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
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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. Not Applicable
Comments:
LOF is not a known disease mechanism
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PS1
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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.
VCEP Specifications:
Previously established variant must be established as pathogenic or likely pathogenic per criteria established by this VCEP. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level Strong
Same amino acid change as a previously established pathogenic or Likely Pathogenic variant regardless of nucleotide change. Previously established variant must be established as pathogenic per criteria established by this VCEP. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.
Modification Type:
No change
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PS2
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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:
This document is also uploaded as “PS2 & PM6 instructions” Very Strong
10 points (per SVI Recommendation for De Novo Criteria (PS2 & PM6) with modifications for GFAP)
Modification Type:
Gene-specific,Strength
Strong
6 points (per SVI Recommendation for De Novo Criteria (PS2 & PM6) with modifications for GFAP)
Modification Type:
Gene-specific,None
Moderate
3 points (per SVI Recommendation for De Novo Criteria (PS2 & PM6) with modifications for GFAP)
Modification Type:
Gene-specific,Strength
Supporting
1.5 points (per SVI Recommendation for De Novo Criteria (PS2 & PM6) with modifications for GFAP)
Modification Type:
Gene-specific,Strength
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PS3
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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.
VCEP Specifications:
Please see instructions in attached document (PS3/BS3 Instructions) (Unsure of what excel file referred to in the comments is. No excel file was submitted and there no “Impaired Enzyme Activity” assays exist) Very Strong
Animal model that replicates human phenotype
Modification Type:
Gene-specific,Strength
Strong
Immunoassay/Western blot that demonstrates GFAP insolubility in non patient-derived cell lines that are transfected (or genome edited) with the variant in question
Modification Type:
Gene-specific
Moderate
Immunocytochemistry/Immunohistochemisty demonstrates abnormal GFAP aggregation (compared to wild-type control with demonstrated intermediate fillament architecture) in non patient-derived cell lines that are transfected (or genome edited) with the variant in question
Modification Type:
Gene-specific,Strength
Supporting
Immunoassay/Western blot, Immunocytochemistry/Immunohistochemisty, or confocal/electron microscopy show protein aggregation/loss of solubility in patient-derived cell lines.
Modification Type:
Gene-specific,Strength
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PS4
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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.
VCEP Specifications:
Follow the instructions in the document provided. Criteria established by VCEP using the Yoshida criteria as guidelines and making appropriate modifications based on discussion with experts on AxD Very Strong
15+ points
Modification Type:
Gene-specific,Strength
Strong
9-14 points
Modification Type:
Gene-specific,No change
Moderate
4-8 points
Modification Type:
Gene-specific,Strength
Supporting
2-3 points
Modification Type:
Gene-specific,Strength
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PM1
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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.
VCEP Specifications:
This includes missense variants and in-frame deletions/duplications in ANY transcript expressed in astrocytes. Moderate
Variant affecting an amino acid at or in between positions 59-88. This region shows variation constraint (p-value 6/45e-4), as well as a high concentration of pathogenic/likely pathogenic variants (24 variants in 30 amino acids in this region vs 41 variants in 402 amino acids outside this region). This includes missense variants and in-frame deletions/duplications
Modification Type:
Gene-specific
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PM2
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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.
VCEP Specifications:
SVI guidance on PM2 recommends decreasing weight of criterion PM2 from moderate to supporting strength level. Supporting
Absent in gnomAD
Modification Type:
Strength
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PM3
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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:
Autosomal Dominant disorder
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PM4
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Original ACMG Summary
Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.
Not Applicable
Comments:
There are some in frame deletions but mechanism is not common enough to assign this criteria
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PM5
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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
Novel missense change at an amino acid residue where one different pathogenic or likely pathogenic variant has been identified.
Modification Type:
None
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PM6
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Original ACMG Summary
Assumed de novo, but without confirmation of paternity and maternity.
VCEP Specifications:
To assign points to the variant, follow instructions in the document provided. Modifications were made to the SVI Recommendation for De Novo Criteria (PS2 & PM6) to adapt to GFAP. (CSPEC editor does not allow to add VeryStrong for PM6) Strong
6 points (per SVI Recommendation for De Novo Criteria (PS2 & PM6) with modifications for GFAP)
Modification Type:
Gene-specific,Strength
Moderate
3 points (per SVI Recommendation for De Novo Criteria (PS2 & PM6) with modifications for GFAP)
Modification Type:
Gene-specific
Supporting
1.5 points (per SVI Recommendation for De Novo Criteria (PS2 & PM6) with modifications for GFAP)
Modification Type:
Gene-specific,Strength
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PP1
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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:
If PP4 is applied, PP1 cannot be applied (per ClinGen Guidance): PMID: 38103548 https://pubmed.ncbi.nlm.nih.gov/38103548/ If PP4 is not met, individuals must meet “consistent”, “probable”, or “definite” diagnosis according to VCEP PS2/PM6 guidelines. Strong
≥5 informative meioses
Modification Type:
Gene-specific,Strength
Moderate
≥3 informative meioses.
