|
PVS1
|
Original ACMG Summary
Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.
Caveats:
• Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7).
• Use caution interpreting LOF variants at the extreme 3’ end of a gene.
• Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact.
• Use caution in the presence of multiple transcripts.
Very Strong
- If a nonsense or frameshift variant introduces a premature stop codon between c.917 and c.1890, which is predicted to trigger nonsense mediated decay, and the exon is present in all 3 biologically-relevant transcripts (NM_181523.3, NM_181504.4, and NM_181524.2),, apply PVS1.
- If a canonical splicing variant is predicted to result in skipping of exon 8, 10, or 12, which would disrupt the frame and be predicted to trigger nonsense-mediated decay, apply PVS1.
- If a nonsense or frameshift variant introduces a premature stop codon between c.4 and c.916, which is predicted to trigger nonsense-mediated decay but the exon is present only in the MANE transcript NM_181523.3 but absent from NM_181504.4 and NM_181524.2, do not apply PVS1 at any strength in the context of this gene-disease relationship (MONDO:1060136). These variants have been published so far only in association with autosomal recessive agammaglobulinemia 7, which is outside the scope of the current specifications.
- Do not apply PVS1 at any strength for initiation codon variants. There are two known alternative start codons located after codon 306 of PIK3R1 in the MANE transcript encoding the p85α isoform (PMID: 28802037). Although there are known disease-causing variants upstream of these alternative start codons, indicating that disruption of the p85α isoform alone is sufficient to cause disease, these variants have so far been published only in association with agammaglobulinemia 7 with autosomal recessive inheritance, and are not considered applicable for the current specifications.
- If a canonical splicing variant is predicted to result in skipping of exon 1 (non-coding), exon 2 (which contains the start codon for the MANE transcript), exon 3 (in-frame), exon 4 (in-frame), exon 5 (in-frame), exon 6 (out-of-frame), or exon 7 (out-of-frame), do not apply PVS1 at any strength in the context of this gene-disease relationship (MONDO:1060136). These variants have been published so far only in association with autosomal recessive agammaglobulinemia 7, which is outside the scope of the current specifications.
- If a missense, synonymous, or intronic variant outside of the canonical splice sites has a SpliceAI score >0.2 and has been confirmed to cause complete or near-complete disruption of splicing within the mRNA in a study of patient RNA or minigene assay, avoid PP3 and PS3_Supporting and evaluate PVS1(RNA) instead (PMID: 37352859).
Modification Type:
Gene-specific
Strong
- If a nonsense or frameshift variant introduces a premature stop between codons 631 and 645, which is predicted to trigger nonsense mediated decay but located 45 nucleotides or less from the NMD prediction boundary, and the exon is present in all 3 biologically-relevant transcripts (NM_181523.3, NM_181504.4, and NM_181524.2), apply PVS1_Strong.
- If a nonsense or frameshift variant introduces a premature stop between codons 646 and 718 that is not predicted to trigger nonsense mediated decay but disrupts the cSH2 domain, apply PVS1_Strong.
- If a canonical splicing variant is predicted to result in skipping of in-frame exon 9 or 11 (which encode the nSH2 domain), in-frame exon 13 or 14, (which encode the iSH2 domain), or either in-frame exon 15 or out-of-frame exon 16, (which encode the cSH2 domain, PMID: 38043374), apply PVS1_Strong.
- If a nonsense or frameshift variant introduces a premature stop codon between c.4 and c.916, which is predicted to trigger nonsense-mediated decay but the exon is present only in the MANE transcript NM_181523.3 but absent from NM_181504.4 and NM_181524.2, do not apply PVS1 at any strength in the context of this gene-disease relationship (MONDO:1060136). These variants have been published so far only in association with autosomal recessive agammaglobulinemia 7, which is outside the scope of the current specifications.
- Do not apply PVS1 at any strength for initiation codon variants. There are two known alternative start codons located after codon 306 of PIK3R1 in the MANE transcript encoding the p85α isoform (PMID: 28802037). Although there are known disease-causing variants upstream of these alternative start codons, indicating that disruption of the p85α isoform alone is sufficient to cause disease, these variants have so far been published only in association with agammaglobulinemia 7 with autosomal recessive inheritance, and are not considered applicable for the current specifications.
