KIT c.1961T>C, p.Val654Ala

Reasoning

Tamborini et al. (PMID:16751810) performed a variant-specific, controlled cellular assay using site-directed KIT V654A constructs in transiently transfected COS1 cells. The constructs were sequenced, KIT/D559 served as an imatinib-sensitive positive control, and antiphosphotyrosine immunoblotting was assessed across multiple imatinib concentrations. KIT/V654A and KIT/D559/V654A retained receptor phosphorylation at 3 micromolar imatinib and were inhibited at 6 micromolar, demonstrating altered KIT activity consistent with acquired imatinib resistance; the paper also reported the variant in patients with clinical progression during imatinib treatment. This is relevant functional evidence for a damaging gain-of-function effect on KIT, but it is a single transient-transfection study rather than independently replicated or validated clinical-laboratory testing, so PS3 is applied at supporting strength.

Rule

PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• Tamborini et al. 2006, Figure 1: COS1 cells were transiently transfected with sequence-verified KIT/V654A and KIT/D559/V654A constructs; KIT/D559 was used as an imatinib-sensitive control and antiphosphotyrosine immunoblotting measured receptor phosphorylation.
• Tamborini et al. 2006, Results and Discussion: KIT/V654A and KIT/D559/V654A remained phosphorylated at 3 micromolar imatinib and were inhibited at 6 micromolar, supporting acquired imatinib resistance and altered KIT function.
• The study examined three GIST patients with clinical progression during imatinib treatment and identified V654A as a secondary KIT mutation; this clinical context supports, but does not replace, the cellular assay evidence.

Still missing

• No independent replication or clinical diagnostic laboratory validation of the functional result was identified.

Sources

Reasoning

PM2 is met at supporting strength under the supplied generic threshold. gnomAD v4.1 reports the variant at AF 6.197046239880223e-07 (1/1,613,672 alleles; 0 homozygotes), which is below the supplied PM2 supporting threshold of <=0.0001. The variant is absent from gnomAD v2.1 and the available non-cancer records. The v4 observation is retained rather than treating the variant as completely absent, so the conclusion is based on extremely low frequency rather than literal absence.

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: total AC=1, AN=1,613,672, AF=6.197046239880223e-07, homozygotes=0; this is below the supplied generic PM2 supporting threshold of <=0.0001.
• gnomAD v2.1: variant absent.
• The available non-cancer gnomAD v2.1 and v3.1 records report the variant as absent; no separate source-registry keys exist for those records, so they are described here without adding non-key values to evidence_refs.
• The supplied calibration threshold is the ClinGen SVI recommendation to use PM2 at supporting strength when allele frequency is <=0.0001 (case-provided threshold block; ACMG/AMP 2015 cited there as PMID:25741868).

Sources

Reasoning

The variant is an exonic missense substitution, NM_000222.3:c.1961T>C (NP_000213.1:p.(Val654Ala)); therefore the governing generic rule selects the REVEL path only, and SpliceAI is not used for this missense PP3/BP4 assessment. The local REVEL lookup reports 0.82, which meets the supplied PP3 moderate threshold of >= 0.773 but not the strong threshold of >= 0.932. BayesDel is not counted because no generic ACMG-strength calibration is established for it. The supplied ClinGen SVI REVEL calibration is from Pejaver et al. 2022, Am J Hum Genet (PMID:36413997).

Rule

PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). ... PP3 (moderate): >= 0.773 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• NM_000222.3:c.1961T>C is an exonic missense variant producing NP_000213.1:p.(Val654Ala); REVEL is therefore the sole applicable PP3/BP4 predictor path.
• The local REVEL lookup reports a score of 0.82 for NM_000222.3:c.1961T>C.
• The supplied ClinGen SVI REVEL calibration assigns PP3 moderate at >= 0.773 and PP3 strong at >= 0.932; REVEL 0.82 supports PP3 at moderate strength (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997).
• SpliceAI max delta 0.029 was not counted because splice prediction is not the applicable path for a missense variant; BayesDel 0.338346 was not counted because no generic ACMG-strength calibration is established.

