PD-EP specifications of the ACMG/AMP guidelines for ITGA2B/ITGB3 variants in relation to GT
| ACMG/AMP criteria . | Original ACMG/AMP criteria summary . | Specification . | Standalone . | Very strong . | Strong . | Moderate . | Supporting . | Comments . |
|---|---|---|---|---|---|---|---|---|
| PVS1 | Null variant in a gene where LOF is a known mechanism of disease | Gene specific | N/A | Per modified PVS1 decision tree (Figure 2) with specified regions critical to protein function | N/A | Three critical regions: (1) the cytoplasmic domain of the β3 subunit, (2) the transmembrane domains of αIIbβ3, and (3) the extracellular domains of αIIbβ3 involved in ligand binding | ||
| PS1 | Same amino acid change as a previously established pathogenic variant regardless of nucleotide change | None | N/A | N/A | Use per original guidelines | N/A | N/A | Both variants must be classified using ITGA2B/ITGB3-specified guidelines |
| PS2 | De novo in a patient (maternity and paternity confirmed) with the disease and no family history | Disease specific, strength | N/A | Use the SVI-recommended point system (supplemental Table 1) based on confirmed vs assumed maternity/paternity status, the number of de novo probands, and the phenotypic consistency | (1) To be scored at phenotype highly specific for gene, the patient must meet the PP4 criteria; (2) proband must harbor an additional pathogenic or likely pathogenic variant with the de novo variant | |||
| PS3 | Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product | Gene specific, strength | N/A | N/A | <5% surface expression of αIIbβ3 (measured by flow cytometry or western blot) and/or significantly reduced function (ie, minimal binding to fibrinogen or ligand mimetic antibodies) | 5%-25% surface expression of αIIbβ3 (measured by flow cytometry or western blot) | N/A | All assays must be performed in a heterologous cell line or animal model with expression of the variant of interest from 1 gene (eg, ITGA2B) and the wild type of the opposite gene (eg, ITGB3) |
| PS4 | The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls | N/A | Rule does not apply because of the rarity of disorder and lack of appropriate studies | |||||
| PM1 | Located in a mutational hotspot and/or critical and well-established functional domain without benign variation | N/A | Rule does not apply because of highly polymorphic nature of genes and no known hotspots | |||||
| PM2 | Absent in population databases (or at extremely low frequency if recessive) | Disease specific | N/A | N/A | N/A | N/A | <1 in 10 000 (0.0001) alleles in all gnomAD continental population cohorts; not present in homozygous state | |
| PM3 | For recessive disorders, detected in trans with a pathogenic variant | Disease-specific, Strength | N/A | Use SVI point recommendations (supplemental Table 2); strength may be adjusted based upon confirmation of variant phasing and classification of the second variant | Both variants must be below the PM2 threshold, and both must be classified using ITGA2B/ITGB3-specified guidelines | |||
| PM4 | Protein length changes as a result of inframe deletions/insertions in a nonrepeat region or stop-loss variants | None | N/A | N/A | N/A | Use per original guidelines | N/A | |
| PM5 | Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before | Strength | N/A | N/A | N/A | Use per original guidelines | Novel missense change at an amino acid residue where a different missense change determined to be likely pathogenic has been seen before | Both variants must be classified using ITGA2B/ITGB3-specified guidelines |
| PM6 | De novo in a patient (maternity and paternity not confirmed) with the disease and no family history | Disease specific, strength | N/A | Use the SVI-recommended point system (supplemental Table 1) based on confirmed vs assumed maternity/paternity status, the number of de novo probands, and the phenotypic consistency | (1) To be scored at phenotype highly specific for gene, the patient must meet the PP4 criteria; (2) proband must harbor an additional pathogenic or likely pathogenic variant with the de novo variant | |||
| PP1 | Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease | Disease specific, strength | N/A | N/A | Segregation in proband plus ≥3 affected relatives | Segregation in proband plus 2 affected relatives | Segregation in proband plus 1 affected relative | Only individuals well documented as having both GT and the variant (phenotype positive, genotype positive) are included when counting segregations; the individuals must be homozygous or compound heterozygous with variants confirmed in trans |
