PLCG2 c.1993C>T, p.Arg665Trp

Reasoning

PS3 is met at strong strength. Two independent, variant-specific studies evaluated PLCG2 p.Arg665Trp (NM_002661.5:c.1993C>T) using complementary functional systems, including PLCG2-deficient and BTK-deficient DT40 cells, transduced HEK293T cells, and primary CLL cells. The assays measured anti-IgM/B-cell-receptor calcium flux, IP3/IP1 production, ERK and AKT phosphorylation, and signaling during ibrutinib exposure. Across models, R665W showed enhanced B-cell-receptor signaling, BTK-independent activity, and resistance to ibrutinib-mediated pathway inhibition; the findings were concordant with its hypermorphic biological effect. This is direct, biologically relevant functional evidence for a damaging gain-of-function effect, although it is not a clinical diagnostic-laboratory assay. The evidence is sufficiently replicated across independent papers, assay modalities, and cellular contexts to support the original PS3 strong code.

Rule

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

Evidence considered

• Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib, Results: PLCG2 p.R665W was identified at relapse and tested in HEK293T and DT40 cells; anti-IgM calcium flux and ERK/AKT phosphorylation assays showed enhanced signaling and reduced ibrutinib inhibition.
• Hypermorphic mutation of phospholipase C, gamma 2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation, Results and Table 1: PLCG2 p.R665W was identified in three relapsed CLL patients and evaluated in PLCG2-deficient and BTK-deficient DT40 cells, transduced 293T cells, and primary CLL cells using calcium-flux, IP3/IP1, immunoblot, and kinase-inhibitor assays; the variant amplified BCR signaling and bypassed BTK.
• The two studies provide concordant, variant-specific gain-of-function results across independent cell models and orthogonal signaling readouts, with direct relevance to PLCG2-mediated B-cell-receptor signaling.

Sources

Reasoning

PM2 is met at supporting strength under the supplied ClinGen SVI generic threshold. The variant is absent from gnomAD v2.1 and present only twice in gnomAD v4.1, with total AF = 1.24e-06 and grpmax FAF = 2.8e-07 (0.000028%), which is below the supplied PM2 supporting threshold of 0.0001. No homozygotes are reported. The v4.1 rare observations do not prevent PM2 under the specified extremely-low-frequency threshold.

Rule

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

Evidence considered

• gnomAD v2.1 reports the variant as absent.
• gnomAD v4.1 reports 2/1,612,904 total alleles, total AF = 1.24e-06, zero homozygotes, and grpmax FAF = 2.8e-07.
• The supplied generic calibration threshold is PM2 supporting at <= 0.0001; the qualifying grpmax FAF of 2.8e-07 is below that threshold.
• The gnomAD v4.1 exome grpmax FAF is 3.0e-07 and the joint grpmax FAF is 2.8e-07; the joint filtering value is used because no governing VCEP specifies an alternate non-cancer or exome-only source.

Sources

Reasoning

The variant is a missense substitution, NM_002661.5:c.1993C>T (p.Arg665Trp), so the governing generic rule requires the missense REVEL path only; SpliceAI is not used for PP3/BP4 in this situation. REVEL is 0.746, which meets the published generic PP3 supporting threshold of >=0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997). BayesDel is not used because no generic ACMG-strength calibration is established.

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_002661.5:c.1993C>T produces NP_002652.2:p.(Arg665Trp), in exon 19.
• Applicable predictor path is REVEL only for a missense variant; SpliceAI is not counted for PP3/BP4 here.
• Local REVEL lookup reports score 0.746.
• The supplied generic calibration assigns PP3 supporting at REVEL >=0.644; threshold source: ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997.
• BayesDel score 0.140822 was not used because the supplied rules state that no generic ACMG-strength calibration is established for BayesDel.

Sources

Reasoning

The queried variant is PLCG2 p.Arg665Trp, and two papers report the same amino-acid change, but they describe it as a somatically acquired resistance mutation in relapsed CLL and do not establish it as a pathogenic germline variant. Therefore the ACMG/AMP PS1 requirement for a previously established pathogenic variant is not met.

