NTRK1 c.1783G>A, p.Gly595Arg

Reasoning

PS3 is met at supporting strength. Three variant-specific publications evaluated NTRK1/TRKA p.G595R, the protein consequence of NM_002529.4:c.1783G>A, in biologically relevant systems. Functional evidence included purified TRKA kinase-domain biochemical assays, engineered cellular pharmacology, patient-derived and engineered tumor models, structural modeling, and longitudinal clinical resistance observations. The variant increased ATP affinity relative to wild-type TRKA (Km 6 µM versus 51 µM), produced an entrectinib IC50 greater than 1000 nM in engineered Ba/F3 cells, and drove resistance to TRK inhibitors; p.G595R was also selected independently in resistant models and suppressed by a next-generation inhibitor in vitro and in xenografts. These reproducible variant-specific results demonstrate altered and damaging protein function, but the assays primarily establish oncogenic drug-resistance biology rather than the physiologic NTRK1 loss-of-function mechanism of hereditary CIPA, and no validated clinical diagnostic-laboratory assay was identified; therefore stronger than supporting strength is not justified.

Rule

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

Evidence considered

• PMID:26546295, Results sections on emergence of NTRK1 mutations and resistance-driving mutations: p.G595R emerged during entrectinib treatment, was found in an entrectinib-resistant xenopatient tumor and independently selected resistant KM12 and ETV6-TRKA/Ba/F3 models, created steric hindrance that abrogated entrectinib binding, and produced an entrectinib IC50 >1000 nM in engineered Ba/F3 cells.
• PMID:28578312, Results on preclinical LOXO-195 development and the adult LMNA-NTRK1 case: structural modeling showed steric interference with larotrectinib, purified TRKA assays showed increased ATP affinity for G595R versus wild type (Km 6 µM versus 51 µM), and LOXO-195 inhibited TRKA-G595R in vitro and suppressed ΔTRKA-G595R tumor models.
• PMID:29466156, Results, Primary and Acquired Resistance and Table S3: NTRK1 G595R was identified as a solvent-front kinase-domain mutation that sterically interferes with larotrectinib binding and reduces inhibitory potency; solvent-front resistance mutations were observed in post-progression tumor or plasma samples from 7 patients.
• OncoKB variant-specific record for NTRK1 G595R: curated biological-effect context is gain-of-function and cites PMID:26546295, PMID:28578312, and PMID:29466156.

Still missing

• The available functional studies primarily assess acquired oncogenic drug resistance in NTRK1-fusion cancer models rather than physiologic NTRK1 function in the germline CIPA mechanism.
• No validated, reproducible clinical diagnostic-laboratory functional assay was identified.

Sources

Reasoning

NTRK1 p.Gly595Arg lies in the receptor tyrosine kinase domain at the solvent front. Variant-specific studies identify G595R as a kinase-domain solvent-front mutation and show structural interference with inhibitor binding; the variant is extremely rare in gnomAD v4.1 (AF 4.96029e-06; 8/1,612,810 alleles; no homozygotes). No VCEP domain table applies, no statistically significant Cancer Hotspots result was found, and the kinase-domain evidence supports the critical, well-established functional-domain route rather than a hotspot route.

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:29466156, Results and Table S3: NTRK1 G595R is described as a solvent-front mutation in the kinase domain that alters a solvent-exposed nucleotide-binding-loop region and interferes with larotrectinib binding.
• PMID:28578312, Introduction and Results: G595R is described in the TRKA solvent front; structural modeling shows steric clash with larotrectinib, and kinase-domain biochemical and cellular studies directly evaluated the variant.
• gnomAD v4.1: 8/1,612,810 alleles, AF 4.96029e-06, with zero homozygotes.
• Cancer Hotspots search for NTRK1 G595 returned no statistically significant hotspot row; PM1 is adjudicated through the critical kinase-domain route.

Sources

Reasoning

PM2 is met at supporting strength under the supplied generic SVI threshold of grpmax FAF <=0.0001. The default all-comers gnomAD v4.1 dataset shows the variant at AF=4.96029e-06 (8/1,612,810 alleles), with grpmax FAF=5.53e-06 and 0 homozygotes. gnomAD v2.1 all-comers is concordant, with AF=2.50855e-05 and grpmax FAF=1.913e-05. The variant is therefore extremely rare, not absent, and meets PM2 supporting rather than a stronger absence claim. No VCEP-specific population source is applicable because no VCEP covers NTRK1.

