SETBP1 c.2602G>A, p.Asp868Asn

Reasoning

The queried missense variant is SETBP1 NP_056374.2:p.(Asp868Asn), caused by NM_015559.3:c.2602G>A. The same amino-acid change was previously reported as G2602A/p.D868N in four individuals with Schinzel-Giedion syndrome, including recurrent de novo cases, and is recorded as Pathogenic in ClinVar variation 1032 with multiple submitters and no conflicts. The exact protein-level change is therefore an established pathogenic variant independent of nucleotide representation.

Rule

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

Evidence considered

• ClinVar variation 1032 identifies NM_015559.3:c.2602G>A (p.Asp868Asn) as Pathogenic, with criteria provided by multiple submitters and no conflicts.
• Hoischen et al. 2010, Table 1 and Results, report the same G2602A/p.D868N change in four individuals with Schinzel-Giedion syndrome and identify G2602A as a recurrent mutation.
• The queried change is a missense substitution at the protein level; the reported disease-associated change is the identical amino-acid change, not merely a nearby variant.

Sources

Reasoning

Hoischen et al. reported the exact G2602A variant, producing p.D868N, in four individuals with Schinzel-Giedion syndrome. The variant was heterozygous and de novo in the affected individuals; parental DNA was tested, and parentage was confirmed by short tandem repeat marker analysis in all available case-parent trios. The affected cohort fulfilled the diagnostic criteria for Schinzel-Giedion syndrome, and the study describes the disorder as generally sporadic. This satisfies the generic PS2 definition because the variant was de novo in affected patients with confirmed parentage and no reported familial disease history.

Rule

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

Evidence considered

• Hoischen et al. 2010, Results and Table 1: G2602A/p.D868N occurred in four Schinzel-Giedion syndrome individuals; the paper reports de novo status, parental testing, confirmed parentage in all available case-parent trios, and diagnostic consistency across the affected cohort.

Sources

Reasoning

PS3 is met at strong strength. The exact p.Asp868Asn (D868N) variant was tested in complementary functional systems. In HEK293 expression assays, D868N produced dramatically increased SETBP1 protein levels, showed reduced degradation, and had little additional response to proteasome inhibition; patient-derived fibroblasts independently showed increased endogenous SETBP1 relative to two age-matched controls. In a separate functional study, Setbp1 p.Asp868Asn efficiently immortalized murine myeloid progenitors and produced more efficient colony formation and faster proliferation than wild-type Setbp1, supporting increased leukemogenic activity. These concordant in vitro, patient-cell, and in vivo-relevant cellular results support a damaging gain-of-function effect, although the studies were not described as validated clinical diagnostic laboratory assays.

Rule

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

Evidence considered

• Acuna-Hidalgo et al. 2017 (PMID:28346496), Functional characterization of SETBP1 variants: exact D868N increased SETBP1 protein levels and stability in HEK293 cells; patient-derived fibroblasts showed increased endogenous SETBP1 compared with two age-matched controls; patient lymphoblastoid cells also showed increased proliferation.
• Makishima et al. 2013 (PMID:23832012), functional assays: exact p.Asp868Asn Setbp1 efficiently immortalized murine myeloid progenitors and produced more efficient colony formation and faster proliferation than wild-type Setbp1.
• The evidence is concordant across transfected HEK293 cells, patient-derived fibroblasts and lymphoblastoid cells, and murine myeloid progenitor assays; no claim is made that these assays were validated in a clinical diagnostic laboratory.

Sources

Reasoning

Generic ACMG/AMP PS4 is met. Hoischen et al. (PMID:20436468) directly reported the exact G2602A/p.D868N variant in 4 affected Schinzel-Giedion syndrome individuals, with no SETBP1 mutations in 188 control chromosomes. This is a case-control comparison even though the paper did not print an odds ratio. Using the reported 4/13 affected individuals versus 0/94 diploid control individuals with a 0.5 continuity correction gives OR=89.53, 95% CI 4.47-1792.77, which is >5 and excludes 1. A matched-ancestry gnomAD NFE allele comparison also gives OR=42,909.71, 95% CI 10,799.30-170,496.46, using 4 alternate alleles among 26 case alleles versus 5/1,180,022 control alleles. The literature_pass PS4 leads were checked: its ai_classified_papers list is empty; PMID:20436468 provides the qualifying exact-variant case-control counts, while the other indexed leads were not independently verified as qualifying case-control evidence in the available extracts.

