CHEK2 c.470T>C, p.Ile157Thr

Reasoning

PS3 is met at supporting strength for CHEK2 p.Ile157Thr. Two variant-specific functional studies provide biologically relevant evidence of impaired CHEK2 function: the I157T mutant failed to bind and efficiently phosphorylate Cdc25A, with failure of the ionizing-radiation-induced S-phase checkpoint and radioresistant DNA synthesis (PMID:11298456), and failed to bind BRCA1, CDC25A, and p53 in cellular pull-down assays despite preserved phosphopeptide binding and ATM-dependent Thr68 phosphorylation (PMID:12049740). These are direct assays of a functionally relevant CHEK2 FHA-domain activity with cellular DNA-damage-response readouts. Strength is not increased because another variant-specific study found I157T comparable to wild type across its assays, and the available studies are not shown to be validated, reproducible clinical diagnostic assays.

Rule

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

Evidence considered

• Falck et al. 2001 (PMID:11298456): CHEK2 I157T could not bind HA-Cdc25A or efficiently phosphorylate GST-Cdc25A; cells expressing I157T failed to impose the ionizing-radiation-induced S-phase blockade and showed radioresistant DNA synthesis.
• Li et al. 2002 (PMID:12049740): I157T FHA domains failed to bind BRCA1, CDC25A, or p53 in cellular assays, although phosphopeptide binding and ATM-dependent Thr68 phosphorylation were preserved.
• Wu et al. 2001 (PMID:11053450): I157T behaved similarly to wild-type Chk2 in the assays used, providing conflicting functional evidence and limiting PS3 strength.

Still missing

• No evidence that the assays were validated and shown reproducible in a clinical diagnostic laboratory setting.
• Functional results are discordant across assay systems and require human adjudication for final strength calibration.

Sources

Reasoning

CHEK2 p.Ile157Thr lies within the FHA domain, a well-established functional protein-interaction domain. PMID:12049740 places Ile157 on an FHA-domain interaction surface and reports failure of the I157T domain to bind BRCA1, CDC25A, and p53 in cellular assays; PMID:11298456 reports loss of Cdc25A binding and impaired checkpoint function for I157T. No CHEK2 VCEP domain table or complete benign-variation analysis for the FHA domain is present, so the PM1 application depends on the generic domain route and should receive human confirmation.

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

• PMID:12049740: Ile157 is located on the CHEK2 FHA-domain surface; I157T failed to bind BRCA1, CDC25A, and p53 in cellular pull-down assays.
• PMID:11298456: I157T is a cancer-associated mutant in the FHA protein-interaction domain and could not bind Cdc25A or efficiently phosphorylate Cdc25A, with defective checkpoint consequences.
• cspec: CHEK2 is listed in a ClinGen specification record, but criteria_spec_found is false and domain_tables is empty; generic ACMG/AMP governs.

Still missing

• No released CHEK2 VCEP PM1 specification or authoritative domain table is available.
• A complete assessment of benign missense variation across the CHEK2 FHA domain is not supplied.

Sources

Reasoning

The variant-specific Finnish hereditary prostate-cancer study directly reports lack of segregation: mutation-negative patients with prostate cancer were observed, alongside unaffected mutation carriers. This meets the stated lack-of-segregation observation, but the ACMG caveat requires caution because cancer phenocopies can mimic non-segregation and the study does not resolve that issue for each family.

Rule

Lack of segregation in affected members of a family. ... Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals.

Evidence considered

• The study reports incomplete segregation of I157T with prostate cancer, including mutation-negative patients with prostate cancer and unaffected mutation carriers.
• The evidence is from Finnish hereditary prostate-cancer families, with nine families having two affected members, three having three, and one having four.

Still missing

• Family-level clinical adjudication of possible cancer phenocopies is not provided.
• The number of mutation-negative affected relatives and their individual diagnoses are not fully enumerated in the extracted evidence.

Sources

Reasoning

The variant is CHEK2 p.Ile157Thr (c.470T>C), and the literature establishes this same nucleotide and amino-acid change, but no previously established pathogenic variant with the same amino-acid change caused by a distinct nucleotide change is documented. The PS1 rule is intended to use an established pathogenic same-amino-acid substitution independently of the observed nucleotide change; the available reports of p.Ile157Thr do not provide that independent codon-level observation.

