MPL c.1544G>T, p.(Trp515Leu)

Reasoning

No MPL-specific ClinGen CSPEC/VCEP framework exists (vcep_materials reports 'no cspec url available'; final framework = generic_acmg_fallback), so generic ACMG/AMP 2015 PS3 was applied. PS3 requires well-established functional studies demonstrating a deleterious effect on the gene or gene product. The exact variant NM_005373.2:c.1544G>T (NP_005364.1:p.(Trp515Leu), MPL W515L) is the best-characterised activating MPL hotspot: expression of the mutant receptor, but not wild-type MPL, conferred cytokine-independent growth to 32D, UT7 and Ba/F3 haematopoietic cells, constitutive phosphorylation of JAK2, STAT3, STAT5, ERK and AKT, and thrombopoietin hypersensitivity, with triplicate cultures, total-protein loading controls and JAK-inhibitor dose-response dependency (PMID:16834459). The same exact-variant study showed the mutant, but not wild-type, receptor produced a fully penetrant lethal myeloproliferative disease in a murine bone-marrow transplant model with thrombocytosis, leukocytosis, splenomegaly, hepatomegaly and myelofibrosis (n=9 mutant vs n=6 wild-type). The activating mechanism (disruption of the amphipathic juxtamembrane motif required to keep the unliganded receptor inactive) and the in vivo phenotype are independently described in the review literature (PMID:18769448), and an independent group reports constitutive pathway activation in a retrovirally expressed MPL W515L MPN cell model (PMID:28823277, abstract level). Gain of function is the established disease mechanism for MPL activation in MPL-associated myeloproliferative disease/thrombocythaemia. Because the assays are variant-specific, include wild-type/normal controls, are reproducible, and use biologically relevant signalling and haematopoietic readouts, PS3 is met at strong strength per the ClinGen SVI PS3/BS3 recommendations (Brnich et al., Genome Med 2020;12(1):3, PMID 31892348); no MPL-specific strength calibration exists.

Rule

generic_acmg_amp_2015_PS3 (no MPL-specific VCEP/CSPEC; strength calibrated per ClinGen SVI PS3/BS3 recommendations, PMID 31892348): well-established functional studies demonstrate a deleterious (gain-of-function) effect of the exact variant on the MPL gene product.

Evidence considered

• PMID:16834459 directly reports and functionally characterises the exact variant (MPL nucleotide 1544 G>T, tryptophan-to-leucine at codon 515, MPLW515L): cytokine-independent growth in 32D, UT7 and Ba/F3 cells expressing MPLW515L but not MPLWT, constitutive phosphorylation of JAK2, STAT3, STAT5, ERK and AKT, thrombopoietin hypersensitivity by growth assay and Western blot, and a fully penetrant lethal myeloproliferative disease in a murine bone-marrow transplant model (n=9 MPLW515L vs n=6 MPLWT) with marked thrombocytosis, leukocytosis, splenomegaly, hepatomegaly and bone-marrow fibrosis.
• Assay quality in PMID:16834459 includes wild-type receptor controls in every system, triplicate cultures with standard-deviation error bars, total-protein loading controls for each phospho-antibody, dose-response thrombopoietin stimulation, and a JAK Inhibitor I dose-response showing the proliferative/signalling phenotype is pathway-dependent; the mutant and wild-type retroviral supernatants infected Ba/F3 and 32D cells at comparable efficiency (40%-60%).
• PMID:18769448 independently describes the mechanism and in vivo phenotype of the same substitution: W515L activates constitutive signalling by disrupting the amphipathic juxtamembrane motif that keeps the unliganded receptor inactive, and TpoR W515L reconstitution produces severe myeloproliferation with marked thrombocytosis, splenomegaly and myelofibrosis within 20-30 days; this is review-level corroboration rather than new primary assay data.
• PMID:28823277 is an independent group's report that retroviral expression of MPL W515L was used to construct an MPN cell model in which constitutive activation of the signalling pathway was detected by Western blot (abstract-level evidence; no full text available in this case).
• PMID:16868251 (17 of 1182 patients carrying MPLW515L) describes the allele as an acquired gain-of-function mutation that induces constitutive, cytokine-independent JAK-STAT activation and identifies codon 515 as a mutation hotspot; its functional statements are background/secondary, with the primary assays in PMID:16834459.
• Strength was calibrated with the ClinGen SVI recommendations for application of the functional evidence PS3/BS3 criterion (Brnich SE et al., Genome Med 2020;12(1):3, PMID 31892348), which allow strong PS3 for variant-specific, reproducible assays with appropriate controls and biologically relevant readouts; no MPL-specific calibration publication was identified.
• PMID:25741868 was used only for the generic ACMG/AMP PS3 definition because no applicable MPL-specific specification was present; it provides no variant-specific functional data.
• Source 'oncokb' curates the exact variant as gain-of-function/oncogenic (OncoKB citations point to PMID:16834459); this was used only as corroboration that the functional direction is gain of function, not as a substitute for the assay data.

Still missing

• No MPL-specific ClinGen VCEP/CSPEC assay calibration or gene-specific strength specification exists; the strong assignment follows the generic ClinGen SVI PS3/BS3 recommendation framework (PMID 31892348) rather than an MPL-validated, clinically calibrated assay with benign-variant controls.
• The exact-variant functional studies model somatic myeloproliferative-neoplasm biology (activating mutation in haematopoietic cells); the bundle contains no proband phenotype, so the gain-of-function direction could not be confirmed as the mechanism relevant to this individual's indication.
• Assays were performed in immortalised haematopoietic cell lines and murine transplant models rather than patient-derived primary material.

Sources

Reasoning

Generic ACMG/AMP PS4 is met for the myeloproliferative-neoplasm phenotype: the exact c.1544G>T change (p.Trp515Leu) is recurrently detected in affected individuals in independent cohorts and is essentially absent from controls. PMID:16834459 detected MPLW515L in 4/45 JAK2V617F-negative myelofibrosis patients (9%) and in 0/50 JAK2V617F-negative essential thrombocythemia patients, 0/10 polycythemia vera patients and 0/270 HapMap samples (one-sided Fisher exact test on the reported counts, 4/45 vs 0/270, P = 3.70e-4). PMID:16868251 found MPL515 mutations in 20/1182 patients with myeloid disorders (MPLW515L n = 17; diagnoses de novo MMM 12, ET 4, post-ET MMM 1, MMM blast crisis 3) with MPL515 positivity confined to MMM (13/290) and ET (4/318) and absent from PV (0/242), MDS (0/88) and CMML (0/118); no carrier was reported among 64 healthy controls, although that comparison alone is not statistically significant given the zero cell and small control group. PMID:21326037 identified W515L in 46/2242 JAK2-mutation-negative patients with suspected myeloproliferative disorders, where it accounted for 67.7% of all MPL mutations. Population data are concordant with near-absence in controls (gnomAD v2.1 2/250,508 alleles, AF 7.98e-6; gnomAD v4.1 20/1,613,182 alleles, AF 1.24e-5, no homozygotes). The criterion is reported at strong rather than the default supporting level because several independent, concordant exact-variant observations in affected individuals are available against negligible control frequencies; no published effect-size calibration for PS4 up-weighting was consulted, so the up-weight rests on the number and consistency of the independent cohorts. The variant is explicitly somatic in the primary study (matched buccal/normal DNA negative), so transfer of this enrichment to a germline classification requires human review.

Rule

Generic ACMG/AMP 2015 PS4: the prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.

