Classification rationale

PS3PM1PM2 BP4 Unclassified

CHEK2 c.1561C>T · exon NC_000022.10

NM_007194.4:c.1561C>T (p.Arg521Trp) is a missense variant in CHEK2 altering the nuclear localization signal (NLS) at amino acids 515-522.¹ Functional studies in PMID:37449874 directly tested p.R521W and demonstrated severely impaired nuclear localization using high-content immunofluorescence microscopy in human RPE1 cells. The variant was classified as functionally impaired alongside protein-truncating CHEK2 variants.² The ENIGMA CHEK2gether consortium reported that functionally impaired CHEK2 missense variants as a group are associated with moderate breast cancer risk (OR 2.83, 95% CI 2.35-3.41), comparable to the risk of truncating variants.³ The variant is present in gnomAD at very low frequency: v2.1 AF=0.0053% (14/264,616 alleles) and v4.1 AF=0.0045% (72/1,595,990 alleles), with zero homozygotes and highest subpopulation frequency in East Asians (0.058%).⁴ In silico predictors are not supportive of pathogenicity: REVEL=0.205, BayesDel=-0.074, SpliceAI delta=0.00.⁵ ClinVar reports this variant as Uncertain Significance with a 1-star review status (15 clinical laboratories), which does not meet the threshold for PP5 or BP6 application.⁶ Two moderate pathogenic criteria (PM1 for NLS domain disruption, PS3 for functional evidence of impaired nuclear localization) and one supporting pathogenic criterion (PM2 for rarity in population databases) are met. One supporting benign criterion (BP4 for in silico predictions) is also met.⁷

PS3 + PM1 + PM2 + BP4 → Unclassified

1 PMID:37449874

2 PMID:37449874

3 PMID:37449874

4 gnomad_v2 ↗gnomad_v4 ↗

5 revelbayesdelspliceai ↗

6 clinvar ↗

7 PMID:37449874gnomad_v2 ↗gnomad_v4 ↗revelbayesdelspliceai ↗

Gene diagram · NM_007194.4 · variants mapped to exon structure

CHEK2 NM_007194.4

Fetching transcript structure from UCSC…

Applied criteria · 4 applied · 16 assessed

Applied · 4

Strength Supporting Moderate Strong Very strong

✓

PS3 moderate Pathogenic

Functional studies in PMID:37449874 directly tested p.Arg521Trp (R521W) in human RPE1 cells using high-content immunofluorescence microscopy. The variant demonstrated severely impaired nuclear localization, disrupting the nuclear localization signal (NLS) at amino acids 515-522. The authors classified p.R521W as functionally impaired alongside truncating variants and included it in the functionally impaired burden group associated with moderate breast cancer risk (OR 2.83). Kinase activity (KAP1 and CHK2 autophosphorylation assays) was discordant (WT-like/intermediate), but the nuclear mislocalization phenotype was unequivocal and mirrors the effect of protein-truncating CHEK2 variants lacking the NLS.

Direct functional testing of p.R521W in RPE1-CHEK2-KO cells: KAP1 phosphorylation assay (WT/intermediate)CHK2 autophosphorylation assay (intermediate/WT)nuclear-to-cytoplasmic ratio (severely impaired — among only two missense variants with this phenotype). Variant classified as functionally impaired by study authors.

✓

PM1 moderate Pathogenic

The variant p.Arg521Trp is located within the nuclear localization signal (NLS) of CHEK2 at amino acids 515-522, a well-characterized critical functional domain. PMID:37449874 explicitly demonstrated that p.R521W causes severely impaired nuclear localization, phenocopying protein-truncating variants that delete the NLS. The NLS is essential for CHEK2 nuclear import and DNA damage response function.

NLS domain defined at amino acids 515-522 (PMID:37449874 Methods). p.R521W at position 521 directly disrupts this domain. Experimental confirmation of aberrant cytoplasmic localization via high-content microscopymatching truncating variant controls lacking the NLS.

✓

PM2 supporting Pathogenic

This variant is present in gnomAD at very low frequency: v2.1 AF=5.29e-05 (14/264,616 alleles, 0 homozygotes), v4.1 AF=4.51e-05 (72/1,595,990 alleles, 0 homozygotes). Highest subpopulation frequency is East Asian: v2.1 AF=0.000409 (8/19,552), v4.1 AF=0.000579 (26/44,874). All frequencies are well below the PM2 threshold of 0.1%. grpmax FAF=0.0004053.

gnomAD v2.1: 14/264616 alleles (AF=0.0053%)gnomAD v4.1: 72/1

gnomad_v2 ↗gnomad_v4 ↗

✓

BP4 supporting Benign

Multiple in silico predictors suggest a benign effect. REVEL score is 0.205, below the typical pathogenic threshold of 0.5. BayesDel score is -0.074, consistent with a benign prediction. SpliceAI delta score is 0.00, predicting no splice impact. HCI prior not applicable (gene not supported). The collective computational evidence favors a benign interpretation.

REVEL=0.205BayesDel=-0.074 (benign)SpliceAI delta=0.00

spliceai ↗

Assessed · not applied

Pathogenic

PS1 No evidence of a different nucleotide change at the same codon previously established as pathogenic.