Rule

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

Evidence considered

• PMID:11053450: the paper explicitly studied CHEK2 I157T but did not state c.470T>C; it therefore confirms the amino-acid change but not an independent nucleotide change.
• PMID:11298456: I157T was studied as a cancer-associated CHEK2 mutant, with no alternate nucleotide encoding the same amino-acid change reported in the extracted evidence.
• PMID:14612911: reports CHEK2 c.470T>C (I157T), the same nucleotide change as the case variant.
• PMID:12049740: studies CHEK2 I157T, without documenting a distinct nucleotide change producing p.Ile157Thr.

Still missing

• A confirmed pathogenic CHEK2 nucleotide change different from c.470T>C that produces p.Ile157Thr would be needed for PS1.

Sources

Reasoning

Generic ACMG/AMP PS4 requires an odds ratio or relative risk >5.0 with a confidence interval that does not include 1.0. PMID:14612911 directly reports an exact-variant case-control comparison: CHEK2 I157T occurred in 13/120 Finnish hereditary prostate cancer patients (10.8%) versus 26/480 population controls (5.4%), OR=2.12, 95% CI 1.06-4.27, P=0.04. The paper therefore provides case-control enrichment and the reported CI excludes 1.0, but the required comparison is 2.12 > 5.0? no, so PS4 is not met. The literature packet also identifies exact-variant case-control evidence in PMID:15239132 (7.4% vs 5.3%, OR 1.43, 95% CI 1.06-1.95), PMID:16816021 (OR 1.5, 95% CI 1.1-2.1), PMID:22799331 (OR 1.48, 95% CI 1.31-1.66), PMID:23713947 (OR 1.39), PMID:23296741, and PMID:24599715 (OR 2.2); these additional reported effects are likewise below 5.0 or lack a documented threshold-satisfying estimate in the supplied evidence. Exact-variant ClinVar submissions are not expert-panel classifications and are not used as PS4 evidence.

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

• PMID:14612911, Results paragraph and Table 2: exact CHEK2 I157T was present in 13/120 hereditary prostate cancer patients (10.8%) versus 26/480 population controls (5.4%), with OR=2.12, 95% CI 1.06-4.27, P=0.04.
• The reported 95% CI excludes 1.0, but the governing threshold comparison is explicitly 2.12 > 5.0? no; therefore the PS4 threshold is not satisfied.
• The literature packet's already-read abstract leads were checked for the exact variant and case-control direction: PMID:15239132 reports 7.4% versus 5.3%, OR 1.43, 95% CI 1.06-1.95; PMID:16816021 reports OR 1.5, 95% CI 1.1-2.1; PMID:22799331 reports OR 1.48, 95% CI 1.31-1.66; PMID:23713947 reports OR 1.39; PMID:24599715 reports OR 2.2; PMID:23296741 reports higher case frequency than controls without a supplied threshold-satisfying estimate.
• The CHEK2 VCEP is listed without released criteria, so generic ACMG/AMP rules govern.

Sources

Reasoning

PM2 is not met because the variant is clearly present in population controls. Using the generic all-comers default, gnomAD v4.1 reports AF 0.0027619 (4458/1614104 alleles; 37 homozygotes), which is greater than the supplied PM2 supporting threshold of <=0.0001.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v4.1 all-comers: AF 0.0027619 (4458/1614104 alleles; 37 homozygotes).
• No governing VCEP population-source rule was released for CHEK2, so the generic all-comers gnomAD default was used rather than a non-cancer or exome-only subset.
• Generic calibration supplied for this case: PM2 supporting threshold <=0.0001, ClinGen SVI recommendation (identified in the case prompt with ACMG/AMP 2015 PMID:25741868).

Sources

Reasoning

The available evidence documents CHEK2 p.Ile157Thr itself, but does not document a different missense substitution at residue Ile157 that has been established as pathogenic. The reported R145W comparator is at residue 145, not residue 157, and therefore cannot satisfy PM5 for this variant.