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 all-comers: AF=4.96029e-06, 8/1,612,810 alleles, homozygotes=0; filtering grpmax FAF=5.53e-06.
• gnomAD v2.1 all-comers: AF=2.50855e-05, 7/279,046 alleles, homozygotes=0; filtering grpmax FAF=1.913e-05.
• Applied the supplied generic PM2 supporting calibration threshold exactly: <=0.0001, identified as the ClinGen SVI recommendation to downgrade PM2 to Supporting (PMID:25741868).
• Because no VCEP covers NTRK1 and no governing non-cancer or exome-only source is specified, the default all-comers GNOMAD_V2_1 and GNOMAD_V4_1 totals were used.

Sources

Reasoning

The variant is a missense substitution, NM_002529.4:c.1783G>A (NP_002520.2:p.(Gly595Arg)); therefore the generic missense path applies and REVEL is the sole applicable predictor. REVEL is 0.926, which meets the generic PP3 supporting threshold of >=0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997). The SpliceAI max delta of 0.057 and BayesDel score of 0.42574 are not used because SpliceAI is not the applicable path for a missense variant and no generic ACMG-strength BayesDel calibration is established.

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.

Evidence considered

• NM_002529.4:c.1783G>A is a missense variant, NP_002520.2:p.(Gly595Arg); under the generic consequence-matched rule, REVEL is the applicable and exclusive predictor path.
• Local REVEL lookup reports 0.926 for this variant (source_registry key: revel).
• REVEL 0.926 meets the generic PP3 supporting threshold >=0.644; threshold supplied from the ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997.
• SpliceAI max delta 0.057 and BayesDel 0.42574 were not counted: SpliceAI is not the missense path, and BayesDel has no established generic ACMG-strength calibration in the governing rules.

Sources

Reasoning

The governing ACMG/AMP PS4 rule requires a significantly increased prevalence in affected individuals compared with controls and, under the supplied generic guidance, an RR or OR >5.0 with a confidence interval that excludes 1.0. The literature_pass PS4 leads were checked against their paper extracts: PMID:24121147 concerns SERPINA1 alpha-1-antitrypsin deficiency and does not mention NTRK1 or this variant; PMID:29419974 has no usable abstract or full-text evidence in the case packet. PMID:26546295, which does mention NTRK1 p.G595R, reports an acquired resistance mutation in one LMNA-NTRK1 colorectal-cancer case and provides no affected-versus-unaffected case-control counts, RR, OR, or confidence interval for this variant. No qualifying case-control comparison or computable counts were found, so PS4 is not met.

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 PS4 lead PMID:24121147 was verified from its extract and is unrelated to NTRK1; it studies SERPINA1 alpha-1-antitrypsin deficiency.
• Literature-pass PS4 lead PMID:29419974 had no usable abstract or full-text evidence available for verification and supplies no case-control data.
• PMID:26546295 reports NTRK1 p.G595R emerging during acquired resistance in a colorectal-cancer case, but not an affected-versus-control comparison; therefore no OR or 95% CI could be computed.
• Exact numeric comparison applied: OR > 5.0 and the confidence interval around OR does not include 1.0; no OR or confidence interval is available to satisfy either requirement.

Still missing

• No variant-specific affected-case and control counts or case-control RR/OR with 95% confidence interval were identified.

Sources

Reasoning

PP5 requires a reputable source to recently report the exact variant as pathogenic when the underlying evidence is unavailable for independent evaluation. ClinVar variation 1055425 contains two clinical-laboratory submissions classifying the exact variant as uncertain significance, with no expert-panel submissions. The reviewed papers that mention NTRK1 p.G595R describe acquired drug resistance in cancer, not a reputable pathogenic classification for this variant in the disease under evaluation. 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 1055425 matches NM_002529.4:c.1783G>A (p.Gly595Arg), but its classification is uncertain significance from two clinical laboratories and its expert-panel submission count is zero.
• PMID:26546295, PMID:28578312, and PMID:29466156 mention p.G595R in acquired resistance or pharmacologic studies; none reports the exact variant as pathogenic for hereditary NTRK1 disease.

Sources

Reasoning

BA1 is not met. The governing generic ACMG/AMP threshold is allele frequency >=0.05, evaluated using the filtering grpmax FAF rather than a population point estimate. The grpmax FAF is 5.53e-06 in gnomAD v4.1 and 1.913e-05 in gnomAD v2.1, both far below 0.05; the highest observed subpopulation point estimates are not used for this decision. No non-cancer or exome-only VCEP source is specified because no VCEP covers NTRK1.