Evidence considered

• PMID:16834459 reports MPL c.1544G>T (MPLW515L, Trp515Leu) in 4/45 JAK2V617F-negative myelofibrosis patients, absent from 50 JAK2V617F-negative essential thrombocythemia patients, 10 polycythemia vera patients and all 270 HapMap samples tested.
• One-sided Fisher exact test computed from the counts reported in PMID:16834459 (4/45 affected vs 0/270 controls) gives P = 3.70e-4; the HapMap samples are population controls rather than disease-matched controls.
• PMID:16868251 screened 1182 patients with myeloproliferative and other myeloid disorders plus 64 healthy controls and reported MPL515 mutations (MPLW515L n = 17; MPLW515K n = 5; 20 patients total) restricted to myelofibrosis with myeloid metaplasia (13/290) and essential thrombocythemia (4/318), with no carriers among polycythemia vera (0/242), myelodysplastic syndrome (0/88) or chronic myelomonocytic leukemia (0/118) patients, and no carrier reported among the 64 healthy controls.
• Because PMID:16868251 reported no positive control, the control comparison cannot reach statistical significance on its own; the quantitative support for PS4 rests on the PMID:16834459 comparison and on the population-database frequencies.
• PMID:21326037 identified W515L (TGG>TTG) in 46 of 2242 patients with suspected JAK2-mutation-negative myeloproliferative disorders (43 heterozygous, 3 homozygous/hemizygous), representing 67.7% of all MPL mutations detected in that screening cohort.
• PMID:20113333 detected MPLW515L in 1 of 100 patients with essential thrombocythemia and 1 of 11 patients with primary myelofibrosis, an additional independent affected-individual observation of the exact variant at low frequency.
• Control frequency data used for comparison: gnomAD v2.1 total AF 7.98378e-06 (2/250,508 alleles, 0 homozygotes) and gnomAD v4.1 total AF 1.23979e-05 (20/1,613,182 alleles, 0 homozygotes; grpmax FAF 1.772e-05).
• PMID:16834459 states that matched buccal (normal) DNA was negative for MPLW515L and that the mutation was absent from 270 HapMap samples, establishing the somatic hematopoietic origin of the enrichment signal; PS4 applicability to a germline classification therefore remains a human-review decision.
• Governing rules: no ClinGen MPL CSPEC/VCEP specification and no local gene-specific framework were present in the bundle, so the generic ACMG/AMP 2015 criteria (PMID:25741868) were applied.

Still missing

• No germline case-control study of this variant is available: the enrichment derives from somatic mutation screening in myeloid neoplasms (blood/bone-marrow DNA), not from germline genotypes in affected versus unaffected individuals.
• No case-level specimen type, tumour/clone context or asserted disease for the individual being interpreted is provided, so it is not established that the affected-individual cohort matches the phenotype being evaluated.
• No published, citable calibration (effect size / odds-ratio thresholds) for weighting PS4 was available, so the strong weighting is based on cohort count and consistency rather than a named calibration publication.

Sources

Reasoning

vcep_materials.json contains no CSPEC/VCEP for MPL and its domain_tables key is empty (no entry for this gene), so PM1 was assessed from the primary literature rather than from a governing VCEP domain table. Residue 515 lies in the juxtamembrane/transmembrane-cytoplasmic hinge region of the thrombopoietin receptor, within the amphipathic KWQFP motif whose deletion constitutively activates downstream signaling in a ligand-independent fashion (PMID:16868251; PMID:18769448). MPL codon 515 is explicitly described in the literature as a hotspot for activating mutations in acquired myeloproliferative disease, with multiple distinct activating missense alleles observed at this residue (W515L in 17 patients and W515K in 5 patients in a 1182-patient screen; PMID:16868251), and W515L is curated by OncoKB as an oncogenic, gain-of-function MPL change. The variant lies in a well-established mutational hotspot/critical functional domain without benign variation, so PM1 is met at its default moderate strength. Of note, the CancerHotspots statistical hotspot lookup performed for this case returned no row for MPL W515, so the hotspot call rests on the primary literature and the residue-level variant data, not on that database.

Rule

PM1 (moderate): located in a mutational hotspot and/or critical and well-established functional domain without benign variation

Evidence considered

• PMID:16868251 (Pardanani et al., Blood 2006; 1182 patients) states that the same-codon MPLW515K mutation 'points to the MPL codon 515 as a hot spot for activating mutations in acquired MPD' and describes the amphipathic KWQFP motif in the transmembrane-cytoplasmic hinge region (residue corresponding to W515) whose deletion constitutively activates downstream signaling without ligand.
• PMID:16834459 identifies c.1544G>T (MPLW515L) in 4/45 JAK2V617F-negative myelofibrosis patients with cytokine-independent growth and constitutive JAK2/STAT3/STAT5/ERK/AKT phosphorylation, placing the variant in a functionally critical region.
• PMID:18769448 states that W515L activates constitutive signaling by disrupting the amphipathic juxtamembrane motif required to keep the unliganded thrombopoietin receptor inactive, and that W515 mutations occur in myelofibrosis and essential thrombocythemia.
• PMID:16868251 additionally cites a highly penetrant gain-of-function MPL variant in the transmembrane domain (MPLS505N) in familial essential thrombocythemia and the murine MPLW508S change (corresponding to human W515S), showing that this region is a recurrent target of activating missense change.
• OncoKB curation for MPL W515L lists the variant as Oncogenic with a Gain-of-function biological effect (variant-specific record; PMID 16834459 cited by the record).
• vcep_materials.json was checked directly: no CSPEC url, no summary files, and domain_tables is an empty object, so no MPL-specific approved domain list was available to test the variant against.

Still missing

• No governing VCEP/CSPEC domain table exists for MPL in vcep_materials.json (domain_tables empty), so the domain boundary was taken from primary literature rather than a curated gene-specific domain table.
• CancerHotspots lookup for MPL W515 returned no result, so the statistical hotspot evidence is derived from published residue-level variant data rather than from that resource.
• No constraint metric (gnomAD missense Z / observed-expected ratio) was retrieved to formally document absence of benign variation at codon 515.

Sources

Reasoning

PM2 (supporting) applies when the variant is absent from, or at extremely low frequency in, population databases (Richards et al. 2015, PMID:25741868); the non-VCEP operating threshold applied by this framework is AF/grpmax FAF < 0.1%. gnomAD v4.1 reports total AF 1.23979e-05 (0.00124%, 20/1,613,182 alleles), joint grpmax FAF 1.772e-05 (0.0018%), exome-only grpmax FAF 2.003e-05, highest subpopulation East Asian 6.683e-05 (0.0067%, 3/44,890), and 0 homozygotes. gnomAD v2.1 reports total AF 7.98378e-06 (0.00080%, 2/250,508 alleles), 0 homozygotes, highest subpopulation African/African American 6.15612e-05 (1/16,244). All metrics are well below 0.1%, and the variant is absent from the control cohorts examined (270 HapMap samples, PMID:16834459; 64 healthy controls, PMID:16868251). PM2 is met at supporting strength. Two caveats are flagged for human review: the variant is not strictly 'absent' from controls (20 alleles in gnomAD v4.1), and MPL W515L is a recurrent somatically acquired clonal driver, so some gnomAD alleles may represent clonal haematopoiesis in blood-derived DNA rather than germline carriers; that would, if anything, make the true germline frequency even lower, but it means the population count is not a clean germline measurement.

Rule

Generic ACMG/AMP PM2 (Supporting): absent from controls or at extremely low frequency in population databases (Richards et al. 2015, PMID:25741868); non-VCEP operating threshold applied by this framework: AF/grpmax FAF < 0.1%.

