CHEK2 c.422A>C, p.Lys141Thr

Reasoning

PS3 is met at supporting strength for CHEK2 NM_007194.4:c.422A>C (p.Lys141Thr). Boonen et al. evaluated the exact p.K141T variant in a Chek2-knockout mouse embryonic stem-cell complementation assay measuring ionizing-radiation-induced KAP1 p.S473 phosphorylation, a biologically relevant downstream readout of CHK2 kinase function. The assay was validated with wild-type, empty-vector, synonymous, and truncating controls; wild-type and synonymous controls retained activity, whereas truncating controls showed marked loss of activity. p.K141T showed an intermediate functional defect, and the result was reproduced with a second EGFP-CHEK2-T2A-mCherry construct and mCherry-positive-cell gating, with concordance between tagged and untagged measurements of R2 = 0.95. This is reproducible, controlled functional evidence supportive of impaired CHEK2 protein function, but the intermediate rather than truncating-like effect and the absence of a clinical diagnostic-laboratory validation setting do not justify the default strong PS3 strength.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• Boonen et al. 2022, Functional Analysis Identifies Damaging CHEK2 Missense Variants Associated with Increased Cancer Risk: exact p.K141T variant classified among intermediate-function variants in the FACS-based KAP1 p.S473 phosphorylation assay.
• Boonen et al. 2022, Results, quantitative FACS assay: wild-type and synonymous controls retained activity, while truncating variants and empty-vector controls showed major or absent activity; p.K141T had an intermediate effect.
• Boonen et al. 2022, Results, Functional analysis of CHEK2 missense VUS, paragraph 2; Fig. 3D and Supplementary Fig. S5B: the p.K141T result was reproduced using EGFP-CHEK2-T2A-mCherry and mCherry-positive-cell gating, with tagged versus untagged assay concordance R2 = 0.95.

Sources

Reasoning

Generic ACMG/AMP rules govern because the listed CHEK2 specification has no released criterion table. gnomAD v4.1 all-comers contains the variant at 5/1,614,184 alleles with total AF 3.09754e-06 and grpmax FAF 1.24e-06; this is at or below the generic PM2 supporting threshold of 0.0001. gnomAD v2.1, v2.1 non-cancer, and v3.1 non-cancer records are absent. Because no governing VCEP specifies a non-cancer or exome-only source, the default all-comers v4.1 figures are used.

Rule

PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v4.1 all-comers record: total AF 3.09754e-06 from 5/1,614,184 alleles, homozygotes 0, and grpmax FAF 1.24e-06.
• The generic PM2 supporting threshold is 0.0001; the filtering frequency 1.24e-06 satisfies it.
• The gnomAD v2.1 all-comers record is absent, and the recorded gnomAD v2.1 non-cancer and v3.1 non-cancer records are also absent. No released CHEK2 criterion specification requires a non-cancer or exome-only source, so gnomAD v4.1 all-comers is the default source.

Sources

Reasoning

The variant is a missense substitution, NP_009125.1:p.(Lys141Thr), so the governing generic rule selects the REVEL path only; SpliceAI is not used for PP3 in this situation. REVEL is 0.738, meeting the supplied generic ClinGen SVI supporting PP3 threshold of >= 0.644 but not the moderate threshold of >= 0.773. PP3 is met at supporting strength. BayesDel is not needed and is not used because no generic ACMG-strength calibration is established for it.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).

Evidence considered

• Variant consequence is missense: NM_007194.4:c.422A>C, NP_009125.1:p.(Lys141Thr).
• Applicable path is REVEL only; SpliceAI is not evidence for PP3/BP4 for a missense variant.
• Local REVEL lookup reports 0.738. The supplied ClinGen SVI calibration gives supporting PP3 at >= 0.644, moderate at >= 0.773, and strong at >= 0.932; therefore PP3 is met at supporting strength.
• BayesDel score 0.140997 was not used because the supplied rules establish no generic ACMG-strength calibration for BayesDel.

Sources

Reasoning

The variant is a missense change, and the supplied CHEK2 gene-context evidence supports loss of function as the germline disease mechanism. The reviewed CHEK2 functional-analysis paper also states that truncating variants in CHEK2 are known to be pathogenic, while missense variants remain challenging to interpret. This supports the BP1 pattern that disease is primarily associated with truncating variants rather than missense variants, although the exact proportion of pathogenic variant classes is not quantified in the supplied evidence.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• Variant consequence: NM_007194.4:c.422A>C is a missense variant producing p.Lys141Thr.
• pvs1_gene_context.json: CHEK2 loss of function is supported as a germline disease mechanism.
• Boonen et al. 2022, PMID:34903604, reports that heterozygous carriers of germline loss-of-function CHEK2 variants are at increased cancer risk and that truncating CHEK2 variants are known to be pathogenic, while missense VUS interpretation is challenging.

