CALR c.1099_1150del, p.Leu367ThrfsTer46

Reasoning

PS4 is met under the ACMG/AMP definition because PMID:24325359 reports the exact CALR L367fs*46 variant, corresponding to this 52-bp exon 9 deletion, in 67 of 148 patients with CALR exon 9 mutations and in none of 550 control samples. This is a case-control comparison even though the paper does not print an odds ratio. Using a 0.5 continuity correction for the zero control cell, the calculated OR is 923.2 with an approximate 95% CI of 56.6-15,059.5; the CI excludes 1. The governing threshold is OR >5.0 with a confidence interval that does not include 1.0: 923.2 > 5.0? yes; 56.6-15,059.5 includes 1.0? no. The paper's broader comparison also found CALR indels in 26/31 affected patients versus 0/120 patients with JAK2 or MPL mutations, but the exact-variant count above is the basis for this criterion. No VCEP-specific PS4 rule applies.

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

• Nangalia et al., PMID:24325359, Results: the exact L367fs*46 variant was one of the two common CALR exon 9 variants and occurred in 67 of 148 patients with CALR exon 9 mutations.
• Nangalia et al., PMID:24325359, Results: CALR exon 9 mutation testing included 52 control samples plus 498 control samples evaluated by exome or genome sequencing, and no CALR mutations were found in control samples; therefore the exact L367fs*46 count was 0/550 controls.
• Computed from the published counts using a 0.5 continuity correction for the zero control cell: OR = (67.5 x 550.5)/(80.5 x 0.5) = 923.2; approximate 95% CI = 56.6-15,059.5.
• Threshold check from the governing rule: OR > 5.0, written as 923.2 > 5.0? yes; the approximate 95% CI 56.6-15,059.5 does not include 1.0.

Sources

Reasoning

PM2 is met at Supporting strength under the supplied ClinGen SVI generic threshold. The variant is observed but extremely rare in population controls: gnomAD v4.1 all-comers AF=2.9149166829984694e-05 (47/1612396 alleles; homozygotes=0), gnomAD v2.1 non-cancer exomes AF=8.604445056316093e-06 (2/232438 alleles; homozygotes=0), and gnomAD v3.1 non-cancer genomes AF=2.028013628251582e-05 (3/147928 alleles; homozygotes=0). The maximum default all-comers frequency is below the supplied PM2 Supporting cutoff of 0.0001; therefore this is not an absent-from-controls claim, but it qualifies as extremely rare PM2 at Supporting strength. The indel calling caveat is noted, with concordant observations across datasets.

Rule

PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v4.1 all-comers: 47/1,612,396 alleles, AF=2.9149166829984694e-05; homozygotes=0.
• gnomAD v2.1 non-cancer exomes: 2/232,438 alleles, AF=8.604445056316093e-06; homozygotes=0.
• gnomAD v3.1 non-cancer genomes: 3/147,928 alleles, AF=2.028013628251582e-05; homozygotes=0.
• Generic ACMG/ClinGen-SVI calibration supplied for this case: PM2 Supporting threshold <=0.0001, attributed in the prompt to the ClinGen SVI 2020 recommendation and PMID:25741868.
• Because the variant is observed at low frequency rather than absent, the conclusion uses the supplied extremely-low-frequency PM2 Supporting threshold rather than the original absent-from-controls wording alone.

Sources

Reasoning

NM_004343.4:c.1099_1150del is a frameshift in exon 9, predicted as NP_004334.1:p.(Leu367ThrfsTer46), and therefore has the structural class of a null variant. However, PVS1 additionally requires that loss of function be a known disease mechanism. The variant-specific CALR literature identifies the exon 9 frameshift L367fs*46 as a recurrent somatic myeloproliferative-neoplasm driver that creates a novel C-terminal sequence and removes the normal KDEL retention signal; related exon 9 CALR indels were shown to produce altered localization and cytokine-independent cellular growth. These findings support an oncogenic altered-function mechanism for this CALR exon 9 frameshift, not disease caused by loss of normal CALR function. Accordingly, the required LOF disease-mechanism condition for PVS1 is not met, so no PVS1 strength is assigned. NMD escape, exon criticality, transcript relevance, repeat-region context, and protein truncation do not override the failed gene-mechanism requirement.

Rule

PVS1: Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.

