Criteria Specification (CSpec) Registry is intended to provide access to the Criteria Specifications used and applied by ClinGen Variant Curation Expert Panels and biocurators in the classification of variants.
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Criteria & Strength Specifications
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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. Stand Alone
Very Strong
Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:
Modification Type:
General recommendation,Gene-specific
Strong
Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with two specifications:
Modification Type:
General recommendation,Gene-specific
Moderate
Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:
Modification Type:
General recommendation
Supporting
Instructions:
See attached PVS1 flowchart. Not Applicable
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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. Stand Alone
Very Strong
Strong
Applicable for a same amino acid change if previously established variant is classified as pathogenic by SCID VCEP specifications for FOXN1. Can also be applied for variants with the same predicted splicing event as a known Pathogenic variant (as classified by the SCID VCEP specifications for FOXN1), only when the strength of the prediction for the variant under assessment is of similar or higher strength than the strength of the prediction for the comparison (Likely) Pathogenic variant (i.e., per in silico splicing tool SpliceAI). See attached instructions (from Table 2 of PMID: 37352859) for determining when PS1 should be applied at PS1_Strong, _Moderate, or _Supporting.
Modification Type:
Gene-specific
Moderate
Applicable for a same amino acid change if previously established variant is classified as likely pathogenic by SCID VCEP specifications for FOXN1. Can also be applied for variants with the same predicted splicing event as a known (Likely) Pathogenic variant (as classified by the SCID VCEP specifications for FOXN1), only when the strength of the prediction for the variant under assessment is of similar or higher strength than the strength of the prediction for the comparison (Likely) Pathogenic variant (i.e., per in silico splicing tool SpliceAI). See attached instructions (from Table 2 of PMID: 37352859) for determining when PS1 should be applied at PS1_Strong, _Moderate, or _Supporting.
Modification Type:
Gene-specific,Strength
Supporting
Can be applied for variants with the same predicted splicing event as a known (Likely) Pathogenic variant (as classified by the SCID VCEP specifications for FOXN1), only when the strength of the prediction for the variant under assessment is of similar or higher strength than the strength of the prediction for the comparison (Likely) Pathogenic variant (i.e., per in silico splicing tool SpliceAI). See attached instructions (from Table 2 of PMID: 37352859) for determining when PS1 should be applied at PS1_Strong, _Moderate, or _Supporting.
Modification Type:
Gene-specific,Strength
Not Applicable
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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. Stand Alone
Very Strong
Use ClinGen SVI recommendations for de novo criteria (see instructions below).
Modification Type:
General recommendation,Gene-specific
Strong
Use ClinGen SVI recommendations for de novo criteria (see instructions below).
Modification Type:
General recommendation,Gene-specific
Moderate
Use ClinGen SVI recommendations for de novo criteria (see instructions below).
Modification Type:
General recommendation,Gene-specific
Supporting
Use ClinGen SVI recommendations for de novo criteria (see instructions below).
Modification Type:
General recommendation,Gene-specific
Instructions:
See attached scoring guide. The following guidelines should be used when determining phenotypic consistency of each proband:
Not Applicable
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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. Stand Alone
Very Strong
Strong
PS3 may be applied at the strong level for evidence from an animal model expressing the variant of interest and recapitulating FOXN1 deficiency (i.e. a mouse model with T cell lymphopenia).
Modification Type:
Gene-specific
Moderate
PS3 may be applied at the moderate level based on a luciferase assay showing reduced (<50%) activity, as part of a validated assay with pathogenic and benign controls (PMID: 37419334).
Modification Type:
Gene-specific,Strength
Supporting
PS3 may be applied at the supporting level based on a luciferase assay, without sufficient validation controls, showing reduced (<50%) activity, such as those reported in PMIDs: 31566583, 33464451, 34860543.
Modification Type:
Gene-specific,Strength
Not Applicable
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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. Stand Alone
Very Strong
Sum of case scores ≥8 points (see instructions below)
Modification Type:
Disease-specific
Strong
Sum of case scores 4-7.75 points (see instructions below)
Modification Type:
Disease-specific
Moderate
Sum of case scores 2-3.75 points (see instructions below)
Modification Type:
Disease-specific
Supporting
Sum of case scores 1-1.75 points (see instructions below)
Modification Type:
Disease-specific
Instructions:
Evaluate each unrelated heterozygous affected individual with the attached Affected Observations Scoring Guide and sum points across all probands.
