XPO1 c.1711G>A, p.Glu571Lys

Reasoning

PS3 is met at supporting strength. The exact XPO1 p.E571K variant was evaluated in a variant-specific functional study using PMBL cell models and transfected U2OS cells. In PMBL cells, the E571K-containing MedB-1 line showed weaker response and apoptosis induction to the XPO1 inhibitor KPT-185 than wild-type XPO1 PMBL cell lines, indicating an altered biologically relevant phenotype. The study also included a REV-GFP nuclear-export assay in transfected U2OS cells, where selinexor inhibited nuclear export similarly for wild-type and E571K XPO1, so the evidence does not justify a stronger level and is not a fully established, reproducible clinical-laboratory assay. No independent replication of the variant-specific 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:27312795: The exact p.E571K variant was studied in PMBL cell models and transfected U2OS cells; the MedB-1 PMBL cell line showed weaker KPT-185 response and apoptosis induction than wild-type XPO1 PMBL cell lines, while selinexor inhibited nuclear export similarly for wild-type and E571K XPO1 in the REV-GFP assay.
• The variant-specific cell-based drug-response phenotype supports altered XPO1-related biology, but the mixed assay results and lack of independent replication limit calibration to supporting strength.

Still missing

• No independent replication of the variant-specific functional result was identified.
• The available evidence is not a validated, reproducible clinical-diagnostic laboratory assay and includes a nuclear-export result similar to wild type.

Sources

Reasoning

The exact XPO1 p.E571K variant was reported in 27/117 PMBL cases and 2/197 DLBCL comparator cases in PMID:27312795. Treating these as a case-control comparison, I calculated the odds ratio from the counts: (27/90)/(2/195) = 29.25, with an approximate log-odds 95% CI of 6.81-125.68; OR >5.0 and the confidence interval excludes 1.0. PMID:27479820 independently reports the exact variant in 22/94 classical Hodgkin lymphoma cases, while PMID:22918138 was checked because literature_pass.json flagged it for PS4 and does not mention XPO1 or this variant.

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:27312795, abstract: XPO1 mutations occurred in 28/117 PMBL cases and 3/197 DLBCL cases; full text specifies the exact p.E571K variant in 27/117 PMBL cases and 2 DLBCL cases (1/81 ABC and 1/33 other/unclassified).
• PMID:27312795, Results, 'XPO1 mutations are highly specific for the PBML subtype': 'The highly recurrent E571K variant (27/28 mutations, NM_003400, chr2:g.61719472C>T)' and exact DLBCL counts at lines 335-336.
• Computed from PMBL 27/117 versus DLBCL 2/197: OR = (27×195)/(90×2) = 29.25; approximate 95% CI = 6.81-125.68. The comparison is 29.25 > 5.0 and the CI does not include 1.0.
• PMID:27479820 reports the exact c.1711G>A/p.E571K mutation in 22/94 classical Hodgkin lymphoma patients (24.2%) using digital PCR.
• PMID:22918138 was explicitly checked because literature_pass.json listed it as a PS4 candidate; its extracted full text contains no XPO1, c.1711G>A, p.E571K, case, or population evidence.

Sources

Reasoning

XPO1 p.E571K is documented as a statistically significant cancer hotspot and a highly recurrent mutation at codon 571. PMID:27312795 places E571K within the H12A heat-repeat domain and hydrophobic nuclear-export-signal-binding cleft; PMID:21642962 describes recurrence at the same codon in a highly conserved region. The reviewed evidence identifies no benign variation at this critical hotspot/domain location; gnomAD shows only 5/1,591,784 alleles in v4.1 (AF 3.14113e-6; grpmax FAF 8e-7).

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 that the variant lies in a statistically significant hotspot.
• PMID:27312795 reports: "The highly recurrent E571K variant (27/28 mutations, NM_003400, chr2:g.61719472C>T) is located within the Heat repeat domain H12A"; the paper places this domain within the hydrophobic NES-binding cleft.
• PMID:21642962 reports four XPO1 cases with p.E571K or p.E571G at the same codon and states that recurrence at the same residue and its highly conserved region indicate an effect on XPO1 activity.
• PMID:27479820 reports recurrent XPO1 c.1711G>A/p.E571K in 22 of 94 classical Hodgkin lymphoma patients (24.2%).
• gnomAD v4.1 reports 5/1,591,784 alleles, AF 3.14113e-6, no homozygotes, and grpmax FAF 8e-7.