Rule

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

Evidence considered

• PMID:24869598 paper extract: PLCG2 p.R665W was identified at relapse in CLL with acquired ibrutinib resistance; the study reports functional gain of signaling but does not establish a germline pathogenic classification.
• PMID:25972157 paper extract: PLCG2 p.R665W was acquired during ibrutinib therapy in three relapsed CLL patients and characterized as hypermorphic; this is somatic cancer evidence rather than an established pathogenic germline record.

Sources

Reasoning

PP5 is not met. ClinVar variation 376213 contains one single-submitter laboratory assertion of uncertain significance with criteria provided and no expert-panel submission; it is not an exact-variant pathogenic or likely pathogenic expert-panel classification. OncoKB labels the variant oncogenic in a somatic/functional context, not as a reputable exact-variant germline pathogenic or likely pathogenic ClinVar expert-panel assertion. Thus the required generic PP5 source classification is absent.

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 376213: NM_002661.5(PLCG2):c.1993C>T (p.Arg665Trp), uncertain significance, criteria provided/single submitter, one submission, zero expert-panel submissions.
• OncoKB reports an oncogenic label and gain-of-function context supported by somatic CLL resistance literature; this is not an exact-variant ClinVar expert-panel pathogenic or likely pathogenic classification.

Sources

Reasoning

BA1 is not met. The governing default is an allele frequency of at least 5%. The gnomAD v4.1 filtering frequency, grpmax FAF, is 2.8e-07 (0.000028%), based on 2 observed alleles in 1,612,904 total alleles; the highest point estimate is only 1.69543e-06 in European non-Finnish individuals. The qualifying filtering frequency is therefore many orders of magnitude below 0.05. gnomAD v2.1 reported the variant as absent. No population-frequency basis supports a stand-alone benign call.

Rule

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

Evidence considered

• gnomAD v4.1 reports total AC/AN = 2/1,612,904, total AF = 1.24e-06, and grpmax FAF = 2.8e-07; the filtering allele frequency is the required figure for BA1.
• The gnomAD v4.1 highest subpopulation point estimate is European (non-Finnish) AF = 1.69543e-06 from 2/1,179,642 alleles, but this point estimate is not used for BA1.
• gnomAD v2.1 reports the variant as absent.
• Generic calibration threshold supplied for this case: BA1 >= 0.05, ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).

Sources

Reasoning

BS1 is not met. The generic default threshold supplied for this case is 0.01, and the required gnomAD filtering frequency is grpmax FAF = 2.8e-07 (0.000028%), based on 2/1,612,904 total alleles. This is far below the default frequency considered greater than expected for the disorder, so no BS1 evidence is present. The low frequency also provides no basis to invoke ancestry-specific or reduced-penetrance exceptions.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v4.1 reports grpmax FAF = 2.8e-07 from 2 observed alleles in 1,612,904 total alleles; this filtering allele frequency is below the generic BS1 default of 0.01.
• The European (non-Finnish) point estimate is 1.69543e-06 from 2/1,179,642 alleles, but popmax point estimates are not used for BS1 under the supplied guidance.
• Generic BS1 assessment does not require a maximum-credible-frequency calculation when the qualifying filtering frequency is many orders of magnitude below the default threshold.
• Generic calibration threshold supplied for this case: BS1 >= 0.01; per-gene derivation is preferred (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule requires the missense REVEL path only; the SpliceAI result is not used to establish BP4 for this variant. REVEL is 0.746, which is above the generic BP4 supporting cutoff of <=0.29 and therefore does not support a benign computational effect. The result is not a gray-zone basis for substituting SpliceAI: the applicable REVEL path simply does not meet BP4. BayesDel is not used because no generic ACMG-strength calibration is established.

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_002661.5:c.1993C>T produces NP_002652.2:p.(Arg665Trp), in exon 19.
• Applicable predictor path is REVEL only for a missense variant; the SpliceAI max delta of 0.008 is not counted for BP4.
• Local REVEL lookup reports score 0.746.
• The supplied generic calibration assigns BP4 supporting at REVEL <=0.29; threshold source: ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997.
• BayesDel score 0.140822 was not used because the supplied rules state that no generic ACMG-strength calibration is established for BayesDel.

Sources

Reasoning

BP5 is not met for this interpretation. PMID:24869598 does report one relapse patient carrying R665W together with low-frequency BTK C481S and other PLCG2 mutations, but the report concerns acquired somatic ibrutinib resistance rather than an alternate molecular basis established for the queried germline disease; it does not establish that BTK C481S explains the relevant disease phenotype instead of the queried variant. PMID:25972157 likewise describes acquired resistance and does not provide a validated alternate molecular diagnosis for this germline interpretation. The available evidence therefore does not satisfy the BP5 statement.