Still missing

• The supplied evidence does not quantify the proportion of disease-causing truncating versus missense variants; human review is retained.

Sources

Reasoning

PP5 is not met. The exact ClinVar record for NM_007194.4:c.422A>C has zero expert-panel submissions; the aggregate record is Uncertain significance, and the only Likely pathogenic submission is a non-expert research submission with no assertion criteria and no validated PMID trail. Ordinary laboratory or non-expert classifications do not satisfy the required reputable-source condition.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 186751 exact match: aggregate classification Uncertain significance; expert_panel_submissions = 0.
• ClinVar submission audit: SCV000987264 is Likely pathogenic but has review status 'no assertion criteria provided', method 'research', is_expert_panel = false, and no validated PubMed trail.
• The reviewed PMID:20301425 and PMID:25356965 entries do not provide an exact-variant reputable-source pathogenic report; PMID:25356965 is a general ACMG policy statement.

Sources

Reasoning

Generic ACMG/AMP rules govern because the listed CHEK2 specification has no released criterion table. gnomAD v4.1 reports 5/1,614,184 total alleles (AF 3.09754e-06), with a grpmax FAF of 1.24e-06 in European non-Finnish data; the relevant population has 1,180,028 observed alleles, exceeding the 2,000-allele minimum. The filtering allele frequency is far below the generic BA1 threshold of 0.05, so BA1 is not met. The ClinVar record is predominantly uncertain significance, with no expert-panel pathogenic consensus creating a frequency-versus-pathogenicity conflict.

Rule

BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v4.1 all-comers record for CHEK2 NM_007194.4:c.422A>C: total AF 3.09754e-06 from 5/1,614,184 alleles, homozygotes 0, and grpmax FAF 1.24e-06; highest observed subgroup was European (non-Finnish), 5/1,180,028 alleles.
• The filtering allele frequency (grpmax FAF), rather than the European non-Finnish point estimate, was used for BA1; 1.24e-06 is below the generic 0.05 stand-alone threshold.
• ClinVar variation 186751: 11 clinical laboratories report uncertain significance, with no expert-panel submission; this does not create a pathogenic-consensus conflict requiring BA1 withholding.

Sources

Reasoning

Generic ACMG/AMP rules govern because the listed CHEK2 specification has no released criterion table. The gnomAD v4.1 filtering allele frequency is grpmax FAF 1.24e-06, based on a population with 1,180,028 observed alleles, which is below the generic SVI default BS1 threshold of 0.01. This low frequency does not support an allele frequency greater than expected for CHEK2-related cancer predisposition, and no per-gene maximum-credible-frequency calculation is needed to reject the criterion at this value.

Rule

BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v4.1 all-comers record: total AF 3.09754e-06 from 5/1,614,184 alleles and grpmax FAF 1.24e-06; the NFE subgroup contributing the observed alleles has 1,180,028 observed alleles.
• The grpmax FAF, not the popmax point estimate, was compared with the generic BS1 threshold; 1.24e-06 is below 0.01 by several orders of magnitude.
• The governing framework identifies CHEK2-related cancer predisposition and autosomal dominant inheritance but provides no released CHEK2 criterion-specific frequency thresholds; generic ACMG/ClinGen-SVI defaults therefore apply.

Sources

Reasoning

BS3 is not met. The exact p.K141T variant did not show preserved CHEK2 function in the available controlled assay; it was classified as an intermediate-function variant based on reduced ionizing-radiation-induced KAP1 p.S473 phosphorylation. The study used appropriate wild-type and synonymous functional controls and reproduced the p.K141T result with a tagged construct, so the available evidence supports impaired rather than normal protein function. No assay result demonstrating that p.K141T has no damaging effect on protein function or splicing was identified.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• Boonen et al. 2022, Results, Functional analysis of CHEK2 missense VUS: p.K141T was explicitly listed among variants with intermediate functional defects in the KAP1 p.S473 phosphorylation assay.
• Boonen et al. 2022, Results, quantitative FACS assay: the assay distinguished wild-type/synonymous functional controls from truncating loss-of-function controls, and p.K141T fell in the intermediate-function group rather than the functional group.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; SpliceAI is not used for BP4 in this situation. REVEL is 0.738, which is above the generic ClinGen SVI supporting BP4 cutoff of <= 0.29 and therefore does not support a tolerated effect. The gray-zone REVEL result also cannot be replaced by the SpliceAI max delta of 0.009. BayesDel is not usable because no generic ACMG-strength calibration is established. BP4 is not met.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)

