KIT c.1924A>G, p.Lys642Glu

Reasoning

PS3 is met at strong strength for NM_000222.3:c.1924A>G (KIT p.Lys642Glu). Two independent, variant-specific functional studies provide concordant evidence of a damaging gain-of-function effect. PMID:11526490 characterized the exact K642E variant in the human GIST882 cell line and primary tumor, demonstrating constitutive KIT kinase activity and tyrosine phosphorylation, with reduced proliferation, reduced focus formation, and increased apoptosis after STI571 treatment. PMID:11073817 independently demonstrated ligand-independent KIT phosphorylation, autonomous Ba/F3-cell growth without IL-3 or SCF, and tumor formation in nude mice for the corresponding KIT642Glu variant. The studies use biologically relevant KIT/GIST and cellular or in vivo tumor models, include functional readouts and treatment/control comparisons, and are mutually concordant; however, they are research assays rather than reproducibility-validated clinical diagnostic assays, so the evidence is calibrated to strong rather than very strong.

Rule

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

Evidence considered

• PMID:11526490: Exact KIT K642E was studied in the human GIST882 cell line and originating primary GIST; the variant showed constitutive kinase activity and tyrosine phosphorylation, while STI571 abolished phosphorylation and reduced proliferation and focus formation and increased apoptotic cell death. Assays included KIT phosphorylation, proliferation, focus formation, and Annexin V flow-cytometry apoptosis measurements.
• PMID:11073817: The corresponding KIT642Glu/c.1924A>G variant caused ligand-independent KIT tyrosine phosphorylation, autonomous growth of Ba/F3 cells without IL-3 or SCF, and tumor formation in nude mice. The study used stable mutant-KIT expression, phosphorylation and MTT proliferation assays, and an in vivo tumorigenicity model.
• The two studies independently examined the exact variant and used concordant cellular and in vivo functional endpoints relevant to KIT gain-of-function biology.

Still missing

• The studies were not described as validated and reproducible clinical diagnostic laboratory assays.

Sources

Reasoning

p.Lys642Glu lies in KIT kinase domain I, specifically the proximal split tyrosine kinase domain, a well-established functional domain. Functional studies show constitutive KIT kinase activation from this region, and the variant is absent from gnomAD v2.1 and v4.1. No VCEP domain table is available, and the generic hotspot route was not used because the hotspot evidence is somatic and no VCEP hotspot route applies.

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

• Isozaki et al. (PMID:11073817) places Lys642 in kinase domain I and demonstrates ligand-independent phosphorylation, autonomous growth, and tumor formation for KIT642Glu.
• Carter et al. (PMID:11526490) places K642E in the proximal split tyrosine kinase domain and demonstrates constitutive kinase activity and tyrosine phosphorylation.
• The variant is reported absent from gnomAD v2.1 and gnomAD v4.1.
• Cancer Hotspots reports a statistically significant K642 hotspot, but this somatic hotspot route was not used because no VCEP names that source or route for KIT.

Sources

Reasoning

PM2 is met at supporting strength. The exact KIT NM_000222.3:c.1924A>G (p.Lys642Glu) variant is absent from gnomAD v2.1 and gnomAD v4.1, and is also absent from the available gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer searches. This satisfies the generic PM2 absence requirement; the supplied ClinGen SVI-based calibration assigns PM2 supporting strength at allele frequency <=0.0001, and the observed frequency is zero in each searched dataset.

Rule

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 reports the exact variant as absent.
• gnomAD v4.1 reports the exact variant as absent.
• The available non-cancer searches report the exact variant as absent from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer; these searches returned no variant-level source record or frequency fields.
• The generic PM2 calibration supplied for this case is <=0.0001 at supporting strength; the observed allele frequency is 0 in all searched datasets.

Sources

Reasoning

The exact KIT c.1924A>G (p.K642E) variant was reported in affected members of three families with germline KIT exon 13 mutations; the report states that the same mutation was identified in all patients and describes 20 affected patients across the families. A separate family report identified the variant in an affected mother and son and confirmed its germline heterozygous state. These observations support cosegregation with familial GIST, but the available extracts do not provide a complete count of tested unaffected relatives or meioses to justify stronger segregation strength.

