CHEK2 c.1420C>T, p.Arg474Cys

Reasoning

PS3 is met at strong strength. Two independent, variant-specific functional studies support a damaging CHEK2 effect. PMID:27900359 tested CHEK2 p.R474C in NIH3T3 cells with wild-type CHEK2 comparison and UV DNA-damage stimulation; the mutant was scarcely expressed or phosphorylated and was unstable and poorly activated. PMID:30851065 tested the exact c.1420C>T/p.Arg474Cys variant in a RAD53/CHEK2-deficient Saccharomyces cerevisiae MMS-induced DNA-damage growth assay, using wild-type CHEK2 and c.1100delC controls, three independent experiments, and four to six replicates per experiment; p.Arg474Cys was categorized as damaging/non-functional. The orthogonal cell-based and in vivo yeast results, relevant DNA-damage assays, explicit controls, and replication provide robust evidence of impaired CHEK2 function, although the studies were not described as validated and reproducible in a clinical diagnostic laboratory setting; therefore no upgrade beyond strong is warranted.

Rule

PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.

Evidence considered

• Kukita et al. (PMID:27900359) tested exact CHEK2 p.R474C in NIH3T3 cells against wild-type CHEK2 with UV exposure; p.R474C was scarcely expressed or phosphorylated and was unstable and poorly activated by DNA damage.
• The same study used two affected siblings, exome sequencing and Sanger confirmation, and a biologically relevant cellular DNA-damage activation readout.
• Functional characterization of CHEK2 variants (PMID:30851065) tested c.1420C>T/p.Arg474Cys in a RAD53/CHEK2-deficient Saccharomyces cerevisiae MMS-induced DNA-damage growth assay against wild-type CHEK2 and c.1100delC controls; the variant was categorized as damaging/non-functional.
• The yeast study reports three independent experiments with four to six replicates each, providing replication and assay controls.

Still missing

• The studies were not described as validated and reproducible in a clinical diagnostic laboratory setting.

Sources

Reasoning

PM2 is met at supporting strength under the supplied generic threshold. The variant is present but extremely rare in the default all-comers gnomAD datasets: v2.1 AF=1.71098e-05 (4/233784 alleles; homozygotes=0) and v4.1 AF=2.00508e-05 (32/1595950 alleles; homozygotes=0). Both are <=0.0001. The non-cancer subsets are concordantly rare, but no released VCEP rule requires them, so the all-comers totals remain the governing default.

Rule

PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 all-comers reports AF=1.71098e-05 from 4/233784 alleles and homozygotes=0.
• gnomAD v4.1 all-comers reports AF=2.00508e-05 from 32/1595950 alleles and homozygotes=0.
• The supplied generic PM2 supporting threshold is <=0.0001; both default all-comers allele frequencies satisfy it.
• The available non-cancer subsets are concordant: gnomAD v2.1 non-cancer exomes reports AF=1.79853e-05 from 4/222404 alleles with homozygotes=0, and gnomAD v3.1 non-cancer genomes reports AF=1.35265e-05 from 2/147858 alleles with homozygotes=0; they were not substituted because no released VCEP criterion requires a non-cancer source.

Sources

Reasoning

PMID:27900359 reports the exact CHEK2 p.R474C variant in two affected siblings from the same Japanese family; both siblings were homozygous for the variant and had multiple primary cancers. This provides co-segregation in multiple affected family members and supports PP1 at supporting strength. No unaffected relatives or additional meioses were reported, so stronger segregation strength is not justified.

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

• Kukita et al. 2016, PMID:27900359: the exact CHEK2 p.R474C variant was identified and confirmed in two affected siblings from a Japanese family; both were homozygous and had multiple primary cancers.
• The report supplies two affected segregating relatives but does not report tested unaffected relatives or additional informative meioses, so only supporting strength is assigned.

Still missing

• Unaffected relatives were not reported as tested.
• Additional informative meioses and quantitative segregation data were not reported.

