Additional variant information

• Annotations
• ACMG criteria
• Quality
• Literature & sources
• Internal variant database details

Annotations

Click on Show more details > Annotations for each variant to see the following groups of selected annotations for each variant, related to Frequency, Evolutionary predictions, Splice and NMD predictions and Protein predictions

By clicking on the annotations, a glossary will appear explaining each annotation in detail.

• AION predictor sub-scores

  • AION amino acid constraint: Indicates the predicted biochemical impact on the protein due to the amino acid change by variant. Values can be NA, High, Moderate or Low. The percentage indicates the confidence in the prediction considering available evidence. Values are calculated using a proprietary machine learning-based model.

  • AION evolutionary constraint: Indicates prediction of evolutionary constraint in the genomic region surrounding the variant. Values are calculated using a proprietary machine learning-based model. Values can be High, Moderate or Low. The percentage indicates the confidence in the prediction considering available evidence. Values are calculated using a proprietary machine learning-based model.

  • AION regional constraint: Predicts variation constraint in the genomic region surrounding the variant (as observed in healthy population individuals). Values can be High, Moderate or Low. The percentage indicates the confidence in the prediction considering available evidence. Values are calculated using a proprietary machine learning-based model.

  • AION splice alteration: Indicates prediction of likelihood of mRNA alterations caused by the variant. Values can be High, Moderate or Low. The percentage indicates the confidence in the prediction considering available evidence. Values are calculated using a proprietary machine learning-based model.

• Amino acids change predictions

  • SIFT score: Values closer to 0 (range from 0 to 1) indicate deleteriousness. Predicts the effect of an amino acid substitution based on sequence homology and amino acid physical properties. If the score value is <= 0.05, the amino acid substitution will be predicted as "deleterious" and if the score is > 0.05, it will be predicted as "tolerated". Values can be deleterious low confidence, tolerated low confidence, tolerated or deleterious. Values may be blank is the variant is not a missense change.

  • Polyphen2 score: Values closer to 0 (range from 0 to 1) indicate higher likelihood of pathogenicity. Predicts the effect of the possible impact of an amino acid substitution on the structure and function of protein. If the score is greater than 0.908, it will predict a "Probably Damaging" effect; if the score is greater than 0.446 and less than or equal to 0.908, it will predict a "Possibly Damaging" effect; and less than or equal to 0.446 will predict a "Benign" effect. Values can be blank if the variant is not a missense change, or if the value is missing from annotation source.

  • Grantham score: Higher values indicate higher likelihood of pathogenicity. Informs on the evolutionary distance between two amino acids. Values range between 0 and 215. Values can indicate a conservative (0-50), moderately conservative (51-100), moderately radical (101-150), or a radical (≥151) change.

  • BLOSUM62: Lower values indicate higher constraint for an amino acid substitution and hence a higher likelihood of pathogenicity. It is a logarithm-of-odds score based on alignments between evolutionarily divergent protein sequences, providing a measure of the amino acid substitution frequency through evolution (i.e. indirect measure for specific amino acid substitutions constraint across evolution).

• Conservation scores

  • phastCons vertebrates (46 species): Values closer to 1 indicate higher conservation (values range from 0 to 1). Estimates the probability that each nucleotide belongs to an evolutionary conserved element in 46 vertebrates, based on multiple sequence alignment. PhastCons values consider not only each individual alignment column, but also its flanking columns. These values are sensitive to "runs" of conserved sites, and are therefore effective for picking out conserved elements.

  • phastCons mammals (33 species): Values closer to 1 indicate higher conservation (values range from 0 to 1). Estimates the probability that each nucleotide belongs to an evolutionary conserved element in 33 placental mammals, based on multiple sequence alignment. PhastCons values consider not only each individual alignment column, but also its flanking columns. These values are sensitive to "runs" of conserved sites, and are therefore effective for picking out conserved elements.