Evidence considered

• NM_004343.4:c.1099_1150del is a 52-bp exon 9 frameshift, predicted as NP_004334.1:p.(Leu367ThrfsTer46).
• Nangalia et al. identify L367fs*46 as a recurrent somatic CALR exon 9 deletion and describe a novel C-terminal sequence with loss of the normal KDEL retrieval signal; functional studies evaluated the mutant protein in cell systems.
• Klampfl et al. report that CALR exon 9 insertions/deletions create an alternative reading frame and describe cytokine-independent growth and altered localization for the recurrent type 1 CALR deletion.
• The ACMG/AMP PVS1 definition requires both a qualifying null-variant class and a gene in which loss of function is a known disease mechanism; the second condition is not supported for this CALR exon 9 oncogenic frameshift context.

Sources

Reasoning

PP5 is not met. The exact ClinVar record is classified Pathogenic/Likely pathogenic, but the record has no expert-panel submissions; it contains ordinary laboratory and literature-only submissions, including OMIM submissions without assertion criteria. Generic PP5 requires a reputable pathogenic report when the evidence is not available for independent evaluation, and the specified rule for this interpretation restricts PP5 to an exact-variant ClinVar expert-panel classification. No qualifying expert-panel classification is present.

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 97006: exact variant record is Pathogenic/Likely pathogenic, but expert_panel_submissions = 0 and the submissions are clinical-laboratory or literature-only assertions rather than an expert-panel classification.
• The exact CALR L367fs*46 variant is independently evaluable from PMID:24325359, so the PP5 premise that evidence is unavailable for independent evaluation is not satisfied.

Sources

Reasoning

BA1 is not met. The highest default population frequency is gnomAD v4.1 AF 2.9149166829984694e-05 (47/1612396 alleles; 0.00291%), with the highest reported v4.1 ancestry frequency 3.3824922202678934e-05 (Ashkenazi Jewish), far below the generic stand-alone threshold of 0.05. The governing generic rule uses all-comers gnomAD because no VCEP specifies a restricted source for this gene; the gnomAD v2.1 non-cancer exome frequency was also only 8.604445056316093e-06. The variant is an indel, so the ACMG/AMP indel calling caveat applies, but the available data do not approach BA1.

Rule

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

Evidence considered

• gnomAD v4.1 all-comers: 47/1,612,396 alleles, AF=2.9149166829984694e-05 (0.00291%); highest listed ancestry AF=3.3824922202678934e-05 in Ashkenazi Jewish individuals; homozygotes=0.
• gnomAD v2.1 non-cancer exomes: 2/232,438 alleles, AF=8.604445056316093e-06 (0.00086%); homozygotes=0.
• Generic ACMG/AMP calibration supplied for this case: BA1 stand-alone threshold >=0.05, attributed to ACMG/AMP 2015 (PMID:25741868).
• The variant is a 52-bp deletion/indel; the governing ACMG/AMP text cautions that population data for indels may be poorly called by next-generation sequencing.

Sources

Reasoning

BS1 is not met under the generic default. The gnomAD v4.1 all-comers AF is 2.9149166829984694e-05, and the highest listed v4.1 ancestry AF is 3.3824922202678934e-05; gnomAD v3.1 non-cancer genomes reached 6.63129973474801e-05 in Admixed American individuals. These are all below the supplied generic BS1 threshold of 0.01, so the allele is not demonstrably more frequent than expected on the available population data. The small gnomAD-Canada Ashkenazi Jewish subgroup singleton (1/832; AF=0.001201923076923077) also remains below 0.01 and does not establish a disease-specific maximum-credible-frequency exceedance. No reliable CALR germline disease prevalence, penetrance, or allelic-heterogeneity inputs were supplied for a Whiffin-based per-gene calculation, but that missing calculation does not change the conclusion because every observed frequency is below the generic default.

Rule

Allele frequency is greater than expected for disorder.

Evidence considered

• gnomAD v4.1 all-comers: AF=2.9149166829984694e-05 overall and maximum listed ancestry AF=3.3824922202678934e-05; 47/1,612,396 alleles; homozygotes=0.
• gnomAD v3.1 non-cancer genomes: AF=2.028013628251582e-05 overall and maximum listed ancestry AF=6.63129973474801e-05 in Admixed American individuals; 3/147,928 alleles; homozygotes=0.
• gnomAD-Canada v1.0: one Ashkenazi Jewish allele among 832 alleles (AF=0.001201923076923077), a small-subgroup singleton still below 0.01.
• Generic ACMG/ClinGen-SVI calibration supplied for this case: BS1 default threshold >=0.01; per-gene derivation is preferred under Whiffin et al. 2017 (PMID:28518168).
• No disease-specific prevalence, penetrance, or allelic-heterogeneity inputs were available to calculate a CALR-specific maximum credible allele frequency under Whiffin et al. 2017 (PMID:28518168).