Evidence considered

• gnomAD v4.1 (source_registry key gnomad_v4): total AF 1.23979e-05 (0.00124%, 20/1,613,182 alleles), joint grpmax FAF 1.772e-05 (0.0018%), exome grpmax FAF 2.003e-05, 0 homozygotes; ancestry breakdown NFE 1.1017e-05 (13/1,179,942), AFR 1.3347e-05 (1/74,922), EAS 6.683e-05 (3/44,890), FIN 1.58e-05 (1/63,282), ASJ XY 6.623e-05 (1/15,098), AMR 0/60,004.
• gnomAD v2.1 (source_registry key gnomad_v2): total AF 7.98378e-06 (0.00080%, 2/250,508 alleles), 0 homozygotes; highest subpopulation African/African American 6.15612e-05 (1/16,244).
• Absence from screened control cohorts: no MPL515 mutation among 64 healthy controls (PMID:16868251); W515L absent from all 270 HapMap samples and from matched buccal DNA (PMID:16834459).
• Rarity in disease cohorts corroborates a low-frequency allele: W515L in 46 of 2,242 JAK2-negative suspected MPN patients (PMID:21326037, 43 heterozygous and 3 homozygous/hemizygous) and in 1/100 essential thrombocythemia plus 1/11 primary myelofibrosis patients (PMID:20113333).
• Somatic-driver artifact (source_registry key oncokb): OncoKB curates MPL W515L as Oncogenic with a gain-of-function biological effect, i.e. an activating change selected for in myeloid neoplasms; clonal haematopoiesis in blood-derived gnomAD samples is therefore a plausible contributor to the observed allele counts.
• PM2 threshold named to a publication: absent from controls or at extremely low frequency per Richards et al., ACMG/AMP 2015 (PMID:25741868); the configured non-VCEP operating threshold applied here is AF < 0.1%, which the variant clears by roughly 80-fold on total AF and ~56-fold on grpmax FAF.
• gnomAD-Canada v1.0 returned no record (API urlopen timeout) so it neither supports nor contradicts the rarity assessment.

Still missing

• Confirm whether the 20 gnomAD v4.1 (and 2 gnomAD v2.1) W515L alleles are germline or clonal-haematopoiesis-derived: the variant is an established somatic activating driver of myeloid neoplasms (PMID:16834459; OncoKB Oncogenic / gain-of-function), and gnomAD genotypes derive from blood-derived DNA, so population counts may overstate true germline carrier frequency.
• PM2 is met on the framework's numeric rarity threshold rather than on strict absence from controls; the literal generic wording ('absent from controls') is not satisfied because the variant is present in gnomAD.
• gnomAD-Canada v1.0 (HostSeq) lookup failed with a network timeout, so no Canadian ancestry-specific frequency data are available for this variant.

Sources

Reasoning

PM5 requires a missense change at an amino acid residue where a different missense change has previously been determined to be pathogenic. A different missense change at the same residue, MPLW515K (TGG>AAG), was identified in 5 of 1182 screened patients with myeloproliferative/myeloid disorders and was characterized as a likely gain-of-function activating mutation at the MPL codon 515 hotspot (PMID:16868251), with W515K and another activating MPL allele (S505N) specifically screened in subsequent MPN cohorts (PMID:20113333). The reference residue is therefore a residue at which a different pathogenic missense change has been observed, so PM5 is met at its default moderate strength. The automated same-residue comparator harvest for this case (pm5_candidates) did not run (found=false, not_applicable recommendation), so the comparator evidence here comes from the primary literature and not from that artifact.

Rule

PM5 (moderate): missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before

Evidence considered

• PMID:16868251 identified MPLW515K in 5 patients in the same screen that detected MPLW515L in 17 patients and argued it is a likely gain-of-function mutation at the same codon 515, i.e. a different amino acid change at the residue under review.
• PMID:20113333 performed allele-specific PCR and sequencing for MPL W515L, W515K and S505N in essential thrombocythemia and primary myelofibrosis, confirming that alternative pathogenic missense alleles at codon 515 are recurrently interrogated in disease cohorts.
• PMID:21326037 detected MPL W515L in 46 JAK2-negative patients (67.7% of all MPL mutations found, including 3 homozygous/hemizygous cases), documenting that residue 515 is a recurrently mutated disease-relevant residue.
• pm5_candidates.json was reviewed: eligibility for classic same-residue PM5 search was not established by the automated step ('Unable to confirm classic same-residue PM5 semantics safely; skipping automatic candidate harvesting'), with candidate_summary total_candidates_seen=0; its own instruction is to review comparators independently rather than apply PM5 from the artifact — that independent review is what is recorded here.

Still missing

• The automated pm5_candidates artifact skipped classic same-residue comparator harvesting (found=false), so the same-residue comparator (W515K) was established from primary literature rather than from an automated ClinVar candidate collection.
• W515K is reported as a previously undescribed, likely gain-of-function activating allele in a cohort study (PMID:16868251) rather than with a formal ACMG classification, so the strength of the comparator is inferred from its recurrent gain-of-function behaviour in MPN.

Sources

Reasoning

PS1 requires a previously established pathogenic variant that yields the SAME amino acid change through a DIFFERENT nucleotide substitution. The amino acid change under review is NP_005364.1:p.(Trp515Leu), encoded here by NM_005373.2:c.1544G>T (TGG>TTG). c.1544G>T is itself the variant that established MPLW515L as a somatic activating mutation in myelofibrosis (PMID:16834459; PMID:21326037), so the only available same-amino-acid comparator is this variant, which would make the criterion circular. Structurally, TGG is the only codon for Trp at residue 515 and TTG (leucine) is the only single-nucleotide derivative of TGG that encodes Leu, so no alternative nucleotide change can produce p.Trp515Leu. There is therefore no independent 'previously established pathogenic variant with the same amino acid change' to anchor PS1, and the criterion is not met.

Evidence considered

• Variant under review is NM_005373.2:c.1544G>T, NP_005364.1:p.(Trp515Leu) (MPLW515L), a somatic/acquired activating MPL variant (case_summary normalization; ClinVar variation 14164).
• PMID:16868251 reports the MPLW515L substitution as the 'TGG to TTG conversion' and the same-codon MPLW515K as the 'TGG to AAG conversion', documenting that p.Trp515Leu in this dataset always derives from the c.1544G>T nucleotide change.
• Codon-level check: TGG is the sole Trp codon; the only single-base change from TGG that encodes leucine is TTG. Thus no alternate nucleotide substitution at c.1544/c.1545/c.1546 can generate p.Trp515Leu, and PS1 (same amino acid change via a different nucleotide change) has no possible comparator.
• No same-amino-acid-change comparator variant distinct from c.1544G>T was found in the case evidence bundle; the automated PM5 comparator search (pm5_candidates) also returned no harvested same-residue comparator collection (found=false).

Sources

Reasoning

PS2 requires a confirmed de novo occurrence (both maternity and paternity confirmed) in a patient whose phenotype is consistent with the gene-disease relationship. No proband-level clinical record, no parental testing result, and no parental-relationship confirmation exist anywhere in this case bundle for NM_005373.2:c.1544G>T (p.Trp515Leu, MPL W515L). Beyond the absence of trio data, the available origin evidence contradicts the premise of a de novo constitutional allele: PMID:16834459 states that matched buccal (normal) DNA from MPLW515L-positive myelofibrosis patients was negative for the substitution, explicitly demonstrating acquired somatic mutation in haematopoietic cells, and the ClinVar record for this variant (variation 14164) carries two OMIM submissions labelled origin=somatic for MYELOFIBROSIS WITH MYELOID METAPLASIA, SOMATIC and THROMBOCYTHEMIA 2, SOMATIC. The variant is therefore better explained as a post-zygotic clonal haematopoietic event than as a de novo germline allele, so PS2 is not met. A conflicting germline clinical-lab submission (SCV000595823, Pathogenic, Thrombocytopenia congenital amegakaryocytic) is the reason for the human-review flag.

