ATM c.4394T>C, p.(Leu1465Pro)

Reasoning

ATM VCEP v1.5 approves three kinase-activity assay papers for PS3/BS3 (Mitui 2009/PMID18634022, Barone 2009/PMID19431188, Scott 2002/PMID11805335), each using stable transfection of ATM cDNA constructs into an ATM-null LCL line with WT-positive and null-negative controls, reading out substrate phosphorylation (S1981 autophosphorylation, SMC1, NBS1, CHK2, p53) by Western blot after irradiation. Barone et al. 2009 (PMID:19431188) directly tested this exact variant, c.4394T>C (p.Leu1465Pro), placing it in their 'Group 3: ATM Sequence Changes That Result in Reduced ATM Kinase Activity' (Table 1) - one of 10 missense changes shown by Figure 2B to retain reduced (not normal, not absent) kinase activity relative to the WT pTAM2 control. The VCEP's own approved-assay calibration table states an abnormal (reduced or no detectable) kinase readout in this assay confers PS3_Supporting. Assay included basic positive control (WT cDNA pTAM2) and negative control (empty cDNA pMEP4), met per the VCEP's own quality table; biological replicates were 'not met' and technical replicate handling was ambiguous ('Y;3;not sure if biological or technical'), and no dedicated benign validation controls were run in this specific paper (0 B/LB), which together with the qualitative/semi-quantitative nature of the readout supports capping strength at Supporting rather than Moderate/Strong. Radiosensitivity (the only other functional readout the VCEP calibrates, up to PS3_Moderate when combined with a supporting kinase result) was not assessed in this or any of the other approved papers for this specific variant, so only the PS3_Supporting kinase-assay strength applies; no moderate escalation is available.

Rule

ATM VCEP v1.5 approved functional assay table: kinase assay abnormal (reduced or absent) readout in an approved assay (Mitui 2009/Barone 2009/Scott 2002) confers PS3_Supporting; radiosensitivity can add PS3_Moderate only in combination with a supporting kinase result (not available here).

Evidence considered

• Barone et al. 2009 (PMID:19431188), Table 1: lists c.4394T4C / p.Leu1465Pro under 'Group 3: ATM kinase reduced mutants'.
• Barone et al. 2009 (PMID:19431188), Results text (Group 3 section): 'Group 3 consists of 10 ATM missense changes (Table 1) that expressed ATM protein with a reduced level of activity when compared with pTAM2... Figure 2B confirmed the presence of reduced ATM kinase activity for all Group 3 ATM mutants modeled.'
• ATM VCEP-approved functional-assay calibration table (clingen_hbop_atm_supplementary_tables_1_and_2_v1.xlsx, 'ATM kinase activity' sheet): Barone 2009 (PMID 19431188) is an approved assay; threshold for abnormal readout is 'no detectable kinase activity (group 2) or reduced level of kinase activity (group 3), (relative to WT pTAM2)'; proposed strength for an abnormal result is PS3_Supporting.

Still missing

• No radiosensitivity assay result available for this variant to allow escalation to PS3_Moderate per the VCEP's kinase+radiosensitivity combination rule.
• Biological replicate status for the Barone assay is 'not met' per the VCEP's own quality assessment, and no benign validation controls were included in that specific paper - both reduce confidence below what a fully validated assay would warrant, consistent with capping at Supporting strength.
• Sun et al. 2025 (PMID 40580951, prime-editing saturation genome editing screen) covers essentially all ATM SNVs and could provide corroborating direct functional data, but it postdates ATM VCEP v1.5 and is not one of the three VCEP-approved assay papers; it was not consulted here to avoid introducing an unapproved functional source, and its inclusion (if desired) would require human review per the VCEP materials index guidance.

Sources

Reasoning

The ATM VCEP specifies PM2_Supporting for gnomAD v4 frequency <=0.001%. The gnomAD v4.1 overall AF is 1.2395e-06 (0.00012395%) and the highest observed population AF is 1.69498e-06 (0.000169498%), both below 0.001%; the variant is observed twice in the large non-Finnish European dataset, with no homozygotes. PM2_Supporting is met.

Rule

ATM HBOP VCEP v1.5: PM2_Supporting if frequency <=0.001% in gnomAD v4.