- If a canonical splicing variant is predicted to result in skipping of exon 1 (non-coding), exon 2 (which contains the start codon for the MANE transcript), exon 3 (in-frame), exon 4 (in-frame), exon 5 (in-frame), exon 6 (out-of-frame), or exon 7 (out-of-frame), do not apply PVS1 at any strength in the context of this gene-disease relationship (MONDO:1060136). These variants have been published so far only in association with autosomal recessive agammaglobulinemia 7, which is outside the scope of the current specifications.
- If a missense, synonymous, or intronic variant outside of the canonical splice sites has a SpliceAI score >0.2 and has been confirmed to cause complete or near-complete disruption of splicing within the mRNA in a study of patient RNA or minigene assay, avoid PP3 and PS3_Supporting and evaluate PVS1(RNA) instead (PMID: 37352859).
Modification Type:
Gene-specific
Moderate
- If a nonsense or frameshift variant introduces a premature stop between codons 719 and 724 that is not predicted to trigger nonsense mediated decay or to disrupt the cSH2 domain, apply PVS1_Moderate.
- If a nonsense or frameshift variant introduces a premature stop codon between c.4 and c.916, which is predicted to trigger nonsense-mediated decay but the exon is present only in the MANE transcript NM_181523.3 but absent from NM_181504.4 and NM_181524.2, do not apply PVS1 at any strength in the context of this gene-disease relationship (MONDO:1060136). These variants have been published so far only in association with autosomal recessive agammaglobulinemia 7, which is outside the scope of the current specifications.
- Do not apply PVS1 at any strength for initiation codon variants. There are two known alternative start codons located after codon 306 of PIK3R1 in the MANE transcript encoding the p85α isoform (PMID: 28802037). Although there are known disease-causing variants upstream of these alternative start codons, indicating that disruption of the p85α isoform alone is sufficient to cause disease, these variants have so far been published only in association with agammaglobulinemia 7 with autosomal recessive inheritance, and are not considered applicable for the current specifications.
- If a canonical splicing variant is predicted to result in skipping of exon 1 (non-coding), exon 2 (which contains the start codon for the MANE transcript), exon 3 (in-frame), exon 4 (in-frame), exon 5 (in-frame), exon 6 (out-of-frame), or exon 7 (out-of-frame), do not apply PVS1 at any strength in the context of this gene-disease relationship (MONDO:1060136). These variants have been published so far only in association with autosomal recessive agammaglobulinemia 7, which is outside the scope of the current specifications.
- If a missense, synonymous, or intronic variant outside of the canonical splice sites has a SpliceAI score >0.2 and has been confirmed to cause complete or near-complete disruption of splicing within the mRNA in a study of patient RNA or minigene assay, avoid PP3 and PS3_Supporting and evaluate PVS1(RNA) instead (PMID: 37352859).
Modification Type:
Gene-specific
Not Applicable
Comments:
In the scope of APDS phenotype, the PVS1 code does not apply. Although loss of function variants in PIK3R1 are associated with APDS, these variants are limited to a single exon (exon 11), which encodes a portion of the inter-SH2 domain critical to the function of the p85α in the inhibition of p110 (PIK3CD) catalytic activity. Based on recommendations from Tayoun et al., 2018 (PMID: 30192042) and the location of known PIK3R1 variants associated with APDS in a single exon, loss of function cannot be considered an established mechanism of disease. In lieu of PVS1, PM4 can be applied to variants leading to in-frame deletion of exon 11 (see PM4 specifications). Although nonsense or frameshift (null) variants in PIK3R1 have been found, they have been associated with other disorders outside the APDS phenotype.
|
|
PS1
|
Original ACMG Summary
Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.
Example: Val->Leu caused by either G>C or G>T in the same codon.
Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.
Strong
- Use PS1 for missense variants when other variant was classified pathogenic by VCEP standards without using PS1. Beware of changes that impact splicing rather than the amino acid (based on RNA data or splicing predictors). Splicing predictions (by SpliceAI) should remain the same for WT and both mutant alleles.
