FBXW7 c.1393C>T, p.Arg465Cys

Reasoning

PS3 is met at strong strength for NM_001349798.2:c.1393C>T (p.Arg465Cys/R465C). Four full-text studies directly evaluated the exact R465C substitution in biologically relevant FBXW7 functional systems. In T-ALL-derived work (PMID:17646408), R465C failed to bind and ubiquitinate NOTCH1 intracellular domain and also failed to bind c-Myc and cyclin E in dominant-negative construct assays; the variant was identified in the CEM T-ALL cell line. In HEK293T binding and HCT116 FBXW7-null cycloheximide-chase experiments (PMID:17909001), R465C failed cyclin E1 binding despite proper localization, and coexpression with wild-type FBXW7 caused marked cyclin E1 accumulation and prolonged its half-life from approximately 1.2 hours to greater than 3 hours. Independent assays showed loss of Aurora B binding for R465C (PMID:23095493) and impaired KLF5 interaction with increased KLF5 protein levels relative to wild-type FBXW7 (PMID:28963353). The studies include wild-type comparator constructs, substrate-binding or ubiquitination readouts, and relevant cellular models, with concordant loss of FBXW7 substrate recognition/degradation across multiple substrates and assay formats. These are direct, variant-specific functional results supporting a damaging effect; no benign functional result was identified.

Rule

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

Evidence considered

• PMID:17646408, Figure 3B-E and Results: dominant-negative FBXW7 R465C failed to bind or ubiquitinate NOTCH1 intracellular domain and failed to bind c-Myc and cyclin E; R465C was identified in the CEM T-ALL cell line.
• PMID:17909001, Functional analysis of Fbxw7 mutants: R465C did not bind cyclin E1 despite proper localization; in HCT116 FBXW7-null cells, coexpression of R465C with wild-type FBXW7 caused marked cyclin E1 accumulation and prolonged its half-life to >3 hours versus approximately 1.2 hours with wild-type FBXW7 alone.
• PMID:23095493, Figure 4D and discussion: co-immunoprecipitation in 293T cells showed loss of Aurora B binding with the R465C mutant.
• PMID:28963353, Figure 4D-E: R465C impaired FBXW7 interaction with KLF5, and unlike wild-type FBXW7, increased KLF5 protein levels in HCT116 cells.
• Across the studies, wild-type FBXW7 comparator constructs and direct substrate-binding, ubiquitination, degradation, or protein-abundance readouts provided assay controls and biologically relevant endpoints.

Still missing

• The available studies are research-laboratory assays rather than validated, reproducible clinical diagnostic laboratory assays.

Sources

Reasoning

Arg465 lies in the FBXW7 substrate-binding region within the WD40 repeats and is repeatedly described as a mutational hotspot. PMID:17909001 identifies Arg465 as a major hotspot and reports recurrent tumor mutations; PMID:28963353 calls R465C/R465H recurrent FBXW7 missense mutations in the WD40 repeat region and demonstrates impaired substrate interaction. The Cancer Hotspots record independently reports FBXW7 R465 as a statistically significant hotspot. gnomAD v4.1 contains only 2/1,592,590 alleles for the observed variant (AF 1.25582e-06), with no homozygotes. Under generic ACMG/AMP, this supports PM1 at moderate strength, although the hotspot extraction is partially parsed and should be confirmed by a reviewer.

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

• Cancer Hotspots reports FBXW7 R465 as a statistically significant hotspot; the extracted record is labeled 'R465 hotspot row partially parsed.'
• PMID:17909001 identifies Arg465 as a major mutational hotspot involved in substrate recognition and reports recurrent R465C/R465H tumor mutations.
• PMID:28963353 describes recurrent FBXW7 missense mutations R465C and R465H in the WD40 repeat region and shows impaired KLF5 interaction.
• PMID:17646408 describes R465 as one of three conserved arginines forming the FBXW7 substrate-binding pocket.
• PMID:23095493 reports loss of Aurora B binding after mutation of R465 and R505.
• gnomAD v4.1: 2/1,592,590 alleles, AF 1.25582e-06, homozygotes 0.

Still missing

• Cancer Hotspots source_data was only partially parsed; confirm the underlying hotspot record and its benign-variation context manually.

