BTK c.1442G>C, p.Cys481Ser

Reasoning

PS3 is met at strong strength. The exact BTK p.Cys481Ser (NM_000061.3:c.1442G>C) variant was evaluated in multiple independent, orthogonal functional systems with wild-type comparators and relevant controls. Recombinant-protein kinase, transfected-cell, BTK-null B-cell, phosphorylation, downstream-signaling, proliferation, binding-affinity, and drug-washout assays consistently showed loss of irreversible ibrutinib binding, reduced inhibition of BTK activity, reversible inhibition, reactivation of B-cell-receptor signaling, and resistant cellular proliferation. These results establish a reproducible abnormal effect on a biologically relevant BTK function; the evidence concerns altered inhibitor binding and signaling rather than loss of basal catalytic activity, but that altered function is directly demonstrated for the exact variant.

Rule

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

Evidence considered

• Woyach et al. 2014 (PMID:24869598), Results section ‘Reversible Inhibition of Mutant BTK by Ibrutinib In Vitro and in Patients,’ paragraph 3 and Table 1: recombinant-protein kinase, HEK293T, BTK-null DT40-cell, phosphorylation/signaling, and washout assays showed that exact BTK C481S retained kinase activity but had reduced ibrutinib affinity, less inhibition of autophosphorylation and downstream signaling, and reversible rather than irreversible inhibition.
• Cheng et al. 2015 (PMID:25189416), Introduction, Results, and Discussion: the exact BTK C481S variant was verified by sequencing and tested with wild-type comparison, immunoblotting, BCR-expression profiling, Ki-67/BrdU proliferation assays, siRNA knockdown, and mutant-versus-wild-type transfection; it disrupted covalent ibrutinib binding, reduced inhibition of BTK enzymatic activity, and supported resistant cellular proliferation and signaling.
• Chiron et al. 2014 (PMID:25082755), longitudinal MCL functional-genomics study: the exact relapse-specific BTK C481S variant was associated with enhanced BTK/AKT activation and proliferation of ibrutinib-resistant cells.
• Estrov et al. 2016 (PMID:27282255), Results ‘BTK variants show differential catalytic activity’ and ‘BTK C481S and C481T confer resistance to ibrutinib’: engineered BTK C481S showed substantial Y223/Y551 phosphorylation and PLCγ2 Y753 phosphorylation, but reduced ibrutinib sensitivity and reversible inhibitor binding, demonstrating an abnormal drug-binding function despite retained basal catalytic activity.

Sources

Reasoning

PM2 is met at supporting strength. The exact BTK NM_000061.3:c.1442G>C (p.Cys481Ser) variant is absent from gnomAD v2.1 and gnomAD v4.1, and the recorded non-cancer gnomAD v2.1 and v3.1 subsets also report absence. For this X-linked disorder, the observed frequency is therefore 0, which is below the supplied PM2 supporting threshold of <=0.0001; no VCEP-specific population source overrides the default.

Rule

PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v2.1: exact variant absent.
• gnomAD v4.1: exact variant absent.
• The evidence record also reports exact-variant absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets; v2.1 non-cancer is represented by exomes and v3.1 non-cancer by genomes as specified for this case.
• Generic PM2 calibration supplied for this case: <=0.0001, identified as the SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020; PMID:25741868).

Sources

Reasoning

The variant is NP_000052.1:p.(Cys481Ser), and the reviewed reports describe the same C481S protein change, but they identify the queried NM_000061.3:c.1442G>C allele; no independently established pathogenic Cys481Ser allele caused by a different nucleotide change was documented. Therefore the PS1 same-amino-acid/different-nucleotide condition is not met.

Rule

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

Evidence considered

• Cheng et al. 2015 (PMID:25189416) explicitly describes BTK C481S and the queried c.1442G>C variant.
• Woyach et al. 2014 (PMID:24869598) reports BTK C481S but does not document a different nucleotide change producing the same amino-acid substitution.
• Burger et al. 2016 (PMID:27199251) reports BTK-C481S in a relapse sample and likewise does not establish a distinct pathogenic Cys481Ser nucleotide allele.

