PS3 Strong.
PS4VCEPNot assessed: ENIGMA PS4 requires a case-control OR >=4 with lower CI excluding 2.0, and no case-control data exist for c.10043del.
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Reasoning
The governing ENIGMA BRCA2 v1.2 PS4 rule requires a case-control comparison in which the variant's prevalence in affected individuals is significantly increased versus ethnicity- and country-matched controls, with p-value <=0.05 and OR >=4 with the lower confidence interval excluding 2.0. Operator check as written in the source: p <= 0.05, OR >= 4, lower CI > 2.0. No case-control study, odds ratio, relative risk or confidence interval exists for BRCA2 c.10043del: the variant is absent from ClinVar (no record), absent from gnomAD v2.1 and v4.1, and none of the five screened full-text papers (PMIDs 10570174, 11239455, 20878484, 22193408, 24312913) reports the variant, a carrier, or any case/control counts. With no OR value, no inequality can be written or evaluated, so the rule is unevaluable rather than contradicted. The VCEP also closes the alternative route: proband counting is explicitly disallowed ('Do not use Proband Counting as originally described'), and Appendix D shows that exon-level case-control/SIR evidence for protein-truncating variants was pre-computed and rolled into the exon-specific PM5 (PTC) weight rather than applied as a per-variant PS4 - so no PS4 weight for a truncating variant in this exon is derivable from the specification's own tables. PS4 is therefore not_assessed.
VCEP rule
The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Case-control studies; p-value ≤0.05 and OR ≥4 (lower confidence interval excludes 2.0). See Appendix F for details.
Evidence considered
ENIGMA BRCA2 v1.2 Specifications, PS4 row: case-control studies with p-value <=0.05 and OR >=4 (lower confidence interval excludes 2.0); case dataset must be ethnicity- and country-matched to controls; proband counting explicitly not to be used as originally described.
ENIGMA BRCA2 v1.2 Appendices Appendix F: PS4 accepted as strong pathogenic evidence only when OR >=4.0 and the confidence interval around the estimate does not include 2.0; Appendix D records that exon-level case-control OR/SIR analyses for PTC variants were used to set the exon-specific PM5 (PTC) weight, not a per-variant PS4.
ClinVar search for BRCA2 c.10043del returned no record (search_status: no_result, 'This variant is absent from ClinVar'), so no affected-individual observations are available there.
gnomAD v2.1 and v4.1 both report the variant absent, so there is no control frequency and no enrichment ratio can be computed.
All five ONCOKB-sourced full texts (PMID:10570174, PMID:11239455, PMID:20878484, PMID:22193408, PMID:24312913) were checked via pre-computed variant extraction: none names c.10043del or p.N3348Ifs*35 and none reports case/control counts.
Still missing
No case-control study, odds ratio or relative risk with confidence interval exists for BRCA2 c.10043del; none of the searched sources reports affected-versus-control counts for this variant.
Variant is absent from ClinVar and from gnomAD v2.1 and v4.1, so no affected/control frequency comparison can be constructed.
Proband counting cannot substitute: ENIGMA v1.2 states 'Do not use Proband Counting as originally described' for PS4_moderate.
PP1VCEPNot assessed: no co-segregation LR (PP1 needs >=2.08:1) exists for this variant in ClinVar, the five papers, or the ENIGMA/Parsons tables.
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Reasoning
Under ENIGMA BRCA2 v1.2, PP1 is a variable-weight code that may only be applied through a quantitative co-segregation analysis (Bayes score using COOL or CAL-Leiden), with supporting weight at co-segregation LR >=2.08:1, moderate >=4.3:1, strong >=18.7:1 and very strong >=350:1. No segregation data of any kind were available for this variant: ClinVar has no record, so no submitter-reported family or segregation information exists; the five retrieved publications (PMIDs 10570174, 11239455, 20878484, 22193408, 24312913) contain no mention of c.10043del or p.N3348Ifs*35, and the one family-based paper (20878484) describes a different BRCA2 frameshift (7525_7526insT, p.Ser2509fs) in an Italian pedigree. Direct greps of the ENIGMA supplementary tables (c.10043del, p.Asn3348IlefsTer35, N3348, 3348), the ENIGMA specifications document, and the Parsons 2019 multifactorial file (HUMU-40-1557-s001) found no row for this variant; the ENIGMA SupplementaryTables posterior-probability/workbook and Parsons SuppT1 are missense/splice-site keyed and contain no frameshift entry at codon 3348. Li et al. 2020 clinical-history LRs are explicitly not a segregation source (index restricts it to PP4/BP5). With zero meioses, no pedigree and no LR, the PP1 threshold cannot be evaluated, so the code is not_assessed rather than not_met.
VCEP rule
Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, as measured by a quantitative co-segregation analysis method. See Appendix I for details. ... Apply weight as per Bayes Score: ... PP1 - LR ≥2.08:1
Evidence considered
ENIGMA BRCA2 specification v1.2, Table 1, PP1_VariableWeight row: 'Co-segregation with disease in multiple affected family members ... Apply weight as per Bayes Score: PP1 - LR >=2.08:1 / PP1_Moderate - LR>=4.3:1 / PP1_Strong - LR>=18.7:1 / PP1_Very Strong - LR>=350:1'.
ENIGMA Appendices v1.2, Appendix I (Co-segregation Analysis (PP1 and BS4)), lines 2847-2861: preferred method is Bayesian analysis; recommend COOL or CAL-Leiden; combined LR thresholds converted to PP1/BS4 strength per Tavtigian et al 2018.
ClinVar search (BRCA2 AND c.10043del): no record found, therefore no submitter-reported segregation or family data.
Greps for 'c.10043del', 'p.Asn3348IlefsTer35', 'N3348', '3348' over all BRCA2 full-text reference files: only numeric coincidences (e.g. row counters, unrelated BRCA1/BRCA2 variants); no entry for this variant in SupplementaryTables_V1.2_2024-11-18.xlsx or HUMU-40-1557-s001.xlsx (Parsons SuppT1 header confirms sheets are keyed to HGVS nucleotide/protein of missense and splice variants).
PMID 20878484 is a family-based Italian study of a different variant (BRCA2 7525_7526insT, p.Ser2509fs) with three tested relatives; it does not report c.10043del and provides no segregations for this variant.
Still missing
No family pedigree, meiosis count, or quantitative co-segregation Bayes score (COOL/CAL-Leiden) is available for c.10043del.
The variant is absent from ClinVar, so no submitter-level family or segregation data exist.
Searched the ENIGMA specifications document, ENIGMA Appendices (Appendix I), SupplementaryTables_V1.2_2024-11-18.xlsx and Parsons et al. 2019 HUMU-40-1557-s001.xlsx for c.10043del / p.Asn3348IlefsTer35 / N3348: no entry for this variant in any of these files.
PP4VCEPNot assessed: ENIGMA PP4 requires a combined multifactorial LR >=2.08, and no clinical-history LR exists for c.10043del in the BRCA2 tables.
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Reasoning
The governing PP4 rule assigns weight only from a combined likelihood ratio derived from multifactorial likelihood clinical data, at supporting strength when LR >=2.08 (moderate >=4.3, strong >=18.7, very strong >=350); ENIGMA states that a combined LR of 1.00 to <2.08 is not informative and PP4 is then not applicable. Operator check as written for this criterion: met requires LR >= 2.08 (direction strengthens as the value goes UP). No combined LR exists for BRCA2 c.10043del. I searched every declared PP4/BP5 source: the Li et al. 2020 BRCA2 clinical-history LR table (2181 BRCA2 rows, keyed by HGVS_Nucleotide) has no entry for c.10043del, and the Parsons et al. 2019 multifactorial table and the ENIGMA Supplementary Tables likewise contain no row for c.10043del, p.Asn3348IlefsTer35 or p.N3348Ifs*35. Because no LR value is available, the comparison 'LR >= 2.08' cannot be instantiated and the criterion is unevaluable rather than refuted. Note that this variant's exon-level PTC evidence was consumed by the specification when it set the exon-specific PM5 (PTC) weight (Appendix D), so no variant-level clinical LR remains to apply here.
VCEP rule
Breast cancer is very common and has a high degree of genetic heterogeneity (caused by pathogenic variants in numerous genes). Use ONLY to capture combined LR towards pathogenicity, based on multifactorial likelihood clinical data. ... PP4 - LR ≥2.08:1
Evidence considered
ENIGMA BRCA2 v1.2 Specifications, PP4 row: use ONLY to capture combined LR towards pathogenicity from multifactorial likelihood clinical data; PP4 supporting LR >=2.08:1, moderate >=4.3:1, strong >=18.7:1, very strong >=350:1; combined LR 1.00 to <2.08 is not informative.
Li et al. 2020 (PMID:31853058) BRCA2 clinical-history LR table, searched for c.10043del, 10043, 3348, N3348Ifs and p.Asn3348IlefsTer35: no matching row (2181 BRCA2 rows present, keyed by HGVS_Nucleotide); e.g. the only entries containing the digit string '3348' are unrelated missense/del rows such as BRCA2 c.6201delC with LOG(LR)=-0.1074, LR=0.781.
Parsons et al. 2019 (PMID:31131967) multifactorial likelihood supplementary tables: no row for c.10043del or p.N3348Ifs*35; the table contains missense and in-frame/splicing entries only, none at codon 3348.
ENIGMA SupplementaryTables_V1.2: no row for c.10043del, p.(Asn3348IlefsTer35) or p.N3348Ifs*35 anywhere in the reference-set or functional-assay sheets.
ENIGMA Appendices Appendix D: exon-level PTC clinical/prevalence analyses (including a >=5 unique PTCs in >=5 CIMBA families criterion) were used to derive the per-exon PM5 (PTC) weight rather than a per-variant PP4.
