CALR c.1154_1155insTTGTC, p.Lys385AsnfsTer47

Reasoning

PM2 is met at supporting strength. Under the supplied generic ClinGen SVI threshold, PM2 supporting applies at allele frequency <=0.0001. The default all-comers gnomAD sources are applicable because no VCEP-specific population source governs this gene. gnomAD v4.1 shows AF=2.6518e-05 from 42/1,583,830 alleles with zero homozygotes and grpmax FAF=1.908e-05; gnomAD v2.1 shows AF=3.0599e-05 from 7/228,766 alleles with zero homozygotes and grpmax FAF=9.5e-06. The variant is therefore present but at an extremely low population frequency below the supplied PM2 threshold. Non-cancer subsets are concordant, including gnomAD v2.1 non-cancer exomes AF=2.65647e-05 from 5/188,220 alleles and gnomAD v3.1 non-cancer genomes AF=2.0281e-05 from 3/147,922 alleles; they do not alter the default-source decision.

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: 42/1,583,830 alleles, AF=2.6518e-05, zero homozygotes, grpmax FAF=1.908e-05.
• gnomAD v2.1: 7/228,766 alleles, AF=3.0599e-05, zero homozygotes, grpmax FAF=9.5e-06.
• Concordant non-cancer data: gnomAD v2.1 non-cancer exomes 5/188,220 alleles, AF=2.65647e-05, zero homozygotes; gnomAD v3.1 non-cancer genomes 3/147,922 alleles, AF=2.0281e-05, zero homozygotes.
• The supplied generic ClinGen SVI calibration threshold is PM2 supporting at allele frequency <=0.0001; the ACMG/AMP 2015 PM2 definition is cited under PMID:25741868.

Sources

Reasoning

ClinVar contains an exact-variant Pathogenic classification, but the submission audit reports zero expert-panel submissions and identifies the Pathogenic assertions as clinical-laboratory submissions or submissions without assertion criteria. Under the specified generic rule, these are not an exact-variant ClinVar expert-panel classification and cannot 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 1028735 exactly matches NM_004343.4(CALR):c.1154_1155insTTGTC and reports Pathogenic, but the submission audit records expert_panel_submissions = 0; the submissions are from clinical laboratories or have no assertion criteria.
• The exact-variant ClinVar record therefore does not meet the required expert-panel source restriction for PP5.

Sources

Reasoning

BA1 is not met. The applicable filtering frequency is the gnomAD v4.1 grpmax FAF of 1.908e-05 (0.001908%), far below the generic stand-alone threshold of 0.05 (5%). The highest population point estimate, Middle Eastern AF 0.000168634 from 1/5930 alleles, is not used for BA1 because the rule requires the filtering allele frequency and the FAF is below threshold. gnomAD v2.1 independently reports grpmax FAF 9.5e-06. The variant is also reported as Pathogenic by four ClinVar laboratories, so a stand-alone benign call from a default frequency threshold would be inappropriate.

Rule

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

Evidence considered

• gnomAD v4.1: 42/1,583,830 total alleles, total AF 2.6518e-05, no homozygotes; filtering grpmax FAF=1.908e-05.
• gnomAD v2.1: 7/228,766 total alleles, total AF 3.0599e-05, no homozygotes; filtering grpmax FAF=9.5e-06.
• The gnomAD v4.1 Middle Eastern point estimate is 1/5,930 alleles (AF 0.000168634), but the required filtering FAF remains 1.908e-05.
• ClinVar variation 1028735: Pathogenic, criteria provided, multiple submitters, no conflicts; four clinical laboratories.

Sources

Reasoning

BS1 is not met. The required filtering frequency is gnomAD v4.1 grpmax FAF=1.908e-05 (0.001908%), which is below the generic BS1 default threshold of 0.01 (1%); gnomAD v2.1 grpmax FAF is even lower at 9.5e-06. The highest population point estimate, 0.000168634 in the Middle Eastern population, is not substituted for FAF. The observed frequency is not greater than expected for the disorder on the supplied generic threshold, and no maximum-credible-frequency calculation is needed to reject this low frequency.

Rule

Allele frequency is greater than expected for disorder.