Rule

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

Evidence considered

• PMID:24869598 full text, Results: 'One patient with a low-frequency C481S mutation in BTK also had three distinct PLCγ2 mutations: the R665W mutation, a leucine-to-phenylalanine mutation at position 845 (L845F)'—this is a somatic acquired-resistance cohort and does not establish an alternate germline disease basis.
• PMID:25972157 paper extract: R665W was somatically acquired in relapsed CLL; one patient also acquired BTK C481S, without an independent germline alternate diagnosis being established.

Still missing

• No case establishes an alternate molecular diagnosis that explains the relevant germline disease phenotype independently of this variant.

Sources

Reasoning

BP6 is not met. ClinVar variation 376213 has only one single-submitter uncertain-significance assertion and no expert-panel submission; it has no exact-variant expert-panel benign or likely benign classification. The available OncoKB oncogenic label is not benign and is not a qualifying expert-panel ClinVar assertion.

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 376213: one laboratory submission, uncertain significance, criteria provided/single submitter, zero expert-panel submissions.
• OncoKB reports an oncogenic rather than benign classification in a somatic/functional context.

Sources

Reasoning

The available case evidence does not document a proband with the relevant disease phenotype, absence of family history, or confirmed maternity and paternity. No germline parental testing, meiosis information, or de novo observation is provided. The two variant-specific papers describe somatically acquired PLCG2 p.Arg665Trp in relapsed CLL, including absence at baseline, which does not establish a germline de novo event for this case.

Rule

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

Evidence considered

• No germline proband-parent segregation data, confirmed maternity and paternity, or family-history assessment is present in the available case evidence.
• PMID:24869598 paper extract: PLCG2 p.R665W was identified at relapse in CLL and was absent at baseline; this is a somatically acquired cancer observation, not evidence of germline de novo status.
• PMID:25972157 paper extract: PLCG2 R665W was acquired during ibrutinib therapy in three relapsed CLL patients and was not detected in baseline samples; this does not establish germline de novo status.

Still missing

• Document the proband's relevant disease phenotype and clinical diagnosis.
• Document family history.
• Provide germline genotype results for the proband and both biological parents with confirmed maternity and paternity.

Sources

Reasoning

PS4 requires a case-control comparison. The two variant-specific papers were verified against full text: PMID:24869598 reports R665W in relapse samples from acquired-ibrutinib-resistance CLL, including Patient 6 and one patient with additional PLCG2 mutations; PMID:25972157 reports R665W as a somatically acquired mutation in three relapsed CLL patients. Neither paper provides unaffected or population control counts for this variant, a comparable germline affected-case frequency, or an odds ratio and confidence interval. The available gnomAD frequency is a population observation but cannot be paired with a defined germline affected-case frequency from these somatic relapse studies, so no valid OR/95% CI can be computed. Governing threshold: OR >5.0 with a confidence interval that does not include 1.0; because the required comparison data are unavailable, PS4 is not assessed rather than inferred from the presence of the variant in affected cancer patients.

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:24869598 full text, Results: R665W was identified in relapse samples from a six-patient cohort with acquired ibrutinib-resistant CLL; this was not an unaffected-control comparison.
• PMID:25972157 full text, Results and Table 1: R665W was identified in three relapsed CLL patients and was absent from baseline samples; no population-control comparison or OR/95% CI was reported.
• gnomAD v4.1 reports 2/1,612,904 alleles overall (AF 1.24e-06; European non-Finnish AF 1.69543e-06), but there is no valid germline affected-case frequency from the cited studies with which to calculate an OR.

Still missing

• A germline case-control dataset for PLCG2 c.1993C>T/p.Arg665Trp with affected-case and matched-control counts is unavailable.
• No valid OR and 95% CI can be calculated from the available somatic relapse reports.

Sources

Reasoning

The case has no PLCG2 VCEP domain table or other authoritative critical-domain specification. Cancer Hotspots returned no statistically significant hotspot for PLCG2 R665, and the available COSMIC record concerns this single somatic variant rather than an established hotspot. The supplied evidence does not establish that residue 665 lies in a critical functional domain without benign variation, so PM1 cannot be assessed.