Still missing
No combined multifactorial likelihood LR exists for BRCA2 c.10043del: the variant is not present in Li et al. 2020 BRCA2 clinical-history LR table, Parsons et al. 2019 multifactorial table (HUMU-40-1557-s001), or the ENIGMA Supplementary Tables, searched under c.10043del, p.Asn3348IlefsTer35 and p.N3348Ifs*35.
No co-segregation, co-occurrence, tumour pathology or family-history LR components are available for this variant in any declared VCEP source, so no combined LR can be assembled.
BS4VCEPNot assessed: no quantitative non-segregation LR (BS4 needs <=0.48:1) exists for this variant in ClinVar, the five papers, or the ENIGMA/Parsons tables.
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Reasoning
ENIGMA BRCA2 v1.2 allows BS4 only for demonstrated lack of segregation in affected family members, measured by a quantitative co-segregation analysis, with supporting weight at LR <=0.48:1, moderate <=0.23:1, strong <=0.05:1 and very strong <=0.00285:1. No family or segregation data exist for this variant: ClinVar has no record; none of the five retrieved publications name c.10043del or p.N3348Ifs*35; the one family study (PMID 20878484) concerns a different frameshift (7525_7526insT). Greps of the ENIGMA specifications, ENIGMA Appendices, ENIGMA SupplementaryTables and Parsons et al. 2019 HUMU-40-1557-s001 matrices found no row keyed to this variant, and none of those files contains a non-segregation LR applicable to it. With no informative meiosis and no computable LR, there is no basis to apply BS4 in either direction, so the code is not_assessed.
VCEP rule
Lack of segregation in affected members of a family, as measured by a quantitative co-segregation analysis method. See Appendix I for details. ... Apply weight as per Bayes Score: ... BS4_Supporting - LR ≤0.48:1
Evidence considered
ENIGMA BRCA2 specification v1.2, Table 1, BS4_VariableWeight row: 'Lack of segregation in affected members of a family. See Appendix I. Apply weight as per Bayes Score: BS4_Supporting - LR <=0.48 to >0.23:1 / BS4_Moderate - LR <=0.23:1 / BS4 - LR <=0.05:1 / BS4_Very strong - LR <=0.00285:1'.
ENIGMA Appendices v1.2, Appendix I, lines 2847-2861: BS4 weights follow Tavtigian et al 2018 LR thresholds; to apply Benign Very Strong, assess the possibility of bi-linearity to explain negative co-segregation.
ClinVar search for BRCA2 c.10043del returned no record, so no family-based non-segregation data are available.
Greps for 'c.10043del', 'p.Asn3348IlefsTer35', 'N3348', '3348' across the BRCA2 full-text reference files returned no entry for this variant in SupplementaryTables_V1.2_2024-11-18.xlsx or HUMU-40-1557-s001.xlsx.
Still missing
No family pedigree or quantitative co-segregation analysis is available to demonstrate lack of segregation for c.10043del.
The variant is absent from ClinVar and from the ENIGMA and Parsons supplementary variant tables, so no segregation LR (positive or negative) exists to convert into PP1/BS4 weight.
BP5VCEPNot assessed: ENIGMA BP5 requires a combined multifactorial LR <=0.48, and no such LR exists for c.10043del in the BRCA2 tables.
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Reasoning
The governing BP5 rule assigns weight only from a combined likelihood ratio derived from multifactorial likelihood clinical data, at supporting strength when LR <=0.48 (moderate <=0.23, strong <=0.05, very strong <=0.00285); ENIGMA states a combined LR of >0.48-1.00 is not informative and BP5 is then not applicable. Operator check as written for this criterion, which runs the opposite way from PP4: met requires LR <= 0.48 (direction strengthens as the value goes DOWN), i.e. for any candidate value the test is 'LR <= 0.48?'. No combined LR exists for BRCA2 c.10043del, so no value can be substituted into that inequality. All declared PP4/BP5 sources were searched for c.10043del, p.Asn3348IlefsTer35 and p.N3348Ifs*35 - the Li et al. 2020 BRCA2 clinical-history LR table (2181 BRCA2 rows), the Parsons et al. 2019 multifactorial table, and the ENIGMA Supplementary Tables - with no entry for this variant. A related but non-qualifying observation: the ONCOKB-sourced full text PMID:10570174 notes the polymorphic Lys3326ter (removing 92 C-terminal residues) occurs 'apparently without consequence' and that a reported Glu3342ter change, downstream of it, was not found in an affected breast cancer family and was considered unlikely to be cancer associated; that is a narrative opinion about neighbouring truncations, not a calibrated variant-specific LR, and it cannot be converted into a BP5 weight under this rule. BP5 is therefore not_assessed.
VCEP rule
Use ONLY to capture combined LR against pathogenicity, based on multifactorial likelihood clinical data. ... BP5 - LR ≤0.48:1
Evidence considered
ENIGMA BRCA2 v1.2 Specifications, BP5 row: use ONLY to capture combined LR against pathogenicity from multifactorial likelihood clinical data; BP5 supporting LR <=0.48:1, moderate <=0.23:1, strong <=0.05:1, very strong <=0.00285:1; combined LR >0.48-1.00 is not informative; not applicable for co-observation with pathogenic variants in other breast-ovarian cancer genes.
Li et al. 2020 (PMID:31853058) BRCA2 clinical-history LR table searched for c.10043del / 10043 / 3348 / p.N3348Ifs*35: no matching row, so no variant-specific clinical-history LR exists to compare against the <=0.48 threshold.
Parsons et al. 2019 (PMID:31131967) multifactorial table and ENIGMA SupplementaryTables_V1.2 searched with the same notations: no row for this variant, no posterior probability of pathogenicity, no IARC class.
PMID:10570174 full text, Results first paragraph: the Lys3326ter polymorphism 'removes 92 residues from the C terminus, apparently without consequence' and a reported Glu3342ter variant 'was not identified as part of an affected breast cancer family and, because it occurs downstream of the Lys3326ter polymorphism, it is unlikely to be cancer associated' - narrative context only, no LR.
Still missing
No combined multifactorial likelihood LR exists for BRCA2 c.10043del: absent from Li et al. 2020 BRCA2 clinical-history LR table, Parsons et al. 2019 multifactorial table (HUMU-40-1557-s001) and ENIGMA SupplementaryTables_V1.2, searched under c.10043del, 10043, 3348, p.Asn3348IlefsTer35 and p.N3348Ifs*35.
No co-segregation, co-occurrence, tumour pathology or family-history LR components are recorded for this variant, so the combined LR required by BP5 cannot be assembled from published data.
PMID:10570174 reports that a truncation downstream of the polymorphic Lys3326ter (Glu3342ter, codon 3342 - 6 residues from this variant's first changed residue) was not found in an affected breast cancer family and was considered unlikely to be cancer associated; this narrative observation is near the variant but is not a calibrated LR and does not satisfy BP5, and a reviewer may wish to note it.
Not applicable · 17· recorded as not applicableShow reasons ▾Hide reasons ▴PVS1N/ANot applicable: BRCA2 Table 4 assigns PVS1_N/A to exon 27 termination codons after p.E3309, and this frameshift stops near codon 3382.
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Reasoning
NM_000059.3:c.10043del (NP_000050.2:p.(Asn3348IlefsTer35)) is an exonic single-nucleotide deletion in BRCA2 exon 27 (exon 27 spans c.9649-c.10254 / p.3217-p.3418). It shifts the reading frame from codon 3348 and introduces a new termination codon 35 residues into the new frame (approximately codon 3382), still within exon 27. Loss of function is an established BRCA2 disease mechanism, but this VCEP does not apply the generic SVI PVS1 tree to protein termination codon (PTC) variants: Specifications Table 1 states that for predicted PTC variants the exon-specific weights derived from the PM5 (PTC) code are applied, and Specifications Table 4 supplies those weights per exon. The exon 27 rows of Table 4 split the exon at codons 3309/3310: 'PVS1' with 'PTC<p.T3310', and 'PVS1_N/A' with 'PTC>p.E3309'. The frameshift begins at codon 3348 and the new termination codon is at about codon 3382, so both the variant position and the termination position lie downstream of p.E3309; the governing pre-assigned code for this variant is therefore PVS1_N/A. Appendix D gives the justification for that boundary: there is no clinical information supporting pathogenicity of nonsense or frameshift variants between positions 3309 and 3325, and termination from p.3309 onward is considered highly unlikely to be clinically important as a high-risk variant, based on mammalian functional assay results that are wild-type-like for terminations from p.3309 compared with the p.Lys3326Ter polymorphism. The one stated caveat concerns frameshifts that begin before position 3309 but end after 3326; here the frameshift begins at 3348, after the boundary, so that caveat does not apply. Loss of NLS3 (residues 3381-3385, per PMID:10570174) does not alter the outcome because the exon 27 weighting already covers termininations from p.3309 onward. The variant is exonic and not in a canonical donor/acceptor +/-1,2 position, so the splice-site rows of Table 4 (and PVS1 (RNA), which requires mRNA assay data) do not apply; SpliceAI max delta 0.205 was noted but no mRNA data exist for this variant and it does not re-route the exon-27 PTC weight. Because the pre-assigned code is PVS1_N/A, the criterion is not applicable rather than met or not met, and strength is left null: no downgrade tier was invoked, no PVS1 evidence is contributed at any strength.
VCEP rule
For predicted protein termination codon (PTC) variants, apply with exon-specific weights derived for the PM5 (PTC) code (see description below). ... There is currently no publicly available clinical information to support pathogenicity of nonsense or frameshift variants located between positions 3309 and 3325. ... That is, a missense or nonsense variant predicted to modify or truncate protein sequence at residues from position p.3309 onwards is considered highly unlikely to be clinically important as a high-risk variant.