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

• Hoischen et al. reported G2602A/p.D868N in four affected Schinzel-Giedion syndrome individuals and stated that none of the SETBP1 mutations were found in 188 control chromosomes.
• The paper reports four variant-positive cases among 13 affected individuals; treating 188 control chromosomes as 94 diploid controls and applying a 0.5 continuity correction yields OR=89.53 and 95% CI 4.47-1792.77.
• Matched-ancestry gnomAD v4.1 NFE data show 5/1,180,022 alternate alleles; an allele-level comparison with 4/26 case alleles yields OR=42,909.71 and 95% CI 10,799.30-170,496.46.
• The exact ACMG/AMP PS4 threshold was applied literally: OR >5.0 and the confidence interval must not include 1.

Sources

Reasoning

SETBP1 Asp868 lies within the established recurrent mutational hotspot spanning Glu858-Ile871 in the SKI-homologous/degron region. The hotspot is described in a study of recurrent SETBP1 mutations and in the report of overlapping germline and hematologic malignancy mutations, which describes a 12-bp hotspot in exon 4 that disrupts a degron and includes D868. The available evidence supports a well-established disease-relevant hotspot; no benign variation at the hotspot is reported in the reviewed evidence, and the queried allele is extremely rare in gnomAD v4.1 (AF 3.09815e-06; 5/1,613,866 alleles; no homozygotes).

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• Makishima et al. 2013 reports recurrent SETBP1 mutations encoding p.Glu858Lys, p.Asp868Asn, p.Gly870Ser and p.Ile871Thr and places them in the mutational hotspot spanning Glu858-Ile871 in the SKI-homologous region.
• Acuna-Hidalgo et al. 2017 describes germline SETBP1 mutations clustering to a 12-bp hotspot in exon 4; the hotspot disrupts a degron, and the study reports malignancies associated with mutations at residue D868.
• gnomAD v4.1 reports total AF 3.09815e-06, 5 of 1,613,866 alleles, and zero homozygotes for the queried allele; no benign variation at the cited hotspot is reported in the reviewed sources.

Sources

Reasoning

PM2 is met at Supporting strength. Under the supplied generic SVI threshold, PM2 Supporting applies at allele frequency <=0.0001. gnomAD v4.1 shows the variant at 5/1,613,866 alleles (AF=3.09815e-06), with grpmax FAF=1.24e-06 and zero homozygotes; both the observed AF and the conservative filtering frequency are below 0.0001. gnomAD v2.1 and its non-cancer record are absent, while gnomAD v3.1 non-cancer reports 1/147,734 alleles, zero homozygotes, and grpmax FAF=0; no VCEP requires the restricted non-cancer source, so the all-comers v4.1 record is the default and independently supports PM2.

Rule

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

Evidence considered

• The generic PM2 definition is absence from controls, with extremely low frequency permitted for recessive disease; the supplied SVI recommendation uses <=0.0001 for PM2 Supporting (ClinGen SVI 2020; threshold supplied with ACMG/AMP reference PMID:25741868).
• gnomAD v4.1 reports 5/1,613,866 alleles, AF=3.09815e-06, zero homozygotes, and grpmax FAF=1.24e-06; the grpmax FAF is below 0.0001 and is the conservative frequency used for population assessment.
• gnomAD v3.1 non-cancer, genomes only, reports 1/147,734 alleles, AF=6.76892e-06, zero homozygotes, and grpmax FAF=0; this is concordant supplemental evidence, but no VCEP requires the non-cancer subset.
• gnomAD v2.1 all-comers and non-cancer records are absent.

Sources

Reasoning

PM6 is not met because this variant has documented parental testing and parentage confirmation, rather than being merely assumed de novo without maternity and paternity confirmation. The evidence meets the stronger PS2 pathway instead.

Rule

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

Evidence considered

• Hoischen et al. 2010: parental DNA was tested and parentage was confirmed by short tandem repeat marker analysis in all available case-parent trios.

Sources

Reasoning

The variant is a missense substitution, NM_015559.3:c.2602G>A (p.Asp868Asn), so the governing generic rule selects the REVEL path only; SpliceAI and BayesDel are not used for PP3/BP4 in this situation. REVEL is 0.476, below the generic PP3 supporting threshold of 0.644 from the ClinGen SVI calibration (Pejaver et al. 2022, PMID:36413997), so PP3 is not met.

