The Desktop apps panel allows you to activate or inactivate connections with your IGV and Alamut desktop applications. This means there won't be any IGV and Alamut windows popping up unless you want them to!

Once you've configured your preferences for the desktop applications connections, they will be saved for your user account and applied to all cases.

The Desktop apps panel appears as a tab in the Variant page sidebar.

Individual case page > Analysis tools tab > Variant table > Variant page

The Variant page showcasing the comprehensive variant information is accessible from the Variant table by selecting the corresponding variant row with a click. Once you're on the Variant page, you can move between variants using left and right arrow keys of your keyboard.

The Variant page is comprised of:

1. Top bar. Displays Case ID, gene symbol, genomic DNA-level description of the variant, variant tags, and a link to your Curate database. If the variant is already in your Curate database, you will see an Open Curate button. Otherwise, you will see an Export to Curate button.

2. Navigation panel (left). Divided into five tabs, leading to the corresponding page sections.

3. Page body:

   1. Summary section. Highlights core variant-related information from other sections

   2. Clinical Significance section. Reports essential variant- and gene-level information and indicates gene-related diseases.

   3. Quality section. Outlines the major variant quality parameters in each sample and demonstrates the family tree with the zygosity for each sequenced sample.

   4. Visualization section. Features the IGV-based BAM file viewer.

   5. Population Statistics section. Addresses alternative allele frequency, alternative allele count, and the number of homozygotes in public and internal databases.

   6. Related Cases section. Displays statistics regarding the pathogenicity and tags assigned to the variant under review, incorporating data from previous cases within both your organization and networks.

   7. Evidence section. Highlights user-selected variant pathogenicity, ACMG class (for a sequence or a genomic variant), and interpretation notes.

4. Variant activity panel (right). Records variant-level user activities, such as tagging a variant, adding comments or evidence notes, or editing the evidence graph. Variant activity panel pops up upon clicking the Activities button.

The Clinical Significance section summarizes essential variant-level and gene-level information and indicates the gene's associated diseases.

This tab is comprised of five different blocks:

1. Variant Info

1. Variant type,

2. Main effect,

3. Zygosity in each sequenced family member,

4. Gene symbol and HGVS descriptions on coding DNA and protein levels. The transcript is marked:

   • with a tick - if it is canonical,

   • with a Curate logo - if it has been selected in your Curate database.

   You may change the reference transcript by selecting one from the dropdown menu, or adding one not listed. For certain variants, such as upstream or downstream gene variants, HGVS descriptions may not be available. In these cases, you have the option to manually input coding change information. The notation should adhere to the format: GENE,NM_123456:c.-123N>N (no spaces are allowed). Once added, this information becomes available for the report.\

1. Exon number and the total number of exons in the transcript chosen,

2. Links to resources, such as UCSC genome browser, GeneCards, PubMed, WikiGenes,

3. dbSNP ID and link (Note: SNV/Indel variants only),

4. SV Type: DEL/DUP (Note: CNVs only),

5. SV Length (Note: CNVs only),

6. Link to DECIPHER (Note: CNVs only).

2. In silico Predictions

Standalone scores for Pathogenicity (Missense) Prediction, Conservation, and Splicing Prediction. These are the summarized indicators computed by our proprietary algorithm based on the in silico prediction tools' output. Click on the dropdown icon next to the score to see the individual scores.

Currently available in silico predictions per variant type:

3. Gene Metrics

from ExAC and gnomAD that resemble clinically relevant gene properties:

1. p(LoF intolerant)

pLI = p(LoF intolerant) is a probability of being loss-of-function intolerant to heterozygous and homozygous LoF variants.

Scale:

• 🔴 pLI ≥ 0.9: extremely LoF intolerant,

• 🟠 pLI > 0.1 & < 0.9: intermediate value,

• 🟢 pLI ≤ 0.1: LoF tolerant.

2. Z missense

The Z missense score indicates intolerance to missense variants based on the deviation of observed missense variants versus the expected number.

Scale:

• 🔴 Z missense ≥ 3: missense intolerant,

• 🟠 Z missense > 2.5 & < 3: intermediate value,

• 🟢 Z missense ≤ 2.5: missense tolerant.

3. p(REC)

p(REC) is a probability of being intolerant to homozygous, but not heterozygous LoF.

Scale:

• 🔴 p(REC) ≥ 0.8: Hom LoF intolerant,

• 🟠 p(REC) > 0.2 & < 0.8: intermediate value,

• 🟢 p(REC) ≤ 0.2: Hom LoF tolerant.