Sources

Reasoning

The variant is a missense substitution, NM_007194.4:c.1420C>T (NP_009125.1:p.(Arg474Cys)); therefore the governing generic rule selects the REVEL path only, not SpliceAI. The REVEL score is 0.903, meeting the generic ClinGen SVI moderate PP3 threshold of >= 0.773 but not the strong threshold of >= 0.932. BayesDel is not used because no generic ACMG-strength calibration is established for it. The REVEL calibration is attributed to Pejaver et al. 2022 (PMID:36413997).

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 (moderate): >= 0.773 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence: missense, NM_007194.4:c.1420C>T, NP_009125.1:p.(Arg474Cys); the generic rule therefore requires REVEL only.
• Local REVEL predictor lookup: score 0.903.
• ClinGen SVI REVEL calibration: PP3 moderate threshold >= 0.773 and strong threshold >= 0.932, Pejaver et al. 2022, American Journal of Human Genetics, PMID:36413997.
• BayesDel score 0.37439 was not used because the supplied generic rules establish no ACMG-strength calibration for BayesDel.

Sources

Reasoning

The governing ACMG/AMP rule requires a significantly increased prevalence in affected individuals and specifies that an RR or OR from a case-control study must be >5.0 with a confidence interval excluding 1.0. The exact-variant report in PMID:27900359 describes two affected siblings but no unaffected or population control comparison, so no OR/RR or 95% CI can be computed. PMID:30269267 reports the variant in 289/289 successfully sequenced thyroid-cancer tumors, but provides no unaffected or population control group and therefore is not a qualifying case-control comparison. PMID:26094658 includes 104 affected probands and 101 matched unaffected controls, but explicitly does not mention c.1420C>T or p.Arg474Cys, so its cohort-level CHEK2 findings cannot establish PS4 for this exact variant. The threshold operator is >5.0; no valid variant-specific OR/RR value is available, so an explicit numeric inequality cannot be evaluated.

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:27900359 reports the exact CHEK2 p.R474C variant in two affected siblings and gives population-frequency information, but does not provide an unaffected/control comparison or an OR/RR with a 95% CI.
• PMID:30269267, Table 2, reports CHEK2 c.1420C>T/p.R474C in 289/289 successfully sequenced thyroid-cancer tumors, all heterozygous; no unaffected or population control group is reported.
• PMID:26094658, Results/Fig. 3A-B, studied 104 familial breast-cancer probands and 101 matched unaffected controls, but the paper does not mention c.1420C>T or p.R474C, so those counts cannot be used for this exact variant.
• The governing threshold operator is >5.0 for RR or OR, but no valid exact-variant RR/OR value is available for substitution.

Still missing

• A variant-specific case-control dataset with affected and unaffected or population-control counts, or a reported OR/RR and 95% CI, is needed.

Sources

Reasoning

The governing rule requires a patient phenotype or family history highly specific for a disease with a single genetic etiology. The exact variant has been reported in a family with multiple primary lung cancers plus breast, prostate, and colon cancer, and in a thyroid-cancer tumor series; these are heterogeneous cancer phenotypes that are not highly specific for CHEK2-related cancer predisposition or a single genetic etiology. No uniquely characteristic phenotype or family-history pattern is documented for this case.

Rule

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

Evidence considered

• PMID:27900359 reports two siblings with multiple primary lung cancers and additional breast, prostate, colon, and other clinical findings; this is not a phenotype specific to one genetic etiology.
• PMID:30269267 reports p.R474C in a thyroid-cancer tumor cohort, without a distinctive phenotype or disease-specific family-history pattern.

Sources

Reasoning

The governing rule concerns a reputable source reporting the variant as pathogenic when the underlying evidence is unavailable for independent evaluation. Generic PP5 is restricted here to an exact-variant ClinVar expert-panel classification. ClinVar reports zero expert-panel submissions for this exact variant; the available Likely pathogenic records are ordinary research or clinical-laboratory submissions and cannot trigger PP5.