Evidence considered
Variant facts: NM_000059.3:c.10043del, NP_000050.2:p.(Asn3348IlefsTer35), exon 27, frameshift (not a canonical splice-consensus change); the frameshift starts at codon 3348 and the new termination codon lies ~35 residues into the new frame (approx. codon 3382), still inside exon 27 (c.9649-c.10254, p.3217-p.3418).
Specifications Table 4, sheet 'Table 4 - Annotated Exons', BRCA2 E27 rows: 'PVS1 | PTC<p.T3310 | PM5_Strong (PTC)' and 'PVS1_N/A | PTC>p.E3309 | PM5_N/A' - i.e. terminations at or after p.3310 in exon 27 carry no PVS1 weight.
Specifications v1.2, Table 1, PVS1 entry: 'For predicted protein termination codon (PTC) variants, apply with exon-specific weights derived for the PM5 (PTC) code (see description below).' - the weight is fixed by exon via Table 4 rather than by the generic SVI PVS1 decision tree.
Appendix C, BRCA2 C-terminal domain table, exon 27 discussion: 'There is currently no publicly available clinical information to support pathogenicity of nonsense or frameshift variants located between positions 3309 and 3325.' and 'That is, a missense or nonsense variant predicted to modify or truncate protein sequence at residues from position p.3309 onwards is considered highly unlikely to be clinically important as a high-risk variant.'
Appendix C caveat, which does not apply here: 'the impact of variants that cause a frameshift before position 3309 but that may end after 3326 is not certain' - this frameshift begins at codon 3348, i.e. after 3309.
PMID:10570174 (Chen et al.) supports the same C-terminal boundary: the p.Lys3326Ter polymorphism removes 92 C-terminal residues 'apparently without consequence', and a further-downstream Glu3342ter was judged unlikely to be cancer associated because it lies downstream of p.Lys3326Ter; the paper maps NLS3 to residues 3381-3385, but ENIGMA's exon-27 weighting already covers terminations from p.3309 onward.
Table 4 exon 27 splice-site entries (PVS1 (RNA) for c.9649-2A variants, PVS1 for c.9649-1G variants) are not applicable: c.10043del is exonic and distant from the canonical donor/acceptor +/-1,2 positions, and no mRNA assay data are available.
Searched all four named VCEP files for c.10043del, p.Asn3348IlefsTer35 and p.N3348Ifs*35; no variant-specific row or mention was found in any of them.
Still missing
No variant-level entry for c.10043del / p.Asn3348IlefsTer35 exists in Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx, Specifications_Table4_V1.2_2024-11-18.xlsx or PMID_22505045_Houdayer_2012_HumMutat.pdf (all searched under c.10043del, p.Asn3348IlefsTer35 and p.N3348Ifs*35); Table 4 is keyed by exon, not by variant, so the PVS1_N/A assignment was derived from the exon 27 rows rather than from a variant-specific row.
No mRNA/minigene splicing data exist for this variant, but the governing exon-27 PTC weight does not require them.
PS1N/ANot applicable: PS1 is restricted to missense substitutions or splice-equivalent variants, and this frameshift has no amino-acid-change comparator at residue 3348.
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Reasoning
NM_000059.3:c.10043del is a single-nucleotide deletion in BRCA2 exon 27 producing the frameshift NP_000050.2:p.(Asn3348IlefsTer35) with a new termination codon at p.Ter3382. The ENIGMA v1.2 PS1 rule is written only for two situations: predicted missense substitutions that lead to the same amino-acid change as a previously classified (likely) pathogenic variant acting via protein change, and exonic/intronic variants with the same predicted splicing impact as a previously classified (likely) pathogenic variant. A frameshift creates no amino-acid substitution at the residue that could match a reference missense variant, so the first limb cannot be evaluated; no predicted splice event of the class contemplated by PS1 was established for this variant (SpliceAI max delta 0.205 arises from a weak alternative AG acceptor gain 33 bp upstream, and there is no previously classified variant at this position whose predicted event it could match). In addition, no comparator exists at all: the variant is absent from ClinVar (no submissions, no review status), and the VCEP tables (SupplementaryTables ST1-ST16, Table 4, Table 9) contain no row for c.10043del or p.Asn3348IlefsTer35. PS1 therefore does not apply to this variant type.
VCEP rule
PS1 may be applied for a variant predicted to lead to the same missense substitution as a known pathogenic/likely pathogenic variant that is due to a different nucleotide change. ... Apply PS1, for predicted missense substitutions, where a previously classified pathogenic variant is considered to act via protein change (no confirmed or predicted effect on mRNA splicing (SpliceAI≤0.1)).
Evidence considered
Specifications_V1.2_2024-11-18, Table 1 (strong criteria), PS1: 'Apply PS1, for predicted missense substitutions, where a previously classified pathogenic variant is considered to act via protein change (no confirmed or predicted effect on mRNA splicing (SpliceAI<=0.1)).' and 'Apply PS1_Variable Weight, for exonic and intronic variants with same predicted impact on splicing, as a previously classified (likely) pathogenic variant.'
Specifications_V1.2_2024-11-18, PS1 footnote: 'For application of PS1 for splicing predictions, see Table 5. The predicted event of the VUA must precisely match the predicted event of the known (likely) pathogenic variant'.
Variant facts: c.10043del in exon 27 (Table 4 exon 27 coding span c.9649-c.10257), protein NP_000050.2:p.(Asn3348IlefsTer35), predicted product 3382 aa (Mutalyzer), i.e. a frameshift, not a missense substitution.
ClinVar search for BRCA2 c.10043del returned no record ('This variant is absent from ClinVar'), so no classified reference variant exists at this codon.
SpliceAI Lookup for NM_000059.3:c.10043del: DS_AG 0.205, DS_AL 0.022, DS_DG 0.0, DS_DL 0.0 (max delta 0.205); no variant with a matching predicted splice event has been classified at this position.
Still missing
Searched SupplementaryTables_V1.2_2024-11-18.xlsx (sheets ST1-ST16), Specifications_Table4_V1.2_2024-11-18.xlsx, Specifications_Table9_V1.2_2024-11-18.xlsx and the Specifications/Appendices text for 'c.10043del', 'p.Asn3348IlefsTer35', 'N3348' and '3348': no entry for this variant in any of them.
No previously classified (likely) pathogenic BRCA2 variant exists at codon 3348 or at this nucleotide position: the variant has no ClinVar record.
PS2VCEPNot applicable: the ENIGMA BRCA2 v1.2 specification explicitly marks PS2 'Do not use' because BRCA1/2 cancers are common, so no de novo weight can be applied.
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Reasoning
The ENIGMA BRCA2 v1.2 specification explicitly disables PS2 for this gene: Table 1 lists the ACMG/AMP description ('De novo (paternity confirmed) in a patient with the disease and no family history') followed by the instruction 'Do not use', justified because BRCA1/2-related cancers occur relatively commonly and there is no information to calibrate the predictive capacity of de novo occurrences. The CSPEC criteria entry for PS2 likewise carries applicability 'Not Applicable'. PS2 therefore cannot be met, and no de novo evidence was sought or applied; the variant (NM_000059.3:c.10043del, p.Asn3348IlefsTer35, exon 27 frameshift) also has no reported de novo occurrence or trio data in this case.
VCEP rule
De novo (paternity confirmed) in a patient with the disease and no family history. ... Do not use ... BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences.
Evidence considered
ENIGMA BRCA2 specification v1.2 (Specifications_V1.2_2024-11-18.docx.txt), Table 1, PS2 row, lines 73-76: 'PS2 / De novo (paternity confirmed) in a patient with the disease and no family history. / Do not use / BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences.'
CSPEC criteria payload for this gene (vcep_materials/prefetch): label PS2, applicability 'Not Applicable'.
Variant identity used throughout: NM_000059.3:c.10043del (NP_000050.2:p.Asn3348IlefsTer35), exon 27, frameshift, per pvs1_variant_assessment.
Still missing
No proband trio/parental testing data exist for this variant, but this is irrelevant because the VCEP disables PS2 outright.
PM1VCEPNot applicable: ENIGMA v1.2 lists PM1 as 'Do not use' (its content folded into PP3/BP4), and residue 3348 lies outside both declared domains (PALB2 aa 10-40, DBD aa 2481-3186).
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Reasoning
The ENIGMA BRCA2 v1.2 specification explicitly disables PM1: Table 1 lists the generic PM1 definition ('Located in a mutational hot spot and/or critical and well-established functional domain') against the instruction 'Do not use', with the justification 'Considered as component of bioinformatic analysis (PP3/BP4)', and the CSPEC entry for PM1 is recorded as 'Not Applicable'. Domain/hotspot evidence is therefore not assignable as a standalone moderate pathogenic code under this VCEP. For completeness the residue was checked against the VCEP's own domain definitions anyway: the specification defines (potentially) clinically important functional domains as the BRCA2 PALB2 binding domain aa 10-40 and the DNA binding domain aa 2481-3186, and residue Asn3348 lies outside both (Table 4 annotates the DBD only through p.3168_3186 in exon 26, with no domain annotation for the exon 27 coding span p.3217_3418). vcep_materials.json has no domain_tables entries for BRCA2, so no separate VCEP domain table could override this. The variant also does not lie in a statistical hotspot (Cancer Hotspots returned no row for BRCA2 N3348).
VCEP rule
Located in a mutational hot spot and/or critical and well-established functional domain. ... Do not use ... Considered as component of bioinformatic analysis (PP3/BP4).
Evidence considered
Specifications_V1.2_2024-11-18, Table 1 (moderate criteria), PM1 row: 'Located in a mutational hot spot and/or critical and well-established functional domain.' / 'Do not use' / 'Considered as component of bioinformatic analysis (PP3/BP4).'
CSPEC criteria list for this gene: PM1 recorded with applicability 'Not Applicable'.