Still missing

• A CALR-specific maximum-credible-frequency calculation with disease prevalence, penetrance, and allelic heterogeneity is unavailable.

Sources

Reasoning

BP5 is not met. The available paper reports CALR L367fs*46 as a somatic driver in myeloproliferative neoplasms and describes CALR, JAK2, and MPL mutations as mutually exclusive in the relevant cohort, but it does not document this patient's case with an alternate molecular basis for disease. No case-specific alternate molecular diagnosis or molecular cause is available for applying BP5.

Rule

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

Evidence considered

• PMID:24325359 reports cohort-level CALR, JAK2, and MPL mutation patterns but does not describe this patient's clinical case or an alternate molecular basis for this patient's disease.

Sources

Reasoning

BP6 is not met. The exact ClinVar record has no expert-panel submission and is not classified benign or likely benign; its reported classifications are Pathogenic/Likely pathogenic from non-expert submissions. Therefore the required exact-variant ClinVar expert-panel benign or likely benign classification is absent.

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 97006: expert_panel_submissions = 0; the record's classifications are Pathogenic and Likely pathogenic, with no benign or likely benign expert-panel assertion.

Sources

Reasoning

No germline proband-level case data document that NM_004343.4:c.1099_1150del occurred de novo with both maternity and paternity confirmed, disease in the proband, and no family history. PMID:24325359 reports the exact CALR L367fs*46 variant as a recurrent somatic mutation in myeloproliferative neoplasms, but provides no germline parental testing or family-history evidence. Therefore PS2 cannot be assessed from the available evidence.

Rule

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

Evidence considered

• PMID:24325359 explicitly reports CALR L367fs*46 as a recurrent somatic mutation in myeloproliferative neoplasms and does not provide germline parental testing, confirmed maternity and paternity, or family-history information.
• No case evidence in the reviewed materials documents a qualifying germline de novo observation.

Still missing

• Germline proband clinical phenotype and diagnosis are not documented.
• Parental testing confirming both maternity and paternity is not documented.
• Family history is not documented.

Sources

Reasoning

The exact CALR L367fs*46 variant was examined in transient-expression and cell-surface studies, but the available evidence does not establish a validated, reproducible assay demonstrating a damaging effect on CALR function. The reported observations—production of the mutant protein, lack of Golgi accumulation, colocalization with nonmutant CALR, and no increase in cell-surface CALR—show altered cellular behavior but do not by themselves define loss or gain of function in a clinically relevant assay with sufficient controls for PS3. No validated diagnostic-laboratory assay, quantitative functional threshold, or reproducibility evidence is supplied. Therefore PS3 is not assessed rather than applied at any strength.

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, exact CALR L367fs*46 functional experiments: transient expression in HEK293T and COS-7 cells, cell-surface expression studies in 32D cells and peripheral-blood leukocytes; the mutant produced a novel C-terminal sequence, did not accumulate in the Golgi, colocalized with nonmutant CALR, and did not increase cell-surface CALR expression.
• These experiments demonstrate cellular differences for the exact variant but do not provide a validated diagnostic-laboratory assay, reproducibility data, or a quantitative disease-relevant functional threshold sufficient to apply PS3.

Still missing

• A validated, reproducible functional assay with appropriate wild-type and disease-relevant controls demonstrating the effect of this exact variant is not available.
• The available cellular observations lack a predefined quantitative threshold linking the result to a damaging CALR functional effect.

Sources

Reasoning

No germline proband-level case data document an assumed de novo occurrence of NM_004343.4:c.1099_1150del without confirmed maternity and paternity. PMID:24325359 reports the exact CALR L367fs*46 variant in a somatic myeloproliferative-neoplasm context and does not provide a germline trio, parental testing, or a qualifying de novo clinical observation. Therefore PM6 cannot be assessed from the available evidence.

Rule

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

Evidence considered

• PMID:24325359 explicitly describes the exact CALR L367fs*46 variant as somatic and supplies no germline de novo or parental-testing evidence.
• No qualifying assumed-de-novo germline observation is documented in the reviewed materials.

Still missing

• A germline proband with a documented disease phenotype is not identified.
• Evidence that the variant was absent from tested parents or otherwise assumed de novo is not documented.
• Parental testing details and family history are not documented.