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

• Cancer Hotspots query for PLCG2 R665: no statistically significant hotspot row was found.
• COSMIC record COSV106062768: PLCG2 c.1993C>T/p.Arg665Trp was reported once in a somatic cancer; this does not by itself establish a recurrent hotspot or critical domain without benign variation.

Still missing

• Authoritative PLCG2 critical-domain boundaries and benign-variation assessment for residue 665 are unavailable.
• A validated hotspot assessment for residue 665 beyond the no-result Cancer Hotspots query is unavailable.

Sources

Reasoning

Not assessed: the available case evidence does not establish that PLCG2-related disease in this case is recessive, does not identify an affected proband carrying NM_002661.5:c.1993C>T with a second pathogenic variant, and provides no parental or offspring testing to establish trans phase. The reported R665W observations are somatic acquired mutations in relapsed CLL, not germline compound-heterozygous or trans observations.

Rule

PM3: For recessive disorders, detected in trans with a pathogenic variant ... Note: This requires testing of parents (or offspring) to determine phase.

Evidence considered

• The case evidence identifies NM_002661.5:c.1993C>T as PLCG2 p.(Arg665Trp) and provides no germline affected-proband, second pathogenic-variant, or parental/offspring phase observation.
• The available R665W literature observations concern somatically acquired mutations in relapsed CLL and do not establish germline trans phase or recessive inheritance for this case.

Still missing

• Confirm the inheritance mode for the relevant PLCG2 disorder.
• Document an affected germline proband carrying NM_002661.5:c.1993C>T and a second pathogenic variant.
• Obtain parental or offspring testing demonstrating that the two variants are in trans.

Reasoning

The available residue-specific literature reports PLCG2 p.Arg665Trp itself but provides no different missense substitution at Arg665 that has been determined to be pathogenic. A complete validated alternate-missense pathogenicity comparison for residue 665 is not available, so PM5 is not assessed.

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:24869598 paper extract: the residue-specific report is Arg665Trp, the queried substitution, with no different Arg665 missense comparator described.
• PMID:25972157 paper extract: the residue-specific report is Arg665Trp, the queried substitution; no alternate Arg665 missense variant determined to be pathogenic is provided.
• pm5_candidates reference artifact: no same-residue comparator candidates were confirmed for classic PM5 semantics.

Still missing

• A validated pathogenic alternate missense comparator at PLCG2 residue Arg665 is not available in the supplied evidence.

Sources

Reasoning

The available case evidence does not document an assumed germline de novo variant in an affected patient. There are no parental genotype results, family-history details, or clinical data establishing the patient's disease. The available variant-specific publications concern somatically acquired PLCG2 p.Arg665Trp in relapsed CLL and therefore do not provide PM6 evidence for this germline case.

Rule

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

Evidence considered

• No germline de novo observation, proband phenotype, family history, or parental testing is documented in the available case evidence.
• PMID:24869598 paper extract describes relapse-acquired PLCG2 p.R665W in CLL, not an assumed germline de novo variant.
• PMID:25972157 paper extract describes PLCG2 R665W acquired during ibrutinib therapy, not a germline de novo event.

Still missing

• Document the proband's relevant disease phenotype and clinical diagnosis.
• Provide evidence that the variant is germline in the proband.
• Provide parental testing or a documented basis for assuming de novo origin.

Sources

Reasoning

No affected family members, genotype-positive relatives, informative meioses, or cosegregation results are documented. The available publications report somatically acquired mutations in relapsed CLL rather than inherited family segregation, so they cannot establish PP1 for this case.

Rule

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

Evidence considered

• No family pedigree, affected relatives, genotype-positive relatives, or informative meioses are present in the available case evidence.

Still missing

• Provide a pedigree and phenotype status for relatives.
• Provide variant testing results for affected and unaffected relatives.
• Provide the number of informative meioses and confirm the disease-gene relationship for the assessed phenotype.

Sources

Reasoning

The supplied evidence does not provide a gene-level benign missense rate or a validated determination that missense variants are a common mechanism of PLCG2 disease. Variant-specific gain-of-function reports cannot substitute for both gene-level PP2 requirements, 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.

Still missing

• Gene-level evidence for a low rate of benign PLCG2 missense variation is unavailable.
• Gene-level evidence that missense variants are a common PLCG2 disease mechanism is unavailable.