Sources

Reasoning

No reviewed source provides a case-control comparison for KIT c.1961T>C (p.Val654Ala), affected-versus-unaffected counts, or an odds ratio/RR with a confidence interval. PMID:18623623 reports V654A in approximately 27% of 53 metastasis samples from 14 GIST patients progressing on imatinib or sunitinib, but this is a treatment-resistance cohort without unaffected or population controls and is not a case-control prevalence comparison. The PS4 rule requires an OR/RR >5.0 with a confidence interval that excludes 1.0; no numeric estimate is available, so the required inequality cannot be satisfied from the evidence reviewed.

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:18623623 reports V654A in approximately 27% of samples after clinical progression on sunitinib, from 53 GIST metastases in 14 patients; the study has no unaffected or population control group and does not provide an OR/RR or confidence interval.
• The exact governing comparison is OR/RR >5.0 with a confidence interval that does not include 1.0; no OR/RR value exists here, so no substituted inequality can be evaluated as satisfied.

Sources

Reasoning

The variant is KIT p.Val654Ala at residue 654. The reviewed residue-specific evidence reports p.Val654Ala itself, but no different missense substitution at residue 654 that has been determined pathogenic. The PM5 comparator requirement is therefore not met.

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:16751810 experimentally studies KIT V654A itself and does not report a different missense change at residue 654 established as pathogenic.
• PMID:18623623 reports KIT V654A itself as a secondary resistance mutation; it does not identify a different pathogenic missense comparator at residue 654.
• The residue-specific comparator collection contains zero same-residue candidates and zero expert-panel or non-expert-majority pathogenic/likely pathogenic candidates.

Sources

Reasoning

The exact-variant ClinVar record for KIT c.1961T>C (p.Val654Ala) has no expert-panel submission and is classified as uncertain significance by clinical-laboratory submissions. PMID:25394175, listed as a PP5 lead, was checked in full text and does not mention KIT c.1961T>C or p.Val654Ala and does not report this variant as pathogenic. The generic PP5 requirement is therefore 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 exact-variant record reports Uncertain significance, with zero expert-panel submissions; the listed submissions are clinical-laboratory assertions rather than expert-panel classifications.
• PMID:25394175 was checked because literature_pass.json listed it for PP5; its full text does not mention KIT, c.1961T>C, or p.Val654Ala and does not report this variant as pathogenic.

Sources

Reasoning

BS2 is not met because the population evidence documents no homozygous observations in gnomAD v4.1 (total homozygotes=0), and no qualifying healthy adult individual is reported as homozygous, heterozygous for a fully penetrant dominant disorder, or hemizygous for an X-linked disorder. One heterozygous allele in gnomAD is not by itself a documented healthy-adult observation meeting the BS2 definition.

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 v4.1 reports total AC=1, AN=1,613,672, total AF=6.197046239880223e-07, and total homozygotes=0.
• No case evidence documents a qualifying healthy adult individual with the genotype required by BS2.

Sources

Reasoning

BP1 is not supported for this KIT missense variant. The reviewed KIT literature describes disease-associated KIT alterations as constitutively activating, mainly gain-of-function mutations, and specifically evaluates V654A as an activating secondary resistance mutation. This is the opposite of a gene in which primarily truncating variants are known to cause disease.

Rule

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

Evidence considered

• PMID:16751810 states that GIST-associated KIT activation is mainly represented by gain-of-function mutations and experimentally evaluates V654A as a resistance-associated KIT mutation.
• OncoKB reports gain-of-function biological-effect context for KIT V654A.

Sources

Reasoning

The variant is an exonic missense substitution, so the governing generic rule selects the REVEL path only; SpliceAI is not used for this missense PP3/BP4 assessment. REVEL is 0.82, exceeding the most permissive supplied BP4 supporting threshold of <= 0.29, so it does not support BP4. This is a gray-zone REVEL result for BP4, and the SpliceAI max delta of 0.029 cannot substitute for the applicable REVEL path. BayesDel is not counted because no generic ACMG-strength calibration is established for it. The supplied ClinGen SVI REVEL calibration is from Pejaver et al. 2022, Am J Hum Genet (PMID:36413997).