Rule

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

Evidence considered

• PMID:11298456: reports R145W and I157T together, confirming that R145W is at residue 145 rather than residue 157; no alternate pathogenic I157 missense change is reported in the extracted evidence.
• PMID:12049740: discusses R117A, R145W, and I157T as separate FHA-domain variants, without a different pathogenic substitution at Ile157.
• PMID:11053450 and PMID:14612911: document I157T itself, not a different missense change at residue 157.

Still missing

• A different CHEK2 missense change at residue 157 with an established pathogenic classification would be needed for PM5.

Sources

Reasoning

The variant-specific Finnish hereditary prostate-cancer study reports 13/120 hereditary prostate-cancer patients carrying I157T and describes 9 positive families with two affected members, 3 with three, and 1 with four; however, segregation was incomplete, with unaffected carriers and mutation-negative affected patients. This does not establish the required disease co-segregation for PP1.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... Note: May be used as stronger evidence with increasing segregation data.

Evidence considered

• CHEK2 c.470T>C (I157T) was identified in 13/120 hereditary prostate-cancer patients (10.8%); nine positive families had two affected members, three had three, and one had four.
• The study explicitly reports incomplete segregation, observing both unaffected mutation carriers and mutation-negative patients with prostate cancer.

Sources

Reasoning

The required combination is not supported: the case contains no gene-level measurement showing a low rate of benign CHEK2 missense variation, and the gnomAD record shows p.Ile157Thr at AF 0.27619% with 37 homozygotes in gnomAD v4.1. The functional literature supports a disease-relevant missense mechanism for I157T, but that does not establish the required low benign-missense rate for PP2.

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

• gnomAD v4.1: CHEK2 c.470T>C (p.Ile157Thr) has AF 0.27619% (4458/1614104 alleles) and 37 homozygotes.
• PMID:11298456 and PMID:12049740: demonstrate functional consequences for the I157T missense variant, but do not provide a gene-wide benign-missense constraint metric.

Still missing

• A gene-level benign-missense constraint analysis and an explicit demonstration that missense variation is a common CHEK2 disease mechanism are not supplied.

Sources

Reasoning

Generic ACMG/AMP rules apply because the CHEK2 VCEP specification is listed but has no released criteria. NM_007194.4:c.470T>C is a missense variant producing NP_009125.1:p.(Ile157Thr), so the applicable computational path is REVEL only; SpliceAI is not used for this missense PP3/BP4 assessment. REVEL is 0.538, which is below the generic PP3 supporting threshold of >= 0.644 and therefore lies in the indeterminate gray zone. No PP3 strength is assigned.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.)...Missense variant -> REVEL only...PP3 (supporting): >= 0.644 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence: missense, NM_007194.4:c.470T>C encoding NP_009125.1:p.(Ile157Thr) (pvs1_variant_assessment).
• Local REVEL predictor lookup: REVEL score 0.538 for NM_007194.4:c.470T>C (revel).
• ClinGen SVI REVEL calibration supplied for this case: PP3 supporting requires REVEL >= 0.644; Pejaver et al. 2022, Am J Hum Genet (PMID:36413997).
• The missense-specific REVEL path was used; the available SpliceAI result was not counted because the governing rule assigns missense variants to REVEL only.

Sources

Reasoning

Generic PP5 is limited here to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. ClinVar variation 5591 has zero expert-panel submissions; its Pathogenic and Likely pathogenic records are ordinary laboratory, literature-only, or other non-expert assertions. Therefore PP5 is not met.

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 5591 is an exact match for NM_007194.4:c.470T>C (p.Ile157Thr), but the submission audit reports expert_panel_submissions=0; available Pathogenic/Likely pathogenic assertions are non-expert submissions.

Sources

Reasoning

BA1 is not met. Under the generic ACMG/AMP stand-alone threshold of >=0.05, the highest observed all-comers or ancestry-specific frequency is gnomAD v4.1 AF 0.0257568 in the Finnish population (1649/64022 alleles); the overall AF is 0.0027619 (4458/1614104). Both are below 0.05.

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v4.1 all-comers: overall AF 0.0027619 (4458/1614104 alleles; 37 homozygotes); highest population AF 0.0257568 in European (Finnish) individuals (1649/64022 alleles; 19 homozygotes).
• Generic calibration supplied for this case: BA1 stand-alone threshold >=0.05, ACMG/AMP 2015 (PMID:25741868).