Sources

Reasoning

PM2 is met at supporting strength. The variant is present only at an extremely low frequency in the default all-comers population sources: gnomAD v4.1 AF=3.1411297010147104e-06 (5/1,591,784 alleles), with homozygotes=0; gnomAD v2.1 AF=4.37395571807231e-06 (1/228,626 alleles), with homozygotes=0. The additional gnomAD v2.1 non-cancer exome subset reports AF=4.6700602437771445e-06 (1/214,130 alleles), and gnomAD v3.1 non-cancer genomes reports the variant as absent. These values are below the supplied PM2 supporting threshold of <=0.0001; the variant is not literally absent, so the extremely-low-frequency path is used.

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=3.1411297010147104e-06 (5/1,591,784 alleles), homozygotes=0; grpmax FAF=8e-07.
• gnomAD v2.1 all-comers: AF=4.37395571807231e-06 (1/228,626 alleles), homozygotes=0.
• gnomAD v2.1 non-cancer exomes: AF=4.6700602437771445e-06 (1/214,130 alleles), homozygotes=0.
• gnomAD v3.1 non-cancer genomes: absent from the queried dataset.
• Generic PM2 calibration threshold supplied for this case: <=0.0001; the variant is present at an extremely low frequency rather than absent.

Sources

Reasoning

The queried variant is XPO1 NM_003400.4:c.1711G>A, p.Glu571Lys (E571K). The reviewed literature documents this exact missense change repeatedly in somatic lymphoma and leukemia cohorts, but no previously established pathogenic variant classification for this amino-acid change is provided. Somatic oncogenicity and recurrent occurrence do not by themselves establish the ACMG/AMP PS1 prerequisite of a previously established pathogenic variant.

Rule

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

Evidence considered

• The normalized variant is NM_003400.4:c.1711G>A, NP_003391.1:p.(Glu571Lys), p.(E571K).
• PMID:27479820 explicitly reports c.1711G>A causing p.E571K in classical Hodgkin lymphoma, and PMID:27312795 reports the recurrent E571K variant in PMBL.
• The available ClinVar record is classified Likely oncogenic with single-submitter review, and OncoKB reports an oncogenic somatic context; neither is a previously established ACMG pathogenic classification for PS1.

Sources

Reasoning

The variant is a missense substitution, so PP3 is evaluated only through the generic missense REVEL path; SpliceAI is not applicable to this path, and BayesDel has no established generic ACMG-strength calibration. REVEL is 0.579, below the generic PP3 supporting threshold of >=0.644 and therefore does not support PP3 at any strength. The threshold is the ClinGen SVI REVEL calibration from Pejaver et al. 2022 (PMID:36413997).

Rule

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

Evidence considered

• NM_003400.4:c.1711G>A is a missense variant producing NP_003391.1:p.(Glu571Lys), so only the REVEL path applies.
• Local REVEL lookup: score 0.579.
• ClinGen SVI REVEL calibration: PP3 supporting requires >=0.644; PP3 moderate requires >=0.773; PP3 strong requires >=0.932 (Pejaver et al. 2022, PMID:36413997).
• SpliceAI was not counted because the generic rule selects REVEL only for missense variants; BayesDel was not counted because no generic ACMG-strength calibration is established.

Sources

Reasoning

The exact ClinVar record is classified Likely oncogenic with one single-submitter somatic submission and criteria-provided, single-submitter review status; no expert-panel Pathogenic or Likely pathogenic classification is present. OncoKB labels the variant Oncogenic, but that is not an exact-variant ACMG/AMP expert-panel P/LP ClinVar assertion, and the available variant-specific literature is independently reviewable.

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 4539776 exactly matches NM_003400.4:c.1711G>A (p.Glu571Lys) but is classified Likely oncogenic, review status criteria provided/single submitter, with one somatic submission and zero expert-panel submissions.
• OncoKB reports a variant-specific Oncogenic label, but this is not an ACMG/AMP expert-panel Pathogenic or Likely pathogenic ClinVar classification and cannot trigger generic PP5.

Sources

Reasoning

BA1 is not met. The governing generic ACMG/AMP threshold is >=0.05. gnomAD v4.1 reports grpmax FAF=8e-07, far below 0.05; its reported highest population point estimate (1.62549e-05) is not used for BA1, and the grpmax FAF is based on a population with 61,520 observed alleles, exceeding the 2,000-allele minimum. The all-comers frequency is therefore nowhere near the stand-alone benign threshold.