Rule

ACMG/AMP 2015 PS2 (de novo, both maternity and paternity confirmed); generic fallback per generic_acmg_combination_rules (PMID:25741868)

Evidence considered

• Case bundle contains no proband clinical record, no parental (trio) testing, no pedigree structure and no stated meioses for MPL c.1544G>T (p.Trp515Leu); the only case-level assertions present are the ClinVar submissions for variation 14164.
• ClinVar variation 14164 contains four submissions: two OMIM submissions flagged origin=somatic with condition labels 'MYELOFIBROSIS WITH MYELOID METAPLASIA, SOMATIC' (SCV000035485) and 'THROMBOCYTHEMIA 2, SOMATIC' (SCV000043957), one clinical-testing submission flagged origin=germline for 'Thrombocytopenia, congenital amegakaryocytic' classified Pathogenic (SCV000595823, Genetic Services Laboratory, University of Chicago, cites only the ACMG guideline PMID:25741868 and provides no criterion-level evidence signals), and one clinical-testing submission classified Uncertain significance from Labcorp Genetics/Invitae (SCV001557092) whose only extracted evidence signal is PM2.
• PMID:16834459 reports MPLW515L in 4/45 JAK2V617F-negative myelofibrosis patients and states that sequencing of matched normal DNA from buccal swabs did not identify the substitution, demonstrating that MPLW515L is a somatic mutation in haematopoietic cells; the same paper found the variant in none of 50 JAK2V617F-negative ET patients, none of 10 PV patients, and none of 270 HapMap samples.
• PMID:16868251 states that MPLW515L is an acquired mutation and reports it in a cohort of 1182 patients with myeloid disorders; among the Mayo Clinic MPL-mutant patients none had a history of familial myeloproliferative disease, so there is no familial (inherited) occurrence to explain, and no de novo germline assertion anywhere in that cohort.
• COSMIC records 277 somatic observations of this variant (COSMIC COSV65243776). COSMIC is not present as a key in source_registry, so this observation is reported here as narrative only and is deliberately not listed as an evidence_ref.
• PMID:18769448 describes TpoR/MPL W515L as disrupting the juxtamembrane inhibitory motif and producing a myeloproliferative phenotype in a murine bone-marrow reconstitution model, and states that W515 mutations occur in patients with myelofibrosis and essential thrombocythemia; it contains no parental, germline or de novo data.
• Governing framework: no ClinGen CSPEC/VCEP and no local MPL-specific specification was retrieved (cspec found=false, gene_vcep_dir=null, vcep_materials note 'no cspec url available'), so the generic ACMG/AMP 2015 definitions of PS2 and the generic combination rules (PMID:25741868) were applied via generic_acmg_combination_rules.

Still missing

• No proband-level clinical/phenotypic record and no statement of the disease under investigation for the tested individual.
• No parental testing results: neither maternity nor paternity is confirmed, and neither parent was genotyped.
• No documentation of the tissue/sample origin for the tested proband (blood/bone marrow vs germline-surrogate tissue), which is decisive for assigning somatic vs germline origin at this locus.
• No family history statement for the tested proband (de novo status also requires absence of a family history).

Sources

Reasoning

No MPL-specific VCEP/CSPEC specification exists for this case (no CSPEC found; generic ACMG/AMP 2015 fallback), so generic PM3 was applied: for a recessive disorder, the variant detected in trans with a pathogenic variant. MPL germline disease (congenital amegakaryocytic thrombocytopenia) is autosomal recessive, but no reviewed source reports MPL c.1544G>T (p.Trp515Leu) in trans with a second (likely) pathogenic MPL allele in an affected individual, and the bundle contains no proband genotype or phase data at all. The exact-variant literature instead characterises W515L as an acquired somatic activating mutation: PMID:16834459 showed it was absent from matched buccal DNA in affected myelofibrosis patients; PMID:21326037 reports 43 heterozygous and 3 homozygous/hemizygous W515L cases in myeloproliferative neoplasms; and PMID:16868251 reports two patients in whom wild-type MPL, W515L and W515K were all detected in a mixed clonal state that the authors explicitly could not resolve into independent clones versus a subclone (phase unresolved). The six PMID:16868251 patients carrying MPLW515L together with JAK2 V617F involve a different gene and are therefore not an allelic trans configuration. No germline trans observation with a pathogenic allele exists for this variant, so PM3 is not met.

Rule

generic_acmg_amp_2015_PM3

Evidence considered

• No MPL-specific VCEP/CSPEC framework was available, so the generic ACMG/AMP PM3 definition (recessive disorder, variant in trans with a pathogenic variant) was used.
• ClinVar ID 14164 contains an exact variant match (NM_005373.3(MPL):c.1544G>T, p.Trp515Leu) with submissions of differing origin (one germline submission for Thrombocytopenia, congenital amegakaryocytic; two somatic-origin submissions for myelofibrosis with myeloid metaplasia / thrombocythemia 2, somatic; one germline submission with uncertain significance), but no submission reports a second MPL pathogenic allele in trans or any phase information.
• PMID:16834459 (Pikman et al.) reports MPLW515L in 4/45 JAK2V617F-negative myelofibrosis patients, absent from matched buccal DNA, demonstrating acquisition in hematopoietic cells rather than a constitutional biallelic configuration, and reports no second pathogenic MPL allele.
• PMID:16868251 (Pardanani et al., 1182 patients) reports all three alleles (wild-type MPL, MPLW515L, MPLW515K) in two de novo MMM patients in a mixed clonal state, and states that the approach could not distinguish independent clones from a JAK2V617F-bearing subclone of the MPL-mutant progenitor; this is somatic clonal co-occurrence with unresolved phase, not germline trans compound heterozygosity.
• PMID:16868251 also screened 64 healthy controls and reports a patient homozygous for MPLW515L (used for assay sensitivity), and six patients carrying MPLW515L and JAK2V617F concurrently; JAK2 is a different gene, so this co-occurrence is intergenic and cannot be counted as allelic trans or cis phase.
• PMID:21326037 (Ma et al.) detected W515L in 46 JAK2-negative myeloproliferative-neoplasm patients (43 heterozygous, 3 homozygous/hemizygous) by RNA-based bidirectional sequencing; no second pathogenic MPL allele or phase assessment in an affected individual is reported.
• PMID:20113333 (Glembotsky et al.) reports MPLW515L in 1/100 ET and 1/11 PMF patients, with a PMF platelet sample showing 100% mutant allele and an ET patient with mutant allele present in granulocytes, monocytes, B cells and platelets at <50%; these are somatic mutant-allele-burden observations and do not establish a germline trans configuration.
• PMID:25741868 (Richards et al. 2015) was consulted only for the generic ACMG/AMP PM3 definition; it provides no variant-specific allelic evidence.

Still missing

• No proband-level genotype: no second MPL variant reported for the individual under review.
• No germline phase data (allele-specific, long-read, or family-based) establishing a trans configuration with a pathogenic MPL allele.
• No documented recessive MPL disease context (e.g., congenital amegakaryocytic thrombocytopenia phenotype) for the individual under review.
• ClinVar ID 14164 mixes germline-origin and somatic-origin submissions, so the interpretation context for PM3 needs human confirmation.

Sources

Reasoning

PM6 requires the variant to be assumed de novo in the proband without confirmation of maternity/paternity - i.e. a clinical, non-tested de novo assertion. This case bundle contains no de novo assertion of any kind, no proband data and no family history: the only case-level evidence is the ClinVar record, which contains two somatic-origin submissions for the exact variant and one germline clinical-testing submission that reports no parental testing. In addition, the variant is a well-documented acquired (somatic) activating mutation of haematopoietic stem cells: matched buccal DNA was negative in PMID:16834459, and the variant is observed recurrently as a somatic event in myeloproliferative neoplasms (PMID:16868251 describes it as an acquired mutation; PMID:21326037 recovered it from RNA of JAK2-negative MPN patients including 3 homozygous cases; PMID:20113333 found it in granulocytes, monocytes, B cells and platelets of an ET patient). Nothing supports an assumed de novo germline origin, so PM6 is not met rather than merely unevaluated.