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 exact-variant submission audit for NM_007194.4:c.1420C>T (p.Arg474Cys) reports expert_panel_submissions=0; available Likely pathogenic assertions are from research or clinical laboratories and are not expert-panel assertions.

Sources

Reasoning

BA1 is not met. The generic stand-alone benign threshold is >=0.05. The required filtering metric, gnomAD grpmax FAF, is 7.33e-06 in v2.1 and 1.568e-05 in v4.1, both far below 0.05; the all-comers allele frequencies are 1.71098e-05 (4/233784 alleles) and 2.00508e-05 (32/1595950 alleles), respectively. The CHEK2 specification is listed but has no released criteria, so no VCEP-specific population threshold or source replaces the generic rule. The frequency does not support a stand-alone benign conclusion.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 all-comers reports AF=1.71098e-05 from 4/233784 alleles, homozygotes=0, and grpmax FAF=7.33e-06.
• gnomAD v4.1 all-comers reports AF=2.00508e-05 from 32/1595950 alleles, homozygotes=0, and grpmax FAF=1.568e-05; the popmax point estimate was not substituted for FAF.
• The CHEK2 specification is listed on CSPEC but has no released criteria; generic ACMG/AMP rules therefore govern and do not provide a different population source or threshold.
• The supplied generic BA1 threshold is >=0.05.

Sources

Reasoning

BS1 is not met on the available population data. The generic default threshold is >=0.01, whereas the filtering allele frequency is only 7.33e-06 in gnomAD v2.1 and 1.568e-05 in gnomAD v4.1. These grpmax FAF values are approximately 1,364-fold and 638-fold below 1%, respectively, and the relevant observed-allele denominators exceed 2000. Because the filtering frequencies are far below the default, the data do not trigger a maximum-credible-frequency calculation for applying BS1; popmax point estimates were not used instead of FAF.

Rule

BS1: Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1 all-comers reports grpmax FAF=7.33e-06 and total AF=1.71098e-05 from 4/233784 alleles.
• gnomAD v4.1 all-comers reports grpmax FAF=1.568e-05 and total AF=2.00508e-05 from 32/1595950 alleles.
• The filtering FAF, not the popmax point estimate, was used; the relevant population allele-count denominators exceed the 2000-allele minimum.
• The supplied generic BS1 default is >=0.01; no released CHEK2 VCEP criterion supplies a different disorder-specific threshold.

Sources

Reasoning

BS3 is not met. The available variant-specific functional evidence does not show no damaging effect; instead, PMID:27900359 reports markedly reduced expression and DNA-damage-induced phosphorylation of p.R474C in NIH3T3 cells, and PMID:30851065 categorizes p.Arg474Cys as damaging/non-functional in a replicated MMS-induced DNA-damage survival assay. These findings are directionally incompatible with applying BS3.

Rule

BS3: Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• PMID:27900359 reports that CHEK2 p.R474C was scarcely expressed or phosphorylated after UV exposure and was unstable and poorly activated by DNA damage.
• PMID:30851065 reports that CHEK2 p.Arg474Cys was categorized as damaging/non-functional in a replicated MMS-induced DNA-damage growth assay.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; SpliceAI cannot be substituted for a gray-zone REVEL result. The REVEL score is 0.903, which is above the BP4 supporting threshold of <= 0.29 and therefore does not support no impact. BayesDel is not used because no generic ACMG-strength calibration is established for it.

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.1420C>T, NP_009125.1:p.(Arg474Cys); the generic rule therefore requires REVEL only.
• Local REVEL predictor lookup: score 0.903.
• ClinGen SVI REVEL calibration: BP4 supporting threshold <= 0.29, Pejaver et al. 2022, American Journal of Human Genetics, PMID:36413997; the score is not within the BP4 range.
• The available SpliceAI max delta of 0.017 was not used because a missense variant must be evaluated through the REVEL path and a gray-zone or non-supportive REVEL result does not license substituting SpliceAI.
• BayesDel score 0.37439 was not used because the supplied generic rules establish no ACMG-strength calibration for BayesDel.

