ALK c.3522C>A, p.Phe1174Leu

Reasoning

The queried variant is NP_004295.2:p.(Phe1174Leu) (F1174L), and the same amino-acid substitution is explicitly reported in multiple ALK neuroblastoma studies as a recurrent activating disease-associated variant. PMID:18923525 reports F1174L as the recurrent exon-23 mutation at codon 1174 and demonstrates transforming activity, constitutive ALK signaling, and inhibitor sensitivity; PMID:18923523 reports constitutive kinase activity for F1174L. The ClinVar record for the exact variant is Pathogenic, but has no assertion criteria and is treated only as corroboration, not as the primary basis.

Rule

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

Evidence considered

• ALK p.F1174L is the same amino-acid change as the queried NM_004304.5:c.3522C>A variant.
• Activating mutations in ALK provide a therapeutic target in neuroblastoma (PMID:18923525) reports F1174L as a recurrent exon-23 mutation and demonstrates Ba/F3 transformation, constitutive signaling, and inhibitor sensitivity.
• Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma (PMID:18923523) reports that ALK F1174L mutants have constitutive ALK kinase activity.
• ClinVar variation 217851 classifies the exact variant as Pathogenic, but the single submission has no assertion criteria and no review stars.

Sources

Reasoning

PS3 is met at strong strength for ALK NM_004304.5:c.3522C>A (p.Phe1174Leu). Multiple independent, variant-specific functional studies show a coherent gain-of-function phenotype: increased or constitutive ALK kinase activity/autophosphorylation, transformation of IL-3-dependent Ba/F3 cells to cytokine-independent growth, downstream STAT3/AKT signaling, and inhibitor-sensitive cellular dependence. PMID:18923523 used in vitro kinase assays and immunoblotting and reported constitutive kinase activity for F1174L; PMID:18923525 demonstrated Ba/F3 transformation, constitutive phosphorylation, downstream signaling, inhibitor sensitivity, apoptosis, and knockdown effects; PMID:20719933 independently reproduced autophosphorylation and Ba/F3 transformation, with faster transformation by F1174L than R1275Q. PMID:18724359 additionally reported constitutive ALK phosphorylation and growth dependence in F1174L-harboring neuroblastoma models. These are direct, biologically relevant assays with wild-type or comparator context and concordant results across biochemical, cellular, and disease-model systems, supporting a damaging functional effect rather than merely relying on a clinical classification or hotspot observation.

Rule

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

Evidence considered

• PMID:18923523, Table 1 and functional characterization: ALK F1174L was evaluated in neuroblastoma cell lines; in vitro kinase assays and immunoblotting showed strong/constitutive kinase activity.
• PMID:18923525, primary-tumor sequencing and functional characterization, Table 1 and Figures 1–3: F1174L supported cytokine-independent Ba/F3 growth, constitutive ALK/STAT3/AKT phosphorylation, TAE684 sensitivity, reduced proliferation, and apoptosis/knockdown effects in neuroblastoma cells.
• PMID:20719933, Results section 'Transforming capacity of ALK hotspot mutations': F1174L induced ALK autophosphorylation and transformed IL-3-dependent Ba/F3 cells to IL-3-independent growth, with faster transformation than R1275Q.
• PMID:18724359, Results sections 'Identification of somatic ALK mutations' and 'Functional consequences of ALK mutations': F1174L was associated with constitutive ALK phosphorylation and growth dependence in neuroblastoma models.
• The evidence is variant-specific and concordant across biochemical kinase activity, phosphorylation/signaling, transformation, inhibitor-response, and knockdown assays; no negative functional study was identified for this variant.

Still missing

• The studies were not described as validated, reproducible, and robust functional testing in a clinical diagnostic laboratory setting.

Sources

Reasoning

The variant lies at ALK residue F1174 within the receptor tyrosine kinase domain. PMID:18923525 explicitly places F1174L within the kinase domain and reports recurrent activating behavior; PMID:20719933 identifies recurrent F1174 mutations as an ALK mutational hotspot and demonstrates transforming activity. The case evidence also records a statistically significant ALK F1174 hotspot in Cancer Hotspots. No ALK VCEP domain table is present, so generic ACMG/AMP guidance applies. The queried variant is absent from gnomAD v2.1 and v4.1, providing no population observation that would contradict the hotspot/domain evidence.

