KIT c.2466T>G, p.Asn822Lys

Reasoning

NM_000222.3:c.2466T>G produces KIT p.Asn822Lys (N822K). Multiple variant-specific functional studies support an activating/damaging effect in biologically relevant systems. In PMID:15790786, engineered Ba/F3 cells expressing N822K acquired IL-3-independent growth, showed constitutive KIT phosphorylation, and had reproducible inhibitor-response data from at least two triplicate experiments, with N822K transforming and showing a PKC412 IC50 of 59 nM versus 138 nM for stem-cell-factor-stimulated wild-type KIT. PMID:17699867 independently evaluated engineered Ba/F3 cells expressing KIT N822K and reported variant-specific proliferation/drug-response and KIT-phosphorylation assays. PMID:16954519 reported N822K in kinase-domain functional studies and biochemical imatinib-sensitivity testing, while PMID:21482694 identified N822K as an activating KIT mutation in Kasumi-1 cells and showed dose-dependent inhibition of KIT phosphorylation and cell viability. The concordant cellular transformation/signaling and drug-response findings, with wild-type or untreated controls where reported, support PS3 at strong strength. These are research functional studies rather than validated/reproducible clinical diagnostic-laboratory assays, so no higher-than-strong calibration is applied.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.

Evidence considered

• Growney et al. 2005 (PMID:15790786), Results/Table 1: engineered Ba/F3 cells expressing KIT N822K transformed to IL-3-independent growth; N822K was constitutively phosphorylated and showed PKC412 IC50 59 nM versus 138 nM for wild-type KIT stimulated with recombinant human stem cell factor; values were representative of at least two experiments performed in triplicate.
• Guo et al. 2007 (PMID:17699867), Ba/F3 KIT mutant system: single KIT N822K was directly tested in proliferation/drug-response and KIT-phosphorylation assays, providing variant-specific functional evidence.
• Heinrich et al. 2008 (PMID:16954519), functional and biochemical testing: KIT N822K was evaluated in kinase-domain mutation studies and imatinib-sensitivity assays, including comparison of isolated N822K with a V560D/N822K context.
• Shabbir et al. 2014 (PMID:21482694), Kasumi-1 leukemic cells harboring activating KIT N822K: ponatinib inhibited KIT phosphorylation dose-dependently and inhibited cell viability, with phosphorylation IC50 values in the reported 0.3-20 nM range across activated receptor tyrosine kinase models and viability IC50 values of 0.5-17 nM.
• The evidence supports altered KIT signaling and oncogenic cellular behavior, not merely a computational prediction or a clinical classification label.

Sources

Reasoning

PM2 is met at supporting strength. The exact variant is absent from gnomAD v2.1 and gnomAD v4.1, and the evidence also reports absence from the available gnomAD v2.1 non-cancer and v3.1 non-cancer subsets. This satisfies the supplied generic PM2 supporting threshold of <=0.0001 because the observed allele frequency is zero in the queried datasets.

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 (exome) reports the exact KIT NM_000222.3:c.2466T>G variant as absent.
• gnomAD v4.1 (exome) reports the exact KIT NM_000222.3:c.2466T>G variant as absent.
• The population evidence additionally reports absence from GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; no governing VCEP-specific source restriction applies because no VCEP covers KIT.
• The supplied generic PM2 calibration threshold is <=0.0001 for supporting strength, identified as the ClinGen SVI recommendation to downgrade PM2 to Supporting (PMID:25741868).

Sources

Reasoning

The variant is a missense substitution, NM_000222.3:c.2466T>G, p.(Asn822Lys), so the governing generic rule selects the REVEL path only; SpliceAI is not used for missense PP3/BP4 assessment. REVEL is 0.682, meeting the generic PP3 supporting threshold of >= 0.644 from the ClinGen SVI calibration (Pejaver et al. 2022, PMID:36413997). BayesDel is not used because no generic ACMG-strength calibration is established.