| PP2 | Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease | N/A | Rule does not apply because benign missense variants are not rare, and the missense constraint z scores for ITGA2B and ITGB3 are <3.09 | |||||
| PP3 | Multiple lines of computational evidence support a deleterious effect on the gene or gene product | Gene specific | N/A | N/A | N/A | N/A | REVEL score of ≥0.7 | |
| PP4 | Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology | Disease specific, strength | N/A | N/A | Meeting criteria (1) and (2) as described for PP4_moderate as well as (3a) absent or reduced (<25% compared with normal) surface expression of αIIbβ3 or (3b) functional flow cytometry to demonstrate lack of fibrinogen-mediated platelet binding to activated αIIbβ3 and (4) full sequencing (all exons and intron-exon boundaries) of both ITGA2B and ITGB3 | (1) At least 1 mucocutaneous bleeding phenotype (eg, epistaxis, petechiae, easy bruising, oral bleeding, gastrointestinal bleeding, or menorrhagia) and (2) normal ristocetin-induced platelet agglutination but minimal to no platelet aggregation with all tested physiological agonists, at a minimum of 2 agonists tested | N/A | Surface expression of αIIbβ3 must be established by either flow cytometry or western blotting |
| PP5 | Reputable source recently reports variant as pathogenic | N/A | Do not use this rule as per SVI recommendations | |||||
| BA1 | Allele frequency is greater than expected for disorder | Disease specific | MAF ≥0.0024 in a gnomAD population cohort | N/A | N/A | N/A | N/A | Cohort must be a continental population with a minimum number of 2000 alleles and the variant present in ≥5 alleles |
| BS1 | Allele frequency is greater than expected for the disorder | Disease specific | N/A | N/A | MAF of 0.00158-0.0024 in a gnomAD population cohort | N/A | N/A | Cohort must have a minimum number of 2000 alleles and the variant present in ≥5 alleles |
| BS2 | 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 | Disease specific | N/A | N/A | Variant observed in >1 unaffected homozygote | N/A | N/A | Unaffected individuals must have been assessed for GT by at least platelet aggregometry |
| BS3 | Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing | Gene specific | N/A | N/A | (1) Normal aggregometry in a transgenic mouse model or (2) both normal surface expression (>75%) and normal fibrinogen binding in either a heterologous cell line or animal model | N/A | N/A | Expression must be measured by flow cytometry or western blot |
| BS4 | Lack of segregation in affected members of a family | Disease specific | N/A | N/A | Lack of segregation in ≥1 affected family member who is well documented as having GT | N/A | N/A | This code should only be applied for phenotype-positive (meeting PP4), genotype-negative family members |
| BP1 | Missense variant in a gene for which primarily truncating variants are known to cause disease | N/A | Rule does not apply because truncating variants do not predominate, and missense variants are a known cause of disease | |||||
| BP2 | Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant | None | N/A | N/A | N/A | N/A | Use per original guidelines | This rule can be applied when the variant of interest is observed in cis with a pathogenic variant |
| BP3 | Inframe deletions/insertions in a repetitive region without a known function | None | N/A | N/A | N/A | N/A | Use per original guidelines | ITGB3 has 3 repetitive microsatellite regions, and there are no known repetitive regions in the ITGA2B gene |
| BP4 | Multiple lines of computational evidence suggest no impact on gene/gene product | Gene specific | N/A | N/A | N/A | N/A | REVEL score ≤0.25 | |
| BP5 | Variant found in a case with an alternative molecular basis for disease | N/A | Rule does not apply because an individual with an alternative basis for disease can still be a carrier of an unrelated pathogenic variant in ITGA2B/ITGB3 | |||||
| BP6 | Reputable source recently reports variant as benign | N/A | Do not use this rule as per SVI recommendations | |||||
| BP7 | A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence or the creation of a new splice site AND the nucleotide is not highly conserved | None | N/A | N/A | N/A | N/A | Use per original guidelines | |