Rule

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

Evidence considered

• PMID:23648119, Results section 3.1: the exact KIT c.1924A>G, p.K642E mutation was identified in all patients in a retrospective analysis of three families; the paper reports 20 affected patients across the kindreds.
• PMID:11073817: a French mother and son with multiple GISTs both carried KIT642Glu, and the variant was present in normal pancreatic tissue from both, indicating familial germline heterozygosity.

Still missing

• The available evidence does not provide a complete tested-relative table, unaffected noncarrier count, or informative meiosis count for formal strength escalation.

Sources

Reasoning

The variant is a missense substitution, NM_000222.3:c.1924A>G (KIT p.Lys642Glu), so the governing generic rule selects the REVEL path only; SpliceAI and BayesDel are not used for PP3 in this situation. The REVEL score is 0.908, meeting the supplied generic PP3 moderate threshold of >= 0.773. The threshold is the ClinGen SVI REVEL calibration from 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

• VariantValidator and normalization evidence identify NM_000222.3:c.1924A>G as the missense variant NP_000213.1:p.(Lys642Glu), with the variant in exon 13.
• Local REVEL lookup reports a score of 0.908 for the variant.
• The supplied generic calibration assigns PP3 moderate at REVEL >= 0.773; this threshold is from the ClinGen SVI REVEL calibration, Pejaver et al. 2022, Am J Hum Genet (PMID:36413997).
• SpliceAI max delta 0.102 was not counted because the governing generic rule selects REVEL only for a missense variant; BayesDel 0.345632 was not counted because no generic ACMG-strength calibration is established.

Sources

Reasoning

The case documents the exact KIT p.Lys642Glu substitution as pathogenic in germline GIST families and functional studies, but no distinct nucleotide change producing the same amino-acid substitution was identified. Therefore the same-amino-acid-change evidence required for PS1 is not demonstrated independently of the observed nucleotide change.

Rule

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

Evidence considered

• ClinVar variation 13866 matches NM_000222.3:c.1924A>G (p.Lys642Glu) and reports Pathogenic submissions, but this is the observed nucleotide change rather than an independent alternate nucleotide change.
• Isozaki et al. (PMID:11073817) reports the exact AAA-to-GAA Lys642Glu substitution in a familial GIST kindred.
• Bachet et al. (PMID:23648119) reports c.1924A>G, p.K642E in patients from three GIST families.

Still missing

• A pathogenic KIT variant caused by a different nucleotide change but producing p.Lys642Glu was not identified.

Sources

Reasoning

The exact variant is reported in patients with familial or multiple gastrointestinal stromal tumors, including a mother-son pair and three families. However, GIST is not highly specific for a disease with a single genetic etiology because it occurs sporadically and has multiple molecular causes, including KIT and non-KIT alterations. The documented phenotype therefore does not satisfy generic PP4.

Rule

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

Evidence considered

• PMID:11073817 documents multiple GISTs in a mother and son carrying germline KIT642Glu.
• PMID:23648119 reports the exact KIT c.1924A>G p.K642E variant in three families with multiple GISTs.
• These reports establish familial GIST but do not establish that the phenotype is highly specific to a single genetic etiology.

Sources

Reasoning

ClinVar variation 13866 is an exact match and has Pathogenic submissions from OMIM and individual clinical laboratories, but the submission audit reports zero expert-panel submissions. The aggregate ClinVar label, ordinary laboratory submissions, and non-expert assertions cannot trigger PP5 under the specified generic rule. 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 13866 exactly matches NM_000222.3:c.1924A>G (p.Lys642Glu) and lists Pathogenic assertions from OMIM and clinical laboratories, with expert_panels empty and expert_panel_submissions equal to 0.
• The aggregate ClinVar label and ordinary laboratory submissions were not treated as expert-panel evidence.

Sources

Reasoning

BA1 is not met. The variant was absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer datasets, so no allele frequency at or above the generic stand-alone threshold of 0.05 was observed. No gnomAD population-specific frequency or grpmax FAF is available that could support a BA1 call. The pathogenic ClinVar consensus and published germline disease reports also provide no basis for ending classification on an unsupported default frequency.