PS2 No de novo evidence was identified in any of the reviewed publications or ClinVar submissions for this variant.

PS4 The variant is present in gnomAD (14/264,616 alleles v2.1; 72/1,595,990 alleles v4.1) at very low frequency but no case-control study has demonstrated statistically significant enrichment of this specific variant in affected individuals versus controls.

PM6 No de novo evidence identified for this variant.

PP1 No cosegregation data available for this variant with disease in families.

PP2 CHEK2 has a substantial number of benign missense variants; missense variation is not a rare/unique mechanism of disease for this gene.

PP3 In silico predictors are not supportive of a pathogenic effect.

PP4 No specific phenotypic data are available for individuals carrying this variant.

Benign

BA1 gnomAD allele frequency is far below the BA1 threshold of 1%.

BS1 Allele frequency is below the BS1 threshold of 0.3%.

BS2 No homozygotes observed in gnomAD.

BS3 While the kinase activity assays (KAP1 phosphorylation and CHK2 autophosphorylation) in PMID:37449874 showed discordant WT-like/intermediate results — suggesting no major catalytic defect — the variant was nonetheless categorized as functionally impaired by the study due to severely disrupted nuclear localization.

BS4 No segregation data available showing lack of cosegregation with disease.

BP1 BP1 applies to missense variants in genes where only truncating variants cause disease.

BP2 No observation of this variant in trans with a known pathogenic CHEK2 variant.

BP5 No observation of this variant in an individual with an alternate molecular basis for disease (e.g., a known pathogenic variant in another gene explaining the phenotype).

N/A · 8 PVS1 · PM3 · PM4 · PM5 · PP5 · BP3 · BP6 · BP7

Research & evidence

Population frequency

gnomAD v4.1

gnomAD v2.1

v4.1

This variant is present in gnomAD v4.1 (AF= 4.51131e-05; MAF= 0.00451%, 72/1595990 alleles, homozygotes = 0) and has highest observed frequency in the East Asian population (AF= 0.0005794; MAF= 0.05794%, 26/44874 alleles, homozygotes = 0); grpmax FAF= 0.0004053.

v2.1

This variant is present in gnomAD v2.1 (AF= 5.29069e-05; MAF= 0.00529%, 14/264616 alleles, homozygotes = 0) and has highest observed frequency in the East Asian population (AF= 0.000409165; MAF= 0.04092%, 8/19552 alleles, homozygotes = 0); grpmax FAF= 0.00022085.

🇨🇦 CA

Absent from gnomAD-Canada v1.0.

Allele frequency by ancestry

three datasets · side by side

gnomAD v4.1

0.0045% · 72 / 1,595,990
0 hom · FAF 0.041%

East Asian 26 / 44,874	0.058%
Remaining individuals 3 / 62,202	0.0048%
South Asian 4 / 90,962	0.0044%
African/African American 3 / 74,954	0.004%
European (non-Finnish) 35 / 1,179,442	0.003%
Admixed American 1 / 59,992	0.0017%

+ 4 not observed (European (Finnish), Amish, Middle Eastern, Ashkenazi Jewish)

gnomAD v2.1

0.0053% · 14 / 264,616
0 hom · FAF 0.022%

East Asian 8 / 19,552	0.041%
African/African American 1 / 23,290	0.0043%
European (non-Finnish) 5 / 123,944	0.004%

+ 5 not observed (Admixed American, Ashkenazi Jewish, European (Finnish), Remaining individuals, South Asian)

gnomAD Canada 🇨🇦

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD v4 ↗ gnomAD v2 ↗ gnomAD 🇨🇦 ↗

ClinVar

In progress — evidence not uploaded yet.

ClinVar ↗

In silico

In progress — evidence not uploaded yet.

SpliceAI ↗

Functional Unknown Oncogenic Effect

In progress — evidence not uploaded yet.

OncoKB ↗

COSMIC

Cancer hotspots

Somatic evidence Not in COSMIC / hotspots

COSMIC

In progress — evidence not uploaded yet.

Hotspots

This variant does not lie in a statistically significant hotspot.

COSMIC ↗ Hotspots ↗

Sources & reference links

9Sources

CSpec VCEP

ClinVar

gnomAD v2.1

gnomAD v4.1

gnomAD-Canada

SpliceAI

OncoKB

COSMIC

Cancer hotspots

Triaged references · 5 PMIDs not cited in assessment

34991090 ↗ Identification of CHEK2 Germline Mutations in BRCA1/2- and PALB2-Negative Breast and Ovarian Cancer Patients. CLINVAR

37449874 ↗ ENIGMA CHEK2gether Project: A Comprehensive Study Identifies Functionally Impaired CHEK2 Germline Missense Variants Associated with Increased Breast Cancer Risk. CLINVAR

27783279 ↗ Frequency of pathogenic germline mutation in CHEK2, PALB2, MRE11, and RAD50 in patients at high risk for hereditary breast cancer. CLINVAR

28580595 ↗ Mutation screening of 10 cancer susceptibility genes in unselected breast cancer patients. CLINVAR

32980694 ↗ Genetic characterization of pancreatic cancer patients and prediction of carrier status of germline pathogenic variants in cancer-predisposing genes. CLINVAR