- Use PS1 for canonical splice variants or variants predicted to disrupt splicing when the predicted impact is the same as a previously classified P / LP variant. The appropriate PS1 strength level is shown in the figure and table below (adapted from Table 2 in PMID: 37352859) based on the locations of the variant under assessment and the comparison variant within the splice donor or splice acceptor motif, as well as the classification of the comparison variant as Pathogenic (A) vs. LIkely Pathogenic (B). Please note that PS1 strength is reduced for variants disrupting a canonical splice site adjacent to an out-of-frame exon because PVS1 (very strong) has already been applied.
Modification Type:
General recommendation,Strength
Moderate
- Use PS1_Moderate for missense variants when the other variant was classified likely pathogenic by VCEP standards without using PS1. Beware of changes that impact splicing rather than the amino acid (based on RNA data or splicing predictors). Splicing predictions (by SpliceAI) should remain the same for WT and both mutant alleles.
- Use PS1 for canonical splice variants or variants predicted to disrupt splicing when the predicted impact is the same as a previously classified P / LP variant. The appropriate PS1 strength level is shown in the figure and table below (adapted from Table 2 in PMID: 37352859) based on the locations of the variant under assessment and the comparison variant within the splice donor or splice acceptor motif, as well as the classification of the comparison variant as Pathogenic (A) vs. LIkely Pathogenic (B). Please note that PS1 strength is reduced for variants disrupting a canonical splice site adjacent to an out-of-frame exon because PVS1 (very strong) has already been applied.
Modification Type:
General recommendation,Strength
Supporting
- Use PS1 for canonical splice variants or variants predicted to disrupt splicing when the predicted impact is the same as a previously classified P / LP variant. The appropriate PS1 strength level is shown in the figure and table below (adapted from Table 2 in PMID: 37352859) based on the locations of the variant under assessment and the comparison variant within the splice donor or splice acceptor motif, as well as the classification of the comparison variant as Pathogenic (A) vs. LIkely Pathogenic (B). Please note that PS1 strength is reduced for variants disrupting a canonical splice site adjacent to an out-of-frame exon because PVS1 (very strong) has already been applied.
Modification Type:
General recommendation,Strength
|
|
PS2
|
Original ACMG Summary
De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.
Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.
VCEP Specifications:
- For PS2, both maternity and paternity must be confirmed, with no family history of disease.
- Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.
- Please note that this code will also be used in place of PM6 for assumed de novo occurrences without confirmation of paternity and maternity.
Very Strong
- The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system:
- (A) If the proband scores greater than or equal to 4 but <6 phenotype points in the PS4 counting rubric and lacks genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.
- (B) If the proband scores greater than or equal to 6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.
- (C) If the proband scores greater than or equal to 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype highly specific for gene”.
Modification Type:
Disease-specific
Strong
- The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system:
- (A) If the proband scores greater than or equal to 4 but <6 phenotype points in the PS4 counting rubric and lacks genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.
- (B) If the proband scores greater than or equal to 6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.
- (C) If the proband scores greater than or equal to 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype highly specific for gene”.
Modification Type:
Disease-specific
Moderate
- The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system:
- (A) If the proband scores greater than or equal to 4 but <6 phenotype points in the PS4 counting rubric and lacks genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.
- (B) If the proband scores greater than or equal to 6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.
- (C) If the proband scores greater than or equal to 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype highly specific for gene”.
Modification Type:
Disease-specific
Supporting
- The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system:
- (A) If the proband scores greater than or equal to 4 but <6 phenotype points in the PS4 counting rubric and lacks genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.
- (B) If the proband scores greater than or equal to 6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.
- (C) If the proband scores greater than or equal to 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out variants in the PIK3CD locus, use the number of de novo points corresponding to “Phenotype highly specific for gene”.
Modification Type:
Disease-specific
|
|
PS3
|
Original ACMG Summary
Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.
Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.
Strong
- PS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and recapitulating the immunodeficiency phenotype (PMID: 26974159, PMID: 28632845).