Evidence considered

• ATM HBOP VCEP v1.5 specifies PM2_Supporting for frequency <=0.001% in gnomAD v4 and states that a single observation in a single subpopulation is sufficiently rare.
• gnomAD v4.1 reports 2/1,613,560 alleles overall (AF 1.2395e-06; 0.00012395%), highest observed population AF 1.69498e-06 (0.000169498%) in non-Finnish European individuals, grpmax filtering AF 2.8e-07, and zero homozygotes.

Sources

Reasoning

Expert panel ClinGen Hereditary Breast, Ovarian and Pancreatic Cancer Variant Curation Expert Panel, ClinGen classified as Likely pathogenic.

Rule

User-specified rule: an exact-variant ClinVar expert-panel Pathogenic or Likely Pathogenic classification supports PP5.

Evidence considered

• ClinVar variation 181996 is an exact transcript-HGVS match to NM_000051.4:c.4394T>C (p.Leu1465Pro).
• The exact record includes the ClinGen Hereditary Breast, Ovarian and Pancreatic Cancer Variant Curation Expert Panel assertion of Likely Pathogenic, with review status 'reviewed by expert panel'.
• Single-submitter laboratory assertions were not used for PP5.
• ClinVar expert panel classification

Sources

Reasoning

ATM VCEP v1.5 PS1 guide (ATM_PS1_1.5.pdf) provides two sub-paths: a standard missense path (same amino acid change, i.e. p.(Leu1465Pro), already established as an existing P/LP variant via a different nucleotide substitution) and a splicing-region table for variants near donor/acceptor sites. NM_000051.4:c.4394T>C is a straightforward exonic missense substitution (p.(Leu1465Pro)) located outside any donor/acceptor +/-1,2 dinucleotide position, so the splicing sub-path prerequisite (PVS1/PP3 splice-position comparison) does not apply; only the standard missense PS1 path is relevant. No other nucleotide change producing the identical p.(Leu1465Pro) substitution and independently classified Pathogenic/Likely Pathogenic was identified anywhere in the case bundle (ClinVar record 181996 IS this exact variant/nucleotide change, not a distinct comparator), and pm5_candidates.json (which also captures same-residue ClinVar search context) returned zero same-residue candidates. Per the VCEP's own instruction ('use for missense changes as long as splicing is ruled-out for both alterations'), a same-amino-acid comparator is required before PS1 can be applied; none exists in the evidence provided.

Rule

ATM VCEP v1.5 PS1: missense sub-path - apply Strong when the variant produces the same amino acid change as an established P/LP variant (via a different nucleotide change), with splicing ruled out for both.

Evidence considered

• ATM VCEP v1.5 PS1 guide (ATM_PS1_1.5.pdf): standard missense PS1 sub-path requires the same amino acid change as an established P/LP variant, arising from a different nucleotide substitution, with splicing ruled out for both alleles.
• case_summary.json cspec block lists PS1 defaultStrength Pathogenic Strong with the missense-change instruction quoted above.
• pm5_candidates.json (same-residue candidate search infrastructure) reports total_candidates_seen=0 and same_residue_candidates=0 for codon 1465, indicating no comparator variant at this residue was located.
• ClinVar variation 181996 corresponds to this exact variant (NM_000051.4:c.4394T>C, p.(Leu1465Pro)) itself, not an independent comparator, so it cannot serve as the PS1 reference variant.

Still missing

• No independent ClinVar/literature comparator variant producing the identical p.(Leu1465Pro) change via a different nucleotide substitution was found in the case bundle; a dedicated ClinVar search restricted to codon 1465 amino-acid-change matches was not performed as part of this bundle and would be needed to fully rule PS1 in or out.

Sources

Reasoning

Variant is missense (NP_000042.3:p.(Leu1465Pro)). ATM VCEP v1.5 PP3 rule requires REVEL >0.7333 for missense variants, or SpliceAI >=0.2 for silent/missense/in-frame variants (splicing sub-path, applicable to any variant type per the VCEP's own instructionsToUse note that any variant can potentially impact splicing). REVEL score for this variant = 0.329, well below the 0.7333 pathogenic threshold. SpliceAI max delta score = 0.008 (DS_AG 0.0, DS_AL 0.003, DS_DG 0.008, DS_DL 0.007), far below the 0.2 threshold - no predicted splice impact. Neither the missense sub-path nor the splicing sub-path meets the PP3 threshold, so PP3 is not met via either mechanism. The lab's BP4 curator override note explicitly states PP3 is unaffected by the override and its splicing sub-path should still be checked for missense variants exactly as the VCEP specifies; this was done and both paths are negative. BayesDel score (0.299728) was also checked but per this pipeline's policy no verified published threshold/PMID is available for BayesDel, so it cannot be used to support or refute PP3.