  • phastCons primates (10 species): Values closer to 1 indicate higher conservation (values range from 0 to 1). Estimates the probability that each nucleotide belongs to an evolutionary conserved element in 10 primates, based on multiple sequence alignment. PhastCons values consider not only each individual alignment column, but also its flanking columns. These values are sensitive to "runs" of conserved sites, and are therefore effective for picking out conserved elements.

  • phyloP vertebrates (46 species): Positive scores indicates a conserved region and so higher conservation constraint, while negative scores indicates accelerated evolution (scores ranging from -14 to 6.4). Specifically, it indicates evolutionary conservation at individual alignment sites for nucleotide/s changes in 46 vertebrates. PhyloP is specially appropriate for evaluating signatures of selection at particular nucleotides or classes of nucleotides (e.g., third codon positions, or first positions of miRNA target sites).

  • phyloP mammals (33 species): Positive scores indicates a conserved region and so higher conservation constraint, while negative scores indicates accelerated evolution (scores ranging from -13.7 to 2.9). Specifically, it indicates evolutionary conservation at individual alignment sites for nucleotide/s changes in 33 placental mammals. PhyloP is specially appropriate for evaluating signatures of selection at particular nucleotides or classes of nucleotides (e.g., third codon positions, or first positions of miRNA target sites).

  • phyloP primates (10 species): Positive scores indicates a conserved region and so higher conservation constraint, while negative scores indicates accelerated evolution (scores ranging from -9 to 0.65). Specifically, it indicates evolutionary conservation at individual alignment sites for nucleotide/s changes in 10 primates. PhyloP is specially appropriate for evaluating signatures of selection at particular nucleotides or classes of nucleotides (e.g., third codon positions, or first positions of miRNA target sites).

• Frequency in gnomAD

  • Allele frequency indicates alternate allele frequency for the variant of interest. This value is also displayed in the main variant card (variants can be filtered based on it).
  • Allele count indicates alternate allele count for the variant of interest.
  • Homozygous individuals indicate the total number of individuals homozygous for the alternate allele in the variant of interest.
  • Hemizygous individuals indicate the total number of individuals hemizygous for the alternate allele in the variant of interest. Notice only variants in sex chromosomes can have a value in this entry.
    

     

• Splice and NMD predictions

  • Distance to splice indicates distance to closest splice site in basepairs (1 to 10).

  • Splice type indicates the nature of the closest splice site (only shown for variants <10bp away from a splice site). Values can be blank (when the variant is not located in a donor or acceptor site), donor (GT) or acceptor (AG).

  • NMD indicates if the variant is predicted to undergo nonsense mediated decay (NMD).

  • NMD boundaries Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that eliminates mRNA transcripts containing premature stop codons. The NMD boundaries are defined as 1) the genomic position 50bp upstream of the last exon-exon junction (at the 3' end of the penultimate coding exon) and 2) the genomic position of the stop-codon. The NMD boundaries are computed only in genes with multiple coding exons. Hence, non-coding exons are discarded. If a LoF variant is located inside of this interval, NMD is not expected to occur. Examples with interpretations:

    • chr19:44677055-44681836(strand=1), where chr19:44677055 is the location of NMD boundary and chr19:44681836 is the location of stop codon.

    • chr2:37398626-37415690(strand=-1), where chr2:37398626 is the location of the stop codon and chr2:37415690 is the location of the NMD boundary.

  • dbscSNV ADA score: Prediction score based on AdaBoost, an ensemble machine learning algorithm. Ranges 0 to 1, indicating the predicted probability that the variant will affect splicing. dbscSNV developers suggest a cutoff for a binary prediction (affecting splicing vs not affecting splicing) of 0.6.

  • dbscSNC RF score: Prediction score based on random forest, an ensemble machine learning algorithm. Ranges 0 to 1, indicating the predicted probability that the variant will affect splicing. dbscSNV developers suggest a cutoff for a binary prediction (affecting splicing vs not affecting splicing) of 0.6.