- PS3 will be applied for an abnormal result in an approved in vitro assay with an OddsPath >18.7 (PMID: 31892348). One example has been found in the literature:
- Ratio of variant enrichment in pS6-high and/or pAKT-high T cells vs. pS6-low and/or pAKT-low T cells (PMID: 40543502)
Modification Type:
Disease-specific
Moderate
- PS3_Moderate will be applied for an abnormal result in an approved in vitro assay with at least 11 known pathogenic or benign variant controls (classified using these specifications, PMID: 31892348).
Modification Type:
Gene-specific
Supporting
- PS3_Supporting can be applied based on an abnormal result in an approved in vitro assay. Approved assay classes and specific assay instances include:
- Lipid kinase activity (PMID: 28167755)
- AKT kinase activity (PMID: 23810379, PMID: 25133428; PMID: 25488983; PMID: 28167755)
- Protein binding (PMID: 25488983)
- Conformational dynamics (PMID: 28167755)
- PS3_Supporting will not be applied to mRNA splicing assays showing exon 11 skipping that affect all or nearly all transcripts (PMID: 27221134, PMID: 25133428, PMID: 25488983), as these can be used in combination with a SpliceAI prediction of disruption of a splice donor / acceptor site to meet PVS1_Strong (RNA) instead.
Modification Type:
Disease-specific
|
|
PS4
|
Original ACMG Summary
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. See manuscript for detailed guidance.
Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.
Strong
- Use PS4 at the default level of strength when four or more probands meet the phenotype scoring criteria in Table 3 and genetic testing requirements described below.
- Please note that a proband used for PP4 or PP4_Moderate cannot be included in PS4.
- Point strength has been determined based on a survey of clinical experts, with the goal of quantifying the degree to which a proband’s phenotypes are specific to PIK3R1-related immunodeficiency. Phenotypes have been grouped into categories such as “respiratory findings” or “gastrointestinal disease” to prioritize the diversity of systems affected. Probands receive full points for at least one reported feature in the category. Points per category have been tailored to reward those considered most characteristic of and prevalent within patients with PIK3R1-related immunodeficiency.
- A proband must reach greater than or equal to 6 points in the phenotype scoring criteria above (Table 3) and, at minimum, a primary immunodeficiency or antibody gene testing panel must have identified no likely pathogenic or pathogenic variants in the PIK3CD locus in order to be counted toward PS4
- Genome or exome sequencing is acceptable in lieu of a gene panel
- For genes associated with autosomal recessive disorders, carrier status is acceptable.
- If no gene testing panel was performed, additional phenotypic features (reaching greater than or equal to 10 points) are required to count the proband toward PS4.
- Scoring of probands with previous genetic testing should be prioritized, particularly in the event of scoring historical or rare probands/variants.
- In order to be evaluated for this criterion, the variant must not meet BS1 or BA1.
Modification Type:
Disease-specific,Strength
Moderate
- Use PS4_Moderate when 2-3 probands meet the phenotype scoring criteria in Table 3 and genetic testing requirements described below.
- Please note that a proband used for PP4 or PP4_Moderate cannot be included in PS4_Moderate.
- Point strength has been determined based on a survey of clinical experts, with the goal of quantifying the degree to which a proband’s phenotypes are specific to PIK3R1-related immunodeficiency. Phenotypes have been grouped into categories such as “respiratory findings” or “gastrointestinal disease” to prioritize the diversity of systems affected. Probands receive full points for at least one reported feature in the category. Points per category have been tailored to reward those considered most characteristic of and prevalent within patients with PIK3R1-related immunodeficiency.
- A proband must reach greater than or equal to 6 points in the phenotype scoring criteria above (Table 3) and, at minimum, a primary immunodeficiency or antibody gene testing panel must have identified no likely pathogenic or pathogenic variants in the PIK3CD locus in order to be counted toward PS4
- Genome or exome sequencing is acceptable in lieu of a gene panel
- For genes associated with autosomal recessive disorders, carrier status is acceptable.
- If no gene testing panel was performed, additional phenotypic features (reaching greater than or equal to 10 points) are required to count the proband toward PS4.
- Scoring of probands with previous genetic testing should be prioritized, particularly in the event of scoring historical or rare probands/variants.
- In order to be evaluated for this criterion, the variant must not meet BS1 or BA1.