Evidence considered

• Variant consequence is missense: NM_007194.4:c.422A>C, NP_009125.1:p.(Lys141Thr).
• Applicable path is REVEL only; SpliceAI is not evidence for PP3/BP4 for a missense variant.
• Local REVEL lookup reports 0.738. The supplied ClinGen SVI calibration gives supporting BP4 at <= 0.29, moderate at <= 0.183, and strong at <= 0.016; 0.738 does not meet any BP4 cutoff.
• SpliceAI max delta is 0.009, but it is not used because this is a missense variant and the one-path rule requires REVEL.
• BayesDel score 0.140997 was not used because the supplied rules establish no generic ACMG-strength calibration for BayesDel.

Sources

Reasoning

BP6 is not met. The exact ClinVar record has no expert-panel submission and no exact-variant reputable-source benign or likely benign report. The aggregate ClinVar classification is Uncertain significance; the audit identifies no expert-panel benign assertion, and ordinary laboratory, research, or non-expert assertions cannot trigger BP6.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 186751 exact match: aggregate classification Uncertain significance and expert_panel_submissions = 0.
• The ClinVar submission audit contains no expert-panel benign or likely benign classification for this exact variant; the only non-VUS classification is a non-expert Likely pathogenic research submission.

Sources

Reasoning

The variant is CHEK2 NP_009125.1:p.(Lys141Thr), but the available evidence does not establish a previously established pathogenic variant producing the same Lys141Thr amino-acid change by another nucleotide substitution. ClinVar reports this exact variant as uncertain significance, and the reviewed papers do not report the exact variant as a pathogenic same-amino-acid comparator. The coding change is missense and SpliceAI predicts no significant splice impact (maximum delta score 0.009), but that does not establish PS1.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• Variant normalization: NM_007194.4:c.422A>C produces NP_009125.1:p.(Lys141Thr).
• ClinVar exact variation record 186751 classifies the variant as uncertain significance; no same-amino-acid pathogenic comparator is documented in the supplied evidence.
• SpliceAI result: maximum delta score 0.009, reported as no significant splice impact.

Still missing

• A validated pathogenic comparator with the same p.Lys141Thr amino-acid change is not available.

Sources

Reasoning

PS2 cannot be assessed because the available case evidence does not document a proband with a disease phenotype and no family history together with confirmed maternity and paternity. No parental testing, de novo status, meiosis information, or relevant variant-specific segregation report is available.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• The case materials provide no documented proband-parent genotypes, confirmed maternity and paternity, de novo observation, or family-history assessment for CHEK2 NM_007194.4:c.422A>C (p.Lys141Thr).

Still missing

• Documented proband disease phenotype and family-history assessment
• Variant testing results for both biological parents
• Confirmed maternity and paternity

Reasoning

No reviewed publication provides exact-variant case and control counts, an odds ratio, or a confidence interval for CHEK2 NM_007194.4:c.422A>C (p.Lys141Thr). The literature_pass.json ai_classified_papers list is empty. PMID:25186627 reports aggregate CHEK2 mutations in breast-cancer cohorts without mentioning c.422A>C; PMID:27616075 reports other CHEK2 variants and does not mention c.422A>C; PMID:34903604 and PMID:37449874 provide functional or group-level risk information without a case-control result for p.Lys141Thr; PMID:25356965 is a general policy statement. gnomAD v4.1 reports 5/1,180,028 European non-Finnish alleles (AF 4.23719e-06), but no affected-case frequency is available, so an OR and 95% CI cannot be computed. PS4 therefore remains not assessed rather than being inferred from gene-level or aggregate findings.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• gnomAD v4.1: European (non-Finnish) population count 5/1,180,028 alleles, AF 4.23719e-06; this is a control-population frequency only and cannot yield an OR or 95% CI without an affected-case frequency.
• PMID:25186627, Results and Table 4: reports aggregate CHEK2 mutations in 2,158 individuals with breast cancer, including 29 and 5 CHEK2-positive patients in two cohorts, but does not mention c.422A>C or p.Lys141Thr.
• PMID:27616075: reports other CHEK2 variants in a 581-patient breast/ovarian cancer cohort, including c.1100delC, c.592+3A>T, and p.Ile157Thr, but does not mention c.422A>C or p.Lys141Thr.
• PMID:34903604: directly studies p.Lys141Thr in a functional assay, but the extracted result is functional and not a case-control prevalence comparison.
• PMID:37449874: reports group-level breast-cancer risk for functionally impaired CHEK2 missense variants, but does not provide a variant-specific case-control result for p.Lys141Thr.
• PMID:25356965: general ACMG policy statement with no variant-specific CHEK2 evidence; literature_pass.json ai_classified_papers is empty.

