NM_002529.3:c.1999G>A (p.Gly667Ser) is a novel missense variant in the NTRK1 gene. It is absent from population databases (gnomAD v2.1, v4.1, gnomAD-Canada; PM2) and is located in the functionally critical xDFG motif of the tyrosine kinase domain immediately N-terminal to the DFG activation loop (PM1). In silico data from two publications (PMID:33004339, PMID:33328556) demonstrate altered kinase inhibitor binding for G667S by molecular docking and molecular dynamics simulations, and G667S has been observed as an acquired somatic resistance mutation in two patients with TRK fusion-positive cancers on larotrectinib therapy (PMID:33004339). No experimental functional characterization of G667S exists, and the variant has not been reported in ClinVar. In silico predictors are discordant (REVEL 0.752, BayesDel 0.262).1 Two moderate pathogenic criteria are met (PM1, PM2). Under the generic ACMG/AMP 2015 framework (Richards et al., PMID:25741868), this does not reach the threshold for Likely Pathogenic (which requires ≥3 moderate criteria, or ≥1 strong + ≥1 moderate, or ≥2 moderate + ≥2 supporting). No benign criteria are met. The variant is classified as a Variant of Uncertain Significance (VUS).2
NTRK1
Final classification
VUS
NTRK1 c.1999G>A · p.Gly667Ser
NTRK1
NM_002529.3:c.1999G>A (p.Gly667Ser) is a novel missense variant in the NTRK1 gene. It is absent from population databases (gnomAD v2.1, v4.1, gnomAD-Canada; PM2) and is located in the functionally critical xDFG motif of the tyrosine kinase domain immediately N-terminal to the DFG activation loop (PM1). In silico data from two publications (PMID:33004339, PMID:33328556) demonstrate altered kinase inhibitor binding for G667S by molecular docking and molecular dynamics simulations, and G667S has been observed as an acquired somatic resistance mutation in two patients with TRK fusion-positive cancers on larotrectinib therapy (PMID:33004339). No experimental functional characterization of G667S exists, and the variant has not been reported in ClinVar. In silico predictors are discordant (REVEL 0.752, BayesDel 0.262).
gene-specific framework lacked a usable explicit final combination framework, so generic ACMG/AMP 2015 final-combination rules were applied as fallback; applied criteria: PM1 moderate, PM2 moderate; combination = 2 moderate, which maps to VUS.
Classification rationale
PM1PM2
VUS
NTRK1 c.1999G>A
PM1 + PM2
→
VUS
1
PMID:33004339 ↗PMID:33328556 ↗revelbayesdelgnomad_v2 ↗gnomad_v4 ↗
2
generic_acmg_combination_rules
Gene diagram
· NM_002529.3 · variants mapped to exon structure
NTRK1
NM_002529.3
Fetching transcript structure from UCSC…
Applied criteria · 2 applied · 20 assessed
Applied · 2
Strength
Supporting
Moderate
Strong
Very strong
✓
PM1
moderate
Pathogenic
The variant alters Gly667, which is the xDFG residue immediately N-terminal to the DFG motif (Asp668-Phe669-Gly670) in the activation loop of the NTRK1 tyrosine kinase domain. The DFG motif is a critical, evolutionarily conserved structural element that governs kinase conformational state (DFG-in active vs DFG-out inactive). PMID:33004339 demonstrates that xDFG substitutions at G667 (including G667S) emerge as clinical resistance mutations to type I TRK inhibitors and alter kinase-inhibitor interactions via in silico modeling. PMID:33328556 confirms altered drug binding for G667S across five kinase inhibitors by molecular docking. This residue lies within a well-characterized, functionally critical domain without known benign variation.
G667 is the xDFG residue in the NTRK1 kinase activation loopcritical for kinase conformation. Altered inhibitor binding demonstrated in silico. Clinical emergence as somatic resistance mutation confirms functional importance.
✓
PM2
moderate
Pathogenic
NM_002529.3:c.1999G>A is absent from gnomAD v2.1 (exomes), gnomAD v4.1 (exomes), and gnomAD-Canada v1.0 (genomes). The variant has not been observed in any population database, consistent with PM2 at the <0.1% allele frequency threshold for a rare disease gene.
Absent from gnomAD v2.1 (AC=0). Absent from gnomAD v4.1 (AC=0). Absent from gnomAD-Canada v1.0 (AC=0AN=0).
Assessed · not applied
Pathogenic
PS2
No de novo observation data are available for this variant.
PS3
No experimental functional data exist for NM_002529.3:c.1999G>A (p.G667S) relevant to germline disease mechanism.
PS4
No case-control or cohort data comparing variant prevalence in affected individuals versus controls are available.
PM5
No missense variant at codon 667 resulting in a different amino acid substitution has been classified as germline pathogenic in ClinVar.
PM6
No de novo observation data are available.
PP1
No segregation data are available for this variant.
PP2
The HCI prior score is not available for NTRK1 (gene_not_supported), and no missense constraint Z-score or benign missense depletion data were provided in the evidence packet.
PP3
In silico predictions are mixed and do not provide multiple concordant lines of evidence for a deleterious effect.
PP4
No patient phenotype or clinical data were provided with this case.
PP5
NM_002529.3:c.1999G>A is absent from ClinVar.
Benign
BA1
The variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada.
BS1
The variant is absent from all population databases.