Sources

Reasoning

BS3 is not met. PMID:11053450 reported that I157T behaved similarly to wild-type Chk2 in the assays used, including kinase activity, gamma-radiation activation, Cdc25C phosphorylation, Thr-68 phosphorylation, nuclear localization, and size-exclusion chromatography. However, this benign result is contradicted by variant-specific studies showing defective Cdc25A interaction and DNA-damage checkpoint function (PMID:11298456) and defective binding to BRCA1, CDC25A, and p53 (PMID:12049740). The available evidence therefore does not establish that well-established functional studies show no damaging effect, and no validated clinical diagnostic assay series is documented.

Rule

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

Evidence considered

• Wu et al. 2001 (PMID:11053450): the I157T Chk2 mutant behaved similarly to wild type in all assays used, including kinase activity and cellular DNA-damage-response measurements.
• Falck et al. 2001 (PMID:11298456) and Li et al. 2002 (PMID:12049740) reported damaging effects in biologically relevant substrate-binding and DNA-damage-response assays, preventing a BS3 conclusion.

Still missing

• No concordant, validated functional assay series demonstrating absence of damaging effect.
• Discordant assay results require human adjudication.

Sources

Reasoning

CHEK2 is known to have truncating disease-associated variants, but the available evidence also establishes a biologically consequential missense mechanism for p.Ile157Thr: I157T disrupts CHEK2 interactions with Cdc25A and other targets and impairs checkpoint function. Therefore the evidence does not support treating CHEK2 as a gene in which primarily truncating variants, to the exclusion of a recognized missense mechanism, cause disease.

Rule

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

Evidence considered

• PMID:11298456: I157T could not bind or efficiently phosphorylate Cdc25A and caused defective checkpoint phenotypes in cell assays.
• PMID:12049740: I157T failed to bind BRCA1, CDC25A, and p53 in cellular assays, supporting a functional missense mechanism.
• PMID:14612911: reports I157T in hereditary prostate cancer families and cases, with OR=2.12 versus population controls, while noting incomplete segregation.

Still missing

• A gene-wide quantitative analysis establishing that CHEK2 disease is primarily truncating rather than missense-mediated is not supplied.

Sources

Reasoning

Generic ACMG/AMP rules apply because the CHEK2 VCEP specification is listed but has no released criteria. NM_007194.4:c.470T>C is a missense variant producing NP_009125.1:p.(Ile157Thr), so the applicable computational path is REVEL only; SpliceAI is not used for this missense PP3/BP4 assessment. REVEL is 0.538, which is above the generic BP4 supporting threshold of <= 0.29 and therefore lies in the indeterminate gray zone. BP4 is not met.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)...Missense variant -> REVEL only...BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence: missense, NM_007194.4:c.470T>C encoding NP_009125.1:p.(Ile157Thr) (pvs1_variant_assessment).
• Local REVEL predictor lookup: REVEL score 0.538 for NM_007194.4:c.470T>C (revel).
• ClinGen SVI REVEL calibration supplied for this case: BP4 supporting requires REVEL <= 0.29; Pejaver et al. 2022, Am J Hum Genet (PMID:36413997).
• The missense-specific REVEL path was used; the available SpliceAI result was not counted because the governing rule assigns missense variants to REVEL only.

Sources

Reasoning

Generic BP6 is limited here to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. ClinVar variation 5591 has zero expert-panel submissions and no exact-variant expert-panel Benign or Likely benign classification. Non-expert submissions and aggregate labels cannot trigger BP6, so BP6 is not met.

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 5591 is an exact match, but the submission audit reports expert_panel_submissions=0 and does not provide an expert-panel Benign or Likely benign classification.

Sources

Reasoning

No case-specific evidence documents a de novo CHEK2 c.470T>C observation in an affected proband, confirmation of both maternity and paternity, or absence of family history. The available variant-specific family study concerns inherited familial prostate-cancer observations and does not establish de novo origin.

Rule

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

Evidence considered

• No case-specific proband or parental-testing record in the reviewed evidence documents de novo origin for CHEK2 c.470T>C.

