KIT c.2447A>T, p.Asp816Val

Reasoning

NM_000222.3:c.2447A>T encodes KIT p.Asp816Val (D816V), and multiple independent variant-specific functional studies demonstrate a damaging gain-of-function effect. In Ba/F3 cells, D816V produced IL-3-independent growth, constitutive KIT and Stat3/Stat5 phosphorylation, and drug-responsive kinase signaling (PMID:15790786). In COS-cell assays, D816V showed increased ligand-independent kinase activity and tyrosine phosphorylation compared with wild-type KIT (PMID:10362788). In an in vivo mast-cell-specific human KIT D816V transgenic mouse model, the variant caused mast-cell hyperplasia, mastocytosis-like disease, and an intestinal mast-cell tumor, with constitutive KIT phosphorylation and growth-factor-independent proliferation (PMID:16352739). Independent Ba/F3 and mast-cell studies also demonstrated increased STAT5 activation and growth associated with D816V (PMID:19893034), while additional assays documented altered kinase inhibitor response and constitutive activation (PMID:12569358; PMID:14645423; PMID:15972446). These are direct variant-specific in vitro and in vivo functional results with relevant cellular controls or wild-type comparisons and are concordant with the established damaging activating mechanism; therefore PS3 is met at strong strength. The studies were not described as validated and reproducible clinical diagnostic-laboratory assays, so no upgrade beyond the standard strong PS3 level is applied.

Rule

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

Evidence considered

• PMID:15790786: In retrovirally transduced murine Ba/F3 cells, KIT D816V caused IL-3-independent growth, constitutive KIT phosphorylation, downstream Stat3/Stat5 phosphorylation, and PKC412-sensitive signaling and growth.
• PMID:10362788: COS-cell transfection and kinase assays showed markedly increased ligand-independent KIT D816V kinase activity and tyrosine phosphorylation compared with wild-type KIT.
• PMID:16352739: Mast-cell-specific transgenic mice expressing human KIT Asp816Val developed mast-cell hyperplasia, systemic mastocytosis-like disease, or an intestinal mast-cell tumor; mutant mast cells showed constitutive KIT phosphorylation and growth-factor-independent proliferation.
• PMID:19893034: Doxycycline-inducible Ba/F3 experiments showed marked KIT D816V-associated STAT5 phosphorylation and growth-related signaling, inhibited by midostaurin.
• PMID:12569358, PMID:14645423, PMID:15972446: Independent cell-based assays documented constitutive D816V activity and characteristic inhibitor-response differences, including resistance to imatinib/STI571 and sensitivity to PKC412 in relevant assays.

Still missing

• The reviewed publications do not establish validation and reproducibility in a clinical diagnostic laboratory setting.

Sources

Reasoning

KIT p.Asp816Val lies at residue 816 in the KIT activation loop. The directly reviewed functional paper states that D816V and D816Y are the most common activating alleles in the relevant disease context and are located in the c-KIT activation loop. The case evidence also reports the variant in a statistically significant hotspot, while gnomAD v4.1 shows only 3/1,612,874 total alleles (AF 1.86003e-06) and no homozygotes. vcep_materials.json has no KIT domain-table entry; therefore there is no authoritative VCEP domain list to extend or override this generic assessment.

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

• Blood 2005, Introduction, states: "the most common activating alleles in the context of SMCD and AML are the D816V and D816Y mutations in the c-KIT activation loop."
• CancerHotspots reports that KIT D816 lies in a statistically significant hotspot; the extracted hotspot record is partially parsed.
• gnomAD v4.1: 3/1,612,874 total alleles, AF 1.86003e-06, zero homozygotes; the maximum group FAF is 6.8e-07.
• vcep_materials.json domain_tables is empty for KIT, so no VCEP domain-table entry exists to check.

Sources

Reasoning

PM2 is met at supporting strength. NM_000222.3:c.2447A>T is absent from gnomAD v2.1 all-comers and non-cancer exomes and from gnomAD v3.1 non-cancer, while gnomAD v4.1 all-comers contains only 3/1612874 alleles (AF 1.86003e-06) and 0 homozygotes. These observations are at or below the supplied generic PM2 supporting threshold of <=0.0001. The variant is a missense substitution, so the indel population-calling caveat does not apply.