Rule

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

Evidence considered

• gnomAD v2.1 reports the exact variant as absent.
• gnomAD v4.1 reports the exact variant as absent.
• The generic BA1 calibration supplied for this case is >=0.05; no population allele frequency or grpmax FAF is reported.

Sources

Reasoning

BS1 is not met. The exact variant was absent from gnomAD v2.1 and v4.1, including the available non-cancer datasets, so there is no observed allele frequency greater than expected for the disorder. The generic 0.01 default cannot be reached, and no population-specific grpmax FAF or maximum-credible-frequency calculation is needed because no elevated frequency is present.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1 reports the exact variant as absent.
• gnomAD v4.1 reports the exact variant as absent.
• The generic BS1 calibration supplied for this case is >=0.01; no allele frequency or grpmax FAF is reported.

Sources

Reasoning

BS3 is not met. No variant-specific study in the reviewed evidence demonstrates that KIT p.Lys642Glu has no damaging effect on protein function or splicing. Instead, the available functional studies show constitutive KIT activation, ligand-independent cellular growth, and tumorigenicity, which are directionally inconsistent with a benign functional effect. The reviewed assays therefore provide evidence against BS3 rather than evidence supporting it.

Rule

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

Evidence considered

• PMID:11073817: KIT642Glu produced ligand-independent phosphorylation, autonomous Ba/F3-cell growth without IL-3 or SCF, and nude-mouse tumors, contradicting a no-damaging-effect conclusion.
• PMID:11526490: K642E showed constitutive KIT kinase activity and tyrosine phosphorylation in GIST882 cells and the primary tumor, with downstream proliferation and apoptosis responses to STI571, contradicting a benign functional interpretation.

Sources

Reasoning

The available disease-focused evidence describes KIT p.Lys642Glu as a germline activating missense variant in familial GIST and demonstrates gain-of-function effects. This is inconsistent with applying BP1 for a gene in which disease is primarily caused by truncating variants.

Rule

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

Evidence considered

• PMID:11073817 reports a germline KIT kinase-domain missense variant causing familial GIST and demonstrates gain-of-function biology.
• PMID:23648119 reports KIT exon 13 p.K642E in three GIST families and describes the familial disease context as associated with this missense variant.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; SpliceAI cannot substitute for a gray-zone or non-supportive REVEL result. The REVEL score is 0.908, which exceeds the most permissive supplied generic BP4 supporting threshold of <= 0.29 and therefore does not support no impact. BayesDel is not usable 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). ... Missense variant -> REVEL only. ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• VariantValidator and normalization evidence identify NM_000222.3:c.1924A>G as the missense variant NP_000213.1:p.(Lys642Glu), with the variant in exon 13.
• Local REVEL lookup reports a score of 0.908 for the variant.
• The supplied generic calibration assigns BP4 supporting at REVEL <= 0.29; this threshold is from the ClinGen SVI REVEL calibration, Pejaver et al. 2022, Am J Hum Genet (PMID:36413997).
• SpliceAI max delta 0.102 was not used because the governing generic rule selects REVEL only for a missense variant; BayesDel 0.345632 was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

ClinVar variation 13866 has no exact-variant benign or likely-benign expert-panel classification; the audit reports zero expert-panel submissions, and the available submissions are Pathogenic or somatic Likely oncogenic. The aggregate record and non-expert assertions cannot trigger BP6, and no qualifying reputable benign report is available. 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 13866 exactly matches the variant and reports no expert-panel submission; the submission audit counts expert_panel_submissions as 0.
• The available exact-variant ClinVar submissions are Pathogenic or somatic Likely oncogenic, not qualifying benign or likely-benign expert-panel evidence.

Sources

Reasoning

The available evidence does not report a de novo occurrence for NM_000222.3:c.1924A>G (p.Lys642Glu), nor does it provide confirmed maternity and paternity testing or establish absence of a family history in the proband. The cited familial reports instead describe germline transmission in affected relatives, so PS2 cannot be assessed from this evidence.