Rule

PP3: 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 (supporting): >= 0.644 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• NM_000222.3:c.2466T>G is a missense variant resulting in NP_000213.1:p.(Asn822Lys), so REVEL is the applicable computational path and SpliceAI is excluded.
• Local REVEL lookup reports a score of 0.682 for chr4:54733174 T>G.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997) supplies the generic PP3 supporting threshold of >= 0.644; 0.682 meets it.
• BayesDel score -0.138449 was not used because the governing generic rules provide no ACMG-strength calibration for BayesDel.

Sources

Reasoning

No qualifying reputable-source assertion for PP5 was identified. ClinVar reports the exact variant as Pathogenic/Likely pathogenic from ordinary laboratory submissions with no expert-panel submissions, and the case instructions exclude ordinary laboratory submissions, aggregate labels, and non-expert assertions from PP5. The cited literature is not an eligible independent PP5 source for this criterion.

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-match record 375932 reports Pathogenic from two clinical laboratories and Likely pathogenic from one clinical laboratory, with review status 'criteria provided, multiple submitters, no conflicts' and zero expert-panel submissions.
• The ClinVar submission audit identifies all usable classifications as non-expert clinical-laboratory submissions; none is an expert-panel assertion eligible for PP5 under the specified rule.

Sources

Reasoning

BA1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, so no filtering allele frequency or grpmax FAF reaches the generic stand-alone threshold of >=0.05. The absence of a qualifying frequency also avoids using a small-population popmax point estimate as a stand-alone benign basis.

Rule

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

Evidence considered

• gnomAD v2.1 (exome) reports the exact KIT NM_000222.3:c.2466T>G variant as absent; no allele count, allele number, AF, or grpmax FAF is available.
• gnomAD v4.1 (exome) reports the exact KIT NM_000222.3:c.2466T>G variant as absent; no allele count, allele number, AF, or grpmax FAF is available.
• The supplied generic BA1 calibration threshold is >=0.05, identified as ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).

Sources

Reasoning

BS1 is not met on the available population evidence. The variant is absent from gnomAD v2.1 and v4.1, so there is no filtering allele frequency or grpmax FAF demonstrating a frequency greater than expected for the disorder. The generic 1% default cannot be reached by an absent variant, and no maximum-credible-frequency calculation is needed because no elevated frequency is observed.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1 (exome) reports the exact KIT NM_000222.3:c.2466T>G variant as absent; no filtering AF or grpmax FAF is available.
• gnomAD v4.1 (exome) reports the exact KIT NM_000222.3:c.2466T>G variant as absent; no filtering AF or grpmax FAF is available.
• The supplied generic BS1 calibration threshold is >=0.01, with per-gene derivation preferred under Whiffin et al. 2017 (PMID:28518168).

Sources

Reasoning

The supplied gene-level and literature evidence describes KIT disease-associated variants as recurrent activating missense substitutions in the kinase domain, including N822K, rather than a gene for which primarily truncating variants are known to cause disease. Therefore the BP1 condition is not met for this missense variant.

Rule

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

Evidence considered

• OncoKB describes KIT as recurrently mutated and identifies a gain-of-function context for KIT N822K.
• PMID:16954519 reports recurrent KIT kinase-domain missense substitutions, including N822K/Y, in GIST.
• PMID:17699867 and PMID:21482694 experimentally study activating KIT missense N822K in cellular systems.

Sources

Reasoning

The variant is a missense substitution, NM_000222.3:c.2466T>G, p.(Asn822Lys), so the governing generic rule selects the REVEL path only; SpliceAI is not evidence for missense BP4. REVEL is 0.682, above the generic BP4 supporting threshold of <= 0.29, so BP4 is not met. BayesDel is not used because no generic ACMG-strength calibration is established.

Rule

BP4: 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

• NM_000222.3:c.2466T>G is a missense variant resulting in NP_000213.1:p.(Asn822Lys), so REVEL is the applicable computational path and SpliceAI cannot substitute for it.
• Local REVEL lookup reports a score of 0.682 for chr4:54733174 T>G.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997) supplies the generic BP4 supporting threshold of <= 0.29; 0.682 does not meet it.
• BayesDel score -0.138449 was not used because the governing generic rules provide no ACMG-strength calibration for BayesDel.