Rule

ACMG/AMP 2015 PM6 (assumed de novo, maternity/paternity not confirmed); generic fallback per generic_acmg_combination_rules (PMID:25741868)

Evidence considered

• No source in this case bundle asserts de novo status for MPL c.1544G>T (p.Trp515Leu) in the tested proband, and no phenotype, parental or family-history data are provided, so there is no basis on which to 'assume' a de novo occurrence as PM6 requires.
• PMID:16834459 explicitly demonstrates somatic acquisition: the MPLW515L substitution in nucleotide 1544 was detected in granulocytes of myelofibrosis patients but 'Sequence analysis of matched normal DNA derived from buccal swabs did not identify the MPLW515L substitution, demonstrating that MPLW515L is a somatic mutation in hematopoietic cells.'
• PMID:16868251 characterised MPLW515L as an acquired mutation and screened 1182 patients with myeloproliferative and other myeloid disorders plus 64 healthy controls; MPL515 mutations were restricted to MMM/ET patients, with 6 patients carrying MPLW515L and JAK2V617F concurrently, which is the expected behaviour of an acquired clonal allele rather than a constitutional de novo variant.
• PMID:21326037 detected MPL W515L in 46 of 2242 JAK2-negative patients with suspected Philadelphia-chromosome-negative myeloproliferative disorders (43 heterozygous, 3 homozygous/hemizygous), i.e. a recurrent acquired mutation in unrelated patients, with no de novo or parental testing reported.
• PMID:20113333 found MPLW515L in 1/100 ET and 1/11 PMF patients, with the mutation present in granulocytes, monocytes, B cells and platelets of the ET patient and the platelet cDNA of the PMF patient being 100% mutant, again consistent with clonal haematopoiesis; no parental or germline testing was reported.
• ClinVar variation 14164: two OMIM submissions are labelled origin=somatic (MYELOFIBROSIS WITH MYELOID METAPLASIA, SOMATIC; THROMBOCYTHEMIA 2, SOMATIC), and the single germline clinical-testing submission (SCV000595823) records no de novo evidence and cites only the ACMG guideline PMID:25741868.
• Governing framework: generic ACMG/AMP 2015 PM6 was used because no ClinGen CSPEC/VCEP or local MPL-specific framework was retrievable (cspec found=false), applied through generic_acmg_combination_rules (PMID:25741868).

Still missing

• No proband-level record stating whether the variant was considered de novo (neither a clinical de novo assertion nor a family-history-negative statement is present).
• No information on whether the tested proband's sample was a haematopoietic (blood/marrow) sample, which would make a somatic clonal explanation more likely than any de novo germline assumption.

Sources

Reasoning

The variant is a missense substitution, MPL p.(Trp515Leu), and no MPL VCEP/CSPEC PP3 rule or pre-assigned lookup rule is available. The required missense-only REVEL path yields 0.328, which is within the generic ACMG operating gray zone of 0.250 to 0.750 and does not meet PP3.

Rule

Generic ACMG/AMP fallback, missense path: REVEL >0.750 for PP3 supporting; score 0.328 is in the gray zone.

Evidence considered

• Variant normalization identifies NM_005373.2:c.1544G>T as the missense substitution NP_005364.1:p.(Trp515Leu).
• No applicable MPL VCEP/CSPEC material or gene-specific PP3/BP4 lookup was available; generic missense calibration was therefore used.
• REVEL score is 0.328. Under the generic missense calibration, PP3 requires REVEL >0.750 and BP4 requires REVEL <0.250; the calibration cutoffs are from the ClinGen SVI REVEL calibration publication, PMID:36413997.

Sources

Reasoning

PP5 requires a reputable source reporting the exact variant as pathogenic. For ClinVar that means an expert-panel (ClinGen/VCEP) classification of the exact variant; ClinVar variation 14164 has no expert-panel submission. Its assertions are ordinary single-submitter laboratory submissions (one Pathogenic, 'criteria provided, single submitter'; one Uncertain significance, 'criteria provided, single submitter'), and the submission audit reports expert_panel_submissions = 0. No ClinGen MPL specification exists for this gene in the bundle. The PP5 precondition is therefore not satisfied.

Rule

PP5 is applied only for an exact-variant ClinVar expert-panel (ClinGen/VCEP) classification of Pathogenic or Likely pathogenic; ordinary laboratory submissions, aggregate labels and non-expert assertions must not trigger it.

Evidence considered

• ClinVar variation 14164 (NM_005373.3(MPL):c.1544G>T, p.Trp515Leu) has overall review status 'criteria provided, single submitter' with a Pathogenic assertion from one clinical laboratory and an Uncertain significance assertion from one clinical laboratory; the submission audit for this record reports expert_panel_submissions = 0 and is_expert_panel = false for every submission.
• Aggregate ClinVar classification labels and ordinary laboratory submissions are not a permitted basis for PP5 under this group's rule; only an exact-variant expert-panel Pathogenic/Likely pathogenic assertion would trigger it.
• The bundle notes no ClinGen CSPEC was found for MPL (cspec found = false, gene_vcep_dir = null, no vcep materials), so no gene-specific PP5 policy exists that could alter this assessment.

Still missing

• No ClinVar expert-panel (ClinGen/VCEP) classification exists for this exact variant; PP5 could only be reconsidered if such an assertion is submitted.

Sources

Reasoning

Generic ACMG/AMP BA1 requires a stand-alone benign allele frequency above 5% (Richards et al. 2015, PMID:25741868); a non-VCEP operating threshold of AF > 1% is applied by this framework. gnomAD v4.1 reports MPL c.1544G>T at total AF 1.23979e-05 (0.00124%, 20/1,613,182 alleles) with joint grpmax FAF 1.772e-05 (0.0018%), and gnomAD v2.1 at total AF 7.98378e-06 (0.00080%, 2/250,508 alleles). These are roughly three to four orders of magnitude below any BA1 threshold, and no ancestry subgroup approaches it (highest: East Asian 6.683e-05, 0.0067%, 3/44,890 alleles; African/African American 6.15612e-05 in v2.1, 1/16,244). BA1 is not met.

Rule

Generic ACMG/AMP BA1: allele frequency > 5% in a population database (Richards et al. 2015, PMID:25741868); non-VCEP operating threshold applied by this framework: AF > 1%.

Evidence considered

• gnomAD v4.1 (GRCh38 joint callset; source_registry key gnomad_v4, captured in evidence.json results.gnomad.GNOMAD_V4_1.source_data): total AF 1.23979e-05 (0.00124%, 20/1,613,182 alleles; exome 20/1,461,002, genome 0/152,180), homozygotes 0, joint grpmax FAF 1.772e-05, exome-only grpmax FAF 2.003e-05; highest subpopulation East Asian AF 6.683e-05 (3/44,890).
• gnomAD v2.1 (GRCh37 exomes; source_registry key gnomad_v2): total AF 7.98378e-06 (0.00080%, 2/250,508 alleles), homozygotes 0; highest subpopulation African/African American AF 6.15612e-05 (1/16,244).
• BA1 threshold named to a publication: stand-alone benign frequency > 5% per Richards et al., ACMG/AMP 2015 standards (PMID:25741868). The configured non-VCEP operating threshold for this framework (BA1 when AF > 1%) is also not remotely approached.
• The variant is 20/1,613,182 alleles (~1 in 80,000) overall in the largest available population dataset, versus a 5% (1 in 20) BA1 requirement; no benign stand-alone frequency argument is available at any ancestry level.
• gnomAD-Canada v1.0 (HostSeq) lookup failed with a network timeout, so no Canadian ancestry-specific data could be added; it is not required for this determination.

Sources

Reasoning

Generic ACMG/AMP BS1 is an allele frequency greater than expected for the disorder (Richards et al. 2015, PMID:25741868); the non-VCEP operating threshold applied by this framework is AF > 0.3%. Every available frequency metric is far below any plausible BS1 floor for this disorder: gnomAD v4.1 joint grpmax FAF 1.772e-05 (0.0018%) and total AF 1.23979e-05 (0.00124%); highest subpopulation East Asian 6.683e-05 (0.0067%, 3/44,890); gnomAD v2.1 total AF 7.98378e-06 (0.00080%). The disease context (MPL-related congenital amegakaryocytic thrombocytopenia and the somatically acquired myeloproliferative neoplasms annotated in ClinVar) is rare, so the frequency expected for the disorder is low, and the observed frequency is lower still. BS1 is not met.

