ALK c.3824G>A, p.Arg1275Gln

Reasoning

PS3 is met at strong strength for NM_004304.5:c.3824G>A (ALK p.Arg1275Gln). Multiple independent, variant-specific studies provide concordant functional evidence across orthogonal systems: R1275Q produced constitutive ALK autophosphorylation and approximately 12-fold increased catalytic activity in biochemical assays, supported cytokine-independent Ba/F3 growth with constitutive ALK/ERK/AKT phosphorylation, accelerated kinase-domain autophosphorylation, transformed cells, and drove tumor growth or regression responses in neuroblastoma xenograft and knock-in mouse models. The studies included wild-type or other-variant comparisons and pharmacologic controls, and the findings are biologically relevant to ALK gain-of-function neuroblastoma. This exceeds a single limited assay and supports the ACMG/AMP strong functional-evidence level, although clinical diagnostic-laboratory validation was not specifically documented. No evidence supports BS3.

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, Results/Figure 2a-b: exact R1275Q was associated with strong ALK expression, phosphorylation at Y1586, and markedly increased in vitro kinase activity; the authors described constitutive ALK kinase activity.
• PMID:18923525, Results/Figures 1b-c and 2a-d: exact R1275Q supported IL-3-independent Ba/F3 growth, showed constitutive ALK/ERK1/2/AKT phosphorylation, and responded to TAE684 in Ba/F3 cells, with neuroblastoma-cell-line resistance demonstrating assay-context differences rather than a benign effect.
• PMID:22072639, Results/Table 1/Figure 5: full-length R1275Q-ALK caused constitutive autophosphorylation in RPE cells, increased unphosphorylated kinase catalytic activity approximately 12-fold, and produced crizotinib-sensitive neuroblastoma cell-line and xenograft phenotypes.
• PMID:25517749, Results/Table 1: exact R1275Q markedly accelerated ALK tyrosine-kinase-domain autophosphorylation; the study used purified kinase assays, peptide phosphorylation, focus formation, and inhibitor comparisons.
• PMID:26554404, Results: exact R1275Q was evaluated in biochemical kinase, cell-viability, NIH 3T3 transformation, and NB-1643 xenograft assays; PF-06463922 produced rapid, sustained complete tumor regression and greater suppression of ALK phosphorylation than crizotinib.

Still missing

• Clinical diagnostic-laboratory validation and formal interlaboratory reproducibility were not specifically documented.

Sources

Reasoning

ALK p.Arg1275Gln lies in the intracellular ALK tyrosine-kinase domain and at the recurrent R1275 mutational hotspot. Bresler et al. report R1275Q in 54 of 1,596 neuroblastoma tumors (43% of ALK-mutated tumors), and the extracted functional literature describes recurrent activating substitutions in this kinase-domain region. No VCEP-specific domain table applies because no VCEP covers ALK; the generic PM1 rule therefore governs.

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

• Bresler et al. describe ALK R1275Q as a recurrent neuroblastoma mutation at the R1275 hotspot, observed in 54 tumors (43% of ALK-mutated tumors), and analyze it within the ALK tyrosine-kinase domain.
• Bresler et al. report that R1275Q and F1174L were the most frequently observed ALK substitutions and that R1275Q greatly accelerated tyrosine-kinase-domain autophosphorylation.
• The Cancer Hotspots record for ALK R1275 reports that the variant lies in a statistically significant hotspot.

Sources

Reasoning

PM2 is met at supporting strength. The variant is absent from the default all-comers gnomAD v2.1 and v4.1 datasets and is also reported absent from the available gnomAD v2.1 non-cancer and v3.1 non-cancer queries. The supplied generic SVI calibration sets PM2 supporting at allele frequency <= 0.0001; absence is below that threshold, with no observed alleles or homozygotes reported.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1: variant absent; no observed allele or homozygote count reported.
• gnomAD v4.1: variant absent; no observed allele or homozygote count reported.
• Available non-cancer queries also reported absence: GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER.
• Generic calibration threshold supplied for PM2: <= 0.0001, identified in the prompt as the ClinGen SVI recommendation to downgrade PM2 to supporting.