Rule

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

Evidence considered

• gnomAD v4.1 all-comers: AF=4.96029e-06, 8/1,612,810 alleles, homozygotes=0; filtering grpmax FAF=5.53e-06.
• gnomAD v2.1 all-comers: AF=2.50855e-05, 7/279,046 alleles, homozygotes=0; filtering grpmax FAF=1.913e-05.
• Applied the published generic BA1 calibration exactly as supplied: >=0.05, identified as the ACMG/AMP 2015 stand-alone benign allele-frequency threshold (PMID:25741868).
• Following ClinGen SVI/Ghosh et al. 2018 guidance supplied for this interpretation, the filtering grpmax FAF was used and not the highest population AF point estimate; the relevant observed population allele counts exceed 2000.

Sources

Reasoning

BS1 is not met. The generic default threshold is >=0.01, and the required filtering grpmax FAF is 5.53e-06 in gnomAD v4.1 and 1.913e-05 in gnomAD v2.1. These values are not greater than expected for NTRK1-related congenital insensitivity to pain with anhidrosis, an early-onset autosomal-recessive disorder; no prevalence, penetrance, or allelic-heterogeneity calculation is needed to resolve an elevated-frequency concern because the observed FAF is orders of magnitude below the default threshold. No population-specific founder signal with a qualifying FAF is present.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v4.1 all-comers: filtering grpmax FAF=5.53e-06 and total AF=4.96029e-06 from 8/1,612,810 alleles; the highest observed ancestry point estimate was 3.33489e-05 in Admixed American from 2/59,972 alleles.
• gnomAD v2.1 all-comers: filtering grpmax FAF=1.913e-05 and total AF=2.50855e-05 from 7/279,046 alleles; the highest observed ancestry point estimate was 0.000139626 in Remaining individuals from 1/7,162 alleles.
• Applied the supplied generic BS1 default calibration exactly: >=0.01; the prompt identifies this as the generic SVI default and recommends per-gene maximum-credible-frequency analysis under Whiffin et al. 2017 (PMID:28518168).
• The highest observed population point estimates were not substituted for FAF; both are from qualifying population counts but remain far below the BS1 threshold.

Sources

Reasoning

BS2 is not met. NTRK1-related congenital insensitivity to pain with anhidrosis is an autosomal-recessive disorder, so the applicable BS2 observation would be a healthy adult homozygote. gnomAD v4.1 reports 0 homozygotes among 1,612,810 alleles, and gnomAD v2.1 reports 0 homozygotes among 279,046 all-comers alleles. No healthy adult homozygous observation is supplied; therefore BS2 cannot be applied.

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

• The case evidence identifies NTRK1-related congenital insensitivity to pain with anhidrosis as autosomal recessive, making homozygous healthy-adult observation the relevant BS2 path.
• gnomAD v4.1: homozygotes=0 among 8/1,612,810 alleles.
• gnomAD v2.1 all-comers: homozygotes=0 among 7/279,046 alleles.
• No individual-level healthy adult homozygote report is present in the supplied evidence.

Sources

Reasoning

BS3 is not met. The variant-specific functional studies do not show no damaging effect on protein function or splicing; instead, they report altered TRKA biochemical behavior, steric interference with inhibitor binding, markedly reduced inhibitor sensitivity, resistance selection in cellular and tumor models, and increased ATP affinity compared with wild-type TRKA. These findings are inconsistent with a benign functional result, although they primarily characterize oncogenic drug resistance rather than the physiologic germline disease mechanism.

Rule

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

Evidence considered

• PMID:26546295: p.G595R was associated with acquired resistance to entrectinib, LOXO-101, and TSR-011, with an entrectinib IC50 >1000 nM in engineered Ba/F3 cells.
• PMID:28578312: TRKA-G595R showed increased ATP affinity versus wild-type TRKA (Km 6 µM versus 51 µM), and G595R tumor models responded to a next-generation inhibitor, demonstrating altered rather than absent functional behavior.
• PMID:29466156: NTRK1 G595R reduced larotrectinib inhibitory potency through solvent-front interference with inhibitor binding.
• OncoKB variant-specific record classifies the biological-effect context as gain-of-function and does not provide evidence of a benign or normally functioning protein.