Rule

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

Evidence considered

• PMID:23648119 reports the same KIT c.1924A>G, p.K642E variant in patients from three families with germline KIT exon 13 mutations, but does not establish a de novo occurrence in an isolated proband.
• PMID:11073817 reports the variant in a mother and son and documents its germline heterozygous nature, which is familial rather than de novo evidence for the reported family.

Still missing

• Proband-specific maternity and paternity confirmation is not reported.
• A proband-specific de novo finding and absence of family history are not documented.

Sources

Reasoning

The exact-variant literature reports affected individuals and familial GIST, but no reviewed study provides variant-positive affected and unaffected or population-control counts, and no matched-ancestry control frequency is available. The literature packet indexed PMID:17824795, PMID:22626674, PMID:22640790, and PMID:25394175 for PS4; their available case-focused records were checked and do not establish a case-control comparison. PMID:11073817 reports a mother and son with multiple GISTs, and PMID:23648119 reports the variant in three families, but neither provides control counts. Therefore no odds ratio or 95% confidence interval can be calculated. The governing PS4 threshold is OR >5.0 with a confidence interval that does not include 1.0, but no OR is available to substitute into that inequality.

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:11073817 reports the exact KIT p.K642E variant in a French mother and son with multiple GISTs; it does not report unaffected-control counts.
• PMID:23648119 reports the exact c.1924A>G p.K642E variant in three families and 20 affected patients; the extracted report does not provide a case-control comparison.
• The literature packet's PS4-indexed records PMID:17824795, PMID:22626674, PMID:22640790, and PMID:25394175 were checked through their available case-focused abstract records; none supplies variant-positive affected and unaffected or population-control counts.
• No OR or 95% CI was calculated because the required case and control counts are absent.

Still missing

• A case-control study or matched-ancestry control frequency with variant counts is needed to calculate an OR and its 95% CI.

Sources

Reasoning

PM3 requires a recessive disorder and detection of the variant in trans with a pathogenic variant, with phase established by parental or offspring testing. The reviewed reports describe this KIT variant as a germline heterozygous variant in an affected mother and son with familial GIST, but do not establish a recessive inheritance context or document a second pathogenic allele and its phase. Therefore PM3 cannot be assessed.

Rule

For recessive disorders, detected in trans with a pathogenic variant

Evidence considered

• Isozaki et al. (PMID:11073817) report KIT642Glu in an affected mother and son and state that the variant was germline and heterozygous; no second pathogenic allele or trans-phase result is reported in the extracted finding.
• Bachet et al. (PMID:23648119) report the same KIT c.1924A>G, p.K642E variant in patients from three GIST families; the extracted finding does not provide a second pathogenic allele or phase result.

Still missing

• Documented inheritance mode for the relevant KIT-associated disorder
• A second pathogenic or likely pathogenic allele
• Parental or offspring testing establishing trans phase

Sources

Reasoning

The variant is a missense change at KIT codon 642, but the available case evidence does not establish a different missense substitution at Lys642 as pathogenic. The collected PM5 candidate summary contains no same-residue candidates and explicitly does not confirm classic PM5 semantics, so a definitive negative adjudication is not justified.

Rule

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

Evidence considered

• The PM5 candidate summary reports zero same-residue candidates and states that classic same-residue PM5 semantics could not be confirmed safely.
• ClinVar variation 13866 documents p.Lys642Glu itself, not a different missense substitution at codon 642.

Still missing

• A verified pathogenic or likely pathogenic KIT missense variant at codon 642 with a different amino-acid substitution is needed.

Sources

Reasoning

The available evidence does not describe an assumed de novo occurrence in a patient. The reports instead describe the variant in familial GIST, including affected relatives and documented germline transmission, and do not provide a proband-specific de novo assessment without parental confirmation.

Rule

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

Evidence considered

• PMID:23648119 describes three families with germline KIT exon 13 mutations and does not identify the variant as de novo.
• PMID:11073817 documents KIT642Glu in both a mother and son and in normal pancreatic tissue, supporting familial germline transmission rather than assumed de novo status.

