Logic behind ACMG classification of SNVs

ACMG score

Since version 32.0, the software calculates the variant score based on a points-based system recommended by the ACMG.

The software computes the ACMG score for SNVs by assigning points to each active criterion based on the strength of evidence provided:

• Supporting evidence: 1 point,

• Moderate evidence: 2 points,

• Strong evidence: 4 points,

• Stand alone/very strong evidence: 8 points.

The total score is determined by summing the points from the pathogenic criteria checked, minus the sum of points from benign criteria. The score is interpreted according to the following thresholds:

• Pathogenic: ≥ 10,

• Likely Pathogenic: 6 to 9,

• Uncertain Significance: 0 to 5,

• Likely Benign: -6 to -1,

• Benign: ≤ -7.

For mtDNA variants, the software excludes the following tags from the score calculation: PM1, PM3, PP2, PP5, BP1, BP3, and BP6.

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ACMG classification

Emedgene implementation of the ACMG variant classification for SNV follows the “Standards and guidelines for the interpretation of sequence variants” published in 2015 by Sue Richards et al. Genetics in medicine 17.5 (2015): 405-423. These guidelines define 28 criteria that address types of evidence for the interpretation of sequence variants.

We implemented a technical automated solution for most criteria based on our scientific advisors’ recommendations and feedback from top clinical customers. For each criterion, we elaborate on the logic employed and the associated underlying thresholds. In addition, we give the user the flexibility to change the weight of specific criteria based on his professional judgment as recommended by ACMG/AMP guidelines.

For mtDNA variants, the software excludes the following tags from the score calculation: PM1, PM3, PP2, PP5, BP1, BP3, and BP6.

Importantly, we have made some adaptations of specific criteria (described below), and several criteria are not automatically calculated and require manual evaluation: PS4, PP4, BP5, BS1 and BS2.

PVS1	PS1(#h_18f3f38fe0) PS2 PS3 PS4	PM1 PM2 PM3 PM4 PM5 PM6	PP1 PP2 PP3 PP4 PP5	BP1 BP2 BP3 BP4 BP5 BP6 BP7	BS1 BS2 BS3 BS4	BA1

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PVS1: “Null variant in a gene where LOF is a known mechanism of disease.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant must be “null variant”: We assess null variants as variants with high severity effect (i.e stop-gain, frameshift etc.). It should be noted that variants with a high splice prediction effect will be included in this tag independently of their main effect.

2. LoF is a known mechanism of disease within the relevant gene: We are checking in ClinVar if there are any submissions of pathogenic or likely pathogenic variants with the following attributes: A review status with minimum 2 stars, LoF variant, variant within the related gene and size less than 51bp.

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PS1: “Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant must be a missense.

2. The splicing prediction for the variant should not be High.

3. A different missense ClinVar pathogenic/likely pathogenic variant (with 2-4 stars) has been previously described leading to the same amino acid change.

PS2: “De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant is de novo variant: A de novo variant is defined as a variant with zygosity heterozygote (on autosome or on chromosome X for female) or HEMI (on chromosome X for male) for the proband and reference for the parents (parents have to be unaffected).

2. The relatedness of the samples has been confirmed: As part of the lab validation service, we are checking the familial relatedness based on the genomic data.

PS3: “Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant was reported in ClinVar as Pathogenic or Likely Pathogenic with 2-4 stars.

2. Supporting evidence from functional studies is available. For this, we are checking the publications associated with ClinVar submission and if any of them contain functional studies.

On the UI interface, the user also has the possibility to add a publication supporting a damaging effect on the gene or gene product.

PS4: “The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.”

This criterion is not currently automated, however the user can manually enter the corresponding information on the UI.

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PM1: “Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant should be a missense variant.

2. The variant should be in a Hotspot region: A Hotspot region is defined as a region of 30 bp surrounding the variant, where the number of missense pathogenic/likely pathogenic variants reported in ClinVar is greater than 70% of the total number of missense variants reported in ClinVar for this region.

3. The Hotspot region should contain at least 10 missense variants reported in ClinVar.

Note: The tag isn't relevant for mtDNA variants and won't be automatically applied to them.

PM2: “Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, ExAC or Gnomad (not comprising Gnomad other) and Local database if >1000.”

For this criterion, we are checking the fulfillment of the following conditions:

1. For dominant disorders, the variant was not reported in any relevant population statistic database.

2. For recessive disorders, the variant was reported with an allele frequency lower than 0.5 % and an hom/hemi count lower than 3 in any relevant population statistic database.

PM3: “For recessive disorders, detected in trans with a pathogenic variant.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The case must contain parental information.

2. The variant must be compound heterozygote.

3. The second variant (in trans) must be reported as pathogenic/likely pathogenic in ClinVar (with 2-4 stars).

4. The disease inheritance mode must be recessive.

Note: The tag isn't relevant for mtDNA variants and won't be automatically applied to them.

PM4: “Protein length changes as a result of in-frame deletions/insertions (in a non-repeat region) and stop losses.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant is an in-frame insertion or deletion variant.

2. The variant is not within a repeat region. The repeat regions used are the ones as defined by Repeat masker data from UCSC.

OR 3. The variant is a stop lost.

PM5: “Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant must be a missense variant.

2. This variant or a different missense pathogenic/likely pathogenic variant has been previously described in ClinVar at the same amino acid position. Please note, we modified this condition to take into consideration the described variant.