Sources

Reasoning

Generic ACMG/AMP PS4 requires significantly increased variant prevalence in affected individuals versus controls; the supplied rule specifies OR or RR >5.0 with a confidence interval excluding 1.0. The literature_pass PS4-indexed papers (PMIDs 28049639, 28418267, and 36029036) were checked against their available abstract material and describe BTK C481S in patients with ibrutinib-resistant malignancy or treatment-related resistance, not a germline disease case-control comparison. The already-read exact-variant papers PMID:24869598 and PMID:27199251 likewise report affected treatment cohorts or relapse observations without unaffected/population control counts, an odds ratio, relative risk, or confidence interval. Therefore no qualifying case-control enrichment estimate can be calculated and PS4 is not met.

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

• The literature_pass PS4-indexed entries PMID 28049639, PMID 28418267, and PMID 36029036 were verified from their available abstract material as studies of BTK C481S/ibrutinib resistance rather than germline case-control prevalence.
• PMID:24869598 reports BTK C481S at relapse in five of six patients with acquired ibrutinib-resistant CLL and gives no unaffected/population control comparison, OR/RR, or confidence interval.
• PMID:27199251 reports BTK-C481S in Patient 4 at relapse in a five-patient longitudinal CLL cohort and gives no control comparison or enrichment estimate.
• No OR or RR was computed because neither affected/control counts nor a matched-ancestry control frequency paired with a case frequency were available.

Still missing

• No germline case-control dataset with affected and unaffected/control counts or an OR/RR and 95% confidence interval was identified.

Sources

Reasoning

Different missense substitutions at Cys481, including C481T and C481F/R/Y, are reported in acquired ibrutinib-resistance studies. These reports do not establish those alternate substitutions as pathogenic germline variants; C481T retained catalytic activity, so no qualifying previously pathogenic different missense change is demonstrated for PM5.

Rule

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

Evidence considered

• Nayar et al. 2016 (PMID:27282255) experimentally studied C481T and described retained catalytic activity; it also cited C481F/R/Y in treated patients, not as established pathogenic germline variants.
• Burger et al. 2016 (PMID:26182309) reports acquired BTK C481F alongside C481S in patients progressing on ibrutinib, without establishing C481F as a pathogenic germline variant.

Sources

Reasoning

Scope was settled first: NM_000061.3:c.1442G>C is a missense variant, so generic PP3 uses the REVEL path only; SpliceAI is not evidence for missense PP3. REVEL is unavailable for this variant, and the available BayesDel score is not usable because no generic ACMG-strength calibration is established. Therefore PP3 is not met.

Rule

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

Evidence considered

• NM_000061.3:c.1442G>C is annotated as missense, p.(Cys481Ser), in exon 15.
• The applicable generic path for a missense variant is REVEL only; the case has no REVEL score.
• SpliceAI max delta is 0.004, but SpliceAI is not applicable to the missense PP3 path.
• BayesDel score is -0.0634965, but the supplied generic rules state that BayesDel has no established generic ACMG-strength calibration and is not available for PP3/BP4.

Still missing

• No REVEL score is available for NM_000061.3:c.1442G>C.

Reasoning

PP5 is limited here to an exact-variant ClinVar expert-panel assertion of pathogenic or likely pathogenic significance. ClinVar variation 376203 exactly matches NM_000061.3:c.1442G>C (p.Cys481Ser), but the record has zero expert-panel submissions and only ordinary laboratory submissions, including conflicting Pathogenic and Uncertain significance assertions. Ordinary submissions, aggregate labels, and non-expert assertions are not eligible for PP5; 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 376203 exactly matches NM_000061.3(BTK):c.1442G>C (p.Cys481Ser).
• ClinVar reports 0 expert-panel submissions and four laboratory submissions, with two Pathogenic and two Uncertain significance submissions; this is not an eligible expert-panel assertion.