Rule

PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868) ... 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 all-comers: NM_000222.3:c.2447A>T was absent, 0/251128 alleles, AF 0, with 0 homozygotes.
• gnomAD v2.1 non-cancer exomes: the variant was absent, 0/236590 alleles, AF 0, with 0 homozygotes.
• gnomAD v3.1 non-cancer: the variant was absent.
• gnomAD v4.1 all-comers: 3/1612874 alleles, AF 1.86003e-06, with 0 homozygotes; highest subpopulation AF 2.54435e-06 in European (non-Finnish).
• Generic calibration threshold supplied for PM2 supporting: <=0.0001, identified as the SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020; PMID:25741868).

Sources

Reasoning

A different missense substitution at the same residue, KIT p.Asp816Tyr (D816Y), is reported as a common activating c-KIT mutation in systemic mast-cell disease and acute myeloid leukemia contexts. The same paper reports D816Y and D816V as activation mutations, satisfying the same-residue alternate-missense requirement for PM5. A separate paper also evaluates KIT p.Asp816His as a constitutively active D816 comparator, but the D816Y evidence is the primary PM5 comparator.

Rule

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

Evidence considered

• Blood 2005, Introduction, identifies D816Y as one of the most common activating c-KIT alleles in SMCD and AML and places it in the activation loop.
• Blood 2005, Abstract, reports that c-KIT D816Y and D816V transformed cells and describes both as activation mutations.
• PMID:10362788 reports D816H as a constitutively active KIT mutant comparator, supporting recurrence of pathogenic activating missense substitutions at residue 816.

Still missing

• The reviewed reports establish disease-associated activating behavior but do not provide a formal germline ClinVar pathogenic assertion for p.Asp816Tyr.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; SpliceAI and BayesDel are not used for PP3 in this situation. REVEL is 0.89, meeting the supplied generic PP3 supporting threshold of >= 0.644 but not the moderate threshold of >= 0.773. Therefore PP3 is met at supporting strength.

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

Evidence considered

• Case normalization identifies NM_000222.3:c.2447A>T as the missense variant NP_000213.1:p.(Asp816Val), so the generic missense path applies.
• Local REVEL lookup reports a score of 0.89.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997) supplies the generic PP3 supporting threshold of >= 0.644 and moderate threshold of >= 0.773; 0.89 meets supporting but not moderate strength.
• SpliceAI max delta 0.008 and BayesDel 0.347289 were not counted because the governing rule requires REVEL only for a missense variant, and no generic BayesDel strength calibration is established.

Sources

Reasoning

The variant is KIT p.Asp816Val caused by NM_000222.3:c.2447A>T. The available literature establishes the p.Asp816Val variant itself, but no distinct nucleotide substitution producing the same Asp816Val amino-acid change was identified. Therefore the specific PS1 requirement for the same amino-acid change caused by a different nucleotide change is not met.

Rule

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

Evidence considered

• ClinVar identifies the observed variant as NM_000222.3(KIT):c.2447A>T (p.Asp816Val), not as an alternate nucleotide change producing the same amino-acid substitution.
• Blood 2005 reports KIT D816V as the observed activation mutation; it does not document a separate nucleotide substitution yielding the same p.Asp816Val change.

Sources

Reasoning

The governing ACMG/AMP rule is: "Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation." This exact variant has no ClinVar expert-panel submission (expert_panel_submissions = 0). The pathogenic and likely pathogenic ClinVar assertions are ordinary laboratory, OMIM/literature-only, or other non-expert submissions, and ClinVar is conflicting. Under the required generic rule, none qualifies for PP5.

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 audit reports 0 expert-panel submissions, conflicting classifications, and pathogenic/likely pathogenic assertions only from non-expert or single-submitter records.
• The OMIM pathogenic records are literature-only/no-criteria submissions and the clinical laboratory pathogenic or likely pathogenic records are not expert-panel assertions; these are excluded from PP5 by the criterion-specific rule.