Sources

Reasoning

PP1 is met at supporting strength. In one neuroblastoma family, the exact ALK R1275Q variant was present in two affected children, providing co-segregation with disease in multiple affected family members. The asymptomatic carrier mother indicates incomplete or age-dependent expression and limits the strength of this segregation observation, so no increased PP1 strength is assigned.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... May be used as stronger evidence with increasing segregation data.

Evidence considered

• Mossé et al. reported one family in which heterozygous ALK R1275Q was observed in two affected children and inherited from their asymptomatic mother.
• The two affected children constitute multiple affected family members carrying the variant; the asymptomatic maternal carrier limits the evidence to supporting strength rather than a stronger segregation level.

Still missing

• Additional independently documented affected relatives and informative meioses are not provided.
• The mother's asymptomatic status at age 39 limits interpretation because penetrance and age-related risk are not established here.

Sources

Reasoning

The variant is a missense substitution, NM_004304.5:c.3824G>A (p.Arg1275Gln), in exon 25. Under the generic missense path, REVEL is the sole applicable predictor; splice prediction is not used for PP3/BP4 in a missense variant. REVEL is 0.885, which meets the supplied generic PP3 moderate threshold of >= 0.773. BayesDel is not used because no generic ACMG-strength calibration is established. The available SpliceAI max delta of 0.002 is not counted for this missense PP3 assessment.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). ... PP3 (moderate): >= 0.773 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence: missense, NM_004304.5:c.3824G>A (NP_004295.2:p.(Arg1275Gln)), exon 25.
• REVEL score 0.885 from the local REVEL predictor lookup; this is above the supplied generic PP3 moderate cutoff of >=0.773.
• The supplied ClinGen SVI REVEL calibration is Pejaver et al. 2022, American Journal of Human Genetics, PMID:36413997.
• SpliceAI max delta 0.002 and BayesDel 0.345833 were not used: the governing generic path for a missense variant is REVEL only, and BayesDel has no generic ACMG-strength calibration.

Sources

Reasoning

PS2 is not met. The exact ALK R1275Q variant was reported in two affected children and was inherited from their asymptomatic mother; the report does not establish confirmed maternity and paternity for a de novo event, and the inheritance finding is inconsistent with de novo occurrence in the children.

Rule

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

Evidence considered

• Mossé et al. reported that heterozygous ALK R1275Q was present in two affected children and inherited from their asymptomatic mother at age 39.
• No evidence in the reviewed report establishes a de novo occurrence with both maternity and paternity confirmed.

Still missing

• No confirmed de novo event is documented for the evaluated patient.
• The report does not document confirmed maternity and paternity for a de novo interpretation.

Sources

Reasoning

PM6 is not met. PM6 concerns an assumed de novo variant without parental confirmation, but the exact variant was reported as inherited from the asymptomatic mother in a family with two affected children; it is therefore not an assumed de novo event.

Rule

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

Evidence considered

• The report states that ALK R1275Q was inherited from the asymptomatic mother by both affected children, which precludes treating this observation as assumed de novo.

Sources

Reasoning

The exact variant is classified as Pathogenic in ClinVar, but the ClinVar audit reports zero expert-panel submissions. The available assertions are ordinary laboratory or other non-expert submissions, so the required exact-variant ClinVar expert-panel condition for PP5 is not satisfied.

Rule

Generic rules for PP5/BP6: apply them only for an exact-variant ClinVar expert-panel classification: Pathogenic/Likely pathogenic supports PP5; Benign/Likely benign supports BP6. Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger either criterion.

Evidence considered

• ClinVar variation 18083 exactly matches NM_004304.5:c.3824G>A and reports Pathogenic classifications, but the submission audit records expert_panel_submissions = 0; the available classifications are not expert-panel assertions.