Modification Type:
Disease-specific,Strength
Supporting
- Use PS4_Supporting when one proband meets the phenotype scoring criteria in Table 3 and genetic testing requirements.
- Please note that a proband used for PP4 or PP4_Moderate cannot be included in PS4_Supporting.
- Point strength has been determined based on a survey of clinical experts, with the goal of quantifying the degree to which a proband’s phenotypes are specific to PIK3R1-related immunodeficiency. Phenotypes have been grouped into categories such as “respiratory findings” or “gastrointestinal disease” to prioritize the diversity of systems affected. Probands receive full points for at least one reported feature in the category. Points per category have been tailored to reward those considered most characteristic of and prevalent within patients with PIK3R1-related immunodeficiency.
- A proband must reach greater than or equal to 6 points in the phenotype scoring criteria above (Table 3) and, at minimum, a primary immunodeficiency or antibody gene testing panel must have identified no likely pathogenic or pathogenic variants in the PIK3CD locus in order to be counted toward PS4
- Genome or exome sequencing is acceptable in lieu of a gene panel
- For genes associated with autosomal recessive disorders, carrier status is acceptable.
- If no gene testing panel was performed, additional phenotypic features (reaching greater than or equal to 10 points) are required to count the proband toward PS4.
- Scoring of probands with previous genetic testing should be prioritized, particularly in the event of scoring historical or rare probands/variants.
- In order to be evaluated for this criterion, the variant must not meet BS1 or BA1.
Modification Type:
Disease-specific,Strength
|
|
PM1
|
Original ACMG Summary
Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.
|
|
PM2
|
Original ACMG Summary
Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.
Caveat: Population data for indels may be poorly called by next generation sequencing.
Supporting
- Used at PM2_Supporting strength.
- Applicable to variants with a total allele frequency <0.00000132 across all populations in gnomAD v4.1.0.
- Population allele frequency threshold has been determined using the Whiffin/Ware calculator (https://www.cardiodb.org/allelefrequencyapp/) and the following estimated parameters (with the prevalence estimated for primary immunodeficiency and the inheritance tailored to the autosomal dominant mode of inheritance):
- Prevalence: 1 in 4000
- Allelic heterogeneity: 1
- Genetic heterogeneity: 1
- Penetrance: 0.95
Modification Type:
Disease-specific,Strength
|
|
PM3
|
Original ACMG Summary
For recessive disorders, detected in trans with a pathogenic variant
Note: This requires testing of parents (or offspring) to determine phase.
Not Applicable
Comments:
Not applicable, as this code is specific to recessive disorders.
|
|
PM4
|
Original ACMG Summary
Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.
Moderate
- This code is mutually exclusive with PVS1 (PMID: 30192042) and PP3 (in order not to double-count in silico predictor data).
- Met at default strength (PM4) by an in-frame deletion resulting in a protein length change greater than or equal to 2 amino acids, if at least one of the deleted nucleotides is highly conserved (PhyloP score greater than or equal to 2.0).
- Met at default strength (PM4) by an in-frame insertion resulting in a protein length change greater than or equal to 2 amino acids, if at least one of the adjacent amino acids is highly conserved (PhyloP score greater than or equal to 2.0).
- Met at default strength (PM4) by a stop-loss variant resulting in the addition of 2 or more amino acids to the C-terminus.
- The region of the protein affected by the variant is key to consider. If only benign or likely benign variants are known to be located within this region, the region is polymorphic and PM4 does not apply.
Modification Type:
Disease-specific,Strength
|
|
PM5
|
Original ACMG Summary
Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.
Example: Arg156His is pathogenic; now you observe Arg156Cys.
Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.
Moderate
- Use PM5 at the default (moderate) strength level for a missense variant when another missense variant encoding a different amino acid change in the same codon was classified as Pathogenic by Antibody Deficiencies VCEP specifications for PIK3R1 without using PM5.
- Neither the variant of interest nor the Pathogenic comparison variant should be predicted to affect splicing (all SpliceAI Δ scores <0.2 for both variants).
- The variant of interest must have a higher Grantham score than the Pathogenic comparison variant (https://en.wikipedia.org/wiki/Amino_acid_replacement#Grantham's_distance).
- Do not apply at codon where any benign variants are known.