Specifications_V1.2_2024-11-18 (and CSPEC BP4/BP7 text): '(potentially) clinically important functional domains are defined as: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186' - Asn3348 is outside both.
Specifications_Table4_V1.2_2024-11-18, BRCA2 rows: domain column annotates 'DBD p.3168_3186' as the last coding DBD segment (exon 26); exon 27 rows (c.9649-c.10257, p.3217-p.3418) carry no domain annotation.
Cancer Hotspots search for 'BRCA2 N3348' returned no hotspot row.
PMID:10570174 (Yang et al. 2001) places the C-terminal functional boundary at the Lys3326ter polymorphism (removes 92 residues 'apparently without consequence') and notes Glu3342ter downstream of it is 'unlikely to be cancer associated', i.e. residue 3348 is not in a critical C-terminal domain - consistent with, though not required for, the VCEP's 'Do not use' instruction.
Still missing
vcep_materials.json domain_tables contains no entry for BRCA2, so the VCEP domain list could not be taken from an extracted domain table; the boundaries quoted are the specification's own definitions (PALB2 binding aa 10-40; DNA binding aa 2481-3186).
No variant-level PM1/hotspot entry exists for c.10043del / p.Asn3348IlefsTer35 in any of the named VCEP files (searched SupplementaryTables_V1.2_2024-11-18.xlsx, Specifications_Table4_V1.2_2024-11-18.xlsx, Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx).
PM2N/ANot applicable: PM2 is excluded for insertion/deletion variants, and this is a 1-bp frameshift deletion.
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Reasoning
The ENIGMA BRCA2 v1.2 PM2_Supporting rule is explicitly restricted in variant type: the specification's application instructions state 'Do not apply for insertion, deletion or delins variants', and Appendix G gives the reason - low recall estimates for this variant class (PMID: 33600021) - while allowing only larger insertions/deletions/indels (>50 bp) to be eligible. BRCA2 c.10043del is a single-base deletion producing a frameshift (p.Asn3348IlefsTer35), i.e. a 1-bp deletion squarely inside the excluded class, so PM2 cannot be applied to this variant at any strength regardless of the fact that it is absent from gnomAD v2.1, gnomAD v4.1 and the non-cancer subsets. The criterion is therefore not applicable rather than met; noting for completeness that even the read-depth guard (region average depth >=25) was not verifiable and the variant being absent would otherwise have satisfied the 'absent from controls' wording, the VCEP's variant-type exclusion governs. No alternative weighting of PM2 (for example moderate strength) is permitted by this VCEP, whose only specified strength is Supporting.
VCEP 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. ... We do not recommend use of PM2_Supporting for insertion, deletion or insertion-deletion variants due to the low recall estimates for variants of this type (PMID: 33600021).
ENIGMA BRCA2 specification v1.2, Table 1 PM2_Supporting row: 'Do not apply for insertion, deletion or delins variants. Do not apply if read depth <25 at region around the variant'; preferred datasets gnomAD v2.1 non-cancer exomes and gnomAD v3.1 non-cancer genomes.
ENIGMA v1.2 Appendix G: 'We do not recommend use of PM2_Supporting for insertion, deletion or insertion-deletion variants due to the low recall estimates for variants of this type (PMID: 33600021). Larger insertions, deletions and indels (>50bp) may be more reliably called and eligible for PM2_Supporting when absent from an appropriate dataset.'
gnomAD v2.1, gnomAD v4.1 and the non-cancer subsets all report the variant absent - recorded but not scored, because the criterion is excluded for this variant type.
Still missing
Regional read depth at this position (>=25 required by the VCEP) was not retrieved; immaterial because PM2 is excluded on variant type.
ENIGMA v1.2 SupplementaryTables_V1.2_2024-11-18.xlsx, Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx and HUMU-40-1557-s001.xlsx were searched for 'c.10043del', 'p.Asn3348IlefsTer35' and 'p.N3348Ifs*35'; no pre-assigned PM2 code or frequency row for this variant exists in any VCEP file.
PM4VCEPNot applicable: the VCEP does not use PM4 for BRCA2, and this frameshift is neither an in-frame indel nor a stop-loss variant.
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Reasoning
PM4 addresses protein length changes due to in-frame deletions/insertions in a non-repeat region, or stop-loss variants. The ENIGMA BRCA2 specification v1.2 does not use this code: Table 1 marks PM4 'Do not use', with the specification note that such length changes are considered as a component of the bioinformatic analysis under PP3/BP4, and the CSPEC entry records applicability 'Not applicable' at every strength tier (stand-alone through supporting). There is a second, independent reason the criterion cannot apply here: c.10043del is a frameshift that introduces a premature termination codon at approximately codon 3382, so it is neither an in-frame deletion/insertion in a non-repeat region nor a stop-loss variant - both variant classes named in the rule. No PM4 evidence is therefore assigned, and no strength is recorded.
VCEP rule
Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. ... Do not use ... Considered as component of bioinformatic analysis (PP3/BP4).
Evidence considered
Specifications v1.2, Table 1, PM4 row: specification 'Do not use', with the note 'Considered as component of bioinformatic analysis (PP3/BP4).'; the CSPEC entry carries applicability 'Not applicable' for the criterion and for every strength tier, so no PM4 weight exists in this framework.
Variant facts: NM_000059.3:c.10043del gives p.(Asn3348IlefsTer35), a frameshift with a premature termination codon (approx. codon 3382), not an in-frame deletion/insertion and not a stop-loss variant, so it also falls outside the variant types PM4 names.
Searched Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx and Specifications_Table4_V1.2_2024-11-18.xlsx for c.10043del, p.Asn3348IlefsTer35 and p.N3348Ifs*35; no variant-level PM4 entry exists.
PM5N/ANot applicable: Table 4 pre-assigns PM5_N/A for BRCA2 exon 27 PTCs terminating after p.E3309, and this frameshift's termination codon is p.Ter3382.
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Reasoning
The VCEP repurposes PM5 as PM5 (PTC): 'Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before', with the weight set per exon by Table 4 and Appendix D. c.10043del lies in BRCA2 exon 27 (Table 4: c.9649-c.10257, p.3217-p.3418) and produces p.(Asn3348IlefsTer35), whose new termination codon is at p.Ter3382 (predicted product 3382 aa). Table 4 gives two BRCA2 exon 27 PTC rows split at the NMD-escape boundary: 'PVS1 / PTC<p.T3310 / PM5_Strong (PTC)' and 'PVS1_N/A / PTC>p.E3309 / PM5_N/A'. Supplementary Table 1 (ST1 PM5_PTC weights) independently confirms the boundary, listing BRCA2 exon 27 (c.9649-c.10257, p.3217-3419) with 'Escape NMD Boundary (p.)' = 3309. Because the termination codon here is p.Ter3382, i.e. beyond p.Glu3309, the pre-assigned governing code for this position is PM5_N/A - PM5 (PTC) is excluded. This is corroborated by Appendix D, which restricts the code to variants that meet PVS1 ('PM5 (PTC) code can only be applied to germline variants that meet PVS1 codes, namely nonsense and frame-shift changes'), and the same exon 27 row that carries PM5_N/A carries PVS1_N/A. No PM5 weight applies to this variant.
VCEP rule
Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before ... Use to justify additional weight for PTC variants annotated as PVS1.
SupplementaryTables_V1.2_2024-11-18, sheet 'ST1 PM5_PTC weights', row BRCA2 exon 27: c.9649-c.10257, p.3217-3419, Escape NMD Boundary (p.) = 3309, PM5_PTC code strength = Strong for truncations within that exon.
Specifications_V1.2_2024-11-18, Table 1, PM5_VariableWeight (PTC): 'Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before' / 'Use to justify additional weight for PTC variants annotated as PVS1. Only applied to genomic PTC changes (not splicing).'
Appendices_V1.2_2024-11-18, Appendix D: 'PM5 (PTC) code can only be applied to germline variants that meet PVS1 codes, namely nonsense and frame-shift changes, including large deletions and tandem duplications. Code weight is determined by the exon in which the nucleotide change occurs.'
Variant protein consequence: NP_000050.2:p.(Asn3348IlefsTer35), predicted product 3382 aa, so the termination codon (p.Ter3382) lies downstream of both p.Glu3309 (Table 4) and the Lys3326 boundary discussed in PMID:10570174.
Still missing
No variant-level row for c.10043del / p.Asn3348IlefsTer35 exists in SupplementaryTables_V1.2_2024-11-18.xlsx (ST1-ST16) or Specifications_Table4_V1.2_2024-11-18.xlsx; the exon-27 rows and the p.3309 NMD-escape boundary were used instead.
The specification does not give a per-nucleotide lookup for PTC position beyond the exon 27 split rows; the boundary value (p.3309) was taken from Specifications_Table4 column 'Vt Allele' and Supplementary Table 1 column 'Escape NMD Boundary (p.)', which agree.
PM6VCEPNot applicable: the ENIGMA BRCA2 v1.2 specification marks PM6 'Do not use', so unconfirmed de novo evidence cannot be scored for this variant.
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Reasoning
The ENIGMA BRCA2 v1.2 specification explicitly disables PM6: Table 1 gives the ACMG/AMP description ('Confirmed de novo without confirmation of paternity and maternity') followed by 'Do not use', for the same stated reason that BRCA1/2-related cancers are relatively common and de novo events cannot be calibrated. The CSPEC entry for PM6 carries applicability 'Not Applicable'. No assumption-based de novo evidence is therefore admissible for this variant under the governing framework, regardless of any parental-testing information.
VCEP rule
Confirmed de novo without confirmation of paternity and maternity. ... Do not use ... BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences.
Evidence considered
ENIGMA BRCA2 specification v1.2 (Specifications_V1.2_2024-11-18.docx.txt), Table 1, PM6 row, lines 169-172: 'PM6 / Confirmed de novo without confirmation of paternity and maternity. / Do not use / BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences.'