Sources

Reasoning

Using the default all-comers gnomAD sources because no VCEP specifies a non-cancer or exome-only alternative, the variant is present only at extremely low frequency: AF 4.324885390537151e-06 in gnomAD v2.1 (1/231220 alleles, 0 homozygotes) and AF 1.2558159978399964e-06 in gnomAD v4.1 (2/1592590 alleles, 0 homozygotes). Both are below the generic PM2 supporting threshold of <=0.0001; the variant is not absent, so the supporting rather than stronger absent-from-controls interpretation applies.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... PM2 (supporting): <=0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v2.1 all-comers exome data: 1/231220 alleles, AF 4.324885390537151e-06, homozygotes 0.
• gnomAD v4.1 all-comers data: 2/1592590 alleles, AF 1.2558159978399964e-06, homozygotes 0.
• No VCEP governs FBXW7, and no non-cancer or exome-only source was specified by a governing rule; therefore the all-comers gnomAD v2.1 and v4.1 entries were used.
• The generic PM2 supporting threshold supplied for this case is <=0.0001; the variant is observed at an extremely low frequency rather than absent.

Sources

Reasoning

The variant is NP_001336727.1:p.(Arg465Cys), c.1393C>T. The reviewed reports describe this same nucleotide change/variant, not a previously established pathogenic variant producing Arg465Cys through a different nucleotide change. Therefore the same-amino-acid-change requirement for PS1 is not met; the reported functional and hotspot evidence is relevant to other criteria, not PS1.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• PMID:17909001 reports FBXW7 c.1393C>T/C1393T causing p.Arg465Cys (R465C).
• PMID:17646408, PMID:23095493, and PMID:28963353 independently discuss R465C, but none supplies a different nucleotide change yielding Arg465Cys.

Still missing

• No independently established pathogenic alternate-nucleotide Arg465Cys variant was identified in the reviewed evidence.

Sources

Reasoning

The variant is a missense substitution, NM_001349798.2:c.1393C>T (p.Arg465Cys), so the governing generic rule selects the REVEL path only; SpliceAI is not applicable to PP3/BP4 for this missense consequence, and BayesDel has no established generic ACMG-strength calibration. REVEL is 0.593, below the generic PP3 supporting threshold of >=0.644 (ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997), so PP3 is not met. The SpliceAI max delta of 0.009 is not used and cannot be combined with REVEL for this criterion.

Rule

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

Evidence considered

• Variant consequence is missense: NM_001349798.2:c.1393C>T, NP_001336727.1:p.(Arg465Cys), exon 11.
• Local REVEL lookup reports score 0.593 for this variant.
• Generic ClinGen SVI REVEL calibration supplied for this case sets PP3 supporting at >=0.644 (Pejaver et al. 2022, PMID:36413997); 0.593 does not meet that threshold.
• The generic rule requires one consequence-matched predictor path: for a missense variant, REVEL only. SpliceAI max delta 0.009 and BayesDel 0.141369 were not used for PP3; the supplied rules state BayesDel has no generic ACMG-strength calibration.

Sources

Reasoning

The exact variant is classified Pathogenic in ClinVar, but the submission is a single-laboratory assertion with criteria provided and no expert-panel review. Under the required generic rule for this interpretation, PP5 is not met because an expert-panel ClinVar classification is absent; ordinary laboratory submissions and aggregate labels do not trigger PP5.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 376414 exactly matches NM_001349798.2(FBXW7):c.1393C>T (p.Arg465Cys), but its review status is criteria provided, single submitter; the audit records zero expert-panel submissions.

Sources

Reasoning

BS3 is not met. No well-established assay showed that FBXW7 p.Arg465Cys has no damaging effect on protein function. Instead, the available variant-specific experiments consistently showed impaired substrate binding, ubiquitination, degradation, or substrate regulation compared with wild-type FBXW7, including effects on NOTCH1, cyclin E1, Aurora B, and KLF5.

Rule

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

Evidence considered

• PMID:17646408: R465C failed NOTCH1 intracellular-domain binding and ubiquitination and failed binding to c-Myc and cyclin E.
• PMID:17909001: R465C failed cyclin E1 binding and caused cyclin E1 accumulation with prolonged half-life when coexpressed with wild-type FBXW7.
• PMID:23095493: R465C caused loss of Aurora B binding in co-immunoprecipitation assays.
• PMID:28963353: R465C impaired KLF5 interaction and increased KLF5 protein levels relative to wild-type FBXW7.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; SpliceAI is not applicable to PP3/BP4 for this missense consequence, and a clean splice prediction cannot substitute for a gray-zone missense REVEL result. REVEL is 0.593, above the generic BP4 supporting threshold of <=0.29 (ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997), so BP4 is not met. The SpliceAI max delta of 0.009 is not used for this criterion.