Sources

Reasoning

The available evidence does not identify multiple affected family members carrying NM_004343.4:c.1099_1150del, does not provide segregation results, and does not report informative meioses. The relevant literature describes this exact CALR variant in somatic myeloproliferative-neoplasm cohorts rather than a familial germline series. 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

• No family pedigree, affected-relative testing, or segregation dataset is documented in the reviewed materials.
• The reviewed exact-variant publication concerns somatic CALR mutations and does not provide familial germline segregation evidence.

Still missing

• No familial germline cases with multiple affected relatives are identified.
• No affected-relative genotypes or segregation results are documented.
• No informative meiosis count is available.

Reasoning

The governing PP4 rule requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The available evidence identifies CALR L367fs*46 in somatic myeloproliferative neoplasms, but it does not provide this patient's phenotype or family history and does not establish a single genetic etiology for the patient's presentation. The required patient-specific phenotype information is therefore unavailable.

Rule

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

Still missing

• Patient-specific phenotype, diagnostic findings, and family history are needed to assess whether the presentation is highly specific for a disease with a single genetic etiology.

Sources

Reasoning

BS2 cannot be assessed from the available evidence. Population databases show no homozygotes for the deletion, but the observed heterozygous alleles are aggregate database observations rather than documented healthy adult individuals with known age, phenotype, and penetrance status. The case evidence does not establish whether the relevant CALR disorder is a fully penetrant early-onset recessive, dominant, or X-linked disorder, so neither the required healthy-individual observation nor the applicability of the inheritance-specific BS2 path can be confirmed.

Rule

BS2

Evidence considered

• gnomAD v2.1: 2/246,350 total alleles and 0 homozygotes; non-cancer exomes 2/232,438 alleles and 0 homozygotes.
• gnomAD v4.1: 47/1,612,396 total alleles and 0 homozygotes; no individual-level age, health, or phenotype documentation is provided for applying BS2.
• The available case evidence does not specify a fully penetrant early-onset CALR germline disorder or a confirmed dominant, recessive, or X-linked inheritance path.

Still missing

• Documented healthy-adult observations with age and phenotype are unavailable.
• The applicable CALR germline disorder inheritance model and expectation of full early-age penetrance are unavailable.

Sources

Reasoning

The exact CALR L367fs*46 variant was tested in cellular expression and localization studies, but those studies did not establish absence of a damaging effect on protein function or splicing. The mutant's altered C-terminal sequence and cellular localization were observed, while no validated normal-function assay, appropriate benign control framework, or reproducible quantitative result demonstrates preserved CALR function. Therefore BS3 is not assessed rather than applied at any strength.

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, exact CALR L367fs*46 functional experiments: the mutant protein did not accumulate in the Golgi, colocalized with nonmutant CALR, and did not increase cell-surface CALR expression.
• Because the study reports altered cellular behavior and does not demonstrate preserved protein function or normal splicing in a validated, reproducible assay, it cannot support BS3.

Still missing

• A validated, reproducible assay showing no damaging effect on protein function or splicing is not available.
• The available study does not provide a quantitative normal-function threshold or sufficient benign controls for BS3.

Sources

Reasoning

The available evidence does not identify an affected family, tested affected relatives, or any affected individual who lacks NM_004343.4:c.1099_1150del. Consequently, there is no documented non-segregation result to evaluate, and BS4 cannot be assessed. The available exact-variant literature is somatic and does not establish a familial segregation analysis.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected-family-member testing or family segregation analysis is documented in the reviewed materials.
• The available exact-variant literature describes somatic disease cohorts and does not report familial non-segregation.

Still missing

• No affected family members with variant testing are documented.
• No family-level genotype-phenotype comparison is available.
• No non-segregation observation can be evaluated.

Reasoning

BP2 requires an observation in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or in cis with a pathogenic variant in any inheritance pattern. The available evidence describes CALR p.(L367Tfs*46) as a somatic MPN mutation and provides no paired pathogenic variant, cis/trans relationship, parental testing, offspring testing, or other phase-resolving observation. The criterion therefore cannot be assessed from the available allelic evidence.

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

• Nangalia et al. (PMID:24325359) reports CALR L367fs*46 as a somatic mutation and describes functional and hematopoietic-compartment studies, but does not report a cis/trans pathogenic partner or phase analysis for this variant.
• ClinVar variation 97006 contains variant submissions and somatic/germline origin annotations but no documented paired-variant phase observation applicable to BP2.

Still missing

• A documented pathogenic partner variant and phase-resolving evidence, such as parental or offspring testing, are needed to assess BP2.

Sources