4. RVIS ratio

RVIS = Residual Variation Intolerance Score is indicative of a gene's intolerance to functional variation based on comparing the overall number of observed variants in a gene to the observed common functional variants.

Scale:

• 🔴 RVIS ≤ 30: functional variation intolerant,

• 🟠 RVIS > 30 & < 50: intermediate value,

• 🟢 RVIS ≥ 50: functional variation tolerant.

5. pLoF o/e

O/E Score is the ratio of the observed/expected number of LoF variants. It is a continuous measure of gene tolerance to LoF variation that incorporates a 90% confidence interval. The closer the O/E is to zero, the more likely the gene is LoF-constrained. If a hard threshold is needed for the interpretation of Mendelian disease cases, use the upper bound of the O/E confidence interval < 0.35.

4. Gene's related diseases

as reported in OMIM, ORPHANET, CGD, ClinVar, and academic papers included in the Emedgene's knowledge graph. Each of the entries is provided with an inheritance mode icon and a link to the source.

5. Clinical significance

highlights previous pathogenicity classifications of the variant under review:

• Manually Classified indicates if the variant has been previously classified in any of the organization's cases by any user.

• Networks Classified indicates if the variant has been previously classified by the partnering organizations in your network.

• Curate indicates if the variant has been previously classified in your Curate variant database.

• ClinVar provides a list of ClinVar submissions for the selected variant.

• ClinGen Regions (only for CNVs) indicates whether a variant overlaps the established dosage-sensitive region defined by ClinGen.

• HGMD provides a link to the HGMD public page for the selected variant.

• Custom database shows a variant class from the variant database(s) curated by your organization. We can easily implement an organization's curated database of classified SNV or CNV variants to facilitate the case review.

• MITOMAP shows a variant's status in MITOMAP. By clicking on the MITOMAP interactive link, you will be taken to MITOMAP: Reported Mitochondrial DNA Base Substitution Diseases: Coding and Control Region Point Mutations.

The Summary section highlights core variant-related information from other Variant page sections:

• Clinical Significance section (Variant Info, In silico Prediction, Gene's related diseases, Clinical significance),

• Quality section (Quality),

• Population Statistics section (Population Summary),

• Evidence section (Variant Interpretation, ACMG Classification, Pathogenicity).

Each of the Summary section cards has a button linking to its original location on the Variant page where you can see more details and/or edit the evidence.

Let's look closer at each of the cards:

1) Variant Interpretation

Notes added automatically or manually in the Evidence section. Shown only if not empty.

2) Variant Info

• Sequence variant (SNV/Indel), mtDNA variant (SNV/Indel): main effect*,* gene symbol, if available - HGVS descriptions on coding DNA and protein levels.

3) Quality

• Sequence variant (SNV/Indel), mtDNA variant (SNV/Indel), STR:

  • variant caller (32.0+),

  • sample name,

  • zygosity,

  • depth of coverage,

  • percentage of alternative allele reads,

  • overall variant quality.

• CNV (DEL/DUP):

  • variant caller (32.0+),

  • sample name,

  • zygosity,

  • overall variant quality.

4a) Population Summary

Population statistics from gnomAD (calculated for the combined gnomAD population including both exome and genome samples):

• Total AF - overall alternative allele frequency,

• Allele count - Counts of alternative allele (or Homoplasmy Count for mtDNA variants),

• Hom/Hemi count - Counts of alternative allele in homozygous or hemizygous state (or Heteroplasmy Count for mtDNA variants),

• Max AF - the highestalternative allele frequency among gnomAD populations;

Population statistics from organization databases (if available):

• Allele count - Counts of alternative allele,

• Hom/Hemi count - Counts of alternative allele in homozygous or hemizygous state.

4b) STR repeats distribution (2.28+)

STR repeats distribution displays allele counts in gnomAD and 1000 Genomes Project, as well as gnomAD pathogenicity ranges.

5) Gene's related diseases

• Number of gene-disease connections for this gene within Emedgene knowledge base,

• Disease name,

• Disease inheritance mode,

• Link to the gene-disease connection source(s),

• Number of patient's phenotypes matching disease phenotypes out of the total. Note: displayed by default for automatically tagged variants; for manually tagged variants you need to first trigger automatic generation of the evidence by entering the variant's Evidence page.