Still missing

• Exact-variant affected-case count and denominator.
• Matched control or population count and denominator suitable for a case-control comparison.

Sources

Reasoning

CHEK2 has no entry in vcep_materials.json's domain_tables. However, the reviewed CHEK2 functional-analysis paper identifies the FHA domain as a characteristic domain spanning residues 92–205, which includes residue 141. The supplied evidence does not establish that this domain is a PM1-qualifying critical functional domain without benign variation, and the supplied hotspot result is negative. Therefore PM1 cannot be concluded from the available evidence.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• vcep_materials.json, domain_tables: no CHEK2 entry; the CHEK2 specification is listed without released criteria.
• Boonen et al. 2022, PMID:34903604, Introduction: CHK2's FHA domain is described as spanning residues 92–205; residue 141 is within that interval.
• Cancer Hotspots result: this variant does not lie in a statistically significant hotspot.
• The supplied CHEK2 CSPEC record has criteria_spec_found=false and provides no PM1 region definition.

Still missing

• A validated PM1 assessment that the FHA domain is critical and lacks benign variation is not available.
• A CHEK2 hotspot route is not documented.

Sources

Reasoning

The target variant changes Lys141 to Thr, but no validated pathogenic or likely pathogenic CHEK2 missense comparator at residue 141 is documented in the supplied PM5 candidate file or paper extracts. The PM5 candidate artifact contains zero same-residue candidates and explicitly cannot confirm classic PM5 semantics, so PM5 cannot be applied.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• pm5_candidates.json, residue K141: total_candidates_seen=0 and same_residue_candidates=0; the artifact recommends review rather than applying PM5.
• ClinVar exact variation record 186751 concerns only CHEK2 c.422A>C:p.Lys141Thr and does not document a qualifying different missense comparator.

Still missing

• A validated pathogenic different-missense comparator at residue 141 is not available.

Sources

Reasoning

PM6 cannot be assessed because the available case evidence does not document an assumed de novo occurrence in the proband. There is no report of parental testing, parentage status, or a variant-specific de novo observation without confirmed maternity and paternity.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• The case materials provide no variant-specific report of an assumed de novo CHEK2 NM_007194.4:c.422A>C (p.Lys141Thr) event and no parental testing data.

Still missing

• Variant-specific de novo observation in the proband
• Available parental testing or parentage information

Reasoning

PP1 cannot be assessed because the available case evidence does not document multiple affected family members carrying the variant, genotype-phenotype concordance, informative meioses, or a segregation analysis in a disease for which CHEK2 is definitively causal. The available literature extracts are functional or aggregate cohort reports and do not provide relevant family segregation data for this variant.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• The available variant-specific paper extract for PMID:34903604 reports functional testing of p.K141T but no family segregation, affected relatives, or meioses.
• The available aggregate cohort paper extracts do not report variant-level family segregation for CHEK2 NM_007194.4:c.422A>C (p.Lys141Thr).

Still missing

• Affected relatives with confirmed disease phenotypes
• Variant genotypes for affected and unaffected relatives
• Informative meioses or a quantitative segregation analysis

Sources

Reasoning

The supplied evidence supports CHEK2 loss of function as a disease mechanism and notes that CHEK2 missense interpretation is challenging, but it does not establish both PP2 requirements: a low rate of benign missense variation and missense variants as a common disease mechanism. No gene-level quantitative benign-missense metric or governing threshold for those requirements is supplied, so PP2 is not assessed.

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.