Rule

Generic ACMG/AMP BS1: allele frequency greater than expected for the disorder (Richards et al. 2015, PMID:25741868); non-VCEP operating threshold applied by this framework: AF > 0.3%.

Evidence considered

• gnomAD v4.1 (source_registry key gnomad_v4): FAF/grpmax metrics 1.772e-05 joint (0.0018%), 2.003e-05 exome-only, total AF 1.23979e-05 (20/1,613,182 alleles), 0 homozygotes; the frequency is ~1 in 56,000 by grpmax FAF.
• gnomAD v2.1 (source_registry key gnomad_v2): total AF 7.98378e-06 (2/250,508 alleles), 0 homozygotes; highest subpopulation 6.15612e-05 (African/African American, 1/16,244).
• Disease-cohort frequencies from the literature are low and do not suggest a common disorder allele: MPLW515L/K in ~5% of myelofibrosis with myeloid metaplasia and ~1% of essential thrombocythemia, absent in polycythemia vera and other myeloid disorders (PMID:16868251, 1,182 patients); W515L detected in 1/100 essential thrombocythemia and 1/11 primary myelofibrosis patients (PMID:20113333).
• BS1 threshold named to a publication: 'allele frequency greater than expected for the disorder' per Richards et al., ACMG/AMP 2015 (PMID:25741868); the configured non-VCEP operating threshold (0.3%) is exceeded by none of the observed metrics.
• Ancestry-specific elevation is based on very small allele counts (3/44,890 East Asian in v4.1; 1/16,244 African/African American in v2.1) and is not interpreted as evidence of a founder or higher-frequency subpopulation.

Still missing

• No prevalence/penetrance-based maximum credible allele frequency calculation for MPL is available in this case bundle, so BS1 was assessed against the framework's default non-VCEP operating threshold rather than a gene-specific published calibration. This does not change the outcome, which fails by more than two orders of magnitude.

Sources

Reasoning

BS2 requires the variant to be observed in a healthy adult individual: homozygous for a recessive disorder, or heterozygous for a dominant disorder with full penetrance expected at an early age (Richards et al. 2015, PMID:25741868). No such observation exists. gnomAD v4.1 contains 0 homozygotes (0/1,613,182 alleles) and gnomAD v2.1 contains 0 homozygotes (0/250,508 alleles); only heterozygous carriers are present. The control cohorts screened in the literature are negative: no MPL515 mutation was detected among 64 healthy controls (PMID:16868251), and W515L was absent from all 270 HapMap control samples and from matched buccal DNA (PMID:16834459), the latter demonstrating that the variant is characteristically somatically acquired in haematopoietic tissue rather than a germline allele. The only homozygous/hemizygous W515L observations reported are in patients with myeloproliferative neoplasms (3 of 2,242 JAK2-negative MPD patients, PMID:21326037), which is the opposite of a healthy-adult observation. Heterozygous gnomAD carriers do not satisfy BS2 because the W515L phenotype is an acquired, late-onset, reduced-penetrance myeloid neoplasm (ClinVar annotations are somatic), not a fully penetrant early-onset dominant condition. BS2 is not met.

Rule

Generic ACMG/AMP BS2: observed in a healthy adult individual with full penetrance expected at an early age (heterozygote, dominant) or in a healthy adult homozygote (recessive) (Richards et al. 2015, PMID:25741868).

Evidence considered

• gnomAD v4.1 (source_registry key gnomad_v4): homozygote count 0 across 1,613,182 alleles; 20 heterozygous alleles total, no homozygous or compound observations.
• gnomAD v2.1 (source_registry key gnomad_v2): homozygote count 0 across 250,508 alleles.
• PMID:16868251 screened 64 healthy controls for MPL515 mutations alongside 1,182 patients and reported carriers only among patients (20 of 1,182), providing a healthy-adult control screen negative for W515L.
• PMID:16834459: matched normal DNA from buccal swabs did not contain the MPLW515L substitution, and the variant was absent from all 270 HapMap samples tested, establishing a somatic rather than germline origin in the reported cases.
• PMID:21326037: 46 W515L-positive cases among 2,242 JAK2-negative patients with suspected Philadelphia chromosome-negative MPN, including 3 homozygous/hemizygous cases - all in affected patients, not healthy adults.
• Disease-context check (source_registry key clinvar): the ClinVar record for this exact variant carries submitter annotations to an autosomal recessive germline condition (Thrombocytopenia, congenital amegakaryocytic) and to two somatically acquired entities (MYELOFIBROSIS WITH MYELOID METAPLASIA, SOMATIC; THROMBOCYTHEMIA 2, SOMATIC). Submitter classifications were not used as evidence for this criterion.
• BS2 definition named to a publication: healthy adult homozygote (recessive) or healthy adult heterozygote for a fully penetrant early-onset dominant disorder, per Richards et al., ACMG/AMP 2015 (PMID:25741868).

Still missing

• BS2 is a negative call based on absence of any healthy adult homozygote or eligible healthy adult carrier: the largest control resources examined are gnomAD homozygote counts (0) and 64 healthy controls screened in PMID:16868251. No phase-resolved germline W515L homozygote data exist in the bundle.

Sources

Reasoning

Generic ACMG/AMP 2015 BS3 requires well-established functional studies demonstrating no deleterious effect. No such study exists for MPL p.(W515L). The only negative-leaning assay data located are patient-derived platelet measurements (PMID:20113333): normal platelet surface and total Mpl protein, MPL transcript levels not different from controls, no constitutive STAT3/STAT5 phosphorylation, and no enhanced thrombopoietin-induced phosphorylation. These are not a validated assay of receptor activation, the authors themselves describe the result as unexpected given experimental-model data showing constitutive receptor activation and TPO hypersensitivity, and they are directly outweighed by exact-variant cytokine-independence, constitutive JAK-STAT/ERK/AKT signalling, TPO-hypersensitivity and murine disease-model data (PMID:16834459, PMID:18769448). BS3 is therefore not met rather than being applied as benign functional evidence.

Rule

generic_acmg_amp_2015_BS3 (no MPL-specific VCEP/CSPEC): well-established functional studies show no deleterious effect on the gene or gene product.

Evidence considered

• PMID:20113333 directly examined MPLW515L-positive patients and reported normal platelet Mpl surface/total protein and MPL transcript levels, absent constitutive STAT3/STAT5 phosphorylation, and no enhanced dose response to TPO-induced phosphorylation (1/100 ET and 1/11 PMF patients, one ET patient with 38% platelet mutant burden and one PMF patient with 100% mutant allele in platelet cDNA).
• The PMID:20113333 negative platelet findings are not a validated functional assay readout of receptor activation (platelets are anucleate and the paper's own conclusion states the lack of spontaneous STAT3/STAT5 activation is unexpected as MPLW515L leads to constitutive receptor activation and TPO hypersensitivity in experimental models).
• PMID:16834459 and PMID:18769448 provide concordant exact-variant gain-of-function and in vivo myeloproliferative-disease data that contradict any interpretation of MPL W515L as functionally neutral; therefore no functional evidence meeting BS3 exists.
• PMID:25741868 was used only for the generic ACMG/AMP BS3 definition because no applicable MPL-specific specification was present.

Still missing

• No benign-variant-controlled, assay-validated functional study demonstrating normal MPL W515L receptor function exists in this case bundle.