Still missing

• No proband-specific assumed de novo observation is documented.
• Parental testing and family-history data for the case proband are not reported.

Sources

Reasoning

The case supports that KIT disease-associated missense variants can be activating, but it does not provide a gene-wide benign-missense rate or a sufficiently comprehensive comparison establishing that KIT has a low rate of benign missense variation. The available gnomAD result is absence of this variant, not a gene-level benign-missense metric.

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:11073817 and PMID:11526490 demonstrate activating effects for KIT p.Lys642Glu, supporting a missense disease mechanism in this context.
• gnomAD v2.1 and v4.1 report the queried variant as absent, but they do not supply the gene-wide benign missense rate required for PP2.

Still missing

• A validated gene-level assessment of benign missense variation and the predominance of missense disease mechanism is required.

Sources

Reasoning

BS2 is not assessed. The available case literature describes the variant in affected germline carriers and does not document an unaffected healthy adult heterozygote, homozygote, or hemizygote. Because the record does not establish a qualifying healthy-adult observation or provide sufficient information about expected early-age full penetrance, the criterion cannot be adjudicated from the available evidence.

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

• Isozaki et al. (PMID:11073817) reported the variant in a mother and son with multiple gastrointestinal stromal tumors and described it as germline heterozygous; this is not a healthy-adult observation.
• Bachet et al. (PMID:23648119) reported the same variant in affected members of three germline KIT families; the source does not provide a qualifying unaffected healthy adult observation for BS2.

Still missing

• A documented healthy adult carrying the variant in the relevant inheritance state is not available.
• The evidence does not establish whether full penetrance is expected at an early age for the relevant disorder.

Sources

Reasoning

No affected family member is reported to have tested negative for KIT c.1924A>G (p.K642E). The available reports describe affected variant carriers but do not provide sufficient affected noncarrier data to establish lack of segregation; therefore BS4 is not assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• PMID:23648119 reports the variant in affected familial GIST cases but does not report an affected tested noncarrier.
• PMID:11073817 reports the variant in affected mother and son and does not provide evidence of an affected relative lacking the variant.

Still missing

• No genotype results for affected noncarriers are reported.
• No complete pedigree with tested affected and unaffected relatives is available.

Sources

Reasoning

BP2 requires either observation in trans with a pathogenic variant for a fully penetrant dominant disorder or observation in cis with a pathogenic variant in any inheritance pattern. The reviewed evidence documents the KIT c.1924A>G, p.K642E variant in familial GIST, including a germline heterozygous mother-son observation, but does not document another pathogenic variant or establish cis/trans phase. Therefore BP2 cannot be assessed.

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

• Isozaki et al. (PMID:11073817) describe an affected mother and son with a germline heterozygous KIT642Glu variant; the extracted finding does not identify a second pathogenic variant or cis/trans phase.
• Bachet et al. (PMID:23648119) report KIT c.1924A>G, p.K642E in three GIST families; the extracted finding does not identify a second pathogenic variant or cis/trans phase.

Still missing

• Identification of a second pathogenic or likely pathogenic variant in the same individual
• Testing that establishes cis or trans phase
• Confirmation that the relevant dominant disorder is fully penetrant if applying the trans pathway

Sources

Reasoning

The reviewed exact-variant reports include familial GIST, acral melanoma, and thymic carcinoma, but none documents that the variant was found in a case with an independent alternate molecular basis for the disease being evaluated. Occurrence in another tumor type is not itself an alternate molecular basis. Therefore BP5 cannot be assessed from the available evidence.

Rule

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

Evidence considered

• PMID:23940219 reports KIT K642E in a metastatic acral melanoma case, but does not document an independent alternate molecular cause of that disease.
• PMID:24855380 reports KIT K642E in a metastatic thymic carcinoma case, but does not document an independent alternate molecular cause of that disease.
• The reports of familial GIST and other tumor types do not establish the BP5 finding of an alternate molecular basis.

Still missing

• A case report documenting the variant together with an independent alternate molecular cause of the relevant disease is needed.

Sources