6) Pathogenicity

Here you can see manually-assigned variant Pathogenicity, change it or select one from the dropdown if it's empty.

7) Clinical significance

This card highlights previous pathogenicity classifications in public and your private variant databases including Curate. Each classification source is represented by one badge. Uncertain and Other classifications are only shown if there are no Benign/Likely Benign and/or Pathogenic/Likely Pathogenic classifications of this variant in a particular database.

8) ACMG Classification

Showcases ACMG tags assigned to the variant and the resulting classification.

• Sequence variant (SNV/Indel), mtDNA variant (SNV/Indel)

  • 32.0+: the final class, the criteria used and the score.

• CNV (DEL/DUP)

9) In silico Prediction

Overall estimations of in silico prediction results.

• Small variant (SNV): Missense Prediction, Conservation, Splicing Prediction;

• Small variant (Indel): Conservation;

• mtDNA (SNV/Indel): Missense Prediction;

• CNV (DEL/DUP), SV, STR: not available.

The Visualization section features an IGV-based tool for the visual review of alignment data for validation and interpretation of variant calls.

Drag visualization tracks, set parameters on the right-hand side and zoom in or out to easily customize your view. To see more details, click on the track of interest.

Visualization tracks

Default tracks:

1. FASTA track displays reference genome sequence;

1. RefSeq Genes track displays gene(s) and transcript(s) affected by the variant;

1. _Test Subject VCF_track (2.28+) represents proband's variants stored in the VCF file. It may come in handy when you're looking for MNVs or large CNVs that may overlap with other variants;

1. Test Subject track showcases read mapping (in a FASTQ case or a VCF case with enabled read alignment view).

Additional tracks:

1. BAM tracks for non-proband samples represent read alignment in patient's relatives;

1. BigWig (2.29+)/ TNS (32.0+) track visualizes output of a systematic noise reducing pipeline - tangent normalized signal (TNS). The TNS track simplifies and increases reliability of CNV analysis. Note: On versions prior to 34.0, BigWig / TNS track is only available for WGS cases run from FASTQ.

1. BAF (32.0+) track presents B-Allele Frequency. The track aids in CNV and LOH analysis. Note: On versions prior to 34.0, BAF track is only available for WES and WGS samples run from FASTQ.

1. ROH (32.0+) track displays runs of homozygosity from whole genome calls on autosomal human chromosomes. The Regions of Homozygosity (ROH) plot is a visualization of homozygosity that may suggest the presence of uniparental isodisomy or partial isodisomy. Multiple ROH in an individual sample can indicate parental relatedness, which may be associated with an increased risk for a recessive disease. Note: On versions prior to 34.0, ROH track is only available for WGS cases run from FASTQ.

When hovering over the region, the ROH score, the number of homozygous SNVs, the number of heterozygous SNVs, and region's start and end positions are displayed.

​

Curated data tracks:

1. ClinVar* track shows short variants submitted to ClinVar.

1. ClinVarSV* track shows structural variants submitted to ClinVar.

1. Curate* track (34.0+)shows short variants that have an entry in the Curate database.

1. CurateSV* (34.0+) track shows structural variants that have an entry in the Curate database.

*Colors indicate variant pathogenicity:

1. Green = Benign/Likely Benign,

2. Yellow = VUS,

3. Red = Pathogenic/Likely Pathogenic,

4. Black = Conflicting interpretation of pathogenicity;

5. Grey = No assertion provided.

Visualization section viewing modes (2.29+)

On versions 2.29+, the Visualization section offers two viewing modes: Simple and Advanced.

Simple viewing mode

By default, the Simple mode displays:

1. RefSeq Genes track;

2. Test Subject track;

3. Test Subject VCF track (2.28+).

Additionally, you can select whether to show:

1. Read alignment tracks for other case samples

(separate feature up to 2.28, part of Additional tracks in 2.29+);

1. All Curated data tracks for all case samples (2.29+);

1. All Additional tracks for all case samples (2.29+).

Advanced viewing mode

In the Advanced mode, you have more control over track visualization, namely, you can specifically select which Curated data tracks and Additional tracks you want to review for each sample.

The Population Statistics section addresses detailed population allele data across various ethnicities in public and internal databases.

• Public databases (SNVs): 1000 Genomes, ESP 6500, ExAC, and gnomAD

• Public databases (CNVs): 1000 Genomes, gnomAD SV, Decipher, DGV

• Internal databases: EmedgeneDB and organization's NoiseDB or other custom databases

By clicking on a particular row of the table, it will provide additional details including alternative allele frequency, alternative allele count, and homozygotes count reported for the selected population by different sources.