CSPEC criteria payload for this gene: label PM6, applicability 'Not Applicable'.
Still missing
No parental testing records exist for this variant, but the VCEP disables PM6, so unconfirmed de novo status is not evaluable as evidence.
PP2VCEPNot applicable: ENIGMA v1.2 marks PP2 'Do not use' (BRCA2 has a high frequency of benign missense variants), and this variant is a frameshift, not missense.
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Reasoning
The ENIGMA BRCA2 v1.2 specification disables PP2 outright. Table 1 lists the generic PP2 definition ('Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease') against 'Do not use', justified by 'High frequency of benign missense variants', and the CSPEC entry for PP2 is recorded as 'Not Applicable'. Independently, the criterion would not apply to this variant in any case because c.10043del is a frameshift, not a missense variant. No PP2 weight is assignable.
VCEP rule
Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease. ... Do not use ... High frequency of benign missense variants.
Evidence considered
Specifications_V1.2_2024-11-18, Table 1 (supporting criteria), PP2 row: 'Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.' / 'Do not use' / 'High frequency of benign missense variants.'
CSPEC criteria list for this gene: PP2 recorded with applicability 'Not Applicable'.
Variant type: NM_000059.3:c.10043del is a frameshift (p.Asn3348IlefsTer35), not a missense change.
PP3N/ANot applicable: PP3 covers only missense/in-frame, silent or intronic variants, and c.10043del is a frameshift (p.N3348Ifs*35) assessed under PVS1.
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Reasoning
NM_000059.3:c.10043del (NC_000013.10:g.32972693del) is a frameshift in BRCA2 exon 27, NP_000050.2:p.(Asn3348IlefsTer35); it is not a missense, in-frame indel, silent or intronic variant and is not at a canonical donor/acceptor dinucleotide. The ENIGMA v1.2 PP3 rule names exactly two scored situations: (i) missense or in-frame insertion/deletion/delins inside a (potentially) clinically important functional domain (BRCA2 PALB2 binding aa 10-40; DNA binding aa 2481-3186) with BayesDel no-AF score >=0.30, and (ii) predicted splicing with SpliceAI >=0.2 for silent, missense/in-frame (irrespective of domain location) or intronic variants outside the donor/acceptor 1,2 positions. A frameshift falls in neither situation, so the criterion is out of scope before any predictor score is considered. The SpliceAI result that exists for this variant (max delta 0.205; DS_AG 0.205 at DP_AG +33 with DS_AL 0.022, DS_DG 0.0, DS_DL 0.0; Pangolin SG 0.126, SL -0.049) therefore does not become PP3 evidence: the ENIGMA splice-prediction arm is not written for truncating variants, and for a frameshift the splice prediction bears on PVS1 (whether the truncating effect might be rescued/redirected), which is adjudicated elsewhere. BayesDel is a missense/in-frame predictor and no score is available for this variant in any case. Independently, the ENIGMA Specifications Table 1 PP3 row carries the stipulation 'Do not apply PP3 if PVS1 is met at any strength', and this variant is the classic PVS1-type null (frameshift) case, so applying PP3 here would additionally be barred by that stipulation.
VCEP rule
Apply PP3 for missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain and predicted impact via protein change (BayesDel no-AF score ≥0.30). ... Apply PP3 for predicted splicing (SpliceAI ≥0.2) for silent, missense/in-frame (irrespective of location in clinically important functional domain) and for intronic variants outside of donor and acceptor 1,2 sites.
Evidence considered
Variant identity from case evidence: NM_000059.3:c.10043del, NP_000050.2:p.(Asn3348IlefsTer35) / p.(N3348Ifs*35), BRCA2 exon 27, consequence frameshift, canonical splice consensus = false.
ENIGMA BRCA2 specification v1.2, Table 1 (Specifications_V1.2_2024-11-18.docx, PP3 row): PP3 is limited to missense/in-frame indels inside a functional domain with BayesDel predicted impact, and to SpliceAI >=0.2 for silent, missense/in-frame or intronic variants outside donor/acceptor +/-1,2; the row also states 'Do not apply PP3 if PVS1 is met at any strength'.
Appendix J, Table 16 (Appendices_V1.2_2024-11-18.docx): 'Apply PP3 for missense or in-frame insertion-deletion inside a (potentially) clinically important functional domain and predicted impact via protein change (BayesDel >=0.28 for BRCA1, >=0.30 for BRCA2). Apply PP3 for predicted splicing (SpliceAI >=0.2) for silent, missense/in-frame (irrespective of location in clinically important functional domain) and for intronic variants outside of the donor and acceptor +/-1,2 positions.' No frameshift or nonsense row is listed; Appendix J states some codes are 'not applicable since they are captured by other codes/criteria'.
Appendix J states the SpliceAI cutpoints were calibrated only 'For variants outside of the donor/acceptor +/-1,2 positions' (<=0.1 BP4; >0.1 and <0.2 not informative; >=0.2 PP3 for predicted splicing impact), i.e. the splice arm is calibrated on the non-truncating/intronic classes named in the rule.
Still missing
No variant-level entry for c.10043del / p.(Asn3348IlefsTer35) / p.N3348Ifs*35 was found in SupplementaryTables_V1.2_2024-11-18.xlsx (searched by coding HGVS, protein change 'N3348'/'3348' and genomic coordinate g.32972693; the neighbouring rows c.10045A>G p.(Thr3349Ala) and c.10184del p.(Glu3395Gly-fs*32) are present but this variant is not), in Specifications_V1.2_2024-11-18.docx, in Appendices_V1.2_2024-11-18.docx, or in PMID_22505045_Houdayer_2012_HumMutat.pdf.
No BayesDel no-AF score exists for this variant in the case evidence (null), consistent with BayesDel being a missense/in-frame predictor; this does not affect the call, which rests on variant-type scope.
PP5VCEPNot applicable: ENIGMA BRCA2 v1.2 specifies PP5 must not be used, and the variant has no ClinVar record in any case.
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Reasoning
The governing ENIGMA BRCA2 v1.2 specification removes this code outright: the PP5 row of the specification table states 'Do not use', and the CSPEC criteria entry records PP5 with applicability 'Not Applicable'. The rule's own applicability therefore excludes it, which is the only basis on which not_applicable may be assigned here. For completeness, the factual precondition is also unmet independently: a ClinVar search for BRCA2 c.10043del returned no record, so there is no reputable-source classification of this variant to weigh even if the code were permitted. No strength is assigned.
VCEP rule
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
Evidence considered
ENIGMA BRCA2 v1.2 CSPEC criteria entry for PP5: applicability 'Not Applicable for this VCEP'; every strength level carries the rule 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.'
ENIGMA BRCA2 v1.2 Specifications table, PP5 row: the criterion description is followed by the instruction 'Do not use'.
ClinVar search for BRCA2 c.10043del returned no record ('This variant is absent from ClinVar'), so no reputable-source pathogenic assertion exists for this variant.
BP1N/ANot applicable: BP1_Strong covers only silent, missense or in-frame variants with SpliceAI <=0.1, and this is a frameshift with SpliceAI max delta 0.205.
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Reasoning
The ENIGMA BP1 rule is a location-plus-prediction benign code limited by variant type: '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)'. c.10043del is a frameshift deletion (p.Asn3348IlefsTer35), which is not among the variant types the rule names, so BP1 is outside its scope for this variant. Two further checks point the same way: the splice condition would independently fail (SpliceAI max delta 0.205 for this variant exceeds the <=0.1 requirement), and although residue 3348 is outside the VCEP's declared (potentially) clinically important domains (PALB2 binding aa 10-40; DNA binding aa 2481-3186), that location fact alone cannot generate BP1_Strong for a frameshift. No BP1 weight is assigned.
VCEP rule
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).
Evidence considered
Specifications_V1.2_2024-11-18, Table 1 (supporting criteria), BP1_Strong: '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). Missense prediction not applicable.'
CSPEC BP1 rule as captured for this gene: same text, with domains defined as 'BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186'.
SpliceAI Lookup for NM_000059.3:c.10043del: DS_AG 0.205, DS_AL 0.022, DS_DG 0.0, DS_DL 0.0; max delta score 0.205, above the <=0.1 splicing condition of the BP1 rule.
Variant type: single-nucleotide deletion causing a frameshift (p.Asn3348IlefsTer35), not a silent, missense or in-frame indel.
Still missing
SpliceAI delta scores were taken from the collected SpliceAI Lookup result for this variant (DS_AG 0.205); the VCEP does not define a BP1 path for frameshift variants, so no further threshold could be applied.
BP2VCEPNot applicable: the ENIGMA BRCA2 v1.2 specification marks BP2 'Do not use', delegating allelic-phase evidence to the BS2 point system.
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Reasoning
The ENIGMA BRCA2 v1.2 specification explicitly prohibits BP2 for BRCA2. Table 1 lists the BP2 row ('Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern') with the instruction 'Do not use' and the qualification 'Applied only in the context of BS2'; the CSPEC entry for this gene likewise records BP2 applicability as 'Not Applicable'. Allelic-phase evidence in BRCA2 is therefore handled entirely inside the BS2 point system (Table 8: VUA in trans with a P/LP, homozygote, or phase-unknown co-occurrence, scored by proband age at cancer onset), not through BP2. Because the governing rule bars the code outright, BP2 is not applicable to this variant irrespective of whether cis/trans data exist — and none was found here anyway (the variant is absent from ClinVar and unnamed in the retrieved literature).
VCEP rule
Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern ... Do not use Applied only in the context of BS2.
Evidence considered
ENIGMA BRCA2 v1.2, Table 1 supporting-criteria block, BP2 row: 'Do not use / Applied only in the context of BS2.'