| ACMG/AMP criteria . | Original ACMG/AMP criteria summary . | Specification . | Standalone . | Very strong . | Strong . | Moderate . | Supporting . | Comments . |
|---|---|---|---|---|---|---|---|---|
| PVS1 | Null variant in a gene where LOF is a known mechanism of disease | Gene specific | N/A | Per modified PVS1 decision tree (Figure 2) with specified regions critical to protein function | N/A | Three critical regions: (1) the cytoplasmic domain of the β3 subunit, (2) the transmembrane domains of αIIbβ3, and (3) the extracellular domains of αIIbβ3 involved in ligand binding | ||
| PS1 | Same amino acid change as a previously established pathogenic variant regardless of nucleotide change | None | N/A | N/A | Use per original guidelines | N/A | N/A | Both variants must be classified using ITGA2B/ITGB3-specified guidelines |
| PS2 | De novo in a patient (maternity and paternity confirmed) with the disease and no family history | Disease specific, strength | N/A | Use the SVI-recommended point system (supplemental Table 1) based on confirmed vs assumed maternity/paternity status, the number of de novo probands, and the phenotypic consistency | (1) To be scored at phenotype highly specific for gene, the patient must meet the PP4 criteria; (2) proband must harbor an additional pathogenic or likely pathogenic variant with the de novo variant | |||
| PS3 | Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product | Gene specific, strength | N/A | N/A | <5% surface expression of αIIbβ3 (measured by flow cytometry or western blot) and/or significantly reduced function (ie, minimal binding to fibrinogen or ligand mimetic antibodies) | 5%-25% surface expression of αIIbβ3 (measured by flow cytometry or western blot) | N/A | All assays must be performed in a heterologous cell line or animal model with expression of the variant of interest from 1 gene (eg, ITGA2B) and the wild type of the opposite gene (eg, ITGB3) |
| PS4 | The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls | N/A | Rule does not apply because of the rarity of disorder and lack of appropriate studies | |||||
| PM1 | Located in a mutational hotspot and/or critical and well-established functional domain without benign variation | N/A | Rule does not apply because of highly polymorphic nature of genes and no known hotspots | |||||
| PM2 | Absent in population databases (or at extremely low frequency if recessive) | Disease specific | N/A | N/A | N/A | N/A | <1 in 10 000 (0.0001) alleles in all gnomAD continental population cohorts; not present in homozygous state | |
| PM3 | For recessive disorders, detected in trans with a pathogenic variant | Disease-specific, Strength | N/A | Use SVI point recommendations (supplemental Table 2); strength may be adjusted based upon confirmation of variant phasing and classification of the second variant | Both variants must be below the PM2 threshold, and both must be classified using ITGA2B/ITGB3-specified guidelines | |||
| PM4 | Protein length changes as a result of inframe deletions/insertions in a nonrepeat region or stop-loss variants | None | N/A | N/A | N/A | Use per original guidelines | N/A | |
| PM5 | Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before | Strength | N/A | N/A | N/A | Use per original guidelines | Novel missense change at an amino acid residue where a different missense change determined to be likely pathogenic has been seen before | Both variants must be classified using ITGA2B/ITGB3-specified guidelines |
| PM6 | De novo in a patient (maternity and paternity not confirmed) with the disease and no family history | Disease specific, strength | N/A | Use the SVI-recommended point system (supplemental Table 1) based on confirmed vs assumed maternity/paternity status, the number of de novo probands, and the phenotypic consistency | (1) To be scored at phenotype highly specific for gene, the patient must meet the PP4 criteria; (2) proband must harbor an additional pathogenic or likely pathogenic variant with the de novo variant | |||
| PP1 | Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease | Disease specific, strength | N/A | N/A | Segregation in proband plus ≥3 affected relatives | Segregation in proband plus 2 affected relatives | Segregation in proband plus 1 affected relative | Only individuals well documented as having both GT and the variant (phenotype positive, genotype positive) are included when counting segregations; the individuals must be homozygous or compound heterozygous with variants confirmed in trans |