Rule

ATM VCEP v1.5 PP3: Missense REVEL >0.7333 OR SpliceAI >=0.2 (silent/missense/in-frame/eligible intronic)

Evidence considered

• ATM VCEP v1.5 PP3 rule (case_summary.json cspec criteria list, label PP3): Missense REVEL >0.7333; Splicing SpliceAI >=0.2 for silent, missense/in-frame and intronic variants outside donor/acceptor 1,2 sites.
• REVEL score = 0.329 (source_registry key 'revel', local REVEL v1.3 lookup) - below the 0.7333 PP3 missense threshold.
• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01)' with raw scores DS_AG=0.0, DS_AL=0.003, DS_DG=0.008, DS_DL=0.007 (source_registry key 'spliceai') - below the 0.2 PP3 splicing threshold.
• ATM VCEP cspec instructionsToUse for PP3: 'The VCEP uses SpliceAI as a sole predictor... This VCEP recommends SpliceAI thresholds set forth by the SVI in applying PP3 and BP4 to non-canonical splice variants: Apply PP3 for SpliceAI scores >=0.2 and apply BP4 for SpliceAI scores <=0.1 (Walker et al., 2023).'
• Curator gene_notes override confirms PP3 is unaffected by the BP4 missense-only restriction and its splicing sub-path should still be applied to missense variants as the VCEP specifies; both sub-paths were checked here.
• BayesDel score (0.299728, source_registry key 'bayesdel') was retrieved but not used for PP3 because no verified published calibration threshold/PMID for BayesDel is available to this pipeline.

Sources

Reasoning

The ATM VCEP BA1 threshold is grpmax filtering AF >0.5% in gnomAD v4. The observed gnomAD v4.1 grpmax filtering AF is 2.8e-07 (0.000028%), far below this threshold.

Rule

ATM HBOP VCEP v1.5: BA1 if grpmax filtering AF >0.5% in gnomAD v4.

Evidence considered

• ATM HBOP VCEP v1.5 specifies BA1 at grpmax filtering AF >0.5% in gnomAD v4.
• gnomAD v4.1 reports 2/1,613,560 alleles overall (AF 1.2395e-06), highest observed population AF 1.69498e-06 in non-Finnish European individuals, grpmax filtering AF 2.8e-07, and zero homozygotes.

Sources

Reasoning

The ATM VCEP BS1 threshold is grpmax filtering AF >0.05% in gnomAD v4. The observed gnomAD v4.1 grpmax filtering AF is 2.8e-07 (0.000028%), far below this threshold.

Rule

ATM HBOP VCEP v1.5: BS1 if grpmax filtering AF >0.05% in gnomAD v4.

Evidence considered

• ATM HBOP VCEP v1.5 specifies BS1 at grpmax filtering AF >0.05% in gnomAD v4.
• gnomAD v4.1 reports 2/1,613,560 alleles overall (AF 1.2395e-06), highest observed population AF 1.69498e-06 in non-Finnish European individuals, grpmax filtering AF 2.8e-07, and zero homozygotes.

Sources

Reasoning

The only ATM VCEP-approved functional assay result available for this variant (Barone et al. 2009, PMID:19431188) showed a clearly abnormal (reduced) kinase-activity readout relative to WT, not a normal readout. Per the VCEP's own calibration table, only a normal (WT-level) kinase readout supports BS3_Supporting; a reduced/abnormal readout is directly inconsistent with BS3 and instead supports PS3. No radiosensitivity data or other approved-assay evidence supporting a normal functional readout for this variant was identified.

Rule

ATM VCEP v1.5 approved functional assay table: BS3_Supporting requires a normal (WT-level) kinase or radiosensitivity readout in an approved assay; this variant's approved assay result was abnormal (reduced), so BS3 is not met.

Evidence considered

• Barone et al. 2009 (PMID:19431188), Table 1 and Group 3 results text: c.4394T>C/p.Leu1465Pro shows reduced (not normal) ATM kinase activity relative to WT pTAM2, ruling out a BS3-supporting normal readout for this variant.