Rule

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

Evidence considered

• Variant consequence is missense: NM_001349798.2:c.1393C>T, NP_001336727.1:p.(Arg465Cys), exon 11.
• Local REVEL lookup reports score 0.593 for this variant.
• Generic ClinGen SVI REVEL calibration supplied for this case sets BP4 supporting at <=0.29 (Pejaver et al. 2022, PMID:36413997); 0.593 does not meet that threshold.
• The generic rule requires one consequence-matched predictor path: for a missense variant, REVEL only. SpliceAI max delta 0.009 was not used for BP4, and its clean result cannot be substituted for the gray-zone REVEL result.

Sources

Reasoning

The exact variant has no ClinVar expert-panel benign or likely-benign classification. ClinVar contains a single-laboratory Pathogenic submission and no expert-panel submission, so BP6 is not met; the non-expert Pathogenic assertion is not evidence for BP6.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 376414 exactly matches NM_001349798.2(FBXW7):c.1393C>T (p.Arg465Cys); the submission audit records one Pathogenic single-laboratory submission and zero expert-panel submissions, with no benign expert-panel classification.

Sources

Reasoning

No clinical proband report or parental testing data were identified documenting a de novo occurrence, and neither maternity nor paternity confirmation is available for this variant.

Evidence considered

• The available case evidence contains no proband-level clinical observation, parental testing result, maternity confirmation, paternity confirmation, or family-history assessment for NM_001349798.2:c.1393C>T (FBXW7 p.Arg465Cys).
• The reviewed variant-specific publications describe somatic cancer-cell experiments and do not provide a germline de novo observation for this variant.

Still missing

• A clinical proband report with disease phenotype is needed.
• Parental variant testing and confirmed maternity and paternity are needed.
• Family-history information is needed.

Reasoning

The governing rule requires significantly increased prevalence in affected individuals compared with controls; its stated case-control threshold is relative risk or odds ratio >5.0 with a confidence interval excluding 1.0. The reviewed literature reports the variant in somatic tumor samples and T-ALL cell lines, including 1,556 pooled tumors and 15 of 95 primary T-ALL samples in study-level analyses, but it does not provide a matched affected-versus-unaffected case-control comparison for the germline FBXW7-related disorder, nor sufficient variant-specific case and control counts to calculate an OR and 95% CI. gnomAD provides population frequency but no affected-case frequency from a comparable germline cohort. Therefore PS4 cannot be assessed; no PS4 threshold inequality can be written from the available evidence.

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

• Akhoondi et al. (PMID:17909001) reports FBXW7 c.1393C>T/p.Arg465Cys among primary tumor samples within a pooled analysis of 1,556 specimens, but does not provide a germline affected-versus-control comparison or variant-specific OR and 95% CI.
• Thompson et al. (PMID:17646408) reports FBXW7 mutations in 4 of 13 T-ALL cell lines and 15 of 95 primary T-ALL samples; R465C was identified in the CEM T-ALL cell line, without unaffected controls or a variant-specific case-control estimate.
• gnomAD v4.1 reports 2/1,592,590 alleles overall and 1/86,736 South Asian alleles, but no affected-case prevalence is available for an OR calculation.

Still missing

• Variant-specific germline affected-case and matched control counts, or a published case-control estimate with its 95% confidence interval, are unavailable.

Sources

Reasoning

The available case evidence does not document a recessive FBXW7 disorder, a pathogenic second allele, or parent/offspring testing establishing that NM_001349798.2:c.1393C>T is in trans with a pathogenic variant. The available germline context describes heterozygous FBXW7 disease variants but does not provide a case-specific phase observation for this variant.

Rule

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

Evidence considered

• The case evidence identifies NM_001349798.2:c.1393C>T as FBXW7 p.Arg465Cys but contains no parent/offspring genotypes, phase result, or pathogenic variant in a second allele.
• The available germline disease context describes heterozygous FBXW7 variants, but it does not document a case-specific recessive inheritance pattern or phase result for this variant.

Still missing

• Confirm the inheritance mode of the relevant FBXW7 disorder.
• Obtain parental or offspring testing and identify any pathogenic second allele with phase resolved.