CSPEC ENIGMA BRCA2 v1.2 criteria list: BP2 recorded with applicability 'Not Applicable'.
ENIGMA BRCA2 v1.2 Table 8 (BS2-related points) is where co-occurrent P/LP variant information for this gene is scored, confirming that cis/trans observations are not assessed under BP2.
No cis/trans or co-occurrence data for c.10043del was available in any case source (absent from ClinVar; not named in PMID:10570174, PMID:11239455, PMID:20878484, PMID:22193408, PMID:24312913), but this does not affect the verdict because the VCEP forbids use of the code.
BP3VCEPNot applicable: the VCEP does not use BP3 for BRCA2, and a frameshift is not an in-frame indel in a repetitive region.
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Reasoning
BP3 addresses in-frame deletions/insertions in a repetitive region without a known function. The ENIGMA BRCA2 specification v1.2 does not use this code: Table 1 marks BP3 'Do not use', stating that such changes are captured by bioinformatic tool prediction and domain analysis (Appendix J), and the CSPEC entry records applicability 'Not applicable' at every strength tier. Separately, c.10043del is a frameshift producing a premature termination codon (p.Asn3348IlefsTer35) rather than an in-frame insertion or deletion, so the variant does not fall within the variant class BP3 describes and there is no repetitive-region indel to evaluate. BP3 contributes no benign evidence and no strength is recorded.
VCEP rule
In-frame deletions/insertions in a repetitive region without a known function ... Do not use ... Captured by bioinformatic tool prediction, and domain analysis.
Evidence considered
Specifications v1.2, Table 1, BP3 row: specification 'Do not use', with the note 'Captured by bioinformatic tool prediction, and domain analysis.' (See Appendix J.); the CSPEC entry carries applicability 'Not applicable' for the criterion and for every strength tier.
Variant facts: NM_000059.3:c.10043del gives p.(Asn3348IlefsTer35), a frameshift with a premature termination codon, not an in-frame deletion or insertion, so BP3's 'in-frame deletions/insertions in a repetitive region' class does not describe this variant.
Searched Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx and Specifications_Table4_V1.2_2024-11-18.xlsx for c.10043del, p.Asn3348IlefsTer35 and p.N3348Ifs*35; no variant-level BP3 entry exists.
BP4N/ANot applicable: BP4 covers missense/in-frame, silent or intronic variants, and c.10043del is a truncating frameshift whose SpliceAI delta (0.205) also fails the <=0.1 rule.
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Reasoning
c.10043del is a frameshift (p.(Asn3348IlefsTer35)) in BRCA2 exon 27, not a missense, in-frame delins, silent or intronic variant. The ENIGMA v1.2 BP4 rule is written for three and only three situations: missense or in-frame indels inside a (potentially) clinically important functional domain with BayesDel no-AF <=0.18 AND SpliceAI <=0.1; silent variants inside a functional domain with SpliceAI <=0.1; and intronic variants outside the native donor/acceptor (+/-1,2) sites with SpliceAI <=0.1. A truncating frameshift lies outside all three, so BP4 is out of scope. This is also the correct logic for a truncating variant: even a 'no predicted splice impact' result would leave the frameshift and its downstream termination codon in place, so it would not be evidence of no impact on the gene product - a splice prediction for such a variant matters only to PVS1 (e.g. a predicted rescue). For completeness, the available SpliceAI result (max delta 0.205, DS_AG 0.205 at DP_AG +33) would in any case not satisfy BP4's <=0.1 requirement. The neighbouring ENIGMA-curated entries in the supplementary tables (e.g. c.10045A>G p.Thr3349Ala with 'No functional impact') are missense/pre-coded benign rows and provide no pre-assigned code for this variant.
VCEP rule
Missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain, and no predicted impact via protein change or splicing (BayesDel no-AF score ≤ 0.18 AND SpliceAI ≤0.1). ... Silent variant inside a (potentially) clinically important functional domain, if no predicted impact via splicing (SpliceAI ≤0.1). ... Intronic variants outside of the native donor and acceptor splice sites (i.e. not +/- 1,2 positions) AND no predicted impact via splicing (SpliceAI ≤0.1).
SpliceAI Lookup (NM_000059.3:c.10043del, hg37): max delta score 0.205 (DS_AG 0.205, DP_AG +33; DS_AL 0.022, DS_DG 0.0, DS_DL 0.0), which exceeds the <=0.1 BP4 requirement in addition to being out of scope.
Appendix J, Table 16 (Appendices_V1.2_2024-11-18.docx, BP4 row): BP4 is applied only for (i) missense or in-frame insertion-deletion inside a (potentially) clinically important functional domain with BayesDel <=0.15 (BRCA1) / <=0.18 (BRCA2) AND SpliceAI <=0.1, (ii) silent variants inside a functional domain with SpliceAI <=0.1, and (iii) intronic variants outside donor/acceptor +/-1,2 with SpliceAI <=0.1.
SupplementaryTables_V1.2_2024-11-18.xlsx: the rows adjacent to the variant position (c.10045A>G p.Thr3349Ala 'No functional impact'; c.10184del p.Glu3395Gly-fs*32 'No functional impact') are listed for other variants; c.10043del itself has no row and hence no pre-assigned BP4 code.
Still missing
No variant-level entry (and therefore no pre-assigned BP4 code) for c.10043del / p.(Asn3348IlefsTer35) / p.N3348Ifs*35 in SupplementaryTables_V1.2_2024-11-18.xlsx, Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx, or PMID_22505045_Houdayer_2012_HumMutat.pdf.
No BayesDel no-AF score exists in the case evidence, and BayesDel is not applicable to a frameshift in any case.
BP6VCEPNot applicable: ENIGMA BRCA2 v1.2 specifies BP6 must not be used, and the variant has no ClinVar record in any case.
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Reasoning
The governing ENIGMA BRCA2 v1.2 specification removes this code outright: the BP6 row of the specification table states 'Do not use', and the CSPEC criteria entry records BP6 with applicability 'Not Applicable'. The rule's own applicability therefore excludes it, which is the only basis on which not_applicable may be assigned here. Independently, the factual precondition is unmet: a ClinVar search for BRCA2 c.10043del returned no record, so there is no reputable-source benign assertion about this variant to weigh even if the code were permitted. No strength is assigned.
VCEP rule
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
Evidence considered
ENIGMA BRCA2 v1.2 CSPEC criteria entry for BP6: applicability 'Not Applicable for this VCEP'; every strength level carries the rule 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.'
ENIGMA BRCA2 v1.2 Specifications table, BP6 row: the criterion description is followed by the instruction 'Do not use'.
ClinVar search for BRCA2 c.10043del returned no record ('This variant is absent from ClinVar'), so no reputable-source benign assertion exists for this variant.
BP7N/ANot applicable: BP7 covers silent, intronic or missense/in-frame variants (RNA assay or BP4 met), and c.10043del is a frameshift with no mRNA assay available.
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Reasoning
c.10043del is a frameshift (p.(Asn3348IlefsTer35)) in BRCA2 exon 27. Both ENIGMA v1.2 BP7 arms are closed to it. The BP7_Strong (RNA) arm requires a well-established in vitro/in vivo functional study showing no damaging effect 'as measured by effect on mRNA transcript profile - mRNA assay only', applied for intronic, silent, or missense/in-frame variants outside a (potentially) clinically important functional domain; no mRNA/minigene assay exists for this variant (no entry in the ENIGMA splicing reference set ST3 or in the Houdayer 2012 dataset), and a frameshift is not among the named variant classes. The supporting arm requires a silent variant inside a functional domain, or an intronic variant at/beyond +7/-21, 'IF BP4 met'; this variant is neither silent nor intronic, and BP4 is itself not applicable here, so that arm cannot be reached. Note also that for a truncating variant a splice prediction indicating no aberrant splicing does not amount to 'no damaging effect', because the frameshift and its downstream termination codon remain; that question belongs to PVS1. The variant's SpliceAI result (max delta 0.205, DS_AG 0.205 at DP_AG +33) is therefore not converted into BP7 evidence in either direction.
VCEP rule
Well-established in vitro or in vivo functional studies shows no damaging effect on protein function as measured by effect on mRNA transcript profile – mRNA assay only. Apply as BP7_Strong (RNA) for intronic and silent variants, as well as missense/in-frame variants located outside a (potentially) clinically important functional domain. ... Silent variant inside a (potentially) clinically important functional domain, IF BP4 met. ... Intronic variants located outside conserved donor or acceptor motif positions (at or beyond positions +7/-21) IF BP4 met.
Evidence considered
Variant identity: NM_000059.3:c.10043del / NP_000050.2:p.(Asn3348IlefsTer35), BRCA2 exon 27, frameshift; the variant is not silent, not intronic, and not a missense/in-frame change.
ENIGMA Specifications v1.2, Table 1 (BP7 and BP7_Strong (RNA) rows): BP7 applies to silent variants inside a functional domain and intronic variants at/beyond +7/-21, each 'IF BP4 met'; BP7_Strong (RNA) requires an mRNA-only assay and applies to intronic, silent and missense/in-frame variants outside a functional domain.
Appendix J, Table 16 (BP7 row) repeats the silent/intronic scope and states nucleotide conservation is not considered relevant.
SupplementaryTables_V1.2_2024-11-18.xlsx, ST3 'Splicing refs' (ENIGMA's splicing calibration set of variant-level patient RNA/minigene results): no row for c.10043del; the curated results listed for BRCA2 exon 27 region concern other variants (e.g. c.9292T>C, c.9905G>A, c.10045A>G, c.10184del).