Not Applicable
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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.
Stand Alone
Very Strong
Strong
Moderate
Applicable for variants in the DNA binding forkhead domain (amino acids 270-367), which is a well-established functional domain (Newman et al., 2020; PMID: 31914405) of FOXN1 with low tolerance for benign variation.
Modification Type:
Gene-specific
Supporting
Not Applicable
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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. Stand Alone
Very Strong
Strong
Moderate
Supporting
gnomAD Grpmax filtering allele frequency ≤0.00002412
Modification Type:
Gene-specific
Instructions:
This value is based on the HMAF of NM_001369369.1(FOXN1):c.1585del (p.Leu529fs), the most common pLOF variant present in gnomADv4.0.0 that classifies as Pathogenic based on specified guidelines. Not Applicable
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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. Stand Alone
Very Strong
Sum of case scores ≥8 points (see instructions below)
Modification Type:
General recommendation
Strong
Sum of case scores 4-7.75 points (see instructions below)
Modification Type:
General recommendation
Moderate
Sum of case scores 2-3.75 points (see instructions below)
Modification Type:
General recommendation
Supporting
Sum of case scores 1-1.75 points (see instructions below)
Modification Type:
General recommendation
Instructions:
Evaluate each unrelated homozygous or compound heterozygous affected individual with the attached Affected Observations Scoring Guide and sum points across all probands.
Not Applicable
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PM4 | ||||
Original ACMG Summary
Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.
Stand Alone
Very Strong
Strong
Moderate
Additional requirement that when applied to deletion variants, the deleted region must contain a known pathogenic or likely pathogenic variant that is not predicted/observed to alter splicing.
Modification Type:
Gene-specific
Supporting
Additional requirement that when applied to deletion variants, the deleted region must contain a known VUS variant that is not predicted/observed to alter splicing.
Modification Type:
Gene-specific,Strength
Not Applicable
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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. Stand Alone
Very Strong
Strong
Moderate
Applicable if previously established variant is classified as pathogenic by SCID VCEP specifications for FOXN1. Previously established variant must be classified by SCID VCEP specifications for FOXN1.
Modification Type:
Gene-specific
Supporting
Applicable if previously established variant is classified as likely pathogenic by SCID VCEP specifications for FOXN1. Previously established variant must be classified by SCID VCEP specifications for FOXN1.
Modification Type:
Gene-specific,Strength
Not Applicable
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PM6 | ||||
Original ACMG Summary
Assumed de novo, but without confirmation of paternity and maternity.
Stand Alone
Very Strong
Strong
Use ClinGen SVI recommendations for de novo criteria (see instructions below).
Modification Type:
General recommendation,Gene-specific
Moderate
Use ClinGen SVI recommendations for de novo criteria (see instructions below).
Modification Type:
General recommendation,Gene-specific
Supporting
Use ClinGen SVI recommendations for de novo criteria (see instructions below).
Modification Type:
General recommendation,Gene-specific
Instructions:
See attached scoring guide. The following guidelines should be used when determining phenotypic consistency of each proband:
Not Applicable
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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. Stand Alone
Very Strong
Strong
Can be applied when there is a 32:1 likelihood ratio (LOD score ≥1.5, summed across all families with segregation evidence) per recommendations from PMID: 30311386 Table 4a. See instructions for calculating the estimated LOD score.
Modification Type:
General recommendation
Moderate
Can be applied when there is a 16:1 likelihood ratio (LOD score 1.2-<1.5, summed across all families with segregation evidence) per recommendations from PMID: 30311386 Table 4a. See instructions for calculating the estimated LOD score.
Modification Type:
General recommendation
Supporting
Can be applied when there is a 4:1 likelihood ratio (LOD score 0.6-<1.2, summed across all families with segregation evidence) per recommendations from PMID: 30311386 Table 4a. See instructions for calculating the estimated LOD score.
Modification Type:
General recommendation
Instructions:
The estimated LOD (Z) score is calculated as follows Z = log10 {1/[(0.25)x(0.5)y]} x is the number of affected relatives with at least one core feature (see top three bullet/sub-bullet points of PP4) harboring biallelic FOXN1 variants (confirmed by genetic analysis), not including the proband y is the number of affected relatives with at least one core feature (see top three bullet/sub-bullet points of PP4) harboring a heterozygous FOXN1 variant (confirmed by genetic analysis or an obligate carrier), not including the proband Not Applicable
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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.