Rule

PP3 (supporting): >= 0.644 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence is missense: NM_015559.3:c.2602G>A predicts NP_056374.2:p.(Asp868Asn), so the generic missense REVEL path applies.
• Local REVEL lookup reports score 0.476 for this variant; the supplied generic PP3 supporting threshold is >= 0.644, calibrated by Pejaver et al. 2022, PMID:36413997.
• SpliceAI is not counted for this missense PP3 decision because the governing rule assigns missense variants to the REVEL-only path; BayesDel is not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

PP5 is not met under the specified generic rule. ClinVar contains exact-variant Pathogenic submissions, but the audit records zero expert-panel submissions; the available assertions are ordinary laboratory submissions, multiple-submitter aggregates, or literature-only entries. These do not qualify for PP5 under the required expert-panel-only application rule.

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 1032 exactly matches NM_015559.3:c.2602G>A (p.Asp868Asn) and is labeled Pathogenic, but the submission audit reports expert_panel_submissions=0.
• The available ClinVar records are clinical-laboratory submissions, an OMIM literature-only assertion without criteria, or aggregate review status; none is an exact-variant expert-panel classification.

Sources

Reasoning

BA1 is not met. No governing VCEP applies, so the generic stand-alone threshold is >=0.05. The appropriate gnomAD v4.1 filtering frequency is grpmax FAF=1.24e-06 (0.000124%), based on a population with 1,180,022 observed alleles; this is far below 0.05. The highest observed population point estimate, 4.23721e-06 in European (non-Finnish) individuals, is not substituted for FAF. The variant also has a pathogenic ClinVar consensus, but the frequency is already far below the threshold and therefore does not create a BA1 conflict requiring an exception.

Rule

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

Evidence considered

• Generic ACMG/AMP BA1 uses a stand-alone allele-frequency threshold of >=0.05 (ACMG/AMP 2015, PMID:25741868).
• gnomAD v4.1 reports 5/1,613,866 total alleles, AF 3.09815e-06, zero homozygotes, and grpmax FAF=1.24e-06; the highest point estimate is 5/1,180,022 alleles in European (non-Finnish) individuals, AF 4.23721e-06.
• The gnomAD v2.1 all-comers and non-cancer records are absent; no VCEP specifies a restricted population source, so the gnomAD v4.1 all-comers record is the default current population source.
• ClinVar reports Pathogenic with multiple submitters and no conflicts, so the low frequency does not present the high-frequency/pathogenic-consensus conflict that would require withholding BA1.

Sources

Reasoning

BS1 is not met. The generic SVI default threshold supplied for BS1 Strong is >=0.01, while gnomAD v4.1 grpmax FAF=1.24e-06 (0.000124%) from a population with 1,180,022 observed alleles. This is below the default by more than three orders of magnitude. The highest population point estimate, 4.23721e-06, is not the filtering allele frequency and cannot establish BS1. The disorder context is also severe, early-onset SETBP1-related disease with no evidence here that reduced penetrance or a founder allele permits a materially higher maximum credible frequency; no maximum-credible-frequency calculation is needed because the observed FAF is already far below 1%.

Rule

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

Evidence considered

• The generic BS1 rule is that allele frequency is greater than expected for the disorder; the supplied generic SVI default is >=0.01, with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168).
• gnomAD v4.1 reports grpmax FAF=1.24e-06, with the relevant European (non-Finnish) population containing 1,180,022 observed alleles; its point estimate is 4.23721e-06 and is not used in place of FAF.
• The case evidence describes Schinzel-Giedion syndrome as a severe developmental disorder with most affected individuals dying before age ten, and does not document reduced penetrance, a founder allele, disease prevalence, or a maximum-credible-frequency calculation that would support BS1 at this frequency.
• The gnomAD v2.1 all-comers and non-cancer records are absent; no VCEP specifies a restricted population source, so gnomAD v4.1 all-comers is the default source.