BS2
The variant has not been observed in any individual, healthy or affected, in population databases or published cohorts.
BS3
No well-established in vitro or in vivo functional studies demonstrate a benign effect for p.G667S.
BS4
No segregation data are available to assess lack of segregation with disease in affected family members.
BP1
NTRK1-related CIPA (congenital insensitivity to pain with anhidrosis) is caused by both missense and truncating loss-of-function variants.
BP2
No phasing or trans-configuration data are available.
BP4
Computational evidence is mixed and does not provide multiple concordant lines suggesting no impact.
BP5
No data are available indicating that the proband has an alternate molecular basis for disease.
BP6
NM_002529.3:c.1999G>A is absent from ClinVar.
N/A · 3
PVS1 · PS1 · BP7
Research & evidence
Population frequency
gnomAD v4.1
gnomAD v2.1
v4.1
Absent from gnomAD v4.1.
v2.1
Absent from gnomAD v2.1.
🇨🇦 CA
Absent from gnomAD-Canada v1.0.
Allele frequency by ancestry
three datasets · side by side
gnomAD v4.1
Absent
· 0 / ?
0 hom
0 hom
Not observed in any ancestry group.
gnomAD v2.1
Absent
· 0 / ?
0 hom
0 hom
Not observed in any ancestry group.
gnomAD Canada 🇨🇦
Absent
· 0 / ?
0 hom
0 hom
Not observed in any ancestry group.
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.16). REVEL score = 0.752. BayesDel score = 0.262014.
Functional
Inconclusive
OncoKB identified variant-specific curated literature and context relevant to functional review; biological-effect context: Inconclusive; curated oncogenicity label: Inconclusive.
COSMIC
Cancer hotspots
Somatic evidence
Not in COSMIC / hotspots
COSMIC
This variant does not lie in a statistically significant hotspot. This variant has not previously been reported in somatic cancers (COSMIC).
Hotspots
This variant does not lie in a statistically significant hotspot.
Literature · how each cited paper was used
2papers cited
Each card is an audit: what was searched, what was found, whether it names the variant, which criteria it fed, and why. 2 further PMIDs triaged but not cited — see Sources & References.
TRK xDFG Mutations Trigger a Sensitivity Switch from Type I to II Kinase Inhibitors.
Searched
G667Sp.Gly667Serc.1999G>AG667
Found
TRKA G667S emerged as an acquired resistance mutation in two patients with TRK fusion-positive cancers on larotrectinib: Patient 4 with TPR-NTRK1 non-small cell lung cancer and Patient 5 with TPM3-NTRK1 thyroid cancer. Both patients showed intrinsic resistance to the second-generation inhibitor selitrectinib. G667S was included in molecular dynamics simulations of TRKA xDFG mutants. In vitro kinase assays were performed on G667C and G667A but not on G667S.
Variant
✓ Names this variant — characterised directly
Applied to
→PM1 supports · met
Why
Variant-specific clinical and in silico data confirm location in functionally critical xDFG motif; supports PM1 domain-level assessment. No experimental functional assay performed on G667S.
Patient 4 with a TPR-NTRK1-positive non-small cell lung cancer had a partial response (PR) on larotrectinib. Sequencing of cfDNA at progression identified the emergence of TRKA G667S.
Location Results, paragraph on TRKA xDFG mutations (Patients 4 and 5); Methods, Structure Preparation section · Context Cell-free DNA sequencing from clinical samples; molecular dynamics simulations; in vitro kinase assays (G667C and G667A only, not G667S) · full text
NTRK kinase domain mutations in cancer variably impact sensitivity to type I and type II inhibitors.
Searched
G667Sp.Gly667Serc.1999G>AG667
Found
NTRK1 G667S was evaluated by molecular docking in both DFG-in (active) and DFG-out (inactive) kinase conformations. Dissociation constants showed reduced larotrectinib affinity in the DFG-in state (Kd 535.9 nM vs 100.9 nM wild-type), reduced entrectinib affinity (Kd 45.3 nM vs 4.6 nM wild-type), and paradoxically enhanced foretinib affinity in the DFG-out state (Kd 17.4 nM vs 54.3 nM wild-type). The study did not perform in vitro kinase assays or cell-based experiments on G667S; experimental work focused on G667C, V573M, and F589L.
Variant
✓ Names this variant — characterised directly
Applied to
→PM1 supports · met
Why
In silico evidence confirms G667S alters kinase inhibitor binding properties compared to wild-type; supports PM1 domain-level assessment. No experimental functional data for G667S.
G667S variant dissociation constants in Table 1: larotrectinib Active=535.9 nM, Inactive=2265.4 nM; entrectinib Active=45.3 nM, Inactive=147.1 nM; altiratinib Active=377.2 nM, Inactive=81.7 nM; cabozantinib Active=689.2 nM, Inactive=116.3 nM; foretinib Active=350.2 nM, Inactive=17.4 nM.
Location Table 1 (Dissociation constants); Results section · Context Molecular docking and homology modeling of NTRK1 kinase domain; Ba/F3 cell viability assays and immunoblotting performed for G667C, V573M, and F589L but not G667S · full text
Sources & reference links
Triaged references · 2 PMIDs not cited in assessment
30624546 ↗
Larotrectinib in adult patients with solid tumours: a multi-centre, open-label, phase I dose-escalation study.
ONCOKB