Sources

Reasoning

BA1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, including the recorded non-cancer subsets, so no filtering allele frequency is available and the observed frequency cannot reach the generic stand-alone threshold of >=0.05. The conflicting ClinVar record is not used to establish BA1, but it further argues against ending interpretation with a frequency-only benign call.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v2.1: exact variant absent; no allele count, allele frequency, or grpmax FAF reported.
• gnomAD v4.1: exact variant absent; no allele count, allele frequency, or grpmax FAF reported.
• The evidence record also reports the exact variant absent from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets; no source-specific frequency is available.
• Generic BA1 calibration supplied for this case: >=0.05, identified as ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).
• ClinVar reports conflicting classifications: Pathogenic from 2 clinical laboratories and Uncertain significance from 2 clinical laboratories; this is contextual and is not used as population evidence.

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, including the recorded non-cancer subsets, so there is no filtering allele frequency or grpmax FAF greater than expected for the disorder. The generic 0.01 default is therefore not reached; no maximum-credible-allele-frequency calculation is needed because the observed frequency is zero in the available population datasets.

Rule

BS1: Allele frequency is greater than expected for the disorder. ... BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v2.1: exact variant absent; no filtering allele frequency, grpmax FAF, allele count, or ancestry-specific count reported.
• gnomAD v4.1: exact variant absent; no filtering allele frequency, grpmax FAF, allele count, or ancestry-specific count reported.
• The evidence record also reports the exact variant absent from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets.
• Generic BS1 calibration supplied for this case: >=0.01; the prompt directs use of filtering allele frequency (grpmax FAF), not the popmax point estimate, and identifies Whiffin et al. 2017 (PMID:28518168) for maximum-credible-frequency assessment.

Sources

Reasoning

BS3 is not met. Although the exact BTK Cys481Ser variant retains basal catalytic activity and phosphorylates PLCγ2 in engineered assays, the same variant consistently causes reduced ibrutinib binding, reversible rather than irreversible inhibition, altered downstream signaling, and resistant cellular proliferation. Therefore, the functional evidence does not show no damaging effect on protein function; it demonstrates a damaging alteration in the biologically relevant inhibitor-binding/signaling function.

Rule

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

Evidence considered

• Estrov et al. 2016 (PMID:27282255), Results: BTK C481S and wild-type both showed substantial Y223/Y551 phosphorylation and PLCγ2 Y753 phosphorylation, establishing retained basal catalytic activity; however, C481S was less inhibited by ibrutinib and bound the drug reversibly.
• Woyach et al. 2014 (PMID:24869598), functional assays: C481S retained kinase activity but had reduced ibrutinib affinity, reversible inhibition after washout, and impaired suppression of BTK autophosphorylation and downstream B-cell-receptor signaling.
• Cheng et al. 2015 (PMID:25189416), functional assays: C481S disrupted covalent ibrutinib binding, reduced inhibition of BTK enzymatic activity, and was associated with reactivated signaling and resistant cell proliferation; these findings preclude concluding that the variant has no damaging effect on protein function.

Sources

Reasoning

Scope was settled first: NM_000061.3:c.1442G>C is a missense variant, so generic BP4 uses the REVEL path only; SpliceAI is not evidence for missense BP4. REVEL is unavailable for this variant, and the available BayesDel score is not usable because no generic ACMG-strength calibration is established. The SpliceAI max delta of 0.004 cannot be substituted across predictor paths. Therefore 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

• NM_000061.3:c.1442G>C is annotated as missense, p.(Cys481Ser), in exon 15.
• The applicable generic path for a missense variant is REVEL only; the case has no REVEL score.
• SpliceAI max delta is 0.004, but the generic rules prohibit using the splice path for a missense BP4 decision.
• BayesDel score is -0.0634965, but the supplied generic rules state that BayesDel has no established generic ACMG-strength calibration and is not available for PP3/BP4.

Still missing

• No REVEL score is available for NM_000061.3:c.1442G>C.