Evidence considered

• gnomAD v4.1: filtering grpmax FAF=1.908e-05; total AF=2.6518e-05 from 42/1,583,830 alleles, with zero homozygotes.
• gnomAD v2.1: filtering grpmax FAF=9.5e-06; total AF=3.0599e-05 from 7/228,766 alleles, with zero homozygotes.
• The gnomAD v4.1 Middle Eastern subgroup has 5,930 observed alleles, satisfying the minimum population-size check, but its point estimate is not used because BS1 requires the filtering FAF.
• Whiffin et al. 2017 (PMID:28518168) is the supplied publication for maximum-credible-frequency guidance; no maximum-credible-frequency calculation was needed because the observed filtering FAF is far below the generic BS1 threshold.

Sources

Reasoning

The exact-variant ClinVar record contains Pathogenic, not benign or likely benign, submissions, and the submission audit reports zero expert-panel submissions. No exact-variant ClinVar expert-panel benign or likely benign classification is available. Ordinary laboratory submissions, aggregate labels, and non-expert assertions cannot trigger BP6 under the specified generic rule.

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 1028735 exactly matches the variant and reports Pathogenic from four submissions, with expert_panel_submissions = 0; no expert-panel Benign or Likely benign assertion is present.
• The available ClinVar record therefore cannot satisfy the required expert-panel benign-source restriction for BP6.

Sources

Reasoning

PS2 cannot be assessed because the available case evidence does not document a proband with a relevant disease phenotype, absence of family history, or confirmed maternity and paternity. The exact-variant literature reviewed describes recurrent somatic CALR mutations in myeloproliferative neoplasms, not a documented germline de novo occurrence, and cannot substitute for parental testing.

Rule

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

Evidence considered

• No proband-level phenotype, family-history, maternity, paternity, or parental-variant-testing result is present in the available case evidence.
• PMID:24325356 and PMID:24325359 describe the exact CALR c.1154_1155insTTGTC/p.K385fs*47 variant as a recurrent somatic mutation in myeloproliferative neoplasms; they do not document a de novo germline case with parental testing.

Still missing

• Document the proband's relevant disease phenotype and family history.
• Obtain and document variant testing in both biological parents with confirmed maternity and paternity.
• Clarify whether the variant is germline in the proband rather than a somatic CALR mutation.

Sources

Reasoning

The exact variant, CALR c.1154_1155insTTGTC (p.(K385Nfs*47)), was expressed in HEK293T, COS-7, and hematopoietic 32D cells. The study confirmed mutant-protein expression and loss of the KDEL-containing C-terminal sequence, but the variant showed no difference from nonmutant CALR in the reported Golgi localization, endogenous CALR pattern, or cell-surface expression assays; the authors stated that partial loss from the endoplasmic reticulum was not excluded. These findings do not establish a validated, reproducible assay result demonstrating a damaging effect on protein function, so PS3 is not assessed.

Rule

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

Evidence considered

• Nangalia et al. 2013, PMID:24325359, Results, 'Mutant Protein with a Novel C-Terminal,' paragraphs 1-3: the exact K385fs*47 insertion was expressed in HEK293T, COS-7, and 32D cells; immunoblotting confirmed expression, while localization and cell-surface assays did not show the required damaging functional phenotype.
• Nangalia et al. 2013, PMID:24325359, the exact variant's altered C-terminal sequence loses the acidic domain and KDEL signal, but the paper described compromised endoplasmic-reticulum retention as a possibility rather than demonstrating it as an established damaging assay result.
• Klampfl et al. 2013, PMID:24325356, identified c.1154_1155insTTGTC as the type 2 CALR mutation, but its functional assays tested the type 1 52-bp deletion rather than this exact variant.

Still missing

• A validated and reproducible functional assay demonstrating that p.(K385Nfs*47) impairs CALR protein function or a disease-relevant cellular phenotype is unavailable.
• Assay controls, replication, quantitative effect sizes, and diagnostic-laboratory validation sufficient for PS3 strength calibration are not documented in the available evidence.