Sources

Reasoning

No qualifying reputable-source benign assertion for the exact variant was identified. ClinVar contains Pathogenic/Likely pathogenic laboratory submissions and no expert-panel benign or likely-benign classification; ordinary submissions, aggregate labels, 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-match record 375932 reports only Pathogenic or Likely pathogenic laboratory classifications, with zero expert-panel submissions and no benign or likely-benign expert assertion.
• The ClinVar submission audit identifies no qualifying reputable-source benign assertion for this exact variant.

Sources

Reasoning

The target variant is KIT p.Asn822Lys (N822K). The reviewed literature documents the same protein-level change, but no independently established pathogenic KIT N822K caused by a different nucleotide change is available in the cited evidence. The target variant's own ClinVar classification and reports of the same nucleotide change cannot independently satisfy PS1.

Rule

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

Evidence considered

• The normalized variant is NM_000222.3:c.2466T>G, KIT p.(Asn822Lys) / N822K.
• PMID:17699867, PMID:16954519, and PMID:21482694 document KIT N822K at the protein level, but the reviewed records do not establish a different nucleotide change for the same amino-acid substitution.
• ClinVar classifies the target NM_000222.3:c.2466T>G variant as Pathogenic/Likely pathogenic, but this is the target nucleotide change rather than an independent alternate-nucleotide N822K record.

Still missing

• An independently established pathogenic KIT N822K record caused by a different nucleotide change is required.

Sources

Reasoning

No case-specific evidence documents that NM_000222.3:c.2466T>G (KIT p.Asn822Lys) is de novo in an affected proband, confirms both maternity and paternity, or establishes absence of family history. The available literature concerns other KIT codon-822 substitutions or functional studies and does not provide this variant's parentage data.

Rule

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

Evidence considered

• No case-specific pedigree or parental-testing result is present for NM_000222.3:c.2466T>G (KIT p.Asn822Lys).
• Thalheimer et al. (PMID:18724244) reports KIT N822Y, not N822K, in an affected father and three affected siblings; this is a different amino-acid substitution and cannot establish de novo status for the assessed variant.
• Wasag et al. (PMID:21689725) reports familial KIT N822I, not N822K; this is a different amino-acid substitution and does not establish de novo status for the assessed variant.

Still missing

• Affected proband phenotype and disease diagnosis are not documented.
• Maternity and paternity confirmation are not documented.
• Parental variant testing and family-history assessment are not documented.

Sources

Reasoning

No qualifying case-control comparison was identified for NM_000222.3:c.2466T>G (KIT p.Asn822Lys). The literature_pass.json PS4 index listed PMID:21262832, PMID:25394175, and PMID:42809666, but verification shows PMID:21262832 is a murine functional model, PMID:25394175 is a referral-practice guideline that does not report this variant, and PMID:42809666 has no usable title or abstract; none provides affected-case and unaffected/population-control counts, an odds ratio, or a confidence interval. The variant is absent from gnomAD v2.1 and v4.1, but no affected-case prevalence is available, so an OR and 95% CI cannot be computed. The governing comparison is OR > 5.0 with a confidence interval that does not include 1.0; because no OR or CI is available, the required inequality cannot be evaluated.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... 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

• literature_pass.json ai_classified_papers contains no paper classified as PS4; the listed PS4 leads were independently checked against the available abstract/metadata.
• PMID:21262832 abstract: the study engineered HyC-KIT N822K and tested transformation in murine 32D cells and mice; it is not a case-control prevalence study and reports no affected-versus-control counts for this exact germline variant.
• PMID:25394175 abstract: this is an ACMG/NSGC practice guideline on referral indications and does not mention NM_000222.3:c.2466T>G or provide variant-specific case-control data.
• PMID:42809666 has no title, abstract, or usable full-text extraction in the case materials, so it cannot establish PS4.
• gnomAD v2.1 and v4.1 both report the exact variant as absent; absence alone supplies no affected-case prevalence and therefore cannot yield an OR or 95% CI.