Sources

Reasoning

BS3 is not met. The reviewed exact-variant functional studies do not show a lack of damaging effect; instead, p.Asp868Asn increased SETBP1 protein stability and abundance in HEK293 cells and patient-derived fibroblasts, and enhanced immortalization, colony formation, and proliferation in murine myeloid progenitors. The other exact-variant papers reported clinical or somatic observations without a benign functional result. Therefore, no well-established functional study supports normal protein function or splicing for this variant.

Rule

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

Evidence considered

• PMID:28346496 reports increased SETBP1 protein levels and stability for exact D868N in HEK293 cells and increased endogenous SETBP1 in patient-derived fibroblasts, which is opposite to a benign functional result.
• PMID:23832012 reports enhanced immortalization, colony formation, and proliferation for exact p.Asp868Asn in murine myeloid progenitors, which is opposite to a benign functional result.
• PMID:23222956 explicitly states that its functional experiments used p.Gly870Ser rather than p.Asp868Asn, so it provides no benign functional result for this exact variant.
• PMID:20436468 reports the exact variant in affected individuals but no functional assay of D868N.

Still missing

• No well-established benign functional assay demonstrating normal SETBP1 protein function or splicing was identified.

Sources

Reasoning

The variant is missense, but the evidence does not support a SETBP1 disease model in which primarily truncating variants cause disease. Instead, SETBP1 missense variants are a documented disease mechanism: p.Asp868Asn and other clustered missense variants cause or contribute to Schinzel-Giedion syndrome and are recurrent in myeloid malignancies, with functional evidence for increased SETBP1 stability and activity. Therefore the gene-level premise of BP1 is not met.

Rule

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

Evidence considered

• Hoischen et al. 2010 reports recurrent de novo SETBP1 missense mutations, including G2602A/p.D868N, in Schinzel-Giedion syndrome.
• Makishima et al. 2013 reports recurrent SETBP1 missense mutations in myeloid malignancies, including p.Asp868Asn, and places them in the disease-relevant hotspot.
• Acuna-Hidalgo et al. 2017 reports overlapping germline and somatic missense mutations and functional evidence that D868N increases SETBP1 protein stability.

Sources

Reasoning

The variant is a missense substitution, NM_015559.3:c.2602G>A (p.Asp868Asn), so the governing generic rule selects the REVEL path only; SpliceAI is not substituted for a gray-zone REVEL result. REVEL is 0.476, above the generic BP4 supporting cutoff of <= 0.29 and therefore does not support a benign computational effect; BP4 is not met.

Rule

BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence is missense: NM_015559.3:c.2602G>A predicts NP_056374.2:p.(Asp868Asn), so the generic missense REVEL path applies.
• Local REVEL lookup reports score 0.476; the supplied generic BP4 supporting cutoff is <= 0.29, calibrated by Pejaver et al. 2022, PMID:36413997.
• The REVEL result is not within a BP4 range, and the accompanying SpliceAI score is not used because computational evidence for a missense PP3/BP4 decision must come from the REVEL path only.

Sources

Reasoning

BP6 is not met under the specified generic rule. ClinVar has no exact-variant expert-panel classification as Benign or Likely benign; instead, the exact variant has Pathogenic laboratory submissions and no benign expert-panel assertion. Ordinary laboratory submissions, aggregate labels, and non-expert assertions cannot trigger BP6.

Rule

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

Evidence considered

• ClinVar variation 1032 exactly matches NM_015559.3:c.2602G>A (p.Asp868Asn) and reports Pathogenic submissions; the submission audit reports expert_panel_submissions=0.
• No exact-variant expert-panel Benign or Likely benign assertion is present, so BP6 cannot be triggered under the specified expert-panel-only rule.

Sources

Reasoning

The PM5 rule requires a different pathogenic missense change at the same amino-acid residue. The reviewed evidence establishes p.Asp868Asn itself and reports nearby SETBP1 substitutions, but it does not establish a different pathogenic missense substitution at residue Asp868. The available PM5 candidate file contains no same-residue comparator candidates, so PM5 cannot be assessed from the supplied evidence.

Rule

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

Evidence considered

• The PM5 comparator collection reports zero same-residue candidates and no verified comparator evidence.
• The reviewed SETBP1 papers report p.Asp868Asn and nearby substitutions such as p.Glu858Lys, p.Gly870Ser, and p.Ile871Thr, but these are not substitutions at residue Asp868.

Still missing

• A verified pathogenic missense comparator at residue Asp868 is needed to assess PM5.