Reasoning

R465H is a different missense change at the same residue and is reported as recurrent; functional assays show impaired substrate interaction. However, the available evidence does not establish R465H as pathogenic under the generic germline ACMG/AMP rule, and no validated pathogenic comparator classification is provided. The evidence is therefore insufficient to apply PM5 without human review.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• PMID:28963353 reports recurrent R465H and shows that R465H impaired interaction with KLF5, but does not provide a germline ACMG pathogenic classification.
• PMID:17909001 reports R465H among recurrent tumor mutations and functional hotspot context, but does not establish R465H as a germline pathogenic comparator.

Still missing

• A validated pathogenic classification or equivalent disease-focused evidence for a different missense variant at Arg465 is required.

Sources

Reasoning

No clinical proband report or parental testing data were identified supporting an assumed de novo occurrence; the available evidence does not establish that the variant arose de novo without parental confirmation.

Evidence considered

• The available case evidence contains no proband-level clinical observation or parental variant results for NM_001349798.2:c.1393C>T (FBXW7 p.Arg465Cys).
• The reviewed variant-specific publications do not report a germline assumed-de-novo case for this variant.

Still missing

• A clinical proband report with disease phenotype is needed.
• Evidence that the variant is absent from both parents, even without identity confirmation, is needed.
• Family-history information is needed.

Reasoning

No affected relatives, informative meioses, or family-based genotype-phenotype observations were identified, so cosegregation cannot be evaluated.

Evidence considered

• The available case evidence contains no pedigree, affected-relative data, family genotypes, or informative meioses for NM_001349798.2:c.1393C>T (FBXW7 p.Arg465Cys).
• The reviewed variant-specific publications report somatic tumor and cell-line studies rather than familial cosegregation.

Still missing

• A pedigree with clinically characterized affected relatives is needed.
• Variant genotypes for affected and unaffected relatives are needed.
• Informative meioses and phenotype consistency are needed.

Reasoning

The available case evidence supports FBXW7 loss of function as a disease mechanism and shows recurrent somatic missense mutations, but it does not provide a reliable gene-level assessment that benign missense variation is low together with evidence that missense variants are a common germline disease mechanism. The PP2 requirements cannot be fully evaluated.

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 materials support FBXW7 loss of function as a germline disease mechanism, but do not supply the two gene-level PP2 elements together: low benign missense variation and common pathogenic missense mechanism.
• Somatic recurrent missense and hotspot observations in PMID:17909001 and PMID:28963353 do not by themselves establish the generic germline PP2 criterion.

Still missing

• Need a validated gene-level benign-missense constraint assessment and disease-focused evidence quantifying the frequency of pathogenic germline missense variants.

Sources

Reasoning

No phenotype or family-history description for the evaluated patient is present in the available case evidence. The reviewed tumor and cell-line publications do not establish that this patient's phenotype is highly specific for a disease with a single genetic etiology. PP4 therefore cannot be assessed.

Rule

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

Still missing

• A documented patient phenotype and relevant family history are needed to evaluate disease specificity.

Reasoning

BA1 cannot be assessed under the required filtering-frequency approach because neither gnomAD v2.1 nor v4.1 reports a grpmax FAF for this variant. The available all-comers total allele frequencies are far below 5% (gnomAD v2.1: 1/231220, AF 4.324885390537151e-06; gnomAD v4.1: 2/1592590, AF 1.2558159978399964e-06), but these total point estimates cannot substitute for the required grpmax FAF. The highest observed South Asian point estimates are also not usable as a replacement for grpmax FAF. BA1 is additionally not an appropriate default override of the available single-submitter Pathogenic ClinVar record without the required high-confidence frequency evidence.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers exome data: total AF 4.324885390537151e-06, 1/231220 alleles, homozygotes 0; grpmax FAF was not reported.
• gnomAD v4.1 all-comers data: total AF 1.2558159978399964e-06, 2/1592590 alleles, homozygotes 0; grpmax FAF was not reported.
• gnomAD v2.1 South Asian point estimate was 3.713882492757929e-05 from 1/26926 alleles, and gnomAD v4.1 South Asian point estimate was 1.1529238147943183e-05 from 1/86736 alleles; these popmax point estimates were not used as filtering AF.
• ClinVar variation 376414: Pathogenic, criteria provided by a single submitter; no expert-panel consensus.

Still missing

• A reported filtering allele frequency (grpmax FAF) from an applicable population dataset is unavailable.
• No validated population-specific BA1 assessment resolving the frequency/classification conflict is available.