Still missing

• A case-control dataset or affected-case and matched-ancestry control counts for the exact variant are required to calculate OR and 95% CI.

Sources

Reasoning

KIT p.Asn822Lys lies in the kinase activation loop, and the case evidence identifies residue N822 as a statistically significant hotspot. However, the generic PM1 definition requires the hotspot or critical domain to be without benign variation, and the supplied evidence does not establish absence of benign variation across the relevant region. No KIT VCEP domain table is present; the target variant's absence from population databases does not establish absence of benign variation in the domain or hotspot.

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

• The Cancer Hotspots result identifies KIT N822 as a statistically significant hotspot.
• PMID:16954519 places N822K/Y in the KIT kinase activation loop and reports recurrent kinase-domain substitutions in that region.
• PMID:17699867 and PMID:21482694 provide functional context for KIT N822K in engineered or leukemic cellular systems.
• No KIT VCEP material or authoritative domain table is present, and the supplied population evidence is for the target variant rather than benign variation across the relevant domain.

Still missing

• A documented assessment showing no benign variation in the relevant KIT activation-loop hotspot or critical domain is required.

Sources

Reasoning

Different missense substitutions at KIT residue 822, including N822Y, are reported. However, the reviewed evidence does not provide an independently auditable determination that an alternate residue-822 missense variant was determined pathogenic, and the target variant's own classification cannot be used for that purpose.

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:16954519 reports KIT N822Y as an alternate missense substitution at the same residue as the target N822K.
• The reviewed extract describes N822Y in a kinase-domain resistance context but does not provide a clear independent pathogenic determination for N822Y under the PM5 rule.
• The target variant's ClinVar classification is not used as evidence that N822Y is pathogenic.

Still missing

• An auditable pathogenic determination for a different KIT missense substitution at residue 822 is required.

Sources

Reasoning

No case-specific evidence documents an assumed de novo occurrence of NM_000222.3:c.2466T>G (KIT p.Asn822Lys) in an affected patient. There is no parental testing result showing the variant absent from either parent, and the available familial reports concern different KIT codon-822 substitutions.

Rule

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

Evidence considered

• No case-specific parental genotypes or de novo assertion are available for NM_000222.3:c.2466T>G (KIT p.Asn822Lys).
• PMID:18724244 concerns familial KIT N822Y, and PMID:21689725 concerns familial KIT N822I; neither paper supplies parental evidence for KIT N822K.

Still missing

• Affected proband phenotype and disease diagnosis are not documented.
• Parental testing is not documented, including whether either parent carries the variant.
• Family history is not documented.

Sources

Reasoning

No case-specific affected relatives, unaffected relatives, variant-positive relatives, or meioses are documented for NM_000222.3:c.2466T>G (KIT p.Asn822Lys). Reports of familial KIT N822Y and N822I show segregation for different substitutions and cannot be counted as cosegregation evidence for N822K.

Rule

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

Evidence considered

• No segregation data for NM_000222.3:c.2466T>G (KIT p.Asn822Lys) are available in the case evidence.
• PMID:18724244 reports KIT N822Y in an affected father and three affected siblings, with a healthy brother carrying wild-type KIT; because N822Y is not N822K, these observations cannot be counted for this variant.
• PMID:21689725 reports KIT N822I in an affected father and two children; because N822I is not N822K, these observations cannot be counted for this variant.

Still missing

• Family pedigree and number of affected relatives are not documented.
• Genotypes and phenotypes of affected and unaffected relatives are not documented.
• Informative meioses and phenotype consistency are not documented.

Sources

Reasoning

The evidence supports recurrent activating KIT missense variants, but it does not establish both gene-level requirements for PP2: a low rate of benign missense variation and missense variants as a common mechanism of disease. Absence of the target variant from gnomAD is variant-level evidence and does not quantify the gene's benign missense rate.

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

• The target variant is absent from gnomAD v2.1 and v4.1, which does not establish a gene-level rate of benign missense variation.
• OncoKB and the reviewed functional and clinical literature document recurrent activating KIT missense variants, but do not provide the required gene-level benign-missense rate and common-mechanism assessment together.