Modification Type:
Gene-specific,Strength
Supporting
≥2 informative meioses.
Modification Type:
Gene-specific,None
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PP2
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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:
Not applicable because missense z score is <3.09.
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PP3
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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. Not Applicable
Comments:
This criterion is not applicable. GFAP has a gain of function mechanism of disease and no individual tool or meta predictor has provided consistent and accurate scores for GFAP variants, especially those variants associated with Type II or adult-onset presentations. Use of this criterion can be revisited in the future if modeling tools specific to this gene or designed to characterize gain of function mutations are shown to more accruately model pathogenicity of GFAP variants.
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PP4
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Original ACMG Summary
Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.
VCEP Specifications:
PP4 is only applicable if individual meets “consistent”, “probable”, or “definite” diagnosis according to VCEP PS2/PM6 guidelines. PP4 and PP1 are mutually exclusive, therefore if PP4 is not met, PP1 should be considered instead. Strong
Elevated GFAP protein levels in plasma, serum, or CSF. For clinical tests, established thresholds are dependent on age and currently only exist for individuals above the age of 18 years. Clinical lab reports clearly indicate if levels are elevated (in pg/mL). For research-based testing, levels must be significantly elevated when compared to age-matched controls.
Modification Type:
Gene-specific,Strength
Moderate
Existence of Rosenthal fibers in addition to gliosis and loss of myelin.
Modification Type:
Gene-specific,Strength
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| PP5 | ||||
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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
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Original ACMG Summary
Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.
Stand Alone
GnomAD (v4.1.0) filtering allele frequency ≥0.01%
Modification Type:
Gene-specific,None
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BS1
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Original ACMG Summary
Allele frequency is greater than expected for disorder.
Strong
GnomAD (v4.1.0) filtering allele frequency ≥0.001%
Modification Type:
Gene-specific,None
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BS2
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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.
VCEP Specifications:
Observed in gnomAD in a homozygous state. Strong
Observed in gnomAD in a homozygous state.
Modification Type:
Gene-specific
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BS3
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Original ACMG Summary
Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
VCEP Specifications:
Please see instructions in attached document (PS3/BS3 Instructions) Strong
Protein expression assay demonstrates loss of function when variant is transfected or genome edited into non patient-derived cell lines
Modification Type:
Gene-specific,None
Moderate
Immunoassay/Western blot that demonstrates GFAP normal solubility in non patient-derived cell lines that are transfected (or genome edited) with the variant in question OR Immunocytochemistry/Immunohistochemisty demonstrates normal GFAP aggregation (compared to wild-type control with demonstrated intermediate fillament architecture) in non patient-derived cell lines that are transfected (or genome edited) with the variant in question OR Immunoassay/Western blot, Immunocytochemistry/Immunohistochemisty, or confocal/electron microscopy show protein normal protein distribution and solubility in patient-derived cell lines.
Modification Type:
Gene-specific,Strength
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BS4
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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.
VCEP Specifications:
To count as affected individuals: Must meet “consistent”, “probable”, or “definite” diagnosis according to VCEP PS2/PM6 guidelines. To count as unaffected they must meet both of the following.
Any individuals not meeting either criteria for affected or unaffected should not be counted as informative. Strong
≥3 informative meioses.
Modification Type:
Gene-specific
Moderate
≥2 informative meioses.
Modification Type:
Gene-specific,Strength
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BP1
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Original ACMG Summary
Missense variant in a gene for which primarily truncating variants are known to cause disease.
Not Applicable
Comments:
GFAP is caused primarily by missense variants.
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BP2
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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.
Supporting
Observed with another variant (regardless of phase) in GFAP that is established as pathogenic or likely pathogenic per these criteria.
Modification Type:
Gene-specific
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BP3
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Original ACMG Summary
In frame-deletions/insertions in a repetitive region without a known function.
Not Applicable
Comments:
No repetitive regions without known function
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BP4
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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. Not Applicable
Comments:
GFAP has a gain of function mechanism of disease and no individual tool or meta predictor has provided consistent and accurate scores for GFAP variants, especially those variants associated with Type II or adult-onset presentations. Use of this criterion can be revisited in the future if modeling tools specific to this gene or designed to characterize gain of function mutations are shown to more accruately model pathogenicity of GFAP variants.
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BP5
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Original ACMG Summary
Variant found in a case with an alternate molecular basis for disease.
Supporting
Variant found in a case with an alternate molecular basis for disease.
Modification Type:
None
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| BP6 | ||||
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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
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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.
Supporting
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. An amino acid is considered highly conserved if the phyloP score is <2.27. PhyloP score can be found in gnomAD in the In Silico Predictors tool.
Modification Type:
None
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