- In order to be evaluated for this criterion, the variant must not meet BS1 or BA1.
Modification Type:
Clarification
Supporting
- Use PM5_Supporting for a missense variant when another missense variant encoding a different amino acid change in the same codon was classified as Likely Pathogenic by Antibody Deficiencies VCEP specifications for PIK3R1 without using PM5.
- Neither the variant of interest nor the Likely Pathogenic comparison variant should be predicted to affect splicing (all SpliceAI Δ scores <0.2 for both variants).
- The variant of interest must have a higher Grantham score than the Likely Pathogenic comparison variant (https://en.wikipedia.org/wiki/Amino_acid_replacement#Grantham's_distance).
- Do not apply at codon where any benign variants are known.
- In order to be evaluated for this criterion, the variant must not meet BS1 or BA1.
Modification Type:
Clarification
Not Applicable
Comments:
Do not apply to missense variants given that missense variation is not a well-described contributor to PIK3R1-related APDS.
|
|
PM6
|
Original ACMG Summary
Assumed de novo, but without confirmation of paternity and maternity.
VCEP Specifications:
Not applicable. When the variant has apparent de novo origin and maternity and paternity are not confirmed but assumed, with no family history of disease, use the PS2 code in lieu of PM6.
Not Applicable
Comments:
Not applicable. When a variant has apparent de novo origin and paternity and maternity are suspected but not confirmed, use the PS2 code instead of PM6.
|
|
PP1
|
Original ACMG Summary
Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.
Note: May be used as stronger evidence with increasing segregation data.
VCEP Specifications:
Use ClinGen SVI recommendations for co-segregation criterion (PMID: 30311386) to determine the appropriate evidence strength based on LOD score calculated by counting the number of affected individuals in a family (minus the proband) that are positive for the variant.
Strong
- PP1 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.
- PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance.
- Met at the PP1_Strong level if the variant co-segregates with the affected phenotype across at least 4 meioses, either in one family or combined across multiple unrelated families.
Modification Type:
General recommendation,Strength
Moderate
- PP1 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.
- PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance.
- Met at the PP1_Moderate level if the variant co-segregates with the affected phenotype across the proband and two affected first degree relatives, across the proband and one affected second degree relative, or across two unrelated probands and one affected first degree relative each (2 meioses total).
Modification Type:
General recommendation,Strength
Supporting
- PP1 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.
- PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance.
- Met at the Supporting level if the variant co-segregates with the affected phenotype across the proband and one affected first degree relative (1 meiosis).
Modification Type:
General recommendation,Strength
|
|
PP2
|
Original ACMG Summary
Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.
Not Applicable
Comments:
Does not apply. The gnomAD v2.1.1 missense Z score for PIK3R1 (Z = 2.72) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in PIK3R1.
|
|
PP3
|
Original ACMG Summary
Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).
Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.
Supporting
- PP3 is met by a missense variant with both REVEL greater than or equal to 0.644 and CADD greater than or equal to 26.0.
- The VCEP will try to determine during the pilot whether higher strength levels of PP3 are appropriate for PIK3R1 variants.
- PP3 is met by missense, synonymous, or intronic variants outside the +/-1,2 dinucleotide positions that are predicted as damaging using SpliceAI (Δ score for donor gain, donor loss, acceptor gain, or acceptor loss score greater than or equal to 0.2, PMID: 37352859).
- Splice impact must be assessed for every missense, small in-frame insertion and/or deletion, synonymous, or intronic variant.
Modification Type:
Disease-specific
|
|
PP4
|
Original ACMG Summary
Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.
Moderate
- PP4_Moderate is met when a proband scores greater than or equal to 10 phenotype points in the PS4 counting rubric AND has genotyping that did not identify an alternative basis for disease at the PIK3CD locus AND has patient cell-based evidence of abnormally high activity of the disease-relevant PI3K delta pathway.
- Patient cell-based experiments generally isolate T cells from affected and unaffected patients and often perform stimulation by anti-CD3 and anti-CD28 antibodies, followed by assessment of PI3K delta pathway function using western blotting or flow cytometry in combination with phospho-specific antibodies. These methods detect levels of AKT phosphorylation at Ser473 or Thr308 (PMID: 25133428, PMID: 25488983) and/or S6 phosphorylation at Ser235/Ser236 or Ser240/Ser244 (PMID: 25488983). Phosphorylation may be upregulated in cells from the affected patient relative to the healthy control.