Still missing

• Gene-level evidence for a low rate of benign KIT missense variation is missing.
• Gene-level evidence establishing missense variants as a common KIT disease mechanism under this rule is missing.

Sources

Reasoning

The case materials do not provide the patient's phenotype, family history, or a disease-specific clinical diagnosis for this variant. Therefore, specificity of the phenotype for a single genetic etiology cannot be judged.

Rule

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

Still missing

• Patient phenotype, family history, and the suspected disease diagnosis are required for PP4 assessment.

Reasoning

BS2 is not assessed. The available case bundle contains no documented observation of this exact variant in a healthy adult, no healthy-adult genotype/phenotype cohort, and no evidence establishing that the relevant KIT disorder has full penetrance with expected early-age expression. Population absence cannot substitute for the healthy-adult observation required by 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.

Still missing

• A documented healthy adult carrying the exact KIT NM_000222.3:c.2466T>G variant, with age and phenotype context, is unavailable.
• The relevant disorder-specific penetrance and age-of-onset context needed for BS2 are unavailable.

Reasoning

No well-established functional study in the reviewed evidence demonstrates that KIT p.Asn822Lys has no damaging effect on protein function or splicing. The available variant-specific studies instead report activating KIT signaling, cellular transformation, altered phosphorylation, or drug-response phenotypes, which cannot establish BS3. A validated benign functional assay with appropriate wild-type and disease-relevant controls is the remaining gap.

Rule

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

Evidence considered

• The reviewed variant-specific functional studies report activating or altered KIT phenotypes rather than a benign functional result; they therefore do not support BS3.
• No source was used as evidence for a benign functional effect.

Still missing

• A well-established, validated functional assay demonstrating no damaging effect on KIT protein function or splicing is not available in the reviewed evidence.

Sources

Reasoning

No case-specific affected family members carrying or lacking NM_000222.3:c.2466T>G (KIT p.Asn822Lys) are documented, so lack of segregation cannot be evaluated. The available familial papers describe different KIT substitutions and do not provide non-segregation evidence for N822K.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected-relative non-segregation data for NM_000222.3:c.2466T>G (KIT p.Asn822Lys) are available in the case evidence.
• PMID:18724244 describes KIT N822Y, and PMID:21689725 describes KIT N822I; neither is evidence that KIT N822K fails to segregate with disease.

Still missing

• Affected relatives and their clinical phenotypes are not documented.
• Variant testing in affected relatives is not documented.
• Potential phenocopies and alternative pathogenic variants in the family are not documented.

Sources

Reasoning

BP2 requires an observed allelic relationship: the variant in trans with a pathogenic variant for a fully penetrant dominant disorder, or in cis with a pathogenic variant in any inheritance pattern. The available case evidence contains no affected-proband observation, second pathogenic variant, parental testing, pedigree phase, or other direct cis/trans determination for KIT p.Asn822Lys. Functional studies and somatic tumor observations do not establish cis or trans phase, so 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

• The reviewed evidence reports KIT p.Asn822Lys in functional cellular and tumor contexts, but does not report a second pathogenic allele in an individual carrying this variant.
• No parental testing, offspring testing, pedigree phase, or affected-proband allelic observation is documented for cis or trans assessment.

Still missing

• No second pathogenic variant is reported for the same individual or family.
• No parental or offspring testing establishes phase.
• The available functional and somatic tumor studies do not provide cis/trans evidence.

Reasoning

The case materials do not document that this variant was found in a case with an alternate molecular basis for disease. The available papers describe functional models, tumor mutations, or unrelated clinical guidance, not an affected case with an independently established alternate molecular diagnosis.

Rule

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

Evidence considered

• The reviewed case and literature materials contain no documented alternate pathogenic molecular diagnosis in a patient carrying NM_000222.3:c.2466T>G.

Still missing

• A clinical case linking the exact variant to disease while establishing an alternate molecular diagnosis is required for BP5.

Sources