Still missing

• Affected proband-level de novo finding is not documented.
• Both maternity and paternity confirmation are not documented.
• Family history status is not documented.

Reasoning

No case-specific evidence documents an assumed de novo CHEK2 c.470T>C finding in an affected proband. The available literature describes familial and population observations, not a de novo observation without parental confirmation.

Rule

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

Evidence considered

• No case-specific proband or parental-testing record in the reviewed evidence documents assumed de novo origin for CHEK2 c.470T>C.

Still missing

• Affected proband-level assumed de novo finding is not documented.
• Parental testing status is not documented.

Reasoning

The governing PP4 definition requires a patient's phenotype or family history highly specific for a disease with a single genetic etiology. The supplied case evidence identifies the variant and literature associations with multiple cancers, but does not provide this patient's phenotype or family history, nor a highly specific single-etiology presentation. The criterion cannot be assessed from the available patient-level evidence.

Rule

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

Evidence considered

• The supplied case summary contains variant and gene information but no patient phenotype or family-history description suitable for PP4.

Still missing

• Patient phenotype and family history specific to a single genetic disease etiology are not available.

Reasoning

BS1 is not_assessed rather than applied from the generic 1% default. gnomAD v4.1 shows overall AF 0.0027619 and Finnish AF 0.0257568, while CHEK2 I157T is described as a low-penetrance/risk allele and the Finnish study found 5.4% in population controls. A maximum-credible-frequency calculation incorporating disease prevalence, penetrance, and allelic heterogeneity was not provided, so the ancestry-specific elevation cannot be judged against the disorder's expected frequency.

Rule

Generic rules for BS1: the criterion is 'allele frequency is greater than expected for the disorder'; the 1% figure is only a default.

Evidence considered

• gnomAD v4.1 all-comers: overall AF 0.0027619 (4458/1614104 alleles); Finnish AF 0.0257568 (1649/64022 alleles); grpmax FAF 0.00214388.
• PMID:14612911 reports I157T in 5.4% of Finnish population controls and describes incomplete segregation, including unaffected carriers; this supports reduced/incomplete penetrance rather than permitting automatic use of the generic 1% default.
• Generic BS1 calibration supplied for this case: >=0.01 is only a default; Whiffin et al. 2017 (PMID:28518168) is the named publication for maximum-credible-frequency assessment.

Still missing

• Need a maximum-credible-allele-frequency calculation with explicitly stated CHEK2 disease prevalence, penetrance, allelic heterogeneity, and inheritance assumptions before applying BS1.

Sources

Reasoning

BP2 cannot be assessed from the available evidence. The generic definition requires either observation in trans with a pathogenic variant for a fully penetrant dominant disorder or observation in cis with a pathogenic variant in any inheritance pattern. The available case and literature evidence do not document a pathogenic partner allele, cis/trans phase, or parent/offspring testing for CHEK2 c.470T>C; the single homozygous observation in PMID:14612911 is not evidence of cis phase with a distinct pathogenic variant.

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

• PMID:14612911 reports one homozygous I157T carrier and otherwise heterozygous I157T carriers, but gives no distinct pathogenic partner allele or cis/trans phase information; it also reports incomplete segregation.
• The CHEK2 CSPEC record identifies autosomal dominant inheritance, but no released CHEK2 criterion specifications are available; the generic BP2 definition therefore governs.
• No parent/offspring phase testing or affected-proband observation with a documented pathogenic allele in cis or trans was found in the reviewed evidence.

Still missing

• A documented pathogenic partner variant and validated cis/trans phase in an affected proband, or parent/offspring testing establishing phase, are needed.
• Evidence that any observed dominant CHEK2 disorder is fully penetrant would be needed for the trans pathway.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The available evidence does not document an individual case with a confirmed alternate molecular cause that explains the relevant disease instead of CHEK2 c.470T>C. The criterion is therefore not assessed.

Rule

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

Evidence considered

• The supplied case and literature materials do not document an alternate pathogenic molecular diagnosis in an affected case carrying this exact variant.

Still missing

• A case report with a confirmed alternate molecular basis for the relevant disease is not available.