Sources

Reasoning

BA1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1; the available population evidence contains no allele frequency, filtering allele frequency, or population count approaching the generic stand-alone threshold of 0.05. The non-cancer gnomAD v2.1 and v3.1 queries were also absent, but no non-cancer-specific governing rule applies because this is a generic ACMG/AMP assessment.

Rule

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

Evidence considered

• gnomAD v2.1: variant absent; no allele frequency, grpmax FAF, popmax AF, or homozygote count reported.
• gnomAD v4.1: variant absent; no allele frequency, grpmax FAF, popmax AF, or homozygote count reported.
• Generic calibration threshold supplied for BA1: >= 0.05, identified in the prompt as the ACMG/AMP 2015 stand-alone benign allele-frequency threshold (PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, so there is no filtering allele frequency (grpmax FAF) to compare with the generic 0.01 default or with a disorder-specific maximum credible allele frequency. No ancestry-specific elevated frequency, founder signal, prevalence estimate, penetrance estimate, or allelic-heterogeneity calculation supports BS1.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1: variant absent; no filtering allele frequency (grpmax FAF), popmax AF, or ancestry-specific count reported.
• gnomAD v4.1: variant absent; no filtering allele frequency (grpmax FAF), popmax AF, or ancestry-specific count reported.
• Generic calibration threshold supplied for BS1: >= 0.01; the prompt identifies Whiffin et al. 2017 (PMID:28518168) as the preferred source for per-gene maximum-credible-frequency derivation.

Sources

Reasoning

BS2 is not met. A heterozygous ALK p.Arg1275Gln carrier was reported as asymptomatic at age 39, but the BS2 definition requires that full penetrance be expected at an early age. The available case evidence does not establish that requirement for ALK-associated neuroblastoma, so the healthy-adult observation cannot support BS2.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• Janoueix-Lerosey et al. 2008 (PMID:18923523), Results/Figure 1d: a heterozygous R1275Q mutation was present in two affected children and inherited from their mother, who was asymptomatic at age 39.
• The observation is insufficient for BS2 because the governing definition additionally requires full penetrance expected at an early age; that prerequisite is not established by this source.

Sources

Reasoning

BS3 is not met. The reviewed exact-variant functional literature does not show a well-established absence of damaging effect on ALK protein function or splicing. Instead, the available biochemical, cellular, and in vivo studies consistently support constitutive kinase activation and oncogenic activity of R1275Q. No negative functional assay with adequate wild-type and benign-control interpretation was identified, and the available splice prediction is not a functional assay.

Rule

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

Evidence considered

• The reviewed exact-variant papers provide positive damaging/activating findings, not benign functional findings; therefore they cannot support BS3.
• SpliceAI max delta 0.002 is a computational prediction, not a validated in vitro or in vivo assay, and does not establish BS3.

Still missing

• No validated negative functional assay demonstrating preserved ALK protein function or normal splicing was identified.

Sources

Reasoning

The reviewed ALK disease literature shows recurrent activating missense variants in the tyrosine-kinase domain, including R1275Q, F1174L, and other substitutions, as a principal disease mechanism. This directly contradicts the BP1 premise that disease in the gene is caused primarily by truncating variants; the available evidence does not support applying BP1 to this missense variant.

Rule

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

Evidence considered

• Janoueix-Lerosey et al. report germline and somatic activating ALK R1275Q and describe constitutive kinase activity.
• Bresler et al. identify F1174L and R1275Q as the most frequently observed ALK substitutions and characterize recurrent tyrosine-kinase-domain missense mutations in neuroblastoma.
• Bresler et al. report recurrent ALK tyrosine-kinase-domain mutations across 1,596 neuroblastoma samples, including R1275Q in 54 tumors.