Evidence considered

• pvs1_gene_context.json states that CHEK2 loss of function is supported as a germline disease mechanism, which addresses neither PP2 requirement by itself.
• Boonen et al. 2022, PMID:34903604, reports that truncating CHEK2 variants are known to be pathogenic and that missense VUS interpretation is challenging; it does not establish the PP2 gene-level prerequisites.

Still missing

• A validated gene-level assessment of benign missense variation is not available.
• Evidence that missense variants are a common CHEK2 disease mechanism is not established.

Sources

Reasoning

The supplied case evidence does not provide a patient phenotype, family history, or a sufficiently specific clinical presentation for this exact variant. The available literature concerns aggregate cancer cohorts or laboratory studies and cannot substitute for the patient's phenotype or family history. No disease-specific phenotype assessment can therefore be made.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• The case summary contains variant normalization and population/literature evidence but no patient phenotype or family-history description.
• The reviewed publications do not provide a phenotype assessment for the individual carrying CHEK2 c.422A>C.

Still missing

• Patient's clinical phenotype and confirmed diagnosis.
• Relevant family history and evidence that the presentation is highly specific for CHEK2-related disease.

Reasoning

The available population data show no homozygotes in gnomAD v4.1, but BS2 for this autosomal-dominant, cancer-predisposition context requires observation in a healthy adult individual. Aggregate gnomAD counts do not document the age, phenotype, or adult health status of any of the five heterozygous carriers, so the criterion cannot be assessed from these data.

Rule

BS2: Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v4.1 all-comers record: 5 total alleles among 1,614,184 observed alleles and zero homozygotes; the record provides aggregate counts only, not carrier-level age or phenotype data.
• The governing framework identifies CHEK2-related cancer predisposition as autosomal dominant, but the available record does not establish the healthy-adult and full-penetrance conditions required by BS2.

Still missing

• No individual-level age and phenotype information is available to establish a healthy adult heterozygous carrier.
• No documented full-penetrance early-onset disease model is available for applying BS2 to this cancer-predisposition context.

Sources

Reasoning

BS4 cannot be assessed because the available case evidence does not identify affected family members who lack the variant. No family pedigree, relative phenotypes, relative genotypes, or informative non-segregation observations are documented; therefore the phenocopy caveat cannot be evaluated.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The case materials provide no pedigree, affected-relative genotypes, or variant-specific non-segregation observation for CHEK2 NM_007194.4:c.422A>C (p.Lys141Thr).

Still missing

• Affected family members with documented clinical phenotypes
• Variant testing results for affected relatives
• Pedigree information sufficient to evaluate phenocopies and informative non-segregation

Reasoning

No affected-proband or family-level evidence documents a pathogenic variant on the other allele, and no parental or offspring testing establishes cis or trans phase for NM_007194.4:c.422A>C (p.Lys141Thr). The available literature extraction includes no variant-specific segregation or allelic-phase observation. Therefore, BP2 cannot be assessed from the current evidence; the cis pathway remains unresolved, and the trans pathway additionally requires a fully penetrant dominant gene/disorder.

Rule

BP2: 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.

Evidence considered

• The case evidence documents CHEK2-related cancer predisposition as autosomal dominant but does not document a fully penetrant dominant disorder for the BP2 trans pathway.
• No case-level affected-proband observation, pathogenic second allele, cis/trans phase result, or segregation result is documented for NM_007194.4:c.422A>C (p.Lys141Thr).
• The reviewed variant-specific paper extraction for PMID:34903604 reports an intermediate functional result for p.K141T but no allelic phase, pathogenic partner, or segregation observation; it therefore does not support BP2.

Still missing

• Documented pathogenic variant in the same individual and its exact phase relative to NM_007194.4:c.422A>C.
• Parental or offspring testing, or equivalent molecular phasing, to establish cis or trans status.
• For a trans-based BP2 path, evidence that the relevant gene/disorder is fully penetrant and dominant.

Sources

Reasoning

No case evidence identifies this exact variant in an affected individual who has an alternate molecular basis for disease. The available breast-cancer cohort papers do not provide an exact-variant BP5 observation, and the functional paper does not establish an alternate molecular diagnosis for a case.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• PMID:25186627 and PMID:27616075 report aggregate or other-variant cancer-panel findings without an exact-variant alternate molecular diagnosis.
• PMID:34903604 evaluates p.Lys141Thr functionally but does not report an affected case with an alternate molecular basis.

Still missing

• A case report or clinical record documenting the exact variant together with an established alternate molecular cause of disease.

Sources