Still missing

• The available studies do not directly assess normal versus variant NTRK1 physiologic function or splicing in a germline disease-relevant system.

Sources

Reasoning

The variant is a missense substitution, so BP4 must be evaluated through the generic missense REVEL path only. REVEL is 0.926, which is above the generic BP4 supporting cutoff of <=0.29 and therefore does not support a benign/no-impact computational conclusion. The SpliceAI max delta of 0.057 is not used for BP4 because SpliceAI is not the applicable path for a missense variant; BayesDel is not available for this criterion 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.

Evidence considered

• NM_002529.4:c.1783G>A is a missense variant, NP_002520.2:p.(Gly595Arg); under the generic consequence-matched rule, REVEL is the applicable and exclusive predictor path.
• Local REVEL lookup reports 0.926 for this variant (source_registry key: revel).
• REVEL 0.926 does not meet the generic BP4 supporting threshold <=0.29; threshold supplied from the ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997.
• SpliceAI max delta 0.057 was not substituted for the missense REVEL path, and BayesDel 0.42574 was not used because no established generic ACMG-strength BayesDel calibration is available.

Sources

Reasoning

BP6 requires a reputable source to recently report the variant as benign when the underlying evidence is unavailable for independent evaluation. ClinVar variation 1055425 has no expert-panel submission and is classified as uncertain significance by two clinical laboratories, not benign or likely benign. No reviewed publication reports the exact variant as benign. BP6 is therefore 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 1055425 exactly matches NM_002529.4:c.1783G>A (p.Gly595Arg), but reports uncertain significance from two clinical laboratories and zero expert-panel submissions.
• No reviewed source reports the exact variant as benign or likely benign.

Sources

Reasoning

The variant is NTRK1 p.Gly595Arg. The reviewed evidence documents this exact amino-acid change, but does not document a different nucleotide change producing the same amino-acid change in a previously established pathogenic variant. A targeted, sufficiently comprehensive same-amino-acid pathogenic-variant comparison is therefore unavailable.

Rule

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

Evidence considered

• The case variant is NM_002529.4:c.1783G>A, NP_002520.2:p.(Gly595Arg).
• The reviewed variant-specific papers describe this exact p.G595R change; they do not establish a different nucleotide change causing the same amino-acid substitution.

Still missing

• A comprehensive search for independently established pathogenic NTRK1 variants producing p.Gly595Arg through a different nucleotide change is required.

Reasoning

No documented de novo observation is available for NM_002529.4:c.1783G>A (NTRK1 p.Gly595Arg). The evidence does not establish that the variant is absent from both parents, confirm maternity and paternity, document a disease-consistent affected proband, or establish no family history.

Rule

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

Still missing

• Documented testing of both biological parents is needed.
• Maternity and paternity confirmation are needed.
• A disease-consistent proband phenotype and family-history assessment are needed.

Reasoning

NTRK1-related congenital insensitivity to pain with anhidrosis is an autosomal recessive disorder, but the available evidence does not report an affected proband carrying NM_002529.4:c.1783G>A with a pathogenic variant in trans. No parental or offspring testing, haplotype result, or other phase determination is provided, so PM3 cannot be assessed.

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 variation 1055425 records two germline submissions for NM_002529.4(NTRK1):c.1783G>A (p.Gly595Arg), but provides no affected-proband, second pathogenic-variant, or phase result relevant to PM3.

Still missing

• Affected-proband data with a second pathogenic NTRK1 variant are not available.
• Parental, offspring, haplotype, or other definitive phase testing is not available.

Sources

Reasoning

The variant is a missense substitution at residue 595, so PM5 is applicable in principle. However, the available comparator collection contains no same-residue candidates and the reviewed papers describe G595R itself, not a different missense substitution at Gly595 established as pathogenic. The required pathogenic alternate-at-the-same-residue evidence is therefore insufficient.

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 available PM5 comparator collection reports zero same-residue candidates.
• PMID:26546295, PMID:28578312, and PMID:29466156 discuss NTRK1 p.G595R itself and do not establish a different pathogenic missense substitution at residue 595.

Still missing

• Search ClinVar, literature, and other validated variant resources for a different NTRK1 missense substitution at residue 595 with an established pathogenic classification.

Sources

Reasoning

No assumed-de-novo observation is documented for NM_002529.4:c.1783G>A (NTRK1 p.Gly595Arg). The available evidence does not report parental testing, parental variant status, or a documented disease-consistent proband from which de novo status could be assumed.