Rule

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

Evidence considered

• gnomAD v4.1 all-comers: total AF=3.1411297010147104e-06 (5/1,591,784 alleles), homozygotes=0, grpmax FAF=8e-07.
• gnomAD v4.1 reports the highest population point estimate as 1.625487646293888e-05 in Remaining individuals (1/61,520 alleles); this popmax point estimate was not used for BA1.
• The reported grpmax population has 61,520 observed alleles, above the 2,000-observed-allele requirement. Generic BA1 calibration threshold supplied for this case: >=0.05 (ACMG/AMP 2015, PMID:25741868).

Sources

Reasoning

BS1 is not met. The generic SVI default threshold supplied for this case is >=0.01, but the appropriate filtering metric is gnomAD v4.1 grpmax FAF=8e-07, not the highest population point estimate of 1.62549e-05. The grpmax FAF is far below 0.01 and comes from a population with 61,520 observed alleles, so the frequency does not support BS1 regardless of disease prevalence, penetrance, or allelic heterogeneity considerations.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v4.1 all-comers: grpmax FAF=8e-07; total AF=3.1411297010147104e-06 (5/1,591,784 alleles).
• The highest population point estimate is 1.625487646293888e-05 in Remaining individuals (1/61,520 alleles), but popmax point estimate is not the filtering metric for BS1.
• The reported grpmax population has 61,520 observed alleles, above the 2,000-observed-allele requirement. Generic BS1 default threshold supplied for this case: >=0.01; a per-disorder maximum-credible-frequency calculation is not needed because the filtering FAF is orders of magnitude below that default.

Sources

Reasoning

BS3 is not met. The exact CRM1/XPO1 E571K mutation was tested using a cellular nuclear-export reporter and in vitro biochemical analyses. The study states that the mutation does not necessarily abrogate nuclear-export activity but may increase affinity for NES sequences with a more negatively charged C-terminal end. This indicates that the assay did not establish an unequivocally nondamaging effect; possible altered substrate affinity is functionally relevant and is inconsistent with using the study as well-established evidence of no damaging effect. The PMBL study likewise found altered drug response rather than a uniformly normal functional phenotype.

Rule

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

Evidence considered

• García-Santisteban et al. 2016, cellular reporter and biochemical analyses of CRM1/XPO1 E571K: the abstract states that the mutation does not necessarily abrogate nuclear-export activity but may increase affinity for NES sequences bearing a more negatively charged C-terminal end.
• PMID:27312795: the variant-specific PMBL assay reported weaker inhibitor response and apoptosis induction in the E571K-containing MedB-1 line than in wild-type XPO1 PMBL cell lines, rather than uniformly normal function.

Still missing

• No independent assay series demonstrating normal XPO1 protein function across relevant substrates was identified.
• The available study reports a possible altered NES-affinity phenotype rather than unequivocally no damaging effect.

Sources

Reasoning

The variant is a missense substitution, so BP4 is evaluated only through the generic missense REVEL path; the SpliceAI result cannot substitute for a gray-zone REVEL result, and BayesDel has no established generic ACMG-strength calibration. REVEL is 0.579, above the BP4 supporting threshold of <=0.29 and therefore does not support BP4. The threshold is the ClinGen SVI REVEL calibration from Pejaver et al. 2022 (PMID:36413997).

Rule

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

Evidence considered

• NM_003400.4:c.1711G>A is a missense variant producing NP_003391.1:p.(Glu571Lys), so only the REVEL path applies.
• Local REVEL lookup: score 0.579, which is indeterminate for benign computational evidence under the supplied generic calibration.
• ClinGen SVI REVEL calibration: BP4 supporting requires <=0.29; BP4 moderate requires <=0.183; BP4 strong requires <=0.016 (Pejaver et al. 2022, PMID:36413997).
• SpliceAI was not counted because the generic rule selects REVEL only for missense variants; a gray-zone REVEL result does not permit substituting the splice prediction, and BayesDel was not counted because no generic ACMG-strength calibration is established.

Sources

Reasoning

No exact-variant ClinVar expert-panel classification of Benign or Likely benign is present. The available ClinVar record is a single-submitter Likely oncogenic somatic assertion, and the available functional literature is independently reviewable rather than an unavailable reputable benign assertion.

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 4539776 exactly matches the queried variant but has one somatic single-submitter Likely oncogenic submission, criteria provided/single submitter, and zero expert-panel submissions; no Benign or Likely benign expert-panel assertion is available.