Sources

Reasoning

The governing rule requires the variant to be found in a case with an alternate molecular basis for disease. The reviewed exact-variant evidence documents CHEK2 p.R474C in affected individuals and tumor samples but does not identify an alternative pathogenic molecular diagnosis explaining the disease in a case carrying this variant.

Rule

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

Evidence considered

• PMID:27900359 identifies CHEK2 p.R474C in two affected siblings but does not report an alternate molecular diagnosis explaining their cancer phenotype.
• PMID:30269267 reports the variant in thyroid-cancer tumors but does not provide an alternate molecular basis for disease.

Still missing

• A case report with an independently established alternate molecular diagnosis in the same affected individual would be needed.

Sources

Reasoning

The governing rule concerns a reputable source reporting the variant as benign when the underlying evidence is unavailable for independent evaluation. Generic BP6 is restricted here to an exact-variant ClinVar expert-panel classification. ClinVar reports zero expert-panel submissions for this exact variant and no exact-variant expert-panel benign or likely benign assertion is available; ordinary laboratory submissions and aggregate labels do not 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 exact-variant submission audit for NM_007194.4:c.1420C>T (p.Arg474Cys) reports expert_panel_submissions=0 and provides no exact-variant expert-panel benign or likely benign assertion.

Sources

Reasoning

The variant is p.Arg474Cys. PMID:27900359 reports this same amino-acid change, but the paper is not an independent prior pathogenic classification and does not establish a different nucleotide change producing the same amino-acid substitution. The supplied evidence therefore does not establish PS1.

Rule

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

Evidence considered

• Kukita et al. (PMID:27900359) explicitly report CHEK2 p.R474C, the same variant under review, but this paper does not document an alternate nucleotide change producing the same amino-acid substitution or independently establish the prior variant as a pathogenic comparator for PS1.

Still missing

• A validated prior pathogenic variant causing the same amino-acid change through a different nucleotide change is needed.

Sources

Reasoning

No case-specific evidence documents a de novo occurrence with both maternity and paternity confirmed, a disease-consistent phenotype, and no family history. The available variant report describes two affected siblings who were homozygous for p.R474C, not a confirmed de novo event in the present proband.

Rule

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

Evidence considered

• The available case evidence does not document parental testing, maternity confirmation, paternity confirmation, or a proband-specific de novo occurrence.
• PMID:27900359 reports two affected siblings with homozygous p.R474C, which is not sufficient to establish PS2 for this proband.

Still missing

• Proband-level parental genotypes and maternity confirmation are unavailable.
• A documented absence of family history for the proband is unavailable.

Reasoning

CHEK2 p.Arg474Cys is reported within the kinase domain, and CHEK2 is a kinase. However, the generic PM1 rule requires a mutational hotspot or critical, well-established functional domain without benign variation; the supplied evidence does not establish absence of benign variation across the kinase domain, and no CHEK2 domain-table entry is available. The cancer-hotspot source reports no statistically significant hotspot for CHEK2 R474.

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

• Stutz et al. (PMID:30851065) place p.(Arg474Cys) in the CHEK2 kinase domain and list it among kinase-domain variants tested in a yeast DNA-damage assay.
• Aloraifi et al. (PMID:26094658) describe CHEK2 missense variants in conserved protein kinase-like and forkhead-associated domains, but do not establish a PM1 domain boundary or absence of benign variation for CHEK2.
• The Cancer Hotspots result for CHEK2 R474 states that no statistically significant hotspot was found.

Still missing

• Evidence that the CHEK2 kinase domain is a qualifying critical, well-established PM1 domain without benign variation is needed.
• No qualifying CHEK2 R474 hotspot was found in the supplied Cancer Hotspots result.

Sources

Reasoning

The supplied evidence reports CHEK2 p.Arg474Cys itself, but does not document a different missense substitution at Arg474 that has been determined to be pathogenic. The same variant cannot serve as the different missense comparator required for PM5.