Rule

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

Still missing

• Parental variant testing or a documented basis for assuming de novo status is needed.
• A disease-consistent proband phenotype should be documented.

Reasoning

No affected relatives, unaffected relatives, genotype results, or informative meioses are documented for NM_002529.4:c.1783G>A (NTRK1 p.Gly595Arg). Therefore, co-segregation cannot be evaluated or assigned a strength.

Rule

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

Still missing

• Pedigree and phenotype data for affected and unaffected relatives are needed.
• Variant testing results for informative relatives are needed.
• The number of informative meioses must be established before assigning strength.

Reasoning

The available material supports NTRK1 loss of function as a germline disease mechanism, but it does not provide a validated gene-wide assessment showing both a low rate of benign missense variation and that missense variants are a common disease mechanism. The two required PP2 components cannot be established from the available evidence.

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 case summary identifies NTRK1 loss of function as a germline disease mechanism, but does not provide the gene-wide missense constraint and mechanism evidence required by PP2.
• The queried variant is rare in gnomAD v4.1 (AF 4.96029e-06), but the frequency of this single variant cannot establish the gene-level PP2 rule.

Still missing

• Provide a validated NTRK1 missense-variant spectrum and benign-missense constraint assessment demonstrating both PP2 requirements.

Reasoning

The rule requires a patient's phenotype or family history highly specific for a disease with a single genetic etiology. The available case evidence identifies NTRK1-related congenital insensitivity to pain with anhidrosis as the disease context, but does not provide the evaluated patient's phenotype, clinical findings, or family history. The criterion therefore cannot be assessed without patient-level phenotype or pedigree information.

Rule

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

Evidence considered

• The case identifies hereditary insensitivity to pain with anhidrosis as a ClinVar condition context, but this does not establish the evaluated patient's phenotype or family history.

Still missing

• Patient-level phenotype and family-history information is missing.

Reasoning

No affected family members with documented absence of NM_002529.4:c.1783G>A (NTRK1 p.Gly595Arg) are reported. The available evidence does not provide an informative family structure, phenotype adjudication, genotype testing, or a basis to exclude phenocopies or another pathogenic variant.

Rule

Lack of segregation in affected members of a family.

Still missing

• An informative family pedigree with affected relatives is needed.
• Variant testing and reliable phenotype assessment in affected relatives are needed.
• Potential phenocopies and additional pathogenic variants should be considered.

Reasoning

The available material supports NTRK1 loss of function as a germline disease mechanism, but does not establish that disease-causing variants in NTRK1 are primarily truncating. A complete, validated disease-variant spectrum distinguishing truncating from missense and other variant classes is not available, so BP1 cannot be adjudicated.

Rule

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

Evidence considered

• The case summary supports NTRK1 loss of function as a germline disease mechanism, which is not equivalent to demonstrating that disease-causing variants are primarily truncating.
• No validated gene-wide count or proportion of truncating versus missense disease-causing NTRK1 variants is supplied.

Still missing

• Provide a validated NTRK1 disease-variant spectrum quantifying whether truncating variants are the primary known disease-causing class.

Reasoning

The available evidence does not document NM_002529.4:c.1783G>A in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant. No family-based phase data or suitably documented allelic observation is provided, 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

• ClinVar variation 1055425 records two germline submissions for NM_002529.4(NTRK1):c.1783G>A (p.Gly595Arg), but provides no cis/trans phase observation with a pathogenic variant relevant to BP2.

Still missing

• Family-based or other definitive phase data are not available.
• No documented cis/trans observation with a pathogenic variant is available.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. PMID:26546295 and PMID:28578312 describe p.G595R in tumors with NTRK1 fusions and acquired drug resistance, but those are unrelated somatic cancer cases and do not establish that the evaluated patient's NTRK1 variant occurred with an alternate molecular basis for the hereditary disease under review. No patient-specific alternate molecular diagnosis is provided, so BP5 cannot be assessed.

Rule

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

Evidence considered

• PMID:26546295 reports p.G595R acquired in an LMNA-NTRK1 fusion-positive colorectal-cancer case; this is not evidence that the evaluated hereditary-disease case has an alternate molecular basis.
• PMID:28578312 reports p.G595R in an LMNA-NTRK1 fusion-positive colorectal-cancer case with acquired resistance; the report is unrelated to the patient's molecular diagnosis.

Still missing

• No patient-specific alternate molecular diagnosis or alternate disease-causing variant is documented.

Sources