Sources

Reasoning

No germline proband-level evidence establishes a de novo occurrence of NM_003400.4:c.1711G>A (p.Glu571Lys), and no confirmed maternity and paternity, parental genotypes, disease phenotype, or family-history information is provided. The reviewed variant-specific publications describe somatic tumor cohorts rather than a germline de novo case. Therefore PS2 cannot be assessed.

Rule

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

Evidence considered

• PMID:27312795 reports XPO1 p.E571K in PMBL tumor cohorts and describes the variant as acquired in two analyzed cases; it provides no germline parental testing or de novo pedigree.
• PMID:27479820 reports XPO1 c.1711G>A/p.E571K in classical Hodgkin lymphoma tumor DNA and plasma cell-free DNA and reports absence from peripheral-blood mononuclear cells in four mutated patients; it provides no germline de novo pedigree.

Still missing

• Documented disease phenotype in the proband
• Parental genotypes with confirmed maternity and paternity
• Explicit absence of family history
• Evidence that the tested variant is germline rather than somatic

Sources

Reasoning

PM3 cannot be assessed. The available evidence does not document an affected germline proband with a second pathogenic variant, parental or offspring testing, or phasing that places NM_003400.4:c.1711G>A (XPO1 p.Glu571Lys) in trans with a pathogenic variant. The variant-specific publications reviewed describe somatic cancer cohorts and do not provide the required recessive-disease phase evidence.

Rule

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

Evidence considered

• No parental, offspring, segregation, or phase data for NM_003400.4:c.1711G>A were identified in the consolidated case evidence.
• PMID:27312795 reports XPO1 p.E571K in primary mediastinal B-cell lymphoma as a somatic tumor variant and provides no germline trans-phase observation.
• PMID:27479820 reports XPO1 p.E571K in a classical Hodgkin lymphoma tumor cohort and provides no germline trans-phase observation.

Still missing

• Documented affected germline proband(s) with a second pathogenic or likely pathogenic variant
• Parental or offspring testing, or equivalent molecular phasing, establishing the variants are in trans
• Relevant inheritance mode and disease context for XPO1

Sources

Reasoning

PMID:21642962 reports a different missense change at residue 571, p.E571G, alongside p.E571K, but the supplied evidence does not establish p.E571G as pathogenic under ACMG/AMP. The available PM5 candidate artifact contains no qualifying comparator. A pathogenic same-residue comparator therefore cannot be confirmed from the supplied evidence.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• PMID:21642962 states that XPO1 p.E571K and p.E571G occurred in the same codon, but does not establish p.E571G as a pathogenic variant.
• The PM5 candidate collection reports zero qualifying same-residue candidates and explicitly does not establish classic PM5 eligibility; it is not sufficient to apply PM5.

Still missing

• A different missense variant at XPO1 residue 571 with an established pathogenic classification is required to assess PM5.

Sources

Reasoning

No germline proband-level evidence establishes an assumed de novo occurrence of NM_003400.4:c.1711G>A (p.Glu571Lys). The available variant-specific publications concern somatic tumor cohorts and do not provide a disease phenotype, parental testing, or a germline family history. Therefore PM6 cannot be assessed.

Rule

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

Evidence considered

• PMID:27312795 reports p.E571K in PMBL tumors and states that the variant was acquired in two analyzed cases; it does not report assumed germline de novo status.
• PMID:27479820 reports p.E571K in Hodgkin lymphoma tumor and plasma samples, with absence from peripheral-blood mononuclear cells in four mutated patients; it does not report assumed germline de novo status.

Still missing

• Documented disease phenotype in the proband
• Evidence that the tested variant is germline
• Parental testing or a documented assumption of de novo origin
• Family-history information

Sources

Reasoning

No affected family members, informative meioses, genotype-phenotype relationships, or segregation results are reported for NM_003400.4:c.1711G>A (p.Glu571Lys). The reviewed literature concerns somatic cancer cohorts and does not establish familial segregation in a disease for which XPO1 is definitively known to be causative. Therefore PP1 cannot be assessed.

Rule

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

Evidence considered

• PMID:27312795 describes recurrent p.E571K in PMBL tumor samples and cell-line experiments, not affected relatives or familial segregation.
• PMID:27479820 describes p.E571K in a 94-patient classical Hodgkin lymphoma tumor cohort and plasma samples, not familial segregation or informative meioses.