ACMG Criteria

AMP evidence codes annotated by AION are displayed in the ACMG tab. Criteria are organised by several properties, such as Segregation, Disorder, Effect, etc. “Selected by AI” means that the criteria is automatic applied by AION.

In order to improve variant classification, the user can manually apply or remove any evidence code, based on the evidence they have for a particular variant in a patient by selecting or unselecting “Apply”. New ACMG/AMP classification will be calculated on the fly. By selecting Reset changes user selected changes can be reset and criteria automatically applied shown again.

AION implementation of ACMG criteria

Pathogenic criteria

Automated

Very strong

• PVS1: null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon) in a gene where LOF is a known mechanism of disease.

Strong

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

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

Moderate

• 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.

• PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium.

• PM3: For recessive disorders, detected in trans to a pathogenic variant.

• PM4: Protein length changes as a result of in-frame deletions/insertions in a non-repeat region or stop-loss variants.

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

• PM6: Assumed de novo, but without confirmation of parental status.

Supporting

• PP1: Co-segregation with disease in multiple affected family members in a well known disease causing gene.

• 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.

• PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product.

Manual

Some ACMG/AMP evidence codes are not annotated automatically by AION because they require information which is challenging to process, or not present in the VCF file. Our team is working to automate these criteria.

• PS3: Well-established in vitro or in vivo functional studies supportive of 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.

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

Benign criteria

Automated

Stand-Alone

• BA1: Allele frequency is >5% in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium.

Strong

• BS1: Allele frequency is greater than expected for disorder.

• 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.

• BS4: Lack of segregation in affected members of a family.

Supporting

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

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

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

• BP7: Synonymous variant has no predicted effect on splice consensus sequence nor is predicted to create a new splice site, and is not in a highly conserved region.

Manual

Some ACMG/AMP evidence codes are not annotated automatically by AION because they require information which is challenging to process, or not present in the VCF file. Our team is working to automate these criteria.

• BS3: No damaging effect on splicing and protein function provided by well-established in vivo or in vitro functional studies.

• 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.

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

Quality

• Genotype (GT)

  • Homozygous ref (0/0): both alleles match the reference allele.

  • Heterozygous (0/1): two different alleles.

  • Homozygous alt (1/1): both alleles are the same but are different than the reference allele.

  • Hemizygous alt* (1/-1), (1): the variant is found on a single allele (e.g. chrX in a male) and the variant present is different than the reference allele.

  • Hemizygous ref* (0/-1), (0): the variant is found on a single allele (e.g. chrX in a male) and the variant present matches the reference allele.

    • *Pseudoautosomal regions in sex chromosomes can lead some variant callers not to be able to distinguish if a variant is in chrX or chrY.

  • Can't make a zygosity call (./.).

• Genotype quality (GQ) represents the Phred-scaled confidence that the genotype assignment (GT) is correct.

• Depth (DP) refers to the filtered depth (the number of filtered reads that support each of the reported alleles). Only reads that passed the variant caller’s filters are included in this number. However, unlike the AD calculation, uninformative reads are included in DP.

• Allele depth (AD) is the unfiltered allele depth, i.e. the number of reads that support each of the reported alleles. Allele depths (ref, alt) are separated by commas. All reads at the position (including reads that did not pass the variant caller’s filters) are included in this number, except reads that were considered uninformative. Reads are considered uninformative when they do not provide enough statistical evidence to support one allele over another.

• Variant Allele frequency (VAF) is the percentage of sequence reads observed matching a specific DNA variant (alternative reads) divided by the overall coverage at that locus.

Literature & Resources

This section currently provides direct links to:

• Mastermind (Genomenon® research engine)
• Disease information source: OMIM (Online Mendelian Inheritance in Man database) or Orphanet
• UCSC Browser to find additional information on the variant of interest
• Google Scholar link to automatically search for the gene and variant of interest
• PubMed link to automatically search for the gene and variant of interest

 

AION DB: Internal variant database details

Please see the dedicated pages:

• AION Database - variant statistics

• AION Database - variant classifications
• AION Database - onboarding