- Please note that a proband used for PP4 or PP4_Moderate cannot be included in PS4.
- In order to be evaluated for this criterion, the variant must not meet BS1 or BA1.
Modification Type:
Disease-specific
Supporting
- PP4 is met when a proband scores greater than or equal to 10 phenotype points in the PS4 counting rubric (Table 3) AND has genotyping that did not identify an alternative basis for disease at the PIK3CD locus.
- Please note that a proband used for PP4 or PP4_Moderate cannot be included in PS4.
- In order to be evaluated for this criterion, the variant must not meet BS1 or BA1.
Modification Type:
Disease-specific
|
|
PP5
|
Original ACMG Summary
Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.
Not Applicable
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
PubMed : 29543229
|
|
BA1
|
Original ACMG Summary
Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.
Stand Alone
- Applicable to variants with a GrpMax filtering allele frequency greater than or equal to 0.00316 in gnomAD v4.1.0.
- If GrpMax filtering allele frequency is not listed for the variant, this code is applicable to the maximum allele frequency among the five major continental populations (African / African-American, East Asian, European non-Finnish, Latino / Admixed-American, or South Asian).
- Maximum credible population allele frequency threshold determined using Whiffin/Ware calculator (https://www.cardiodb.org/allelefrequencyapp/) and the following estimated parameters:
- Inheritance: biallelic (in order to generate a higher / more stringent threshold that would rule out a variant as a credible cause of either autosomal dominant or autosomal recessive disease)
- Prevalence: 1 in 100,000 (calculated at 1 in 7,500,000 based on the USIDNET cohort, PMID: 34352450, but adjusted based on expectation that cases are actually more common, resulting in a much more frequent / more aggressive prevalence estimate of 1 in 100,000 in order to generate a more conservative estimate)
- Allelic heterogeneity: 1
- Genetic heterogeneity: 1
- Penetrance: 1
- Please note that the values cited above (e.g. complete penetrance) are not asserted to be true for this condition. Rather, they are used in order to derive the most rigorous thresholds possible for a disease for which there are incomplete population epidemiology data.
Default Point Value:
Not Applicable
Modification Type:
Disease-specific
|
|
BS1
|
Original ACMG Summary
Allele frequency is greater than expected for disorder.
Strong
- Applicable to variants with a GrpMax filtering allele frequency greater than or equal to 0.000316 in gnomAD v4.1.0.
- If GrpMax filtering allele frequency is not listed for the variant, this code is applicable to the maximum allele frequency among the five major continental populations (African / African-American, East Asian, European non-Finnish, Latino / Admixed-American, or South Asian).
- The BS1 threshold was derived by decreasing the BA1 cutoff (greater than or equal to 0.00316) by one order of magnitude.
Modification Type:
Disease-specific
|
|
BS2
|
Original ACMG Summary
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.
Not Applicable
Comments:
Does not apply due to incomplete penetrance and variable expressivity of disease.
|
|
BS3
|
Original ACMG Summary
Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
Strong
- BS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and failing to recapitulate the phenotype. Animal models will be reviewed on a case-by-case basis by the VCEP to determine the appropriate strength level.
- BS3 will be applied for a non-damaging result in an approved in vitro assay with an OddsPath <0.053 (PMID: 31892348). No such assays have yet been identified in the PIK3R1 literature.
Modification Type:
Disease-specific
Moderate
- BS3_Moderate will be applied for a non-damaging result in an approved in vitro assay with at least 11 known pathogenic or benign variant controls (classified using these specifications, PMID: 31892348).
- While one such in vitro assay has been identified in the PIK3R1 literature (PMID: 40543502), the authors have recommended a non-damaging result in their assay of the ratio of enrichment of the variant in pS6-high and/or pAKT-high T cells vs. pS6-low and/or pAKT-low T cells for BS3_Supporting, based on their calculated OddsPath of 0.25.