Houdayer et al. 2012 (PMID:22505045) searched in full text for c.10043del, p.Asn3348IlefsTer35, p.N3348Ifs*35, N3348 and exon 27: the variant is not reported; the paper's 272 BRCA1/BRCA2 VUSs/327 transcript analyses contain no result for it. Its calibration is for MES/SSF, whereas the ENIGMA v1.2 thresholds name SpliceAI.
SpliceAI Lookup (NM_000059.3:c.10043del, hg37): max delta score 0.205 (DS_AG 0.205, DP_AG +33; other deltas <=0.022).
Still missing
No mRNA/minigene transcript assay result exists for c.10043del: no entry in the ENIGMA splicing reference set (SupplementaryTables_V1.2_2024-11-18.xlsx, ST3 Splicing refs) and no mention of c.10043del / p.N3348Ifs*35 in PMID_22505045_Houdayer_2012_HumMutat.pdf, so a BP7 (RNA) arm could not be built even if the variant class were in scope.
No variant-level row for this variant in Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx or SupplementaryTables_V1.2_2024-11-18.xlsx (searched as c.10043del, p.Asn3348IlefsTer35, p.N3348Ifs*35, N3348, g.32972693).
Every interpreted variant in BRCA2, mapped onto the exon structure of NM_000059.3. Select a row to find it on the plot.
BRCA2NM_000059.3
Fetching transcript structure from UCSC…
Exons
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Transcript span
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Strand
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Variants mapped
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Source
UCSC ncbiRefSeqCurated
All variants in BRCA2—Select a row to find it on the plot; use the link to open its page.
Protein
Location
Classification
Link
gnomAD v4.1Allele count data not recorded
gnomAD v2.1Allele count data not recorded
gnomAD-Canada v1.0Allele count data not recorded
Statements
gnomAD v4.1
Absent from gnomAD v4.1.
gnomAD v2.1
Absent from gnomAD v2.1.
gnomAD-Canada
Not available in gnomAD-Canada v1.0.
SpliceAI and Pangolin
SpliceAI acceptor lossat -415 bp
0.022
SpliceAI acceptor gainat +33 bp
0.205
SpliceAI donor lossat -376 bp
0.000
SpliceAI donor gainat +394 bp
0.000
Pangolin splice lossmagnitude shown
-0.049
Pangolin splice gain
0.126
Other predictors
REVEL
Not available
BayesDel
Not available
How the predictions were used
PP3N/ANot applicable: PP3 covers only missense/in-frame, silent or intronic variants, and c.10043del is a frameshift (p.N3348Ifs*35) assessed under PVS1.
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Reasoning
NM_000059.3:c.10043del (NC_000013.10:g.32972693del) is a frameshift in BRCA2 exon 27, NP_000050.2:p.(Asn3348IlefsTer35); it is not a missense, in-frame indel, silent or intronic variant and is not at a canonical donor/acceptor dinucleotide. The ENIGMA v1.2 PP3 rule names exactly two scored situations: (i) missense or in-frame insertion/deletion/delins inside a (potentially) clinically important functional domain (BRCA2 PALB2 binding aa 10-40; DNA binding aa 2481-3186) with BayesDel no-AF score >=0.30, and (ii) predicted splicing with SpliceAI >=0.2 for silent, missense/in-frame (irrespective of domain location) or intronic variants outside the donor/acceptor 1,2 positions. A frameshift falls in neither situation, so the criterion is out of scope before any predictor score is considered. The SpliceAI result that exists for this variant (max delta 0.205; DS_AG 0.205 at DP_AG +33 with DS_AL 0.022, DS_DG 0.0, DS_DL 0.0; Pangolin SG 0.126, SL -0.049) therefore does not become PP3 evidence: the ENIGMA splice-prediction arm is not written for truncating variants, and for a frameshift the splice prediction bears on PVS1 (whether the truncating effect might be rescued/redirected), which is adjudicated elsewhere. BayesDel is a missense/in-frame predictor and no score is available for this variant in any case. Independently, the ENIGMA Specifications Table 1 PP3 row carries the stipulation 'Do not apply PP3 if PVS1 is met at any strength', and this variant is the classic PVS1-type null (frameshift) case, so applying PP3 here would additionally be barred by that stipulation.
VCEP rule
Apply PP3 for missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain and predicted impact via protein change (BayesDel no-AF score ≥0.30). ... Apply PP3 for predicted splicing (SpliceAI ≥0.2) for silent, missense/in-frame (irrespective of location in clinically important functional domain) and for intronic variants outside of donor and acceptor 1,2 sites.
Evidence considered
Variant identity from case evidence: NM_000059.3:c.10043del, NP_000050.2:p.(Asn3348IlefsTer35) / p.(N3348Ifs*35), BRCA2 exon 27, consequence frameshift, canonical splice consensus = false.
ENIGMA BRCA2 specification v1.2, Table 1 (Specifications_V1.2_2024-11-18.docx, PP3 row): PP3 is limited to missense/in-frame indels inside a functional domain with BayesDel predicted impact, and to SpliceAI >=0.2 for silent, missense/in-frame or intronic variants outside donor/acceptor +/-1,2; the row also states 'Do not apply PP3 if PVS1 is met at any strength'.
Appendix J, Table 16 (Appendices_V1.2_2024-11-18.docx): 'Apply PP3 for missense or in-frame insertion-deletion inside a (potentially) clinically important functional domain and predicted impact via protein change (BayesDel >=0.28 for BRCA1, >=0.30 for BRCA2). Apply PP3 for predicted splicing (SpliceAI >=0.2) for silent, missense/in-frame (irrespective of location in clinically important functional domain) and for intronic variants outside of the donor and acceptor +/-1,2 positions.' No frameshift or nonsense row is listed; Appendix J states some codes are 'not applicable since they are captured by other codes/criteria'.
Appendix J states the SpliceAI cutpoints were calibrated only 'For variants outside of the donor/acceptor +/-1,2 positions' (<=0.1 BP4; >0.1 and <0.2 not informative; >=0.2 PP3 for predicted splicing impact), i.e. the splice arm is calibrated on the non-truncating/intronic classes named in the rule.
Still missing
No variant-level entry for c.10043del / p.(Asn3348IlefsTer35) / p.N3348Ifs*35 was found in SupplementaryTables_V1.2_2024-11-18.xlsx (searched by coding HGVS, protein change 'N3348'/'3348' and genomic coordinate g.32972693; the neighbouring rows c.10045A>G p.(Thr3349Ala) and c.10184del p.(Glu3395Gly-fs*32) are present but this variant is not), in Specifications_V1.2_2024-11-18.docx, in Appendices_V1.2_2024-11-18.docx, or in PMID_22505045_Houdayer_2012_HumMutat.pdf.
No BayesDel no-AF score exists for this variant in the case evidence (null), consistent with BayesDel being a missense/in-frame predictor; this does not affect the call, which rests on variant-type scope.
BP4N/ANot applicable: BP4 covers missense/in-frame, silent or intronic variants, and c.10043del is a truncating frameshift whose SpliceAI delta (0.205) also fails the <=0.1 rule.
Details ▾Hide ▴
Reasoning
c.10043del is a frameshift (p.(Asn3348IlefsTer35)) in BRCA2 exon 27, not a missense, in-frame delins, silent or intronic variant. The ENIGMA v1.2 BP4 rule is written for three and only three situations: missense or in-frame indels inside a (potentially) clinically important functional domain with BayesDel no-AF <=0.18 AND SpliceAI <=0.1; silent variants inside a functional domain with SpliceAI <=0.1; and intronic variants outside the native donor/acceptor (+/-1,2) sites with SpliceAI <=0.1. A truncating frameshift lies outside all three, so BP4 is out of scope. This is also the correct logic for a truncating variant: even a 'no predicted splice impact' result would leave the frameshift and its downstream termination codon in place, so it would not be evidence of no impact on the gene product - a splice prediction for such a variant matters only to PVS1 (e.g. a predicted rescue). For completeness, the available SpliceAI result (max delta 0.205, DS_AG 0.205 at DP_AG +33) would in any case not satisfy BP4's <=0.1 requirement. The neighbouring ENIGMA-curated entries in the supplementary tables (e.g. c.10045A>G p.Thr3349Ala with 'No functional impact') are missense/pre-coded benign rows and provide no pre-assigned code for this variant.
VCEP rule
Missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain, and no predicted impact via protein change or splicing (BayesDel no-AF score ≤ 0.18 AND SpliceAI ≤0.1). ... Silent variant inside a (potentially) clinically important functional domain, if no predicted impact via splicing (SpliceAI ≤0.1). ... Intronic variants outside of the native donor and acceptor splice sites (i.e. not +/- 1,2 positions) AND no predicted impact via splicing (SpliceAI ≤0.1).
SpliceAI Lookup (NM_000059.3:c.10043del, hg37): max delta score 0.205 (DS_AG 0.205, DP_AG +33; DS_AL 0.022, DS_DG 0.0, DS_DL 0.0), which exceeds the <=0.1 BP4 requirement in addition to being out of scope.
Appendix J, Table 16 (Appendices_V1.2_2024-11-18.docx, BP4 row): BP4 is applied only for (i) missense or in-frame insertion-deletion inside a (potentially) clinically important functional domain with BayesDel <=0.15 (BRCA1) / <=0.18 (BRCA2) AND SpliceAI <=0.1, (ii) silent variants inside a functional domain with SpliceAI <=0.1, and (iii) intronic variants outside donor/acceptor +/-1,2 with SpliceAI <=0.1.
SupplementaryTables_V1.2_2024-11-18.xlsx: the rows adjacent to the variant position (c.10045A>G p.Thr3349Ala 'No functional impact'; c.10184del p.Glu3395Gly-fs*32 'No functional impact') are listed for other variants; c.10043del itself has no row and hence no pre-assigned BP4 code.