PM6: “Assumed de novo, but without confirmation of paternity and maternity.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant is a de novo variant: A de novo variant is defined as a variant with zygosity heterozygote (on autosome or on chromosome X for female) or HEMI (on chromosome X for male) for the proband and reference for the parents (parents have to be unaffected).

2. The relatedness of the samples has not been confirmed.

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PP1: “Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The case should contain at least 2 affected members.

2. The variant segregates with the disease within the pedigree.

3. The variant is in a gene known to cause a disease with a phenotypic match.

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.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant must be a missense variant.

2. The gene has a low rate of benign missense variation and in which, missense variants are a common mechanism of disease. This condition is evaluated by comparing the number of missense pathogenic/likely pathogenic variants reported in ClinVar (with 2-4 stars) to the number of missense benign/likely benign variants reported in ClinVar (with 2-4 stars). The condition is fulfilled if the number of pathogenic/likely pathogenic missense variants is higher or equal to twice the number of benign/likely benign missense variants.

3. At least 10 missense variants with 2-4 stars were submitted to ClinVar for this gene.

Note: The tag isn't relevant for mtDNA variants and won't be automatically applied to them.

PP3: “Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact).”

For this criterion, we are checking the fulfillment of at least 2 out of the 3 following conditions:

1. The conservation prediction score is HIGH.

2. The splicing prediction score is HIGH.

3. The variant effect is predicted to be damaging.

PP4: “Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.”

This criterion is not currently automated, however, the user can manually enter the corresponding information on the UI.

PP5: “Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant was reported in ClinVar as Pathogenic or Likely Pathogenic with 2-4 stars.

2. Supporting evidence from functional studies is not available. For this, we are checking the publications associated with ClinVar submissions and if any of them contain functional studies.

Note: The tag isn't relevant for mtDNA variants and won't be automatically applied to them.

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BA1: “Allele frequency is >5% in Exome Sequencing Project, 1000 Genomes Project, ExAC or Gnomad (not comprising Gnomad other) and Local database if >1000.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant has an allele frequency > 5% in the relevant population statistic database.

2. The variant is not part of the BA1 exception list created by ClinGen.

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BS1: “Allele frequency is greater than expected for the disorder.”

This criterion is not currently automated, however the user can manually enter the corresponding information on the UI.

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.”

This criterion is partially automated, however the user can manually enter the corresponding information on the UI.

1. The variant has been observed in a population statistics database.

2. The observed zygosity for the variant is similar to the one described in the population statistics database.

3. The associated disease should occur at an early age (age of onset < 10 years old).

4. The disease should have 100% penetrance.

BS3: “Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant was reported in ClinVar as Benign or Likely Benign with 2-4 stars.

2. Supporting evidence from functional studies is available. For this, we are checking the publications associated with ClinVar submission and if any of them contains functional studies.

On the UI interface the user also has the possibility to add a publication supporting no damaging effect on the gene or gene product.

BS4: “Lack of segregation in affected members of a family.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The case is not a singleton.

2. The variant is not segregating with the disease within the pedigree.

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BP1: “Missense variant in a gene for which primarily truncating variants are known to cause disease.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant must be a missense.

2. Primarily truncating variants are known to cause disease for this gene. This condition is evaluated by comparing the number of missense pathogenic/likely pathogenic variants reported in ClinVar (with 2-4 stars) to the number of missense benign/likely benign variants reported in ClinVar (with 2-4 stars). The condition is fulfilled if the number of benign/likely benign missense variants is higher or equal to 5 times the number of pathogenic/likely pathogenic missense variants.

3. At least 10 missense variants with 2-4 stars were submitted to ClinVar for this gene.

Note: The tag isn't relevant for mtDNA variants and won't be automatically applied to them.

BP2: “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.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The case must contain the parents.

2. The variant must be compound heterozygote.

3. The second variant (in trans) must be reported as pathogenic/likely pathogenic in ClinVar (with 2-4 stars).

4. The disease inheritance mode must be dominant.

Or the fulfillment to the following conditions:

1. The case must contain the parents.

2. There is a variant in cis which has been reported pathogenic or likely pathogenic in ClinVar (with 2-4 stars).

BP3: “In-frame deletions/insertions in a repetitive region without a known function.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant is an in-frame insertion or deletion variant.

2. The variant is within a repeat region. The repeat regions used are the ones as defined by Repeat masker data from UCSC.

Note: TheThe tag isn't relevant for mtDNA variants and won't be automatically applied to them.

BP4: “Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.)”

For this criterion, we are checking the fulfillment of the following conditions:

1. The conservation prediction score is not HIGH.

2. The splicing prediction score is LOW or Unknown.

3. The variant effect is predicted to be neutral.

BP5: “Variant found in a case with an alternate molecular basis for disease.”

This criterion is not currently automated, however the user can manually enter the corresponding information on the UI.

BP6: “Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant was reported in ClinVar as benign/likely benign with 2-4 stars.

2. Supporting evidence from functional studies is not available. For this, we are checking the publications associated with ClinVar submission and if any of them contain functional studies.

Note: The tag isn't relevant for mtDNA variants and won't be automatically applied to them.

BP7: “A synonymous (silent) 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.”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant must be a synonymous variant.

2. The splicing prediction score is LOW or Unknown.

3. The conservation prediction score is not HIGH.