Sources

Reasoning

BP1 applies only to genes in which disease is caused primarily by truncating variants, i.e. where missense change is not an established mechanism. MPL does not meet that precondition: missense variation is an established disease mechanism in both contexts relevant to this gene. In the germline/MPN literature, a highly penetrant gain-of-function MPL missense change (MPLS505N) was identified in a familial essential thrombocythemia pedigree and missense alleles MPLW515L and MPLW515K are recurrent activating changes in myelofibrosis/essential thrombocythemia (PMID:16868251; PMID:16834459), and W515L is curated as an oncogenic gain-of-function variant (OncoKB). Because missense variants are well documented causes of disease in MPL, BP1 (missense variant in a gene where primarily truncating variants cause disease) is not met.

Evidence considered

• PMID:16868251 states that a highly penetrant gain-of-function MPL mutation in the transmembrane domain (MPLS505N) was identified in a Japanese pedigree with familial essential thrombocythemia and that codon 515 missense changes (W515L, W515K) cause myeloproliferative disease — i.e. missense, not truncating, alleles are established causes of MPL-related disease.
• PMID:16834459 shows MPLW515L (a missense change) confers cytokine-independent growth and constitutive JAK2/STAT3/STAT5 signaling and causes a myeloproliferative phenotype in a murine bone marrow transplant model, confirming a gain-of-function missense disease mechanism.
• OncoKB curates MPL W515L as Oncogenic with gain-of-function effect, consistent with missense-mediated disease rather than a truncating-only mechanism.
• Because missense variation is disease-causing in MPL, the precondition of BP1 (primarily truncating variants cause disease) fails and no benign evidence can be taken from it.

Sources

Reasoning

No MPL-specific VCEP/CSPEC specification exists for this case, so generic ACMG/AMP BP2 was applied: the variant 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. No reviewed source reports MPL c.1544G>T (p.Trp515Leu) in cis with any pathogenic variant. The only same-gene second-allele observation available (W515K alongside W515L in two de novo MMM patients, PMID:16868251) is in trans within a somatic mixed clonal state that the authors state cannot be resolved into independent clones versus a subclone, is not germline allelic evidence, and would in any case not be benign evidence for a variant that is itself an activating driver. The reported co-occurrence of MPLW515L with JAK2 V617F in six patients (PMID:16868251) is intergenic (JAK2 is on chromosome 9), so it is neither cis nor trans allelic phase at the MPL locus. With no cis pathogenic partner and no informative phase or inheritance data, BP2 is not met.

Rule

generic_acmg_amp_2015_BP2

Evidence considered

• No MPL-specific VCEP/CSPEC framework was available, so the generic ACMG/AMP BP2 definition (trans with a pathogenic variant for a fully penetrant dominant disorder, or cis with a pathogenic variant in any inheritance pattern) was used.
• ClinVar ID 14164 provides an exact variant match and submission-level classifications only; no submission documents a second pathogenic variant in the same individual or its phase relative to c.1544G>T.
• PMID:16868251 reports wild-type MPL, MPLW515L and MPLW515K together in two de novo MMM patients, described as a mixed clonal state, with the authors stating that their approach could not distinguish independent clones from a subclone; no cis configuration is reported, so this cannot support BP2.
• PMID:16868251 reports six patients carrying MPLW515L and JAK2V617F concurrently, with the MPL515 mutant allele in excess of the JAK2V617F allele; these variants are in different genes, so the observation is not a cis or trans allelic phase relationship and does not support BP2.
• PMID:16834459 and PMID:21326037/PMID:20113333 report W515L in somatic myeloproliferative disease, including homozygous/hemizygous cases (PMID:21326037) and a PMF platelet sample with 100% mutant allele (PMID:20113333); none of these reports identifies a pathogenic variant in cis with W515L.
• PMID:25741868 (Richards et al. 2015) was consulted only for the generic ACMG/AMP BP2 definition; it provides no variant-specific allelic evidence.

Still missing

• No phase-resolved observation of a cis configuration with any pathogenic variant.
• No proband-level genotype, pedigree, or inheritance-mode documentation needed to place BP2 in the correct dominant/recessive frame.
• No family or molecular phasing data (allele-specific or long-read sequencing) for the individual under review.

Sources

Reasoning

The variant is a missense substitution, MPL p.(Trp515Leu), and no MPL VCEP/CSPEC BP4 rule or pre-assigned lookup rule is available. The required missense-only REVEL path yields 0.328, which is within the generic ACMG operating gray zone of 0.250 to 0.750 and does not meet BP4.

Rule

Generic ACMG/AMP fallback, missense path: REVEL <0.250 for BP4 supporting; score 0.328 is in the gray zone.

Evidence considered

• Variant normalization identifies NM_005373.2:c.1544G>T as the missense substitution NP_005364.1:p.(Trp515Leu).
• No applicable MPL VCEP/CSPEC material or gene-specific PP3/BP4 lookup was available; generic missense calibration was therefore used.
• REVEL score is 0.328. Under the generic missense calibration, PP3 requires REVEL >0.750 and BP4 requires REVEL <0.250; the calibration cutoffs are from the ClinGen SVI REVEL calibration publication, PMID:36413997.

Sources

Reasoning

BP6 requires a reputable source reporting the exact variant as benign. For ClinVar that means an expert-panel (ClinGen/VCEP) classification of the exact variant as Benign or Likely benign; ClinVar variation 14164 has no expert-panel submission and no benign or likely benign assertion of any kind (submissions are one Pathogenic and one Uncertain significance, both 'criteria provided, single submitter'; expert_panel_submissions = 0). The BP6 precondition is therefore not satisfied.

Rule

BP6 is applied only for an exact-variant ClinVar expert-panel (ClinGen/VCEP) classification of Benign or Likely benign; ordinary laboratory submissions, aggregate labels and non-expert assertions must not trigger it.

Evidence considered

• ClinVar variation 14164 shows no benign or likely benign submission: the only submissions are one Pathogenic (University of Chicago, condition label 'Thrombocytopenia, congenital amegakaryocytic') and one Uncertain significance (Labcorp Genetics/Invitae), both with review status 'criteria provided, single submitter'.
• The submission audit for this record reports expert_panel_submissions = 0 and is_expert_panel = false for every submission, so no expert-panel benign assertion exists for the exact variant.
• Aggregate ClinVar labels and ordinary laboratory submissions are not a permitted basis for BP6 under this group's rule.

Still missing

• No ClinVar expert-panel (ClinGen/VCEP) benign or likely benign classification exists for this exact variant; BP6 could only be reconsidered if such an assertion is submitted.

Sources

Reasoning

PP1 requires co-segregation of the variant with disease in multiple affected family members, i.e. genotype data from at least two affected relatives and an informative pedigree. Nothing in this case bundle provides that: there is no pedigree, no family member genotypes, no meioses count and no family history for the tested proband. All retrieved variant-level evidence comes from unrelated patient cohorts of an acquired somatic myeloproliferative mutation (PMID:16834459, PMID:16868251, PMID:20113333, PMID:21326037), which cannot supply germline co-segregation information; PMID:16868251 notes only the absence of familial myeloproliferative disease among MPL-mutant patients, which is a cohort-level statement and not an informative segregation study. With no segregation evidence in either direction, PP1 is not_assessed rather than not_met.

Rule

ACMG/AMP 2015 PP1 (co-segregation with disease in multiple affected family members); generic fallback per generic_acmg_combination_rules (PMID:25741868)

Evidence considered

• ClinVar variation 14164 was the only case-level record available; its submissions contain no pedigree, no relative genotypes, and no segregation statement, and the four submissions cover somatic-origin OMIM entries plus single clinical-laboratory assertions without family data.
• PMID:16868251 was checked specifically for familiality: it reports that among the 13 Mayo Clinic patients with MPL mutations, none had a history of familial myeloproliferative disease, and no family genotypes or meioses are presented anywhere in the paper, so it neither supports nor refutes co-segregation.
• The retrieved literature for this variant (PMID:16834459, PMID:16868251, PMID:18769448, PMID:20113333, PMID:21326037) consists of unrelated-patient cohort and model-system studies of a somatically acquired myeloproliferative neoplasm allele; none contains pedigree or segregation data, which is why PP1 could not be evaluated.
• Governing framework: no ClinGen CSPEC/VCEP or local MPL-specific specification was available (cspec found=false, vcep_materials note 'no cspec url available'), so the generic ACMG/AMP 2015 PP1 definition and generic rules (PMID:25741868) apply via generic_acmg_combination_rules.