Reasoning

BP6 is limited here to an exact-variant ClinVar expert-panel assertion of benign or likely benign significance. ClinVar variation 376203 exactly matches NM_000061.3:c.1442G>C (p.Cys481Ser), but the record has zero expert-panel submissions and no benign or likely benign expert-panel assertion. Ordinary laboratory submissions and aggregate ClinVar labels are not eligible for BP6; therefore 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 376203 exactly matches NM_000061.3(BTK):c.1442G>C (p.Cys481Ser).
• ClinVar reports 0 expert-panel submissions; the available laboratory submissions are Pathogenic or Uncertain significance, not an eligible benign/likely benign expert-panel assertion.

Sources

Reasoning

No case-level evidence documents a proband with the disease, absence of family history, or confirmed maternity and paternity for NM_000061.3:c.1442G>C. The available literature concerns acquired BTK C481S in treated hematologic malignancies and does not provide germline de novo testing.

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, family history, or a germline de novo observation.
• PMID:25189416, PMID:24869598, and PMID:27199251 describe BTK C481S in acquired ibrutinib-resistance studies rather than germline de novo segregation evidence.

Still missing

• A documented affected proband with the variant is needed.
• Maternity and paternity confirmation are needed.
• A documented absence of family history is needed.

Reasoning

BTK Cys481 is described as part of the kinase domain and as an invariant residue, supporting functional importance. However, the available evidence does not establish that the complete relevant domain lacks benign variation, and no VCEP domain table is available; the generic PM1 requirement cannot be confirmed.

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

• Substitution scanning study (PMID:27282255), Discussion: C481 is described as invariant in BTK sequence alignments and the study evaluates substitutions at the BTK ibrutinib-binding/kinase-domain site.
• The queried variant is absent from gnomAD v2.1 and gnomAD v4.1, but absence of this single variant does not establish absence of benign variation across the entire domain.

Still missing

• Domain-wide benign-variation assessment for the relevant BTK kinase domain is unavailable.

Sources

Reasoning

No germline affected-proband observation documents this variant with a pathogenic variant on the homologous chromosome, and no parental or offspring testing establishes phase. The available variant-specific literature concerns acquired BTK C481S in cancer and does not provide the required recessive allelic or phase evidence.

Rule

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

Evidence considered

• The case evidence contains no documented germline affected proband carrying this variant with a pathogenic variant in trans.
• The case evidence contains no parental or offspring testing establishing phase.
• Variant-specific publications describe acquired BTK C481S in cancer and do not report the required recessive allelic or phase observation.

Still missing

• A germline affected-proband observation is needed.
• Parental or offspring testing is needed to establish phase with a pathogenic variant.

Reasoning

No case-level evidence documents an assumed germline de novo occurrence of NM_000061.3:c.1442G>C in an affected patient. The available literature does not report parental testing or a germline de novo observation for this variant.

Rule

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

Evidence considered

• The available case evidence does not document an assumed germline de novo occurrence or parental testing for this variant.
• The available variant-specific publications concern acquired somatic BTK C481S in treated malignancies and do not establish PM6 evidence.

Still missing

• A documented affected patient carrying the variant is needed.
• Evidence that the variant was assumed de novo is needed.
• The parental-testing status must be documented as unconfirmed or incomplete.

Reasoning

No affected family members, genotype-phenotype co-segregation results, or meiosis count are documented for NM_000061.3:c.1442G>C. Although BTK is associated with X-linked agammaglobulinemia in the case context, the required family segregation observations are absent.

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

• The case evidence contains no family pedigree, affected-relative testing, unaffected-relative testing, or informative meiosis count for this variant.
• The reviewed variant-specific papers describe acquired resistance in malignancy cohorts and do not report germline family segregation.

Still missing

• Affected relatives and their clinical phenotypes are needed.
• Variant genotypes for affected and unaffected relatives are needed.
• The number of informative meioses and segregation results are needed.