Sources

Reasoning

The governing ACMG/AMP rule requires that the prevalence of the variant in affected individuals be significantly increased compared with controls, with RR or OR >5.0 and a confidence interval that does not include 1.0. The reviewed exact-variant papers report the CALR c.1154_1155insTTGTC / K385fs*47 insertion in myeloproliferative-neoplasm cohorts, but they do not provide an unaffected or population-control comparison for this exact variant. Therefore no case-control counts, odds ratio, or 95% confidence interval can be calculated. The required numeric comparison is OR > 5.0; explicit substitution is "OR unavailable > 5.0? cannot evaluate", so an inequality cannot be evaluated.

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:24325356 reports the exact 5-bp insertion c.1154_1155insTTGTC among patients with myeloproliferative neoplasms and gives its fraction among CALR-mutated cases, but does not report unaffected or population controls for the exact variant.
• PMID:24325359 reports the exact K385fs*47 5-bp insertion in patients with essential thrombocythemia or myelofibrosis lacking JAK2 or MPL mutations, but does not provide a control group for this exact variant.
• The packet's literature_pass.json has no packet.ai_classified_papers entries requiring additional PS4 verification.

Still missing

• A matched-control case-control dataset or exact-variant case and control counts are required to calculate OR and its 95% CI.

Sources

Reasoning

PM3 cannot be assessed because the available evidence does not establish a recessive CALR disorder for this case and provides no affected-proband observation, pathogenic allelic partner, parental testing, or other phase determination showing this variant in trans with a pathogenic variant.

Rule

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

Evidence considered

• The case evidence documents the CALR c.1154_1155insTTGTC variant and somatic CALR cohort observations, but contains no affected-proband report with a second pathogenic variant and no parental, offspring, or other phase testing.
• The available evidence does not establish recessive inheritance for the disorder being evaluated.

Still missing

• Establish whether the relevant CALR disorder is recessive.
• Obtain affected-proband data and a pathogenic variant on the other allele.
• Obtain parental or offspring testing, or equivalent phasing, to demonstrate trans configuration.

Reasoning

PM6 cannot be assessed because the available case evidence does not document an affected proband with a relevant phenotype or an assumed de novo occurrence. No parental testing, maternity/paternity information, or germline de novo case report is available; somatic reports of this CALR variant do not establish PM6.

Rule

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

Evidence considered

• No assumed de novo observation, parental testing result, or germline case report is present in the available case evidence.
• PMID:24325356 and PMID:24325359 report the exact variant in somatic myeloproliferative-neoplasm cohorts and do not provide evidence for PM6.

Still missing

• Document the proband's relevant disease phenotype.
• Provide evidence supporting a de novo occurrence in the proband.
• Clarify whether the variant is germline rather than somatic.

Sources

Reasoning

PP1 cannot be assessed because no affected family members, variant-positive relatives, phenotype concordance, or informative meioses are documented. The available exact-variant publications describe somatic myeloproliferative-neoplasm cohorts rather than familial segregation, so no segregation strength can be 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

• No family pedigree, affected-relative testing, cosegregation data, or informative meioses are present in the available case evidence.
• The reviewed exact-variant publications PMID:24325356 and PMID:24325359 describe somatic CALR mutations in patient cohorts and do not report familial segregation.

Still missing

• Provide a pedigree with affected relatives and disease phenotypes.
• Provide variant-testing results for affected and unaffected relatives.
• Provide the number of informative meioses and assess phenotype consistency.

Sources

Reasoning

The governing rule requires a patient's phenotype or family history that is highly specific for a disease with a single genetic etiology. The available exact-variant literature places CALR c.1154_1155insTTGTC in essential thrombocythemia or myelofibrosis cohorts, but the evidence does not provide patient-specific phenotype or family-history details for this case, and myeloproliferative neoplasms are not shown here to have a single genetic etiology. The available evidence is therefore insufficient to assess PP4.

Rule

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

Evidence considered

• PMID:24325356 describes the exact insertion in patients with essential thrombocythemia and primary myelofibrosis, but this cohort-level association does not establish a highly specific phenotype for the individual case.
• PMID:24325359 describes the exact insertion in patients with essential thrombocythemia or myelofibrosis, without case-specific phenotype or family-history evidence establishing a single genetic etiology.