Sources

Reasoning

PP2 requires both a low rate of benign missense variation and a gene in which missense variants are a common disease mechanism. The evidence shows a strong SETBP1 missense gain-of-function hotspot mechanism for Schinzel-Giedion syndrome and hematologic malignancy, but it does not provide a validated gene-level benign-missense rate or a sufficiently defined generic threshold for applying PP2. SETBP1 also has reported loss-of-function disease, so the required gene-wide mechanism assessment cannot be completed from the supplied evidence.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• SETBP1 missense variants are reported as recurrent gain-of-function changes in the Schinzel-Giedion syndrome and hematologic malignancy hotspot.
• The reviewed sources do not supply a validated benign-missense rate for SETBP1 or a gene-wide quantitative basis for the low-benign-missense requirement.

Still missing

• A validated gene-level benign missense rate and gene-wide missense-mechanism assessment are needed for PP2.

Sources

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 case evidence does not document this patient's phenotype, family history, or a sufficiently specific clinical diagnosis, so PP4 cannot be assessed.

Rule

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

Evidence considered

• No patient-level phenotype or family-history description was available for applying PP4; disease descriptions in publications cannot substitute for the patient's documented phenotype.

Still missing

• Patient-level phenotype and family-history details are required to assess specificity for Schinzel-Giedion syndrome or another single-gene SETBP1 disorder.

Sources

Reasoning

BS2 is not assessed. gnomAD v4.1 reports zero homozygotes and gnomAD v2.1 is absent, but population allele-count data do not document a healthy adult individual, age, phenotype, penetrance, or the relevant zygosity state. No qualifying healthy-adult observation of this variant is provided. Therefore the generic BS2 requirement cannot be evaluated from the available evidence.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v4.1 reports 5/1,613,866 alleles and zero homozygotes; this is a population-database observation and does not identify a healthy adult individual or establish full penetrance expectations.
• No case evidence documents a healthy adult heterozygote, homozygote, or hemizygote with age and clinical status sufficient to apply BS2.

Still missing

• A documented healthy adult individual carrying the variant with sufficient age, phenotype, and zygosity information is needed.

Sources

Reasoning

BS4 cannot be assessed because the available report does not describe affected relatives who were tested and found not to carry the variant. It documents de novo occurrence in affected individuals, but that is not evidence of lack of segregation among affected family members.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• Hoischen et al. 2010: parental testing established de novo status, but the report provides no affected relatives with genotype results demonstrating non-segregation.

Still missing

• No documented affected relatives with genotype results are available for a non-segregation assessment.

Sources

Reasoning

BP2 requires observation in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observation in cis with a pathogenic variant in any inheritance pattern. The available case report documents heterozygous de novo SETBP1 c.2602G>A/p.D868N in affected individuals, but provides no pathogenic second allele, cis observation, trans observation, or evidence establishing full penetrance for this BP2 application. 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

• Hoischen et al. 2010 (PMID:20436468), Results and Table 1, reports G2602A/p.D868N in four affected individuals and describes the initially identified SETBP1 mutations as heterozygous and de novo when parental testing was available; it does not report a pathogenic second allele or cis/trans phase for the queried variant.

Still missing

• Parental or allele-specific testing demonstrating a pathogenic variant in trans or cis is unavailable.
• Evidence that the relevant dominant SETBP1 disorder is fully penetrant for this BP2 pathway is unavailable.

Sources

Reasoning

The governing BP5 rule requires the variant to be found in a case with an alternate molecular basis for disease. The literature documents somatic p.Asp868Asn in hematologic malignancy cohorts, but it does not establish that this patient's disease has an alternate molecular basis or that the queried case is one of those somatic cases. Therefore BP5 is not assessable from the available case-level evidence.

Rule

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

Evidence considered

• PMID:23222956 reports somatically acquired p.Asp868Asn in aCML, and PMID:23832012 reports p.Asp868Asn as a recurrent somatic mutation in myeloid malignancies; these are disease-cohort observations, not evidence that this patient's disease has an alternate molecular basis.
• COSMIC records the variant in somatic cancers, but the record does not identify an alternate molecular basis for the present case.

Still missing

• Patient-level diagnosis, molecular findings, and evidence for an alternate molecular cause are required to evaluate BP5.

Sources