Still missing

• No pedigree or family structure for the tested proband.
• No genotypes for any relative (affected or unaffected), so no informative meioses exist.
• No family-history statement in the case bundle to establish whether any relatives are affected with an MPL-related phenotype.
• Because the variant is documented as a somatically acquired allele in haematopoietic cells, any future segregation-style assessment would need to specify germline-surrogate tissue, since blood-derived genotypes can carry the somatic clone independently of inheritance.

Sources

Reasoning

PP2 requires BOTH that the gene has a low rate of benign missense variation AND that missense variants are a common mechanism of disease. The second condition is supported for MPL (recurrent activating missense alleles such as W515L/W515K drive myeloproliferative neoplasms and a germline gain-of-function missense change, MPLS505N, has been reported in familial essential thrombocythemia; PMID:16868251, PMID:16834459), but the first condition could not be evaluated because no gene-level missense constraint metric (gnomAD missense Z-score or observed/expected ratio) was retrieved anywhere in the case evidence bundle. Without a documented low rate of benign missense variation, PP2 cannot be applied and is returned as not_assessed rather than met or not met.

Evidence considered

• MPL missense variation is disease-relevant: activating missense alleles at codon 515 (W515L in 4/45 JAK2V617F-negative myelofibrosis patients, PMID:16834459) and at codon 505 (germline MPLS505N in familial essential thrombocythemia, PMID:16868251) cause myeloproliferative disease, supporting the 'missense is a common mechanism' half of PP2.
• The gene-level context file for this case (pvs1_gene_context.json) documents a germline MPL loss-of-function disease mechanism and is flagged literature_only with no official CSPEC/VCEP, but it contains no missense constraint metric and therefore cannot resolve PP2.
• final_classification_framework.json confirms framework_mode is generic ACMG fallback (no gene-specific MPL specification), so generic ACMG/AMP 2015 PP2 wording applies.
• No gnomAD gene constraint table was collected in this bundle (only variant-level gnomAD allele frequencies), leaving PP2's benign-missense-rate precondition undetermined.

Still missing

• Gene-level missense constraint for MPL (gnomAD missense Z-score and/or observed/expected missense ratio) is absent from the evidence bundle, so the 'low rate of benign missense variation' precondition of PP2 could not be tested.
• No gene-specific (VCEP/CSPEC) PP2 specification exists for MPL in this case; only generic ACMG/AMP 2015 PP2 wording is available (PMID:25741868).

Sources

Reasoning

No phenotype, clinical diagnosis, laboratory findings, family history, or specimen context for the individual being interpreted is present anywhere in the consolidated case bundle, so the patient's phenotype cannot be compared with the specificity expected for a disease with a single genetic etiology. Disease-level observations that MPL515 variants cluster in myelofibrosis/essential thrombocythemia cohorts are not a substitute for proband-level phenotype information.

Rule

Generic ACMG/AMP 2015 PP4: the patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• No proband phenotype, indication for testing, or family-history field is present in the case bundle reviewed for this group.
• PMID:16868251 shows that MPL515 mutations in its cohort occurred only in patients with myelofibrosis with myeloid metaplasia or essential thrombocythemia, and PMID:18769448 notes that W515 mutations occur in patients with myelofibrosis and essential thrombocythemia; these are disease-level (cohort) associations for a somatically acquired change and do not establish that any individual's phenotype is highly specific for a single-gene disorder.
• PMID:16834459 detected MPLW515L only in JAK2V617F-negative myelofibrosis samples and not in JAK2V617F-negative essential thrombocythemia or polycythemia vera samples, indicating phenotype-dependent detection in tumour tissue rather than proband phenotype specificity.

Still missing

• Provide the individual's clinical phenotype, diagnosis and relevant laboratory findings (for example blood counts, bone-marrow findings, thrombopoietin level) plus family history.
• Provide the specimen type and germline confirmation for the tested sample (somatic versus germline origin), since the published phenotype associations for this variant are for acquired myeloproliferative neoplasms.
• Provide the diagnostic workup excluding phenocopies and alternative molecular causes, which is normally required before PP4 can be applied.

Sources

Reasoning

BS4 requires an informative family study showing lack of segregation of the variant with the disease (typically the variant absent in an affected relative, with the family phenotype attributable to another cause). No family testing exists in this case: there is no pedigree, no affected relative genotyped, and no family history statement for the tested proband. The only familiality statement retrieved is cohort-level absence of familial myeloproliferative disease among MPL-mutant patients in PMID:16868251, which is not an informative non-segregation study. With no family genotype data, BS4 can be neither met nor excluded and is therefore not_assessed.

Rule

ACMG/AMP 2015 BS4 (lack of segregation in affected family members); generic fallback per generic_acmg_combination_rules (PMID:25741868)

Evidence considered

• Reviewed ClinVar variation 14164 for any family-based assertion: the record contains only single-submitter clinical-laboratory and literature-only (OMIM) submissions, with no family member testing reported in either direction.
• PMID:16868251 was reviewed for non-segregation evidence and contains none; its only relevant statement is that no Mayo Clinic patient with an MPL mutation had a history of familial myeloproliferative disease, which is a cohort-level frequency observation rather than a family non-segregation study of this variant.
• No other source in the bundle (PMID:16834459, PMID:18769448, PMID:20113333, PMID:21326037) reports testing of relatives, so there is no basis to apply BS4.
• Governing framework: generic ACMG/AMP 2015 BS4 was applied because no ClinGen CSPEC/VCEP or local MPL-specific specification was retrieved for MPL (cspec found=false), per generic_acmg_combination_rules (PMID:25741868).

Still missing

• No genotyped relatives, so absence of the variant in an affected relative cannot be demonstrated or excluded.
• No pedigree/phenotype data for relatives of the tested proband.
• No information about the tested proband's own phenotype, which would be needed to judge whether the family phenotype is even attributable to MPL-related disease.

Sources

Reasoning

BP5 requires variant-level evidence that the variant was found in a case whose phenotype is explained by an alternative molecular basis (for example a pathogenic variant in another gene). The bundle contains no individual-level genotype, phenotype or diagnostic information for the case, so no alternate molecular basis can be established or excluded. Reports of concurrent somatic MPLW515L and JAK2V617F in the same myeloproliferative-neoplasm patients are co-mutations in a single disease context, not an alternative molecular explanation of a phenotype, and are therefore not BP5 evidence.

Rule

Generic ACMG/AMP 2015 BP5: the variant is found in a case with an alternate molecular basis for disease.

Evidence considered

• PMID:16868251 reports that 6 of the 20 patients carrying MPL515 mutations also carried JAK2V617F (2 essential thrombocythemia, 3 de novo myelofibrosis with myeloid metaplasia, 1 post-essential thrombocythemia myelofibrosis) and interprets these alleles as possibly functionally complementary in myeloproliferative disease; this concurrent somatic co-mutation is not an alternate molecular basis explaining an individual's phenotype.
• No proband-level genotype, phenotype or diagnostic summary is present in the consolidated case bundle, so BP5 cannot be evaluated for the individual case.

Still missing

• Provide individual-level genotype and phenotype data, including whether a pathogenic variant in another gene (for example JAK2, CALR or a germline congenital amegakaryocytic thrombocytopenia gene) fully explains the reported phenotype.
• Provide the specimen type and germline confirmation for the tested sample, since co-occurring somatic driver mutations in myeloid neoplasms cannot be evaluated as an alternate diagnosis without case-level context.

Sources