Rule

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

Still missing

• A different pathogenic missense variant at amino-acid residue Arg474 is needed.

Reasoning

No case-specific evidence documents an assumed de novo occurrence in the proband. The available literature describes two affected siblings who were homozygous for p.R474C, without establishing that either occurrence was de novo.

Rule

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

Evidence considered

• The available case evidence does not document an assumed de novo origin for the proband.
• PMID:27900359 reports homozygous p.R474C in two affected siblings but does not establish de novo inheritance.

Still missing

• Proband-level evidence supporting a de novo origin is unavailable.
• Parental genotypes and family-history information are unavailable.

Reasoning

The supplied evidence does not establish both components required for PP2: a low rate of benign missense variation in CHEK2 and missense variants as a common disease mechanism. The available CHEK2 loss-of-function context does not quantify benign missense constraint or establish the required missense mechanism.

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.

Still missing

• A gene-level assessment of benign missense variation in CHEK2 is needed.
• Evidence that missense variation is a common CHEK2 disease mechanism is needed.

Reasoning

BS2 cannot be established from the available evidence. The rule requires observation in a healthy adult individual in the relevant inheritance state with full penetrance expected at an early age. gnomAD reports zero homozygotes in v2.1 and v4.1, but absence of homozygotes is not an eligible healthy-adult observation. PMID:27900359 reports p.R474C in two affected siblings and states that no homozygous p.R474 variant was found in healthy individuals; this does not provide a BS2-positive observation. No eligible healthy adult heterozygote or homozygote with sufficient age and phenotype information was identified.

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 v2.1 reports homozygotes=0 among 233784 alleles, and gnomAD v4.1 reports homozygotes=0 among 1595950 alleles.
• Kukita et al. (PMID:27900359) reports the exact p.R474C variant in two affected siblings and states that no homozygous p.R474 variant was found in healthy individuals; this is not a healthy adult carrier observation.
• No healthy adult heterozygote or homozygote observation with age, phenotype, and relevant inheritance-state information was available for this assessment.

Still missing

• A documented healthy adult carrier observation in the relevant CHEK2 inheritance state, with sufficient age and phenotype information, is required to assess BS2.

Sources

Reasoning

No affected family member is documented to lack CHEK2 p.R474C. The available family report describes two affected siblings carrying the variant, while testing of additional affected or unaffected relatives is not reported; therefore lack of segregation cannot be assessed.

Rule

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

Evidence considered

• PMID:27900359 reports the variant in two affected siblings but does not report an affected non-carrier or sufficient broader family testing to establish lack of segregation.
• Because cancer phenocopies can mimic non-segregation, absence of documented segregation data is not treated as BS4 evidence.

Still missing

• Genotypes of additional affected relatives are unavailable.
• The family structure and testing status of unaffected relatives are unavailable.

Sources

Reasoning

The supplied evidence supports CHEK2 loss of function as a disease mechanism, but it does not establish that primarily truncating variants are known to cause disease. Therefore the gene-level premise required for BP1 is not demonstrated.

Rule

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

Still missing

• Gene-level evidence showing that CHEK2 disease is caused primarily by truncating variants is needed.

Reasoning

The available evidence does not establish that CHEK2 NM_007194.4:c.1420C>T (p.Arg474Cys) is in trans with a pathogenic variant, or in cis with a pathogenic variant. PMID:27900359 reports homozygous p.R474C in two affected siblings, but does not identify a different pathogenic allele or provide phase information. Parent, offspring, or allele-specific testing data are therefore needed to assess BP2.

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 final framework records CHEK2-related cancer predisposition as autosomal dominant; no criterion-specific phase rule or phase result is provided.
• Kukita et al. (PMID:27900359) reports homozygous CHEK2 p.R474C in two affected siblings and Sanger confirmation, but no distinct pathogenic variant in cis or trans and no parent/offspring phase testing.

Still missing

• Phase-resolved testing of parents, offspring, or the affected individuals is needed to determine whether a different pathogenic variant is in trans or cis with p.Arg474Cys.

Sources