Sources

Reasoning

Variant is missense. Under the standing lab curator override (gene_notes, applied in addition to the official ATM VCEP v1.5 text): for missense variants, BP4 must be evaluated using ONLY the missense sub-path (REVEL <=0.249), not the splicing sub-path (SpliceAI <=0.1), even though the VCEP's own text would technically permit the splicing sub-path for any variant type. REVEL score = 0.329 falls in the 0.249-0.7333 gray zone (above the BP4 benign threshold of 0.249 but below the PP3 pathogenic threshold of 0.7333). Per the override, when REVEL is in this gray zone, BP4 is not_met regardless of the clean SpliceAI result (max delta 0.008, well under 0.1). This override changes the outcome from what the VCEP's literal text alone would produce: taken literally, the VCEP's splicing sub-path (SpliceAI <=0.1, applicable to 'any variant' per its own instructionsToUse note) would have been met and BP4 could have been called on splicing grounds alone; the override blocks that because a clean SpliceAI score does not clear this variant's own missense/protein-level effect, which REVEL (gray-zone, inconclusive) fails to rule out.

Rule

ATM VCEP v1.5 BP4 (missense sub-path only, per curator override): REVEL <=0.249

Evidence considered

• ATM VCEP v1.5 BP4 rule (case_summary.json cspec criteria list, label BP4): Missense REVEL score <=0.249; Splicing SpliceAI <=0.1 (no predicted splice impact).
• REVEL score = 0.329 (source_registry key 'revel') falls above the 0.249 BP4 missense threshold, i.e. in the 0.249-0.7333 gray zone, not benign-supporting.
• SpliceAI max delta score = 0.008 (source_registry key 'spliceai'), below the 0.1 BP4 splicing threshold on its own, but this sub-path is not applicable to missense variants per curator override.
• Curator gene_notes override (BP4 missense/splicing policy): 'Missense variants: use ONLY the missense sub-path (REVEL <=0.249). Do NOT apply BP4 from the splicing sub-path (SpliceAI <=0.1) for a missense variant... If REVEL is in the 0.249-0.7333 gray zone or unavailable, BP4 is not_met/not_assessed for this variant regardless of SpliceAI. State explicitly in the BP4 rationale when this override changes the outcome from what the VCEP's literal text alone would produce.' - this override was decisive here.
• BayesDel score (0.299728, source_registry key 'bayesdel') was retrieved but not used for BP4 because no verified published calibration threshold/PMID for BayesDel is available to this pipeline.

Sources

Reasoning

No exact-variant ClinVar expert-panel Benign or Likely Benign assertion was identified. The only expert-panel assertion on the exact matched ClinVar record is Likely Pathogenic; ordinary laboratory assertions and aggregate labels were not considered for BP6.

Rule

User-specified rule: an exact-variant ClinVar expert-panel Benign or Likely Benign classification supports BP6.

Evidence considered

• ClinVar variation 181996 exactly matches NM_000051.4:c.4394T>C (p.Leu1465Pro).
• Its expert-panel assertion is Likely Pathogenic, not Benign or Likely Benign.
• Ordinary laboratory submissions and aggregate ClinVar labels were excluded from BP6 assessment.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely Benign classification would be required to support BP6.

Sources

Reasoning

No exact-variant case-control enrichment result was identified. The ATM VCEP PS4 rule requires a case-control study with p<=0.05 and an odds ratio, hazard ratio, or relative risk >=2 (or lower 95% CI >=1.5).

Rule

ATM VCEP v1.5: PS4 Strong requires case-control p<=0.05 and OR/HR/RR >=2 or lower 95% CI >=1.5.

Evidence considered

• ATM VCEP v1.5 specifies PS4 at Strong only for qualifying case-control evidence: p<=0.05 plus OR/HR/RR >=2 or a lower 95% confidence bound >=1.5.
• The reviewed bundle contains no qualifying case-control statistic for this exact variant.

Still missing

• An exact-variant case-control analysis for NM_000051.4:c.4394T>C (p.Leu1465Pro), including effect estimate, confidence interval, and p-value.