| PP2 | Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease | N/A | Rule does not apply because benign missense variants are not rare, and the missense constraint z scores for ITGA2B and ITGB3 are <3.09 | |||||
| PP3 | Multiple lines of computational evidence support a deleterious effect on the gene or gene product | Gene specific | N/A | N/A | N/A | N/A | REVEL score of ≥0.7 | |
| PP4 | Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology | Disease specific, strength | N/A | N/A | Meeting criteria (1) and (2) as described for PP4_moderate as well as (3a) absent or reduced (<25% compared with normal) surface expression of αIIbβ3 or (3b) functional flow cytometry to demonstrate lack of fibrinogen-mediated platelet binding to activated αIIbβ3 and (4) full sequencing (all exons and intron-exon boundaries) of both ITGA2B and ITGB3 | (1) At least 1 mucocutaneous bleeding phenotype (eg, epistaxis, petechiae, easy bruising, oral bleeding, gastrointestinal bleeding, or menorrhagia) and (2) normal ristocetin-induced platelet agglutination but minimal to no platelet aggregation with all tested physiological agonists, at a minimum of 2 agonists tested | N/A | Surface expression of αIIbβ3 must be established by either flow cytometry or western blotting |
| PP5 | Reputable source recently reports variant as pathogenic | N/A | Do not use this rule as per SVI recommendations | |||||
| BA1 | Allele frequency is greater than expected for disorder | Disease specific | MAF ≥0.0024 in a gnomAD population cohort | N/A | N/A | N/A | N/A | Cohort must be a continental population with a minimum number of 2000 alleles and the variant present in ≥5 alleles |
| BS1 | Allele frequency is greater than expected for the disorder | Disease specific | N/A | N/A | MAF of 0.00158-0.0024 in a gnomAD population cohort | N/A | N/A | Cohort must have a minimum number of 2000 alleles and the variant present in ≥5 alleles |
| BS2 | 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 | Disease specific | N/A | N/A | Variant observed in >1 unaffected homozygote | N/A | N/A | Unaffected individuals must have been assessed for GT by at least platelet aggregometry |
| BS3 | Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing | Gene specific | N/A | N/A | (1) Normal aggregometry in a transgenic mouse model or (2) both normal surface expression (>75%) and normal fibrinogen binding in either a heterologous cell line or animal model | N/A | N/A | Expression must be measured by flow cytometry or western blot |
| BS4 | Lack of segregation in affected members of a family | Disease specific | N/A | N/A | Lack of segregation in ≥1 affected family member who is well documented as having GT | N/A | N/A | This code should only be applied for phenotype-positive (meeting PP4), genotype-negative family members |
| BP1 | Missense variant in a gene for which primarily truncating variants are known to cause disease | N/A | Rule does not apply because truncating variants do not predominate, and missense variants are a known cause of disease | |||||
| BP2 | Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant | None | N/A | N/A | N/A | N/A | Use per original guidelines | This rule can be applied when the variant of interest is observed in cis with a pathogenic variant |
| BP3 | Inframe deletions/insertions in a repetitive region without a known function | None | N/A | N/A | N/A | N/A | Use per original guidelines | ITGB3 has 3 repetitive microsatellite regions, and there are no known repetitive regions in the ITGA2B gene |
| BP4 | Multiple lines of computational evidence suggest no impact on gene/gene product | Gene specific | N/A | N/A | N/A | N/A | REVEL score ≤0.25 | |
| BP5 | Variant found in a case with an alternative molecular basis for disease | N/A | Rule does not apply because an individual with an alternative basis for disease can still be a carrier of an unrelated pathogenic variant in ITGA2B/ITGB3 | |||||
| BP6 | Reputable source recently reports variant as benign | N/A | Do not use this rule as per SVI recommendations | |||||
| BP7 | A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence or the creation of a new splice site AND the nucleotide is not highly conserved | None | N/A | N/A | N/A | N/A | Use per original guidelines | |
MAF, minor allele frequency; N/A, not applicable.