Sources

Reasoning

The variant is a missense substitution, so the generic BP4 assessment uses REVEL only; SpliceAI is not an alternative BP4 path for this variant. REVEL is 0.885, far above the supplied generic BP4 supporting threshold of <= 0.29, so BP4 is not met. BayesDel is not used because no generic ACMG-strength calibration is established.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc). ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence: missense, NM_004304.5:c.3824G>A (NP_004295.2:p.(Arg1275Gln)), exon 25.
• REVEL score 0.885 from the local REVEL predictor lookup; this exceeds the supplied generic BP4 supporting cutoff of <=0.29.
• The supplied ClinGen SVI REVEL calibration is Pejaver et al. 2022, American Journal of Human Genetics, PMID:36413997.
• SpliceAI max delta 0.002 was not used because the governing generic path for a missense variant is REVEL only; BayesDel 0.345833 was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

No exact-variant ClinVar expert-panel classification as Benign or Likely benign is present. ClinVar instead reports Pathogenic submissions and zero expert-panel submissions; ordinary submissions and aggregate labels cannot trigger BP6.

Rule

Generic rules for PP5/BP6: apply them only for an exact-variant ClinVar expert-panel classification: Pathogenic/Likely pathogenic supports PP5; Benign/Likely benign supports BP6. Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger either criterion.

Evidence considered

• ClinVar variation 18083 exactly matches NM_004304.5:c.3824G>A and reports Pathogenic classifications; the audit records expert_panel_submissions = 0 and no Benign/Likely benign expert-panel assertion.

Sources

Reasoning

The variant is ALK p.Arg1275Gln (c.3824G>A). The reviewed evidence documents this exact amino-acid and nucleotide change, but does not document a previously established pathogenic ALK p.Arg1275Gln caused by a different nucleotide change. The available papers and ClinVar-linked records therefore do not establish the alternate-codon-change comparison required for PS1.

Rule

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

Evidence considered

• ClinVar variation 18083 identifies the queried variant as NM_004304.5:c.3824G>A (p.Arg1275Gln), with no alternate nucleotide change for the same amino-acid substitution documented in the reviewed record.
• Janoueix-Lerosey et al. report ALK R1275Q in germline and tumor contexts, but the extracted record identifies the same R1275Q change rather than a different nucleotide change producing R1275Q.
• George et al. report ALK R1275Q as c.3824G>A; no distinct nucleotide change producing p.Arg1275Gln is reported in the extracted finding.

Still missing

• A verified pathogenic ALK p.Arg1275Gln record caused by a different nucleotide substitution is not documented.

Sources

Reasoning

PS4 requires a statistically significant case-control comparison: the ACMG/AMP note specifies an odds ratio or relative risk >5.0 with a confidence interval that does not include 1.0. PMID:25517749 reports the exact R1275Q variant in 54 of 1,596 diagnostic neuroblastoma tumors, and PMID:18923523 reports affected families, but the available reports do not provide a control count or matched-ancestry control frequency from which an OR/RR and 95% CI can be calculated. The variant is absent from gnomAD v2.1 and v4.1, but no allele count/denominator or ancestry-matched frequency is available, so a valid OR/RR and confidence interval cannot be computed. The literature_pass packet's papers classified as PS4 were checked; its ai_classified_papers contains no paper classified as PS4, and the reviewed exact-variant extracts do not establish the required thresholded case-control statistic.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: 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

• Bresler et al. 2014 (PMID:25517749), Results/Table 1: exact ALK R1275Q was observed in 54 of 1,596 diagnostic neuroblastoma samples; the paper describes two germline cases but does not provide an unaffected control comparison for this exact variant.
• Janoueix-Lerosey et al. 2008 (PMID:18923523), Results/Figure 1d: exact R1275Q was present in two affected children and inherited from an asymptomatic mother; this is familial observation, not a case-control estimate.
• gnomAD v2.1 and gnomAD v4.1 report the exact variant as absent, but the available records provide no allele count/denominator or ancestry-matched control frequency for calculation of an OR/RR and 95% CI.
• The governing PS4 comparison was not numerically completed because the required control data are unavailable; therefore no substituted inequality is applicable.

Still missing

• A control cohort or matched-ancestry population allele count and denominator for the exact variant is required.
• A calculable odds ratio or relative risk with a 95% confidence interval is required.