Sources

Reasoning

BA1 is not met. NM_000222.3:c.2447A>T has an observed allele frequency of 0 in gnomAD v2.1 all-comers (0/251128 alleles) and 1.86003e-06 in gnomAD v4.1 all-comers (3/1612874 alleles), far below the generic stand-alone BA1 threshold of >=0.05. The available non-cancer records are also below this threshold: 0/236590 alleles in gnomAD v2.1 non-cancer exomes and absent from gnomAD v3.1 non-cancer. The variant is a missense substitution, so the indel-calling caveat does not apply.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers: NM_000222.3:c.2447A>T was observed at 0/251128 alleles, AF 0, with 0 homozygotes.
• gnomAD v4.1 all-comers: NM_000222.3:c.2447A>T was observed at 3/1612874 alleles, AF 1.86003e-06, with 0 homozygotes; the highest subpopulation AF was 2.54435e-06 in European (non-Finnish).
• gnomAD v2.1 non-cancer exomes: 0/236590 alleles, AF 0, with 0 homozygotes; gnomAD v3.1 non-cancer reported the variant as absent.
• Generic calibration threshold supplied for BA1: >= 0.05, identified as ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).

Sources

Reasoning

BS1 is not met. The observed frequency is 0 in gnomAD v2.1 all-comers and 1.86003e-06 in gnomAD v4.1 all-comers, with no population frequency near the generic BS1 default of >=0.01. The non-cancer subsets are likewise 0 or absent. Although a disease-specific maximum credible allele frequency would be preferred when prevalence, penetrance, and allelic heterogeneity are known, the observed frequency is already far below the supplied default threshold and provides no BS1 evidence.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers: NM_000222.3:c.2447A>T was observed at 0/251128 alleles, AF 0.
• gnomAD v4.1 all-comers: NM_000222.3:c.2447A>T was observed at 3/1612874 alleles, AF 1.86003e-06; the highest subpopulation AF was 2.54435e-06 in European (non-Finnish).
• gnomAD v2.1 non-cancer exomes: AF 0 at 0/236590 alleles; gnomAD v3.1 non-cancer reported the variant as absent.
• Generic calibration threshold supplied for BS1: >= 0.01, identified as the generic SVI default with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

The reviewed variant-specific functional studies do not show a benign or non-damaging effect. Instead, KIT p.Asp816Val produces constitutive kinase and downstream signaling, IL-3-independent or growth-factor-independent proliferation, altered drug response, and mastocytosis-like disease in vivo. Therefore, the evidence is directionally inconsistent with BS3 and does not support applying this benign functional criterion. No assay in the reviewed evidence demonstrated preserved KIT function or normal splicing for this variant.

Rule

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

Evidence considered

• The reviewed functional evidence is contrary to a benign functional interpretation: KIT D816V showed constitutive activation, downstream signaling, growth-factor-independent proliferation, or disease phenotypes in the cited assays.
• No reviewed study demonstrated normal KIT function, absence of damaging effect, or normal splicing for KIT p.Asp816Val.

Still missing

• No well-established variant-specific assay demonstrating no damaging effect on KIT protein function or splicing was identified.

Sources

Reasoning

BP1 is not supported because the available KIT evidence describes the gene as an oncogene with recurrent activating missense mutations, including D816V and D816Y, rather than a gene in which disease is primarily caused by truncating variants. No evidence was found that truncating variants are the primary known disease mechanism for KIT.

Rule

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

Evidence considered

• OncoKB classifies KIT as an oncogene and identifies the variant-specific biological effect as gain-of-function.
• Blood 2005 describes recurrent activating KIT missense mutations D816V and D816Y in disease-associated contexts.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; the SpliceAI score cannot be substituted for a gray-zone REVEL result. REVEL is 0.89, which is above the supplied generic BP4 supporting cutoff of <= 0.29 and therefore does not support no impact. BP4 is not met.