Modification Type:
Disease-specific
Supporting
- BS3_Supporting can be applied based on a normal result in at least two different approved in vitro assays. Approved assay classes and specific assay instances include:
- Lipid kinase activity (PMID: 28167755)
- AKT kinase activity (PMID: 23810379, PMID: 25133428; PMID: 25488983; PMID: 28167755)
- Protein binding (PMID: 25488983)
- Conformational dynamics (PMID: 28167755)
- BS3_Supporting can be applied based on a normal result in the following approved in vitro assay:
- Ratio of variant enrichment in pS6-high and/or pAKT-high T cells vs. pS6-low and/or pAKT-low T cells (PMID: 40543502, OddsPath 0.25)
Modification Type:
Disease-specific
|
|
BS4
|
Original ACMG Summary
Lack of segregation in affected members of a family.
Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation.
Strong
- Use caution in case the phenotype is not highly specific. Each of the affected family members lacking the variant must reach at least 6 points in the PS4 scoring table to be considered for this code.
- BS4 is met at the default level of strength when at least 2 affected family members do not harbor the variant.
Modification Type:
Disease-specific,Strength
Supporting
- Use caution in case the phenotype is not highly specific. The affected family member lacking the variant must reach at least 6 points in the PS4 scoring table to be considered for this code.
- BS4_Supporting is met if only 1 affected family member does not harbor the variant.
Modification Type:
Disease-specific,Strength
|
|
BP1
|
Original ACMG Summary
Missense variant in a gene for which primarily truncating variants are known to cause disease.
Not Applicable
Comments:
Not applicable, as pathogenic PIK3R1 variants are not limited to truncating variants, but can be missense as well.
|
|
BP2
|
Original ACMG Summary
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.
Not Applicable
Comments:
Do not use this criterion.
BP2 is considered not applicable, as the field at present does not understand all of the potential allelic mechanisms associated with PIK3R1 variants, so that the possibility of diverse combinatorial variant effects cannot be excluded. This has been described for other inborn errors of immunity genes.
|
|
BP3
|
Original ACMG Summary
In frame-deletions/insertions in a repetitive region without a known function.
Not Applicable
Comments:
Not applicable, as repetitive regions of unknown function are not known within PIK3R1.
|
|
BP4
|
Original ACMG Summary
Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)
Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.
Supporting
- BP4 is met by a missense variant with both REVEL less than or equal to 0.290 and CADD less than or equal to 21.5, as well as SpliceAI Δ scores for donor gain, donor loss, acceptor gain, and acceptor loss <0.1.
- The VCEP will try to determine during the pilot whether higher strength levels of BP4 are appropriate for PIK3R1 variants.
- BP4 is met by synonymous or intronic variants not predicted to impact splicing by SpliceAI (Δ scores for donor gain, donor loss, acceptor gain, and acceptor loss <0.1 (PMID: 37352859)).
- BP4 can be used in combination with BP7 for synonymous or intronic variants without being considered double-counting (PMID: 37352859).
Modification Type:
Disease-specific
|
|
BP5
|
Original ACMG Summary
Variant found in a case with an alternate molecular basis for disease.
Supporting
At least 2 cases with an alternative molecular basis for disease are required to mitigate the reliance on assertions of variant pathogenicity in genes outside of the purview of the Antibody Deficiencies VCEP.
Modification Type:
Disease-specific
|
|
BP6
|
Original ACMG Summary
Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.
Not Applicable
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
PubMed : 29543229
|
|
BP7
|
Original ACMG Summary
A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.
Strong
- BP7_Strong (RNA) is met by variants with experimental evidence of no impact on splicing.
Modification Type:
Clarification
Supporting
- Met by synonymous variants (except those located in the first nucleotide of an exon or last 3 nucleotides of an exon) not predicted to impact splicing by SpliceAI (all Δ scores <0.1, PMID: 37352859).
- Met by intronic variants (outside of the +1 to +6 and -1 to -20 positions relative to the exon) not predicted to impact splicing by SpliceAI (all Δ scores <0.1, PMID: 37352859).
- BP7 can be used in combination with BP4 for synonymous or intronic variants without being considered double-counting (PMID: 37352859).
Modification Type:
Clarification
|