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

• Activating mutations in ALK provide a therapeutic target in neuroblastoma (PMID:18923525) states that F1174L is within the kinase domain and is a recurrent activating mutation.
• Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification (PMID:20719933) reports recurrent F1174 mutations and functional transformation by ALK F1174L.
• Cancer Hotspots reports ALK F1174 as a statistically significant hotspot; the extraction is partially parsed but the hotspot result is present.
• The exact variant is absent from gnomAD v2.1 and gnomAD v4.1.

Sources

Reasoning

PM2 is met at supporting strength. NM_004304.5:c.3522C>A (ALK p.Phe1174Leu) was absent from gnomAD v2.1 and gnomAD v4.1. The case also reports absence from the available gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets. This satisfies the supplied ClinGen SVI generic PM2 threshold of <=0.0001 because the observed allele frequency is effectively zero in the reported reference populations.

Rule

PM2: 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): NM_004304.5:c.3522C>A was absent.
• gnomAD v4.1: NM_004304.5:c.3522C>A was absent.
• The case evidence also reports absence from GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; no governing VCEP rule requires a subset, so the all-comers gnomAD results remain the default evidence.
• Generic calibration threshold: PM2 is supporting at <=0.0001, supplied as the ClinGen SVI recommendation to downgrade PM2 to supporting (Richards et al. 2015, PMID:25741868, as specified in the case threshold block).

Sources

Reasoning

The variant is a missense substitution, NP_004295.2:p.(Phe1174Leu), so the governing generic rule requires the missense REVEL path only; splice prediction is not used for this criterion. REVEL is 0.752, which meets the supplied generic ClinGen SVI supporting threshold of >=0.644 for PP3. BayesDel is not used because no generic ACMG-strength calibration is established for it. The evidence supports PP3 at supporting strength without double-counting another computational predictor.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).

Evidence considered

• Local REVEL lookup reports a score of 0.752 for NM_004304.5:c.3522C>A / NP_004295.2:p.(Phe1174Leu).
• The supplied generic ClinGen SVI REVEL calibration assigns PP3 supporting at REVEL >=0.644; the threshold publication is Pejaver et al. 2022, PMID:36413997.
• The missense-specific REVEL path was used exclusively; SpliceAI was not counted for this missense criterion, and BayesDel was not used because the supplied rules state that no generic ACMG-strength calibration is established.

Sources

Reasoning

The generic PP5 definition concerns a reputable source reporting the variant as pathogenic when the underlying evidence is unavailable for independent evaluation. Under the required generic rule for this interpretation, PP5 is restricted to an exact-variant ClinVar expert-panel classification. ClinVar variation 217851 has one clinical-laboratory Pathogenic submission, no assertion criteria, zero review stars, and zero expert-panel submissions. The ClinVar-linked PMID:20301782 therefore cannot substitute for an expert-panel classification. 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 217851 exactly matches NM_004304.5(ALK):c.3522C>A (p.Phe1174Leu), but the record has one clinical-laboratory Pathogenic submission, review status 'no assertion criteria provided,' zero review stars, and zero expert-panel submissions.
• The ClinVar submission audit identifies SCV000256816 as a clinical-testing submission with is_expert_panel=false and usable_for_criterion_leads=false.
• The generic rule for this adjudication permits PP5 only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification; that prerequisite is absent.

Sources

Reasoning

BA1 is not met. NM_004304.5:c.3522C>A (ALK p.Phe1174Leu) was absent from gnomAD v2.1 and gnomAD v4.1, so no allele frequency reaches the generic stand-alone threshold of >=0.05. The available evidence also provides no conflicting high-frequency population signal that would require a reduced-penetrance or disease-association exception analysis.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 (exome): NM_004304.5:c.3522C>A was absent; no allele frequency or grpmax FAF was reported.
• gnomAD v4.1: NM_004304.5:c.3522C>A was absent; no allele frequency or grpmax FAF was reported.
• Generic calibration threshold: BA1 stand-alone requires >=0.05, supplied as the ACMG/AMP 2015 stand-alone benign allele-frequency threshold (Richards et al. 2015, PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant was absent from gnomAD v2.1 and gnomAD v4.1, with no filtering allele frequency or grpmax FAF above the generic default threshold of >=0.01. Because there is no elevated ancestry-specific frequency, a maximum-credible-allele-frequency calculation is not needed to reject BS1 on the available population evidence.