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

• Case normalization identifies NM_000222.3:c.2447A>T as the missense variant NP_000213.1:p.(Asp816Val), so the generic missense path applies.
• Local REVEL lookup reports a score of 0.89.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997) supplies the generic BP4 supporting threshold of <= 0.29; 0.89 does not meet it.
• SpliceAI max delta 0.008 was not used for BP4 because the governing rule selects REVEL only for a missense variant; the REVEL result is not in the BP4 range.

Sources

Reasoning

The governing ACMG/AMP rule is: "Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation." No exact-variant ClinVar expert-panel classification of benign or likely benign is present. The available ClinVar record is conflicting and contains no expert-panel submission; therefore the required source does not exist for 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 audit reports 0 expert-panel submissions and no benign or likely benign expert-panel classification; the record is conflicting.

Sources

Reasoning

A ClinVar submission states that the variant likely occurred de novo in the proband, but the available evidence does not document confirmed maternity and paternity, parental variant testing, or the absence of family history. Therefore, the required PS2 evidence cannot be established.

Rule

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

Evidence considered

• ClinVar exact-variant submission SCV007519758 reports that the variant likely occurred de novo in the current proband, while explicitly noting that parental germline mosaicism cannot be excluded; no maternity, paternity, parental-testing, or family-history results are provided.
• The available literature extracts concern functional studies or somatic disease and do not provide a germline family study for this variant.

Still missing

• Primary proband report or laboratory record documenting the de novo observation
• Confirmed maternity and paternity
• Parental testing results
• Documented absence of family history

Sources

Reasoning

The governing ACMG/AMP rule is: "The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls." The specified generic threshold is: "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." The reviewed literature documents KIT D816V in affected patients and disease models, including six systemic mastocytosis patients in PMID:12569358, but does not provide a usable affected-versus-unaffected case-control count or a matched-ancestry case frequency with which to compute an OR and 95% CI. The gnomAD frequency alone (3/1,612,874 alleles overall; 3/1,179,082 European non-Finnish alleles) cannot establish the affected-case numerator or denominator. Therefore the required comparison cannot be calculated and PS4 is not assessed.

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

• PMID:12569358 reports D816V in bone-marrow cells from all six examined patients with systemic mastocytosis, but does not provide an unaffected-control comparison or a case-control OR/95% CI.
• PMID:19893034 studies 40 patients with systemic mastocytosis and KIT D816V-associated signaling, but the extracted evidence does not provide the D816V-positive case count needed for a prevalence comparison.
• PMID:16352739 is a transgenic mouse disease model comparing transgenic and nontransgenic mice; it is not a human case-control prevalence study for this variant.
• PMID:15725473 and PMID:15972446 are individual clinical reports, not case-control studies.
• gnomAD v4.1 reports 3/1,612,874 total alleles and 3/1,179,082 European non-Finnish alleles; without the affected-case frequency, an OR and 95% CI cannot be calculated.

Still missing

• A case-control study or affected-case cohort must report the number of KIT c.2447A>T/p.Asp816Val carriers and total affected individuals, with comparable controls or matched-ancestry control frequency.
• The OR and 95% CI cannot be computed from the available patient reports and gnomAD frequency alone.

Sources

Reasoning

PM3 cannot be assessed from the available evidence. The governing definition applies to recessive disorders and requires the variant to be detected in trans with a pathogenic variant; this case does not document the relevant inheritance pattern, an affected germline proband, parental testing, or a pathogenic allele in trans.

Rule

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

Still missing

• Confirm whether the relevant KIT-associated disorder is recessive.
• Document an affected germline proband and a second pathogenic variant.
• Perform or provide parental/offspring testing establishing the variants are in trans.

Reasoning

The only available de novo information is an unsupported ClinVar submission assertion that the variant likely occurred de novo. No primary proband report, parental testing details, or clinical documentation establishes an assumed de novo occurrence sufficient for PM6.

Rule

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

Evidence considered

• ClinVar exact-variant submission SCV007519758 states that the variant likely occurred de novo in the current proband, but this is not accompanied by the underlying proband or parental data.
• The available variant-specific paper extracts do not describe a germline de novo case, parental testing, or family segregation.