Sources

Reasoning

The reviewed evidence establishes ALK p.Arg1275Gln, but does not provide a verified different missense substitution at residue Arg1275 that has independently been determined to be pathogenic. The documented comparator substitutions F1174L and other ALK variants occur at different residues and cannot satisfy PM5.

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 PM5 candidate record contains zero same-residue candidates and explicitly does not confirm classic same-residue PM5 semantics.
• Bresler et al. distinguish R1231Q and R1275Q as different substitutions; the other recurrent comparator F1174L is also at a different residue, so these are not PM5 comparators for residue 1275.

Still missing

• A verified pathogenic different missense change at ALK residue 1275 is not documented.

Sources

Reasoning

The evidence supports that activating missense variants are an important ALK disease mechanism, but it does not establish the gene-level low rate of benign missense variation required by PP2. Variant-level absence from gnomAD cannot substitute for a gene-level benign-missense-rate assessment, and no applicable threshold or validated gene-level metric 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

• Functional studies establish recurrent activating ALK missense variants, including R1275Q, as a disease mechanism.
• The queried variant is absent from gnomAD v2.1 and v4.1, but those variant-level observations do not quantify the gene's overall benign missense variation rate.

Still missing

• A validated gene-level assessment showing that ALK has a low rate of benign missense variation is unavailable.

Sources

Reasoning

PP4 requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The available evidence documents that ALK R1275Q occurs in neuroblastoma, including familial neuroblastoma, but it does not provide the current patient's phenotype, phenotype specificity, or family history. The criterion cannot be adjudicated from variant-associated disease reports alone.

Rule

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

Evidence considered

• PMID:18923523 reports exact R1275Q in familial and sporadic neuroblastoma contexts, but does not describe the current case's phenotype.
• PMID:25517749 reports R1275Q in diagnostic neuroblastoma samples, but population-level disease association does not establish the current patient's phenotype specificity.

Still missing

• The patient's clinical phenotype and diagnostic features are required.
• The patient's family history and assessment of phenotype specificity are required.

Sources

Reasoning

BS4 is not assessed. The available family report documents variant carriage in two affected children and an unaffected mother, but it does not document an affected family member who lacks the variant. An unaffected carrier is not evidence of lack of segregation in affected members, and the caveat concerning phenocopies cannot be evaluated from the available information.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The report documents two affected children carrying ALK R1275Q and an asymptomatic mother carrying the variant, but does not report an affected non-carrier.
• The reported unaffected carrier status does not satisfy the BS4 definition of lack of segregation in affected family members.

Still missing

• No affected family member without the variant is documented.
• Phenocopy status and the possibility of more than one pathogenic variant in the family are not evaluated.

Sources

Reasoning

BP2 requires either observation in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observation in cis with a pathogenic variant in any inheritance pattern. The available familial report documents inheritance of ALK R1275Q from an asymptomatic mother but does not report another pathogenic variant, cis/trans phase, or a cis pathogenic haplotype; therefore BP2 cannot be assessed.

Rule

BP2: 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

• Janoueix-Lerosey et al. 2008 (PMID:18923523): the report describes heterozygous ALK R1275Q inherited from an asymptomatic mother but provides no cis/trans observation with another pathogenic variant.
• No affected-proband phase data, parental testing for a second pathogenic allele, or cis pathogenic-variant observation was found for NM_004304.5:c.3824G>A.

Still missing

• Testing or phased results for additional pathogenic variants in the reported individuals and their parents are not documented.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The available exact-variant literature reports neuroblastoma cases and functional studies, but it does not establish that this case carried an alternate pathogenic molecular basis that explains the disease instead of the ALK variant. No case-specific alternate molecular diagnosis is available.

Rule

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

Evidence considered

• The reviewed exact-variant reports document ALK R1275Q in neuroblastoma and functional models, but do not provide a case-specific alternate molecular basis for disease.

Still missing

• A case-specific alternate pathogenic variant or molecular diagnosis, with evidence that it explains the phenotype, is required.

Sources