Rule

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

Evidence considered

• NM_000222.3:c.1961T>C is an exonic missense variant; REVEL is therefore the sole applicable PP3/BP4 predictor path.
• The local REVEL lookup reports a score of 0.82 for NM_000222.3:c.1961T>C.
• The supplied ClinGen SVI REVEL calibration sets BP4 supporting at <= 0.29, BP4 moderate at <= 0.183, and BP4 strong at <= 0.016; REVEL 0.82 meets none of these thresholds (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997).
• SpliceAI max delta 0.029 was not used because splice prediction is not the applicable path for this missense variant; BayesDel 0.338346 was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The exact-variant ClinVar record has no expert-panel submission and reports uncertain significance from clinical-laboratory submissions. No reputable source reviewed here reports KIT c.1961T>C (p.Val654Ala) as benign or likely benign. Ordinary laboratory submissions, the aggregate uncertain-significance label, and non-expert assertions cannot 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 record reports Uncertain significance and zero expert-panel submissions; the available submissions are clinical-laboratory assertions and do not classify the variant as benign or likely benign.
• No reviewed source provides a reputable benign or likely-benign report for KIT c.1961T>C (p.Val654Ala).

Sources

Reasoning

The variant is KIT p.Val654Ala (c.1961T>C). PMID:16751810 and PMID:18623623 report the same p.Val654Ala change, but the available records do not establish a previously pathogenic comparator for this amino-acid change; ClinVar classifies the exact variant as uncertain significance. The evidence therefore does not establish PS1, and a validated pathogenic variant record with the same amino-acid substitution is still required.

Rule

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

Evidence considered

• The case variant is NM_000222.3:c.1961T>C, NP_000213.1:p.(Val654Ala).
• PMID:16751810 reports V654A as an acquired-resistance mutation in KIT; it does not establish the variant as a pathogenic germline ACMG comparator.
• PMID:18623623 reports KIT V654A as a recurrent secondary mutation in GIST, not as a separately established pathogenic same-amino-acid comparator.
• ClinVar variation 375921 lists the exact variant as Uncertain significance with two clinical-laboratory submissions.

Still missing

• A validated pathogenic classification for a KIT variant producing the same p.Val654Ala amino-acid change is not available.

Sources

Reasoning

No case-level documentation establishes that the variant occurred de novo in an affected proband, that there was no family history, or that both maternity and paternity were confirmed. The available variant-specific literature concerns somatic or treatment-resistant GIST observations and does not provide germline parent testing or a de novo assessment.

Rule

PS2: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• No case-level proband, family-history, or parental-testing evidence was available for evaluation.
• PMID:18623623 reports V654A in metastatic GIST samples after treatment progression, but does not provide germline parent testing or de novo evidence.

Still missing

• Affected proband phenotype and diagnosis are not documented.
• Family history is not documented.
• Maternity and paternity confirmation results are not documented.
• Parental testing results for NM_000222.3:c.1961T>C are not documented.

Sources

Reasoning

PMID:16751810 places residue V654 in KIT kinase domain 1 and the ATP-binding domain, supporting a critical functional-domain location. However, the available evidence does not establish that the domain lacks benign variation, and the cancer-hotspot record is a somatic hotspot source not named by a VCEP route. PM1 cannot be concluded from the available generic evidence.

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:16751810 states that residue V654 is in KIT kinase domain 1 and the ATP-binding domain, and describes structural interactions involving V654 and imatinib.
• gnomAD v4.1 reports the exact variant at AF 6.19705e-07, one of 1,613,672 alleles, with zero homozygotes; this does not by itself demonstrate absence of benign variation throughout the domain.
• Cancer Hotspots reports a statistically significant KIT V654 hotspot, but the record is somatic and the supplied instructions do not provide a qualifying VCEP-named hotspot route for this generic interpretation.

Still missing

• A documented assessment showing no benign variation in the relevant KIT functional domain is required.
• The available somatic hotspot record does not provide a qualifying generic PM1 route under the supplied instructions.

Sources

Reasoning

PM3 cannot be assessed because no affected-proband observation documents NM_000222.3:c.1961T>C (p.Val654Ala) in trans with a pathogenic variant, and no parent or offspring testing establishes phase. ClinVar records the variant as a germline submission, but does not provide a second pathogenic allele or phase information. The available KIT V654A literature describes somatic secondary mutations in GIST metastases, not a germline recessive case with demonstrated trans configuration.