Still missing

• Primary case report or laboratory evidence supporting de novo occurrence
• Parental testing results or explicit documentation that maternity and paternity were not confirmed
• Clinical phenotype and family-history information for the proband

Sources

Reasoning

No affected family members, informative meioses, or variant-positive relatives are documented. The available publications describe unrelated functional or somatic observations rather than cosegregation in a disease family, so PP1 cannot be assessed.

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

• No family segregation dataset is present in the available case evidence.
• Variant-specific literature extracts describe functional assays, somatic disease, or isolated clinical observations and do not report cosegregation with disease in multiple affected family members.

Still missing

• Pedigree with affected and unaffected relatives
• Genotypes and phenotypes of informative family members
• Number of informative meioses and segregation results

Reasoning

The available case evidence does not establish both required gene-level facts: a low rate of benign KIT missense variation and missense variants as a common mechanism of disease in the applicable disease context. Variant-level rarity and activating tumor biology are insufficient to establish the PP2 gene-level rule.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Still missing

• Gene-wide analysis of benign KIT missense variation and disease mechanism is required.

Reasoning

The governing ACMG/AMP rule is: "Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology." The case materials identify the variant as KIT p.Asp816Val but do not provide this patient's phenotype, family history, or a disease diagnosis sufficiently specific for a single genetic etiology. Published reports of mastocytosis associated with D816V describe other patients and cannot substitute for the current patient's phenotype. PP4 therefore cannot be assessed.

Rule

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

Still missing

• Current-patient phenotype, clinical diagnosis, and relevant family history are required.
• The phenotype must be evaluated for specificity to a disease with a single genetic etiology.

Reasoning

BS2 cannot be assessed from the available evidence. gnomAD reports no homozygotes and three total alleles in v4.1, but the records do not establish that any carrier was a healthy adult, provide the required zygosity for a dominant, recessive, or X-linked disorder, or establish full penetrance at an early age.

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 all-comers and non-cancer exomes report 0 alleles and 0 homozygotes.
• gnomAD v4.1 all-comers reports 3 alleles and 0 homozygotes, but does not provide the healthy-adult, disease-penetrance, and inheritance context required for BS2.
• No reviewed source documents this exact variant in a healthy adult with the required zygosity and full-penetrance early-age disorder context.

Still missing

• A reliable healthy-adult observation for this exact variant is needed, including zygosity and confirmation that full penetrance is expected at an early age.

Sources

Reasoning

No affected relatives who lack the variant are documented, and no pedigree or family-testing results are available. Therefore, lack of segregation cannot be established; the phenocopy and multiple-pathogenic-variant caveats also cannot be evaluated.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected non-carriers or other non-segregation observations are reported in the available case evidence.
• The available literature extracts do not provide a family study suitable for BS4 assessment.

Still missing

• Pedigree identifying affected relatives
• Variant testing results for affected relatives
• Phenotype assessment and alternative-etiology review for non-carrier affected relatives

Reasoning

BP2 cannot be assessed from the available evidence. The governing definition requires observation in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or in cis with a pathogenic variant in any inheritance pattern; no such pathogenic allele or phase relationship is documented for this case.

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.

Still missing

• Identify a qualifying pathogenic variant in the same individual, if present.
• Establish whether the variants are in cis or in trans using parental, offspring, or equivalent phase testing.
• For a trans observation, confirm a fully penetrant dominant KIT-associated disorder context.

Reasoning

The governing ACMG/AMP rule is: "Variant found in a case with an alternate molecular basis for disease." The available reports describe KIT D816V-associated disease or experimental models, but do not establish that the current case has an alternate molecular basis explaining the disease instead of this variant. No qualifying alternate pathogenic variant, molecular diagnosis, or case-level exclusion of KIT causality is documented. BP5 is therefore not assessed.

Rule

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

Still missing

• A current-case diagnosis and an independently established alternate molecular basis are required to evaluate BP5.