Still missing

• A disease phenotype and pedigree for the proband
• Genotypes and phenotypes of affected relatives
• Informative meioses and segregation results
• Established germline disease relationship for XPO1

Sources

Reasoning

The supplied evidence does not provide a gene-level assessment demonstrating both a low rate of benign missense variation and that missense variants are a common mechanism of disease for the relevant generic ACMG disease context. Somatic hotspot recurrence and oncogenicity evidence do not establish both PP2 requirements.

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 evidence documents somatic XPO1 hotspot recurrence and a rare gnomAD observation, but does not provide the required gene-level benign-missense rate or a validated generic disease-mechanism assessment.

Still missing

• Gene-level benign-missense constraint and disease-mechanism evidence meeting both PP2 requirements are unavailable.

Reasoning

No individual case phenotype or family history is provided for this somatic XPO1 variant, and the available papers describe cohorts of lymphoma patients rather than a highly specific phenotype for a single genetic etiology. The required phenotype information is unavailable.

Rule

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

Still missing

• Individual patient's phenotype and family history are not available.

Reasoning

BS2 is not assessed. gnomAD shows zero homozygotes for the variant, but that is not evidence that a healthy adult individual was observed with the required genotype. The case contains no documented healthy adult carrier or homozygous/hemizygous observation and no established inheritance, penetrance, or age-of-onset framework for an XPO1 disorder against which the BS2 applicability conditions can be evaluated.

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 all-comers: homozygotes=0 (1/228,626 alleles).
• gnomAD v4.1 all-comers: homozygotes=0 (5/1,591,784 alleles).
• Zero observed homozygotes cannot be converted into a documented healthy-adult observation; no qualifying individual-level BS2 observation is provided.

Still missing

• A documented healthy adult observation with the relevant genotype and a defined XPO1 disorder inheritance, penetrance, and early-onset framework are unavailable.

Sources

Reasoning

No affected family members have been tested for NM_003400.4:c.1711G>A (p.Glu571Lys), and no pedigree documents lack of segregation. The available variant-specific reports are somatic cancer studies and do not provide familial non-segregation data or information needed to evaluate phenocopies. Therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• PMID:27312795 reports somatic p.E571K in PMBL cohorts and does not report affected-family testing or familial non-segregation.
• PMID:27479820 reports somatic p.E571K in classical Hodgkin lymphoma tumors and plasma and does not report affected-family testing or familial non-segregation.

Still missing

• A documented affected family and pedigree
• Genotypes of affected relatives
• Phenotype assessment of affected relatives
• Evaluation of phenocopies and alternative pathogenic variants

Sources

Reasoning

The supplied evidence does not establish that XPO1 is a gene for which primarily truncating variants are known to cause disease. The available material focuses on somatic recurrent missense oncogenicity and does not provide a sufficiently established truncating-variant disease spectrum for BP1.

Rule

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

Evidence considered

• The reviewed XPO1 evidence concerns recurrent somatic missense changes, especially p.E571K, and does not establish the gene-level predominance of truncating disease-causing variants required by BP1.

Still missing

• A validated gene-level disease spectrum showing primarily truncating pathogenic variants is unavailable.

Reasoning

BP2 cannot be assessed. The available evidence does not document NM_003400.4:c.1711G>A (XPO1 p.Glu571Lys) in trans with a pathogenic variant in a fully penetrant dominant disorder, or in cis with a pathogenic variant under any inheritance pattern. The reviewed variant-specific reports are somatic cancer studies and do not establish cis/trans relationships with a pathogenic variant.

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 cis/trans, parental, offspring, segregation, or paired-pathogenic-variant data for NM_003400.4:c.1711G>A were identified in the consolidated case evidence.
• PMID:27312795 describes p.E571K in somatic primary mediastinal B-cell lymphoma samples and does not report a phased pathogenic germline allele.
• PMID:27479820 describes p.E571K in somatic classical Hodgkin lymphoma samples and does not report a phased pathogenic germline allele.

Still missing

• Documented pathogenic variant paired with this variant in the same individual
• Molecular phasing establishing either a trans or cis relationship
• For the trans pathway, confirmation of a fully penetrant dominant gene/disorder and relevant germline clinical context

Sources

Reasoning

The available reports establish that p.E571K is a recurrent somatic mutation in lymphoma cohorts, but they do not provide an individual case-level conclusion that this variant occurred with an alternate molecular basis for that patient's disease. Co-occurring mutations mentioned in some cancer studies are insufficient without a documented alternate molecular basis for the specific case.

Rule

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

Still missing

• Case-level clinical and molecular data establishing an alternate disease basis are unavailable.