Rule

BS1: Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1 (exome): NM_004304.5:c.3522C>A was absent; no filtering allele frequency or grpmax FAF was reported.
• gnomAD v4.1: NM_004304.5:c.3522C>A was absent; no filtering allele frequency or grpmax FAF was reported.
• The generic BS1 default is >=0.01, supplied in this case as the generic SVI default; no higher ancestry-specific frequency requiring a Whiffin et al. 2017 maximum-credible-frequency analysis was present.
• When a frequency is available, BS1/BA1 assessment would use filtering AF, specifically grpmax FAF, rather than popmax point estimate; here no AF or FAF was reported.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule requires the missense REVEL path only; SpliceAI cannot substitute for a gray-zone REVEL result. REVEL is 0.752, which is above the supplied generic BP4 supporting cutoff of <=0.29 and therefore does not support no impact. BayesDel is not used because no generic ACMG-strength calibration is established for it.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)

Evidence considered

• Local REVEL lookup reports a score of 0.752 for NM_004304.5:c.3522C>A / NP_004295.2:p.(Phe1174Leu).
• The supplied generic ClinGen SVI REVEL calibration assigns BP4 supporting at REVEL <=0.29; the threshold publication is Pejaver et al. 2022, PMID:36413997.
• The missense-specific REVEL path was used exclusively; the SpliceAI score was not substituted for REVEL, and BayesDel was not used because the supplied rules state that no generic ACMG-strength calibration is established.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The case materials contain no evidence that this exact variant was found in a case with an independently established alternate molecular cause. Reports of somatic ALK p.F1174L in neuroblastoma tumors, ALK amplification, or other tumor alterations do not by themselves establish an alternate molecular basis for the clinical case. BP5 is not met.

Rule

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

Evidence considered

• The reviewed literature reports ALK p.F1174L in neuroblastoma tumors and, in one tumor, co-occurring ALK amplification, but does not establish an alternate molecular basis for the submitted clinical case.
• No case-level molecular diagnosis independent of this variant is documented in the available evidence.

Sources

Reasoning

The generic BP6 definition concerns a reputable source reporting the variant as benign when the underlying evidence is unavailable for independent evaluation. Under the required generic rule for this interpretation, BP6 is restricted to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. ClinVar variation 217851 has only one non-expert clinical-laboratory Pathogenic submission, no assertion criteria, and no expert-panel submission; it contains no qualifying benign expert-panel classification. 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 217851 exactly matches NM_004304.5(ALK):c.3522C>A (p.Phe1174Leu), but reports one clinical-laboratory Pathogenic submission, no assertion criteria, zero review stars, and zero expert-panel submissions.
• The generic rule for this adjudication permits BP6 only for an exact-variant ClinVar expert-panel Benign or Likely benign classification; no such classification is present.

Sources

Reasoning

No proband-level de novo assessment is available: the case materials do not document an affected patient with confirmed maternity and paternity, a no-family-history assessment, or parental genotypes. The available variant-specific literature describes tumor or cell-line observations and does not establish constitutional de novo origin.

Rule

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

Evidence considered

• No case-level parental testing, maternity confirmation, paternity confirmation, or family-history assessment is present in the supplied case evidence.
• The variant-specific paper extraction for PMID:18923523 reports cell-line functional studies and explicitly states that segregation data for F1174L are not provided; this does not establish de novo origin.

Still missing

• Affected-proband clinical diagnosis and phenotype are not documented for this criterion.
• Maternal and paternal identity or genotype confirmation is not documented.
• Family history and parental genotypes are not documented.

Sources

Reasoning

The governing PS4 definition requires a significant increase in variant prevalence among affected individuals compared with controls, with the ACMG/AMP note specifying an RR or OR >5.0 and a confidence interval that excludes 1.0. The reviewed variant-specific literature reports ALK p.F1174L in neuroblastoma tumor cohorts, including 4/93 primary tumors in PMID:18923525, 14/167 high-risk tumors in PMID:18724359, and broader tumor frequencies in PMID:20719933, but these reports do not provide an unaffected or population-control comparison for this exact variant. Therefore an odds ratio and 95% CI cannot be computed, and PS4 cannot be assessed. The literature_pass PS4 candidate PMID:20301782 has no abstract or full-text evidence available in the case materials, so it cannot establish case-control enrichment.