Reasoning

The case materials do not provide a patient phenotype or family history for this germline variant that is highly specific for a disease with a single genetic etiology. The verified papers concern somatically acquired R665W in ibrutinib-resistant CLL and functional resistance mechanisms, not a phenotype-specific germline presentation. Therefore the phenotype-specificity evidence required for PP4 is unavailable.

Rule

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

Evidence considered

• The case ClinVar record concerns Familial cold autoinflammatory syndrome 3 but classifies the variant as uncertain significance from one single-submitter laboratory; it does not provide a phenotype or family-history description sufficient for PP4.
• PMID:24869598 and PMID:25972157 describe acquired somatic mutation in relapsed CLL, not a phenotype-specific germline disease presentation.

Still missing

• A documented patient phenotype or family history for the queried germline variant is unavailable.
• No evidence establishes that the reported phenotype is highly specific for a single genetic etiology.

Sources

Reasoning

BS2 is not assessed. The available evidence contains no observation of this variant as a germline allele in a healthy adult individual, and no adequately characterized healthy-adult cohort, inheritance context, disease penetrance, or age-of-onset information is provided. The two cited reports describe somatically acquired R665W in relapsed CLL, not healthy adult germline observations; those reports cannot establish BS2.

Rule

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

• PMID:24869598 reports R665W acquired at relapse in a six-patient CLL cohort; it does not report a healthy adult germline carrier.
• PMID:25972157 reports R665W as acquired during ibrutinib therapy in three relapsed CLL patients; baseline samples did not show the mutation.
• No case evidence documents a healthy adult carrying this variant in the homozygous, heterozygous, or X-linked state relevant to the disorder.

Still missing

• A documented healthy adult carrying the variant in the relevant inheritance state is needed.
• The disorder's expected penetrance and age of onset are not sufficiently characterized for BS2.
• Germline versus somatic status and clinical phenotype are not established for the reported cancer observations.

Sources

Reasoning

No variant-specific functional study in the reviewed evidence demonstrates that PLCG2 p.Arg665Trp has no damaging effect on protein function or splicing. The available studies instead report enhanced signaling and a hypermorphic effect, so they cannot establish BS3; a benign functional result using validated controls and an assay relevant to PLCG2 function is lacking.

Rule

BS3

Evidence considered

• Both reviewed variant-specific studies reported increased or persistent PLCG2 signaling for p.Arg665Trp rather than normal protein function or absence of damaging effect.
• The available assay results therefore do not provide evidence satisfying the BS3 definition.

Still missing

• No variant-specific benign functional assay result with appropriate controls and biological relevance was identified.

Sources

Reasoning

No affected relatives with documented absence of the variant are identified, and no pedigree, phenotype assessment, or familial genotype data are available. Therefore lack of segregation cannot be evaluated. The ACMG/AMP caveat regarding phenocopies and families with more than one pathogenic variant further requires family-level clinical and genetic information that is not provided.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected-relative genotype results or family-level phenotype assessments are documented in the available case evidence, so non-segregation cannot be assessed.

Still missing

• Provide a family pedigree and identify affected relatives.
• Provide reliable variant testing results for affected relatives.
• Assess phenocopy risk and whether another pathogenic variant could contribute to the familial phenotype.

Reasoning

The supplied evidence does not establish that PLCG2 disease is caused primarily by truncating variants. The available variant-specific reports concern a somatic missense gain-of-function change, but that observation alone cannot determine the gene-wide relative contribution of truncating versus missense disease variants, so BP1 is not assessed.

Rule

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

Still missing

• A gene-level disease-mechanism assessment comparing truncating and missense PLCG2 variants is unavailable.

Reasoning

Not assessed: the available case evidence does not document NM_002661.5:c.1993C>T in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant. No affected-proband genotype, parental testing, offspring testing, or other phase-resolved germline observation is provided.

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 provides no phase-resolved germline observation pairing NM_002661.5:c.1993C>T with a pathogenic variant in cis or trans.
• The available R665W literature observations are somatic acquired CLL findings and do not satisfy the required germline allelic configuration for BP2.

Still missing

• Document a phase-resolved germline observation with a pathogenic variant in trans or cis.
• For a trans observation, establish that the relevant dominant disorder is fully penetrant.