Rule

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

Evidence considered

• ClinVar exact-match record NM_000222.3(KIT):c.1961T>C (p.Val654Ala) contains two germline clinical-testing submissions but no reported second pathogenic allele, affected-proband genotype, or parent/offspring phase result.
• Heterogeneity of kinase inhibitor resistance mechanisms in GIST, PMID:18623623, reports V654A as a somatic secondary KIT mutation in 53 GIST metastases from 14 patients; this is not evidence of germline trans phase or recessive inheritance.

Still missing

• Documented recessive KIT-related disorder context
• Affected proband with NM_000222.3:c.1961T>C
• A second pathogenic variant
• Parent or offspring testing establishing trans phase

Sources

Reasoning

No case-level documentation establishes an assumed de novo occurrence in an affected proband. There is no documented parental testing, family history, or proband phenotype, and the available variant-specific literature does not describe a germline de novo observation.

Rule

PM6: Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• No case-level proband, family-history, or parental-testing evidence was available for evaluation.
• PMID:18623623 reports V654A in metastatic GIST samples after treatment progression, but does not describe a germline assumed-de novo case.

Still missing

• Affected proband phenotype and diagnosis are not documented.
• Family history is not documented.
• Parental testing results are not documented.
• A report establishing assumed de novo status is not documented.

Sources

Reasoning

No affected relatives, informative meioses, variant testing in family members, or phenotype-consistent segregation results are documented. The available variant-specific literature describes tumor samples and treatment resistance rather than familial germline cosegregation.

Rule

PP1: Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• No family-based segregation dataset was available for evaluation.
• PMID:18623623 reports V654A in 53 GIST metastases from 14 patients, with secondary mutations assessed by tumor-based methods, but provides no familial germline cosegregation analysis.

Still missing

• Affected relatives and their diagnoses are not documented.
• Family variant-testing results are not documented.
• Informative meioses and segregation counts are not documented.
• Phenotype consistency among relatives is not documented.

Sources

Reasoning

The available evidence describes recurrent activating KIT missense mutations in cancer, but it does not establish both required gene-level elements for PP2: a low rate of benign missense variation and missense variants as a common disease mechanism in the applicable disease context. No validated gene-level missense constraint or benign-missense rate is available.

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

• PMID:16751810 describes KIT-associated GIST as involving constitutively activated KIT and mainly gain-of-function mutations, but it does not quantify benign missense variation across KIT.
• OncoKB describes KIT as recurrently mutated and gives gain-of-function context for V654A, but does not supply the gene-level benign-missense requirement for PP2.
• The available gnomAD record is a frequency observation for this single variant, not a validated gene-level benign-missense rate.

Still missing

• A validated gene-level assessment of benign missense variation is unavailable.
• The applicable disease context has not been documented as having missense variants as a common mechanism under PP2.

Sources

Reasoning

The supplied case evidence does not provide the patient's phenotype, family history, or a defined disease presentation that can be assessed for high specificity to a single-gene disorder. Therefore PP4 cannot be adjudicated.

Rule

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

Still missing

• Patient phenotype and family history are not supplied.
• The specific disease being evaluated and its phenotypic specificity are not supplied.

Reasoning

BA1 cannot be applied because the required filtering frequency, gnomAD grpmax FAF, is not reported (null). The available gnomAD v4 data show only 1/1,613,672 total alleles (AF 6.197046239880223e-07) and no homozygotes, while the highest observed subpopulation point estimate is 1/1,179,646 (8.477119407008543e-07); however, the point estimate is not the required FAF for a stand-alone BA1 decision. The available values are far below the generic 0.05 threshold, but the missing grpmax FAF leaves the specified BA1 frequency metric unavailable.

Rule

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

Evidence considered

• gnomAD v4.1: total AC=1, AN=1,613,672, AF=6.197046239880223e-07, homozygotes=0; grpmax FAF=null.
• gnomAD v2.1: variant absent.
• The generic BA1 threshold supplied for this case is >=0.05 under ACMG/AMP 2015 (Richards et al., PMID:25741868). The required filtering metric is unavailable, so the observed popmax point estimate was not substituted for FAF.