Still missing

• Patient-specific phenotype and family-history data are required, together with evidence that the phenotype is highly specific for a disease with a single genetic etiology.

Sources

Reasoning

BS2 is not assessed because the available population data provide aggregate allele and homozygote counts, not an identified healthy adult carrying this variant. The gnomAD v2.1 and v4.1 datasets report zero homozygotes, but that does not establish the presence or absence of a healthy adult heterozygote, hemizygote, or homozygote. The required disease inheritance, expected age-related penetrance, and individual-level healthy-adult observation are not documented in the supplied evidence.

Rule

BS2

Evidence considered

• gnomAD v4.1 reports 42/1,583,830 alleles and zero homozygotes; these are aggregate population counts without individual phenotype or age data.
• gnomAD v2.1 reports 7/228,766 alleles and zero homozygotes; these are aggregate population counts without individual phenotype or age data.

Still missing

• No individual-level observation of this variant in a healthy adult was identified.
• Inheritance model, expected age of onset, and full-penetrance status for the relevant CALR disorder are not established in the supplied evidence.

Sources

Reasoning

The exact-variant study did not demonstrate a damaging effect in its reported localization or cell-surface expression assays, but those negative findings do not establish that the protein is functionally normal or that splicing is unaffected. The study also noted that partial loss of mutant CALR from the endoplasmic reticulum was not excluded. Therefore, the available evidence is insufficient to apply BS3.

Rule

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

Evidence considered

• Nangalia et al. 2013, PMID:24325359, Results, 'Mutant Protein with a Novel C-Terminal,' paragraphs 2-3: K385fs*47 showed no difference from nonmutant CALR in the reported Golgi/endogenous localization and cell-surface assays, but the authors explicitly stated that partial loss from the endoplasmic reticulum was not excluded.
• The study did not provide a validated assay establishing normal CALR protein function or normal splicing for the exact variant.

Still missing

• A well-established assay demonstrating no damaging effect on CALR protein function or splicing for p.(K385Nfs*47) is unavailable.
• The available negative results are limited to selected localization and cell-surface measurements and do not exclude other functional defects.

Sources

Reasoning

BS4 cannot be assessed because no affected family members or family-level variant and phenotype data are documented. There is therefore no demonstrated lack of segregation, and the available somatic cohort literature cannot establish BS4.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No family-level non-segregation observation is present in the available case evidence.
• The reviewed literature concerns somatic CALR mutations in myeloproliferative neoplasms and does not provide affected-family non-segregation data.

Still missing

• Provide a family pedigree identifying affected members.
• Provide variant-testing results and phenotype assessments for affected relatives.
• Consider phenocopies and other pathogenic variants before interpreting any non-segregation.

Sources

Reasoning

BP2 cannot be assessed because the available evidence provides no phase-resolved observation showing this variant in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant in any inheritance pattern.

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

• The case evidence contains no parental, offspring, tumor-normal, family, or other phase-resolved observation placing this variant in cis or trans with a pathogenic variant.
• No fully penetrant dominant CALR disorder is established in the available case evidence for applying the trans pathway.

Still missing

• Obtain affected-proband or family-level genotype data identifying a pathogenic allelic partner.
• Obtain parental, offspring, or equivalent phasing data to establish cis or trans configuration.
• For a trans observation, establish that the relevant dominant disorder is fully penetrant.

Reasoning

The governing rule requires the variant to be found in a case with an alternate molecular basis for disease. The reviewed reports describe the exact CALR insertion as a somatic myeloproliferative-neoplasm driver, including cases lacking JAK2 or MPL mutations; absence of those alternative drivers is not evidence that another molecular basis explains the disease. No exact-variant case with an independent alternate molecular diagnosis is documented, so BP5 cannot be assessed from the available evidence.

Rule

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

Evidence considered

• PMID:24325359 reports K385fs*47 in myeloproliferative-neoplasm patients lacking JAK2 or MPL mutations; those findings describe mutual driver status, not an alternate molecular basis causing the disease instead of CALR.
• No source reviewed documents this exact variant in a case where another molecular diagnosis explains the phenotype.

Still missing

• An exact-variant case with a documented independent molecular cause of the disease is required to evaluate BP5.

Sources