Stand Alone
Very Strong
Strong
Moderate
Supporting
Not Applicable
Comments:
Does not apply, FOXN1 does not have a low rate of benign missense variation, with a missense constraint score of Z=0.66.
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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. Stand Alone
Very Strong
Strong
Moderate
Moderate evidence can be applied for a REVEL score of ≥0.932, downgraded from the recommendation of Strong in Pejaver et al., 2022 (PMID: 36413997).
Modification Type:
General recommendation
Supporting
Modification Type:
General recommendation
Not Applicable
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PP4 | ||||
Original ACMG Summary
Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.
Stand Alone
Very Strong
Strong
Moderate
Patient score of ≥2 points (see instructions below)
Modification Type:
Disease-specific,Gene-specific
Supporting
Patient score of 1-<2 points (see instructions below)
Modification Type:
Disease-specific,Gene-specific
Instructions:
PP4 applicability and strength is determined by the total points accumulated by a single affected individual according to the list below:
*Absent NK cells would not be consistent with a FOXN1 specific phenotype, however if absence/presence of NK cells is not noted, points may still be awarded if SCID gene panel or exome/genome sequencing has ruled out alternative causes Not Applicable
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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
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BA1 | ||||
Original ACMG Summary
Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.
Stand Alone
gnomAD Grpmax filtering allele frequency >0.00447
Modification Type:
Gene-specific
Very Strong
Strong
Moderate
Supporting
Instructions:
Maximum credible population allele frequency threshold determined using Whiffin/Ware calculator (https://www.cardiodb.org/allelefrequencyapp/) and the following parameters:
Not Applicable
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BS1 | ||||
Original ACMG Summary
Allele frequency is greater than expected for disorder.
Stand Alone
Very Strong
Strong
gnomAD Grpmax filtering allele frequency >0.00141 OR a bottle-necked population with a MAF >0.00141 may be used for this criterion. Caveat: If the variant is known to be a founder variant in the bottle-necked population do not consider the frequency in that population for BS1.
Modification Type:
Gene-specific
Moderate
Supporting
Instructions:
Maximum credible population allele frequency threshold determined using Whiffin/Ware calculator (https://www.cardiodb.org/allelefrequencyapp/) and the following parameters:
Not Applicable
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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.
Stand Alone
Very Strong
Strong
Moderate
Supporting
Not Applicable
Comments:
Does not apply due to reduced penetrance.
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BS3 | ||||
Original ACMG Summary
Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
Stand Alone
Very Strong
Strong
Moderate
Supporting
Not Applicable
Comments:
There is not a well-established functional study which can rule out all damaging effects on protein function.
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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. Stand Alone
Very Strong
Strong
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.
Modification Type:
None
Moderate
Supporting
Not Applicable
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BP1 | ||||
Original ACMG Summary
Missense variant in a gene for which primarily truncating variants are known to cause disease.
Stand Alone
Very Strong
Strong
Moderate
Supporting
Not Applicable
Comments:
Does not apply.
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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.
Stand Alone
Very Strong
Strong
Moderate
Supporting
Applicable only when observed in cis with a pathogenic variant in any inheritance pattern, with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for FOXN1.
Modification Type:
Disease-specific
Not Applicable
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BP3 | ||||
Original ACMG Summary
In frame-deletions/insertions in a repetitive region without a known function.
Stand Alone
Very Strong
Strong
Moderate
Supporting
Not Applicable
Comments:
Does not apply.
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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. Stand Alone
Very Strong
Strong
Moderate
Supporting
•Supporting evidence can be applied for a REVEL score of <0.290 based on recommendations of Pejaver et al., 2022 (PMID: 36413997). •Also applicable to synonymous or intronic variants not predicted to impact splicing by SpliceAI ∆ score ≤0.1
Modification Type:
General recommendation
Not Applicable
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BP5 | ||||
Original ACMG Summary
Variant found in a case with an alternate molecular basis for disease.
Stand Alone
Very Strong
Strong
Moderate
Supporting
Use with no specification.
Modification Type:
None
Not Applicable
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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
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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.
Stand Alone
Very Strong
Strong
Moderate
Supporting
A synonymous variant, or deep intronic variant affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions, for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site (SpliceAI ∆ score ≤0.1) AND the nucleotide is not highly conserved.
Modification Type:
General recommendation
Not Applicable
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