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:18923525, Results/Table 1: ALK F1174L was reported in 4 of 93 primary neuroblastomas (4.3%); the study used matched-normal analysis but did not report an unaffected/control comparison for this exact variant.
• PMID:18724359, Results: F1174L was reported among ALK mutations identified in 14 of 167 high-risk primary neuroblastoma tumors; no unaffected/control comparison for the exact variant was reported in the extracted finding.
• PMID:20719933, Results: the study analyzed 709 neuroblastoma tumors and reported F1174 mutation frequencies and MYCN-associated enrichment, but not an unaffected/control comparison for the exact variant.
• PMID:20301782 was listed as a PS4 candidate in literature_pass, but its packet contains no abstract or full-text finding that can verify case-control counts.
• No OR or 95% CI was computed because the reviewed sources lack the required control count or control frequency.

Still missing

• A case-control comparison for ALK NM_004304.5:c.3522C>A (p.Phe1174Leu), including affected and unaffected/control counts, is required.
• The literature_pass candidate PMID:20301782 lacks an available abstract or full text for verification.

Sources

Reasoning

The available evidence does not establish that the disorder context is recessive, does not identify a pathogenic variant in the same affected proband, and provides no parental or offspring testing to establish phase. The variant-specific literature reviewed concerns tumor or cell-line observations and does not provide the required in-trans evidence. PM3 therefore cannot be assessed under the generic rule.

Rule

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

Evidence considered

• No case-level observation identifies a second pathogenic variant in the same affected proband.
• No parental or offspring testing, haplotyping, or other phase evidence is reported.
• The variant-specific literature reviewed reports tumor and cell-line observations rather than a recessive affected-proband trans configuration; PMID:18923523 explicitly states that it does not provide segregation data for F1174L.

Still missing

• Document the inheritance mode for the relevant ALK disorder.
• Identify any pathogenic variant in the same affected proband and obtain parent or offspring testing to establish phase.

Sources

Reasoning

The variant is a missense substitution at residue F1174, so PM5 is applicable in principle. However, the available evidence does not document a different missense change at residue 1174 that has been established as pathogenic. The case's PM5 candidate artifact contains no candidates and explicitly states that classic same-residue PM5 semantics could not be confirmed safely; the available papers report F1174L itself rather than a qualifying alternate F1174 missense 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

• The variant is NP_004295.2:p.(F1174L), a missense substitution at residue 1174.
• pm5_candidates.json reports zero same-residue candidates and says classic PM5 semantics could not be confirmed safely.
• The reviewed ALK papers document F1174L, not a different pathogenic missense substitution at F1174.

Still missing

• A validated pathogenic alternate missense variant at residue F1174 is needed for PM5.

Sources

Reasoning

No case-level evidence shows that this variant was assumed de novo. The available records do not provide an affected proband, parental genotypes, maternity or paternity information, or a family-history assessment; therefore an unconfirmed de novo event cannot be established.

Rule

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

Evidence considered

• No case-level parental testing or clinical pedigree information is present in the supplied case evidence.
• The variant-specific paper extraction for PMID:18923523 reports tumor cell-line studies and states that segregation data for F1174L are not provided; it does not support an assumed de novo classification.

Still missing

• An affected proband and relevant phenotype are not documented.
• Parental genotypes are not documented.
• The basis for assuming de novo status and the family history are not documented.

Sources

Reasoning

No affected relatives, tested family members, meioses, or segregation results are documented. The available variant-specific literature contains tumor or cell-line observations rather than a familial pedigree demonstrating co-segregation.

Rule

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

Evidence considered

• No family segregation data are present in the supplied case evidence.
• The variant-specific paper extraction for PMID:18923523 reports ALK F1174L in neuroblastoma cell lines and explicitly states that segregation data are not provided; this is not co-segregation evidence.

Still missing

• No familial pedigree or affected relatives are documented.
• No genotypes and phenotypes for informative relatives are documented.
• No number of informative meioses or co-segregation result is documented.

Sources

Reasoning

The available case evidence supports an activating missense mechanism for this ALK variant, but it does not establish both required gene-level conditions for PP2: a low rate of benign missense variation and missense variants as a common disease mechanism for the relevant inherited disorder. The available gnomAD results concern the queried variant, not the gene-wide benign missense rate, and no validated gene-level missense constraint or mechanism dataset is supplied.

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

• ALK F1174L has activating functional evidence in PMID:18923525, but that is variant-specific and does not establish the PP2 gene-level prerequisites.
• The queried variant is absent from gnomAD v2.1 and v4.1, but no gene-wide benign missense rate or validated missense-constraint metric is provided.