Sources

Reasoning

BS1 cannot be assessed conclusively because the required gnomAD filtering frequency, grpmax FAF, is not reported for this variant, and no disorder-specific maximum credible allele frequency calculation with prevalence, penetrance, and allelic heterogeneity inputs is available. The observed all-comers frequencies are low (gnomAD v2.1: 4.324885390537151e-06; gnomAD v4.1: 1.2558159978399964e-06) and do not by themselves support an allele frequency greater than expected for FBXW7-related disease, but total AF cannot replace grpmax FAF for this decision.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers exome data: AF 4.324885390537151e-06 from 1/231220 alleles; grpmax FAF was not reported.
• gnomAD v4.1 all-comers data: AF 1.2558159978399964e-06 from 2/1592590 alleles; grpmax FAF was not reported.
• The generic BS1 default is >=0.01, but no disorder-specific maximum credible allele frequency calculation was available; Whiffin et al. 2017 (PMID:28518168) is the named source for using prevalence, penetrance, and allelic heterogeneity in such a calculation.

Still missing

• A reported grpmax FAF from an applicable population dataset is unavailable.
• A maximum credible allele frequency calculation using FBXW7-related disease prevalence, penetrance, and allelic heterogeneity is unavailable.

Sources

Reasoning

No documented healthy adult individual carrying this variant was provided. gnomAD reports zero homozygotes in v2.1 and v4.1, but aggregate population data do not establish that a carrier was a healthy adult, the relevant inheritance state, or full early-age penetrance; 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

• gnomAD v2.1 reports 0 homozygotes among 231220 alleles.
• gnomAD v4.1 reports 0 homozygotes among 1592590 alleles.
• Neither aggregate dataset supplies an individual-level phenotype, age, or verified healthy-adult status for carriers.

Still missing

• No documented healthy adult individual carrying the variant was identified.
• Inheritance model and evidence for full penetrance at an early age were not established for this observation.

Sources

Reasoning

No affected family members with reliable variant testing were identified, so lack of segregation cannot be distinguished from absent family data or possible phenocopies.

Evidence considered

• The available case evidence contains no affected family members, family genotypes, or non-segregation observations for NM_001349798.2:c.1393C>T (FBXW7 p.Arg465Cys).
• The reviewed variant-specific publications do not provide family-based non-segregation evidence.

Still missing

• A pedigree with affected relatives is needed.
• Reliable variant testing in affected relatives is needed.
• Phenotype assessment and consideration of phenocopies are needed.

Reasoning

FBXW7 loss of function is supported as a disease mechanism, but the available evidence does not establish that disease-causing variants in this gene are primarily truncating rather than missense or otherwise distributed. Recurrent functional missense variants are documented, but their presence alone does not quantify the gene-wide variant spectrum required for BP1.

Rule

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

Evidence considered

• The case materials support FBXW7 loss of function as a disease mechanism, but do not quantify truncating variants as the primary disease-causing class.
• PMID:17646408, PMID:17909001, PMID:23095493, and PMID:28963353 document recurrent functional missense changes, but do not provide a complete gene-wide germline variant spectrum.

Still missing

• Need a gene-wide disease-variant spectrum demonstrating that truncating variants are the primary known cause of disease.

Sources

Reasoning

The available evidence does not document NM_001349798.2:c.1393C>T in trans with a pathogenic variant in a fully penetrant dominant disorder, or in cis with a pathogenic variant in any inheritance pattern. The cited FBXW7 publications describe somatic tumor observations and functional assays, not a germline phase observation for this case.

Rule

Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• No case-specific parent/offspring testing, haplotype data, or other phase evidence establishes a cis or trans relationship involving NM_001349798.2:c.1393C>T and another pathogenic variant.
• The available variant-specific publications concern somatic tumor samples or in-vitro functional assays and do not provide the germline allelic observations required for BP2.

Still missing

• Obtain phased genotype data for this variant and any additional pathogenic variant.
• For a trans observation, establish that the relevant disorder is fully penetrant and dominant.

Reasoning

No evidence identifies the evaluated case as having an alternate molecular basis for disease. The available publications concern somatic tumor biology and functional assays rather than a clinical case demonstrating an independent molecular cause. BP5 therefore cannot be assessed.

Rule

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

Still missing

• A clinical case with a documented alternate molecular diagnosis is needed to evaluate BP5.