Still missing

• gnomAD filtering grpmax FAF is not reported for this variant

Sources

Reasoning

BS1 cannot be fully assessed under the specified frequency procedure because gnomAD v4 reports no filtering grpmax FAF (null), the required figure for BS1; a popmax point estimate must not replace it. Available data show only 1/1,613,672 total alleles (AF 6.197046239880223e-07), no homozygotes, and absence from gnomAD v2.1, but the generic 0.01 default cannot be formally tested with the required FAF metric. No prevalence, penetrance, allelic-heterogeneity, or maximum-credible-allele-frequency calculation is supplied to establish a gene/disease-specific BS1 threshold.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v4.1: total AC=1, AN=1,613,672, AF=6.197046239880223e-07, homozygotes=0; grpmax FAF=null.
• gnomAD v2.1: variant absent.
• The generic BS1 default supplied for this case is >=0.01; per-gene maximum credible allele-frequency assessment is preferred (Whiffin et al. 2017, PMID:28518168). Because the required FAF and disease-specific inputs are unavailable, BS1 was not formally assessed.

Still missing

• gnomAD filtering grpmax FAF is not reported for this variant
• No disease-specific prevalence, penetrance, allelic-heterogeneity, or maximum-credible-frequency calculation is available

Sources

Reasoning

No well-established assay demonstrating that KIT V654A has no damaging effect on protein function or splicing was identified. The available variant-specific study tested imatinib resistance and supports altered KIT function rather than a benign result; computational splice prediction is not a functional assay for BS3.

Rule

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

Evidence considered

• The available variant-specific functional study evaluated imatinib response and receptor phosphorylation, not absence of damaging effect.
• SpliceAI's maximum delta score of 0.029 is computational prediction evidence and does not establish BS3.

Still missing

• A validated assay demonstrating preserved KIT protein function or normal splicing is unavailable.

Sources

Reasoning

No affected family members with negative testing for NM_000222.3:c.1961T>C are documented. Therefore, lack of segregation cannot be assessed, and no family-level evidence is available to distinguish true non-segregation from phenocopy or another pathogenic variant.

Rule

BS4: Lack of segregation in affected members of a family.

Evidence considered

• No family-level non-segregation dataset was available for evaluation.
• PMID:18623623 is a tumor/metastasis study and does not provide affected-relative testing or family non-segregation evidence.

Still missing

• Affected family members are not documented.
• Variant-testing results in affected relatives are not documented.
• Phenocopy assessment is not documented.
• The possibility of another pathogenic variant in the family is not assessed.

Sources

Reasoning

BP2 cannot be assessed because no affected-proband observation documents this variant in trans with a pathogenic variant in a fully penetrant dominant KIT disorder, or in cis with a pathogenic variant in any inheritance pattern. ClinVar provides no phase-resolved allelic observation. The available KIT V654A paper concerns somatic secondary mutations and does not establish germline cis or trans phase.

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

• ClinVar exact-match record NM_000222.3(KIT):c.1961T>C (p.Val654Ala) contains germline submissions but no phase-resolved observation with another pathogenic variant.
• Heterogeneity of kinase inhibitor resistance mechanisms in GIST, PMID:18623623, describes somatic V654A secondary mutations with primary KIT exon 11 mutations but provides no germline cis/trans phase evidence or fully penetrant dominant-disease observation.

Still missing

• Affected proband with a documented pathogenic KIT allele pair
• Parent or offspring testing, tumor-normal phasing, or equivalent phase evidence
• Pathogenic variant identified in cis or trans
• Evidence that any dominant KIT disorder is fully penetrant for the trans path

Sources

Reasoning

The reviewed evidence does not establish that this variant was found in a case with an alternate molecular basis for disease. The available V654A literature concerns somatic KIT resistance in GIST and does not provide a germline case with an independently established alternate molecular cause for the relevant disease.

Rule

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

Still missing

• A clinical case with the variant and an independently established alternate molecular diagnosis is not supplied.