Reasoning

The available case evidence does not provide a gene-wide assessment demonstrating both a low rate of benign BTK missense variation and that missense variants are a common disease mechanism. Variant-specific functional and clinical reports cannot establish these gene-level prerequisites.

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-wide missense disease-mechanism and benign-missense-rate assessment is unavailable.

Reasoning

The generic PP4 definition requires a patient's phenotype or family history that is highly specific for a disease with a single genetic etiology. The case materials provide the BTK variant and database/literature records but no clinical phenotype, family history, or patient-level diagnostic description for this case, so PP4 cannot be assessed.

Rule

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

Evidence considered

• The case materials contain no patient-level clinical phenotype or family-history description suitable for PP4.

Still missing

• Patient-level phenotype and family-history information are absent.

Reasoning

BS2 is not assessed. The available evidence establishes absence from population databases but does not document the exact variant in a healthy adult individual as a hemizygous carrier of an X-linked disorder, nor provide age, phenotype, or penetrance information sufficient to satisfy the full-penetrance early-age requirement. Somatic reports of BTK p.Cys481Ser resistance are not evidence of a healthy adult germline observation and are not used for 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

• gnomAD v2.1 and v4.1 report the exact variant as absent, so they provide no healthy-carrier or homozygote observation.
• ClinVar includes germline and somatic submissions but does not provide a qualifying healthy adult hemizygous observation for this criterion.
• Somatic BTK p.Cys481Ser reports concern acquired ibrutinib resistance and cannot establish a healthy adult germline observation.

Still missing

• A verified observation of the exact germline variant in a healthy adult hemizygous individual, with age and phenotype information, is unavailable.

Sources

Reasoning

No affected family members tested negative for NM_000061.3:c.1442G>C, and no family segregation analysis is documented. Therefore, lack of segregation cannot be evaluated; the phenocopy caveat is also not assessable without family phenotype and genotype data.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The available case evidence does not document affected relatives who lack the variant or any family-level non-segregation analysis.
• No family data are available to assess the phenocopy caveat or the possibility of more than one pathogenic variant contributing to an autosomal dominant disorder.

Still missing

• Affected relatives with reliable disease phenotyping are needed.
• Variant testing results for affected relatives are needed.
• Potential phenocopies and alternative genetic causes in non-carriers must be evaluated.

Reasoning

The available evidence does not provide a disease-variant spectrum showing that BTK disease is caused primarily by truncating variants. A general loss-of-function context is insufficient to establish the primarily truncating requirement for BP1.

Rule

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

Still missing

• A curated BTK disease-variant spectrum comparing truncating and missense mechanisms is unavailable.

Reasoning

No germline observation documents the variant in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant. The available variant-specific literature concerns acquired somatic cancer mutations and does not establish either BP2 allelic configuration.

Rule

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 contains no documented germline observation placing this variant in trans with a pathogenic variant in a fully penetrant dominant disorder.
• The case evidence contains no documented germline observation placing this variant in cis with a pathogenic variant.
• Variant-specific publications describe acquired BTK C481S in cancer and do not establish a BP2 allelic configuration.

Still missing

• A germline family or proband record documenting the relevant allelic configuration is needed.
• Phase testing or an equivalent validated phase observation is needed.

Reasoning

The generic BP5 definition requires the variant to be found in a case with an alternate molecular basis for disease. The available BTK C481S reports concern acquired ibrutinib resistance in somatic malignancy cohorts and do not establish that this germline variant was found in a disease case with an alternate molecular diagnosis; the case also supplies no such patient-level molecular diagnosis. BP5 therefore cannot be assessed.

Rule

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

Evidence considered

• PMID:24869598 describes acquired BTK C481S in ibrutinib-resistant CLL and does not establish an alternate molecular cause for the disease being evaluated here.
• PMID:27199251 describes BTK C481S at relapse in a treatment-resistance cohort and does not provide a qualifying alternate molecular diagnosis for a germline disease case.

Still missing

• A patient-level disease diagnosis and independent alternate molecular cause in the same case are not provided.

Sources