Still missing

• Validated gene-level evidence for benign missense constraint and predominance of missense disease variants is needed.

Sources

Reasoning

The case materials do not provide a patient phenotype or family history that can be evaluated for high specificity to a disease with a single genetic etiology. Tumor-cohort reports of ALK p.F1174L establish disease-associated observations but do not supply the individual clinical phenotype required for PP4. Therefore PP4 is not assessed.

Rule

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

Evidence considered

• The case materials identify ALK p.Phe1174Leu and neuroblastoma tumor studies but do not provide an individual phenotype or family history for PP4 assessment.

Still missing

• Individual clinical phenotype and family history for the case are required.
• The disease-specificity and whether a single genetic etiology is appropriate must be documented.

Reasoning

BS2 cannot be assessed from the available evidence. The gnomAD records establish absence of the variant but do not report an observed healthy adult carrier, homozygote, or hemizygote, and no clinical cohort evidence documents a healthy adult with the variant. Therefore the required healthy-adult observation and the applicable inheritance/full-penetrance context are unavailable.

Rule

BS2

Evidence considered

• gnomAD v2.1 and v4.1 report the variant as absent, but provide no individual-level healthy-adult observation for BS2.
• No population or clinical source in the reviewed case evidence documents a healthy adult carrying NM_004304.5:c.3522C>A.

Still missing

• A documented healthy adult observation with genotype and relevant inheritance context is unavailable.

Reasoning

BS3 is not assessed because the reviewed variant-specific studies report activating ALK kinase activity, constitutive signaling, cellular transformation, and inhibitor-sensitive dependence; they do not provide well-established evidence showing no damaging effect on ALK protein function or splicing. The available SpliceAI prediction is not a functional assay and cannot establish the BS3 requirement.

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 evaluated activating kinase activity, phosphorylation, transformation, signaling, and inhibitor response; none tested or demonstrated a normal/no-damaging functional result.
• The available computational splice prediction was not used as BS3 evidence because BS3 requires well-established in vitro or in vivo functional studies.

Still missing

• A validated variant-specific assay demonstrating preserved ALK protein function or normal splicing is not available in the reviewed evidence.

Sources

Reasoning

No affected family members with negative variant testing are documented. Without a family pedigree, affected-relative genotypes, and phenotype verification, lack of segregation cannot be evaluated and cannot be distinguished from phenocopy or an alternative familial variant.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No family pedigree, affected-relative genotypes, or non-segregation findings are present in the supplied case evidence.
• The available variant-specific literature describes tumor or cell-line findings rather than affected-family-member testing.

Still missing

• No affected relatives and their clinical phenotypes are documented.
• No variant-testing results for affected relatives are documented.
• No assessment of phenocopy or another pathogenic familial variant is documented.

Reasoning

The available evidence does not establish that ALK disease is caused primarily by truncating variants. Although the case materials contain a literature-based statement supporting an ALK loss-of-function germline context, the directly relevant ALK literature for this variant emphasizes recurrent activating missense disease biology, and no curated variant-spectrum analysis demonstrating that truncating variants are the primary known disease mechanism is supplied.

Rule

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

Evidence considered

• The relevant ALK literature reports recurrent activating missense variants, including F1174L, rather than establishing a primarily truncating disease mechanism.
• The available case materials do not provide a curated ALK variant-spectrum analysis showing that truncating variants primarily cause disease.

Still missing

• A validated ALK disease-spectrum source showing predominance of truncating pathogenic variants is needed.

Sources

Reasoning

The available evidence does not show this variant in trans with a pathogenic variant in an affected proband with a fully penetrant dominant disorder, nor does it show this variant in cis with a pathogenic variant. No parental, offspring, haplotyping, or other phase data are provided. BP2 therefore cannot be assessed under the generic rule.

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

• No case-level observation identifies a second pathogenic variant in cis or in trans with NM_004304.5:c.3522C>A (NP_004295.2:p.(F1174L)).
• No parental or offspring testing, haplotyping, or other phase evidence is reported.
• The variant-specific literature reviewed reports tumor and cell-line observations rather than an allelic cis or trans configuration; PMID:18923523 does not provide segregation data for F1174L.

Still missing

• Obtain case-level phase data for any additional pathogenic variant, including parental or offspring testing or validated haplotyping.
• Establish whether the relevant dominant disorder is fully penetrant if an in-trans observation is identified.

Sources