Sources

Reasoning

The ATM HBOP VCEP v1.5 PM3/BP2 specification governs this case. It awards PM3 points only for unrelated individuals with A-T, according to phenotype confidence, the classification of a co-occurring ATM variant, and trans/unknown phase or homozygosity. The consolidated bundle provides no affected individual carrying c.4394T>C with a second ATM variant, no co-occurring ATM P/LP allele classified under the VCEP framework, and no phase, parental, segregation, consanguinity, or A-T phenotype information. No PM3 points can be assigned; PM3 is therefore not_assessed rather than not_met.

Rule

ATM HBOP VCEP v1.5 PM3: sum points from unrelated A-T probands. Confident phenotype: confirmed trans with a VCEP-classified P/LP ATM variant = 4.0, phase unknown = 2.0, second variant unidentified/VUS = 1.0, homozygous = 2.0 (maximum two individuals). Consistent phenotype earns half these values. PM3 Supporting = 1.0 point, Moderate = 2.0, Strong = 4.0, Very Strong = 8.0.

Evidence considered

• The governing ATM HBOP VCEP v1.5 PM3 table assigns 4.0, 2.0, 1.0, or 2.0 points per unrelated confidently diagnosed A-T proband according to confirmed trans, phase unknown, second variant unidentified/VUS, or homozygosity, respectively; lower points apply to a consistent phenotype. PM3 strength requires at least 1.0 point.
• The consolidated case-evidence bundle identifies the variant as ATM NM_000051.4:c.4394T>C (p.Leu1465Pro), but contains no individual-level A-T observation, co-occurring ATM allele, phase result, inheritance result, or family data applicable to PM3.

Still missing

• An affected A-T proband carrying c.4394T>C with a second ATM variant is not documented.
• Classification of a co-occurring ATM allele under the ATM VCEP specifications is not available.
• Cis/trans phase, parental testing, segregation, homozygosity, and phenotype-confidence information are not available.

Sources

Reasoning

No case-specific segregation data are available to meet the ATM VCEP AR PP1 thresholds.

Rule

ATM VCEP v1.5 PP1 (AR): Supporting = 1 affected relative; Moderate = 2; Strong = 3 or more.

Evidence considered

• ATM VCEP v1.5 specifies PP1 only for autosomal-recessive disease: Supporting for segregation in 1 affected relative, Moderate for 2, and Strong for 3 or more affected relatives.
• The consolidated case-evidence bundle contains no case-specific pedigree, relative genotype, phenotype, or meiosis data for this variant.

Still missing

• Genotypes and A-T phenotypes for affected relatives, including confirmation that both ATM variants are identified in the proband and relatives, are unavailable.
• The number of informative affected relatives and segregation observations is unavailable.

Sources

Reasoning

The ATM HBOP VCEP v1.5 PM3/BP2 specification governs this case. BP2 requires points from an unaffected non-A-T individual carrying the variant with a VCEP-classified ATM P/LP variant in trans, or from a homozygous unaffected individual under the specified laboratory/database conditions. The consolidated bundle contains no unaffected individual-level co-occurrence, no co-occurring ATM P/LP allele, no phase evidence, and no homozygous observation. No negative points can be assigned; BP2 is therefore not_assessed rather than not_met.

Rule

ATM HBOP VCEP v1.5 BP2: for an unaffected non-A-T individual with an ATM P/LP allele, confirmed trans = -4.0 points and phase unknown = -2.0 points. Qualifying homozygous observations contribute -2.0 in a laboratory setting or -1.0 in a database setting, with a maximum of -2.0. BP2 Supporting = -1.0 point, Moderate = -2.0, Strong <= -4.0.

Evidence considered

• The governing ATM HBOP VCEP v1.5 BP2 table assigns -4.0 points for a confirmed-trans co-occurrence with an ATM P/LP variant in an unaffected non-A-T individual, -2.0 for phase unknown, and -2.0 or -1.0 for qualifying homozygous observations; BP2 Supporting, Moderate, and Strong require -1.0, -2.0, and <=-4.0 points, respectively.
• The consolidated case-evidence bundle contains no unaffected non-A-T co-occurrence, no phased ATM P/LP allele, and no qualifying homozygous observation for c.4394T>C.

Still missing

• An unaffected non-A-T individual carrying c.4394T>C with a co-occurring ATM P/LP allele is not documented.
• Cis/trans phase and VCEP-based classification of any co-occurring ATM allele are not available.
• No homozygous observation in an unaffected individual, including its laboratory or database setting, is available.

Sources