In 2013, ACMG group members came together to develop a recommendation for using standard terminology for classifying variants using applicable rules. Different rules/criteria are assigned to classify variants bearing the said rule’s feature. As mentioned below, each rule has unique characteristics relating to various forms of information, such as frequency and functionality. [1]
Two sets of criteria are specified according to the guideline: one for benign and likely benign classification, and the other for pathogenic and likely-pathogenic classification. There are a total of 28 rules set by ACMG. While 20 of these have been integrated in Massive Analyser, eight of them are still unavailable due to the need for more patient-specific data. Unimplemented rules are:
PS4, PM3, PM6, PP1, PP4, BS4, BP2, BP5
1. Benign Rules
These rules bring the variant which are assigned closer to the benign classification.
1.1. BA1 (Benign, Stand Alone)
Rule BA1 is applied if the total allele frequency is greater than 0.05 threshold in exome sequencing project, gnomAD, 1000Genomes or Exome Aggregation Consortium (ExAC).
Exception list is integrated to our system according to ACMG guideline. You can see the related paper from here. The variant defined as an exception will be shown with warning sign.
1.2. BS1 (Benign, Strong)
While assigning this rule to variants, ClinVar likely benign or benign submissions are checked and gnomAD frequency database is used. Here we find the highest GnomAD allele frequency for the variant according to total allele frequency and compare this to the benign cut-off frequency derived from the gene statistics. For rare diseases, if there are too few known variants (fewer than 6), we use 0.015 as a threshold value.
In order to avoid double-counting, rule BS1 is not assigned if either rules BA1 or PM2 were assigned first.
1.3. BS2 (Benign, Strong)
Initially, the mode of inheritance of the gene is determined according to the data come from Clinical Genomics Database (CGD), then the allele count is compared to these thresholds:
- For recessive or X-linked genes: allele count greater than 3
- For dominant genes: allele count greater than 5.
1.4. BS3 (Benign, Strong)
This rule checks the in vitro or in vivo functional studies in the literature.
If functional studies indicate that the variant is benign, this rule is assigned. Massive Analyzer obtains the data about this rule from ClinGen Evidence Repository.
1.5. BP1 (Benign, Supporting)
Missense variant in a gene for which primarily truncating variants are known to cause disease.
Massive Analyzer utilizes MVP score obtained from dbNFSP database.
MVP score: A pathogenicity prediction score for missense variants using deep learning approach. The MVP score ranges from 0 to 1. The larger the score, the more likely the variant is pathogenic. The authors suggest thresholds of 0.7 and 0.75 for separating damaging vs tolerant variants. You can see the details here.
- Gene statistics are also used in this rule.
The missense variants and ClinVar clinicaly significancy results of these variants were counted for each gene. If benign and likely benign missense variants and total missense variants ratio is greater than 0.5 and MVP score is less than 0.75, BP1 is assigned to related variant.
1.6. BP3 (Benign, Supporting)
In-frame deletions/insertions in a repetitive region without a known function.
- This rule is assigned according to the consequence and repetitive regions information.
- Massive Analyzer obtains repetitive region information from UCSC database simpleRepeat database.
Note: For hg38-based analysis, hg19 regions were lifted-over to hg38 using UCSC [LiftOver](https://genome.ucsc.edu/cgi-bin/hgLiftOver) tool.
If variant consequences are one or more of; inframe insertion, inframe deletion or stop lost, and the variant location is in the repetitive regions, BP3 is assigned to variant.
1.7. BP4 (Benign, Supporting)
Multiple lines of computational evidence suggest no impact on gene or gene product.
This rule is assigned according to the silico pathogenity scores. The softwares that are used for this rule are:
- Polyphen2
- SIFT
- SIFT4G
- MetaLR
- CONDEL
- DEOGEN2
- FATHMM
- PROVEAN
- MutationTaster
- MutationAssessor
- LRT
- REVEL
While pathogenicity results are counted as -1, benign reults are counted as 1. These scores are summed for each variant. If total score is GREATER than 0, BP4 is assigned.
1.8. BP6 (Benign, Supporting)
Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.
If ClinVar clinical interpretation of variant is benign or likely benign, BP6 rule is assigned to variant.