Still missing
No variant-level entry (and therefore no pre-assigned BP4 code) for c.10043del / p.(Asn3348IlefsTer35) / p.N3348Ifs*35 in SupplementaryTables_V1.2_2024-11-18.xlsx, Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx, or PMID_22505045_Houdayer_2012_HumMutat.pdf.
No BayesDel no-AF score exists in the case evidence, and BayesDel is not applicable to a frameshift in any case.
BP7N/ANot applicable: BP7 covers silent, intronic or missense/in-frame variants (RNA assay or BP4 met), and c.10043del is a frameshift with no mRNA assay available.
Details ▾Hide ▴
Reasoning
c.10043del is a frameshift (p.(Asn3348IlefsTer35)) in BRCA2 exon 27. Both ENIGMA v1.2 BP7 arms are closed to it. The BP7_Strong (RNA) arm requires a well-established in vitro/in vivo functional study showing no damaging effect 'as measured by effect on mRNA transcript profile - mRNA assay only', applied for intronic, silent, or missense/in-frame variants outside a (potentially) clinically important functional domain; no mRNA/minigene assay exists for this variant (no entry in the ENIGMA splicing reference set ST3 or in the Houdayer 2012 dataset), and a frameshift is not among the named variant classes. The supporting arm requires a silent variant inside a functional domain, or an intronic variant at/beyond +7/-21, 'IF BP4 met'; this variant is neither silent nor intronic, and BP4 is itself not applicable here, so that arm cannot be reached. Note also that for a truncating variant a splice prediction indicating no aberrant splicing does not amount to 'no damaging effect', because the frameshift and its downstream termination codon remain; that question belongs to PVS1. The variant's SpliceAI result (max delta 0.205, DS_AG 0.205 at DP_AG +33) is therefore not converted into BP7 evidence in either direction.
VCEP rule
Well-established in vitro or in vivo functional studies shows no damaging effect on protein function as measured by effect on mRNA transcript profile – mRNA assay only. Apply as BP7_Strong (RNA) for intronic and silent variants, as well as missense/in-frame variants located outside a (potentially) clinically important functional domain. ... Silent variant inside a (potentially) clinically important functional domain, IF BP4 met. ... Intronic variants located outside conserved donor or acceptor motif positions (at or beyond positions +7/-21) IF BP4 met.
Evidence considered
Variant identity: NM_000059.3:c.10043del / NP_000050.2:p.(Asn3348IlefsTer35), BRCA2 exon 27, frameshift; the variant is not silent, not intronic, and not a missense/in-frame change.
ENIGMA Specifications v1.2, Table 1 (BP7 and BP7_Strong (RNA) rows): BP7 applies to silent variants inside a functional domain and intronic variants at/beyond +7/-21, each 'IF BP4 met'; BP7_Strong (RNA) requires an mRNA-only assay and applies to intronic, silent and missense/in-frame variants outside a functional domain.
Appendix J, Table 16 (BP7 row) repeats the silent/intronic scope and states nucleotide conservation is not considered relevant.
SupplementaryTables_V1.2_2024-11-18.xlsx, ST3 'Splicing refs' (ENIGMA's splicing calibration set of variant-level patient RNA/minigene results): no row for c.10043del; the curated results listed for BRCA2 exon 27 region concern other variants (e.g. c.9292T>C, c.9905G>A, c.10045A>G, c.10184del).
Houdayer et al. 2012 (PMID:22505045) searched in full text for c.10043del, p.Asn3348IlefsTer35, p.N3348Ifs*35, N3348 and exon 27: the variant is not reported; the paper's 272 BRCA1/BRCA2 VUSs/327 transcript analyses contain no result for it. Its calibration is for MES/SSF, whereas the ENIGMA v1.2 thresholds name SpliceAI.
SpliceAI Lookup (NM_000059.3:c.10043del, hg37): max delta score 0.205 (DS_AG 0.205, DP_AG +33; other deltas <=0.022).
Still missing
No mRNA/minigene transcript assay result exists for c.10043del: no entry in the ENIGMA splicing reference set (SupplementaryTables_V1.2_2024-11-18.xlsx, ST3 Splicing refs) and no mention of c.10043del / p.N3348Ifs*35 in PMID_22505045_Houdayer_2012_HumMutat.pdf, so a BP7 (RNA) arm could not be built even if the variant class were in scope.
No variant-level row for this variant in Specifications_V1.2_2024-11-18.docx, Appendices_V1.2_2024-11-18.docx or SupplementaryTables_V1.2_2024-11-18.xlsx (searched as c.10043del, p.Asn3348IlefsTer35, p.N3348Ifs*35, N3348, g.32972693).
SpliceAI predicts possible splice impact for this variant (max delta score = 0.20).
5 paper records listed. No publication is explicitly linked to an applied criterion in the recorded metadata. None of the listed paper records explicitly indicates that the variant is named.
Recorded publications
Truncated BRCA2 is cytoplasmic: implications for cancer-linked mutations.
This paper maps BRCA2 nuclear localization signals to the extreme C-terminus (NLS1 3263-3269, NLS3 3381-3385) and shows that truncated BRCA2 is cytoplasmically excluded, but its variant-level content bears directly on where truncations stop mattering: the p.Lys3326Ter polymorphism removes 92 C-terminal residues 'apparently without consequence', and a reported Glu3342ter change, occurring downstream of Lys3326Ter, was not found in an affected breast cancer family and was judged unlikely to be cancer associated. The most C-terminal cancer-associated truncation the authors list is 9808delCC (termination codon 3195). This supports treating a terminal truncation at ~codon 3382 as not clinically important at the high-risk threshold, consistent with the ENIGMA exon-27 PVS1_N/A assignment, even though NLS3 is disrupted.
Demarcating the C-terminal end of this critical region is the Lys3326ter polymorphism, which removes 92 residues from the C terminus, apparently without consequence (25). In the Breast Cancer Information Core database (18), a variant has been reported that is even further downstream (Glu3342ter). However, it was not identified as part of an affected breast cancer family and, because it occurs downstream of the Lys3326ter polymorphism, it is unlikely to be cancer associated
Results, first paragraph ('BRCA2 NLSs Reside in the Extreme C Terminus'), p. 13921; see also Fig. 1 and Table 1
BRCA2 is required for homology-directed repair of chromosomal breaks.
A functional study of homology-directed repair showing that a mouse Brca2 exon 27 deletion (removing the extreme C-terminal Rad51-interacting domain and conserved NLS, without perturbing the BRC repeats) produced only a 5- to 6-fold HDR reduction and ~1.8-fold reduced gene targeting, whereas the highly truncated 6174delT protein gave a >100-fold defect. The variant itself is not reported. The relevant content for this case is that the magnitude of repair defect tracks with the extent of truncation, with extreme C-terminal-only truncations having a much milder effect than mid-gene truncations - context consistent with no PVS1 weight being assigned to a terminal truncation at ~codon 3382.
Thus, the severity of the defect was significantly greater in cells that express a highly truncated BRCA2 protein than in cells in which only the most C-terminal Rad51-interacting domain is perturbed.
Discussion, opening paragraphs (pp. 267-268)
A new mutation of BRCA2 gene in an Italian healthy woman with familial breast cancer history.
Reports a different BRCA2 frameshift, 7525_7526insT (exon 15, premature stop at codon 2538, ~880 aa shorter than the 3418-aa protein), segregating in an Italian family with five breast cancers, and argues that such truncating frameshifts are non-functional because they remove the two C-terminal NLSs. The variant under review is not mentioned or tested. Gene/mechanism-level context only: this is a mid-gene truncation, far upstream of codon 3348, so its loss-of-function argument does not transfer to a truncation in the final ~36 residues of the protein.
Therefore, the 7525_7526insT permits to conserve the N-terminal region, that includes the transcriptional activation domain, the central region, called BRC domain, that binds Rad51, but it causes the loss of the two nuclear localization signals (NLSs); this event provides to explain why this nuclear truncated protein is non-functional: because it really cannot perform a translocation into the nucleus.
Results, paragraph 1 (variant description and family segregation); Discussion, paragraphs on NLS/nuclear translocation; Conclusion
BRCA1 and BRCA2: different roles in a common pathway of genome protection.
A general review of BRCA1/BRCA2 biology (homologous recombination, RAD51 recruitment, BRC repeats, DNA-binding domain, C-terminal RAD51-binding region and CDK site S3291) that establishes loss-of-function of BRCA2 as an autosomal-dominant cancer mechanism and gives the 3418-aa protein length. It does not name c.10043del or any variant-specific data, and it makes no statement about the pathogenicity of terminal truncations, so it supports only the gene-level premise that LoF is the disease mechanism.
Although human and mouse cells expressing a BRCA2 loss-of-function truncation mutant display some defects in replication and checkpoint control, BRCA2 is not essential for these processes
Section 'BRCA2, HR and replication fidelity', first paragraph
A comprehensive focus on global spectrum of BRCA1 and BRCA2 mutations in breast cancer.
A review of BRCA1/BRCA2 variant spectra in world populations (data to April 2013) stating that most BRCA2 disease alleles are frameshifts creating premature stop codons and that the C-terminal DBD/NLS/Rad51-binding region is critical. It gives no data on codon 3348 or exon 27 and does not name this variant; its general claim that C-terminal truncation is critical is not specific enough to override the VCEP's exon-27 weighting, which places the clinically important boundary at p.3308.
In BRCA2 gene, most of mutations occur in exons 10 and 11 and usually include insertions or deletions which raise the missense alterations and premature stop codon ending in truncated and nonfunctional protein. It was shown that the segments of BRCA2 that were separated out contain double strand break domain (DBD), nuclear localization signal (NLS), and Rad-51 binding motif are located in C-terminal which are critical for BRCA2 function [20].
Section 3, 'BRCA1 and BRCA2 Mutations', paragraph 2
Screenshots represent source records captured during evidence collection. Select one to view the recorded source content.