1.9. BP7 (Benign, Supporting)
This rule investigates the variant consequences and splice-site regions. If the variant is not located in the splice site region and it is synonymous, non-coding or intronic variant, BP7 is assigned to variant.
2. Pathogenic Rules
These rules bring the variant they are assigned closer to the pathogenic classification.
2.1. PVS1 (Pathogenic, Very Strong)
Null variant (nonsense, frameshift, canonical \u00b11 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where LOF (Loss Of Function) is a known mechanism of disease.
In this rule, we check the consequence column. If variant has one or more of the consequences:
- nonsense variant
- frameshift variant
- exon deletion variant
- start loss variant
- stop loss variant
- splice acceptor variant
- splice donor variant
Massive Analyzer determines that loss of function is a Known Mechanism of Disease from consequence information. If the total count of pathogenic variants bearing one or more of the consequences above for the related gene is greater than 3, PVS1 rule is assigned to variant.
2.2. PS1 (Pathogenic, Strong)
Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.
Regardless of the nucleotide change, if the amino acid change found in the variant is established in another pathogenic variant before, PS1 rule is assigned to the variant.
2.3. PS2 (Pathogenic, Strong)
De novo variant
De novo variant is defined as a genetic alteration that is present for the first time in a child due to a variant in the fertilized egg itself during early embryogenesis. Massive Analyser includes this rule only at trio analysis because the data about paternity and maternity information is only used in trio analysis, and de novo variants are detected in trio analysis.
2.4. PS3 (Pathogenic, Strong)
This rule checks the in vitro or in vivo functional studies in the literature. If functional studies indicate that the variant is pathogenic, PS3 rule is assigned.
Massive Analyzer obtains the PS3 rule information from ClinGen Evidence Repository.
2.5. PM1 (Pathogenic, Moderate)
Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation.
If the variant is located in a mutational hot spot and/or critical and well-established functional domain without benign variation, PM1 is assigned to variant. Massive Analyzer obtains the data about this rule from ClinGen Evidence Repository.
2.6. PM2 (Pathogenic, Moderate)
In this rule, Clinical Genomics Database is utilized to obtain information concerning variant recessiveness or dominance.
The rule will trigger if the gnomAD allele frequency information is not found;
- For dominant genes, we check that the allel count is less than 20
- For recessive genes, we check that the allel count is less than 12 PM2 is assigned.
2.7. PM4 (Pathogenic, Moderate)
Protein length changes as a result of in-frame deletions/insertions in a non-repeat region or stop-loss variants.
- This rule is assigned according to consequence and repetitive region information.
- Massive Analyzer obtains the repetitive regions database from UCSC database simpleRepeat database.
This rule will be trigger if variant IS NOT located in the repetitive region and the variant consequence is any of: inframe insertion, inframe deletion, stop lost.
2.8. PM5 (Pathogenic, Moderate)
Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.
- This rule is weaker than PS1. While In PS1 rule, the equilevent aminoacid change is checked, PM5 rule investigates all possible aminoacid changes in the same location.
2.9. PP2 (Pathogenic, Supporting)
Missense variant in a gene for which primarily truncating variants are known to cause disease.
- This rule is the opposite form of BP1.
We utilize MVP score that came from dbNFSP database.
MVP score: A pathogenicity prediction score for missense variants using deep learning approach. The MVP score ranges from 0 to 1. The larger the score, the more likely the variant is pathogenic. The authors suggest thresholds of 0.7 and 0.75 for separating damaging vs tolerant variants. You can see the details from here.
- Gene statistics are also used in this rule.
The missense variants and ClinVar clinicaly significancy results of these variants were counted for each gene. If pathogenic and likely pathogenic missense variants to total missense variants ratio is greater than 0.5 and MVP score is equal or greater than 0.75, PP2 is assigned to related variant.
2.10. PP3 (Pathogenic, Supporting)
Multiple lines of computational evidence suggest no impact on gene or gene product.
- This rule is the opposite form of BP4.
The pathogenicity scores are listed in BP4 rule and pathogenic results counted as -1, benign results are counted as 1. These scores are summed for each variant. If total score is less than 0, PP3 is assigned.
2.11. PP5 (Pathogenic, Supporting)
Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.
- This rule is the opposite form of BP6.
If ClinVar interpretation of variant is pathogenic or likely pathogenic, PP5 is assigned to variant.