Population Statistics for mtDNA variants

The Population Statistics section displays population allele data across various ethnicities in:

• Public databases: gnomAD and MITOMAP

• Internal databases: EmedgeneDB and organization's NoiseDB or other custom databases

By clicking on a particular row of the table, it will provide additional details including the highest homoplasmy frequency, heteroplasmy count, homoplasmy count, and the total number of samples.

The Variant activity panel on the righthand side of the Variant page records variant-level user activities, such as:

• a variant,

• Adding comments,

• Drafting Variant Interpretation notes,

• Editing evidence graph, etc.

The Variant activity panel appears as a tab in the .

The Quality section:

1. Demonstrates the family tree with zygosity status and the overall variant quality indicated for each sample. Zygosity (HET, HOM, HEMI, or REF) is marked inside the symbol, and variant quality grade (H, M, or L) is denoted on the side.

The variant quality score in the proband is highlighted in the section title. Upon 32.0+, the title also features variant caller notation.

1. Outlines the major variant quality parameters underlying the general grade in each sequenced individual:

• SNV/Indel variants:

  • Base Quality,

  • Depth,

  • Mapping Quality,
• CNVs:

  • Copy Number,

  • CNV Quality,

  • Size,
• STRs:

  • Depth;

  • Repeat Number,

1. Illustrates the allele fraction per sample in a pie chart. Not relevant for CNVs.

To change the sample in review for the particular variant, click on the corresponding icon in the family tree.

The Variant page Top bar displays:

1. Case ID,

2. Gene symbol,

3. ,

4. Genomic location of the variant,

5. notes if available,

6. Variant tag field.

   If the variant has been manually (besides or instead of automatic tagging by the AI Shortlist), the user-selected tag will be shown. Otherwise, you'll see an automatically selected tag or N/A for no tag. Clicking on the Variant tag field lets you review a record of tagging in a dropdown, in the Assigned tags section. 🆕 34.0+: the Variant tag menu also includes Viewed by section. Note: After a case , all variants appear as not viewed.

The Variant page > section offers a as well as an column for CNVs in the data table. This percentage is computed by dividing the length of a common region between CNVs by the size of the CNV under study.

Examples of CNV overlap percentage calculation (see legend below):

• Orange represents the current CNV;

• Violet blue represents a previously reported CNV;

• Darker shade indicates the region of overlap between the current and previously reported CNV.

Where can I review and edit the ACMG Classification of a CNV?

The ACMG CNV Classification wizard is located in the Evidence section of the Variant page. Itis available for tagged genomic variants.

The tool automatically scores sections 1, 2, 3, and partially scores sections 4 and 5 of the ACMG/Clingen guidelines, including the full PVS1 calculation required for intragenic variants. All the relevant data is summarized in an accessible table.

This tool is highly accurate and can save 75-90% of manual review time for CNVs (ASHG 2020 abstract).

How do I review and edit the ACMG Classification of a CNV?

The ACMG CNV Classification wizard features:

1. Automatically calculated ACMG class | ACMG score

1. _ACMG score slider_depicting ranges of ACMG score values for each ACMG class and where the current classification falls: Benign: ≤-0.99; Likely Benign: -0.98...-0.90; VUS: -0.89...0.89; Likely Pathogenic: 0.90...0.98; Pathogenic: ≥0.99.

1. Reclassify button that enables Edit mode

1. Gene Number:

   • Number of protein-coding RefSeq genes overlapped by the CNV; of these:

     • Number of established ClinGen genes, i.e., genes with sufficient evidence of dosage sensitivity (defined by having ClinGen's Haploinsufficiency and/or Triplosensitivity scores of 3) or dosage insensitivity (scores of 40),
2. Genes affected by breakpoints - protein-coding RefSeq genes affected by CNV's breakpoints, and positions of breakpoints relative to the canonical transcript of each affected gene. Keep in mind that sometimes a breakpoint falls into more than one gene because genes may overlap.

1. Gene table that provides a summary of the affected protein-coding genes:

   • Gene description:

     • Name - HGNC gene symbol,

     • Strand orientation;

   • Overlap info:

     • Gene - percentage of a gene involved in a CNV,

     • CNV - percentage of a CNV that overlaps with a gene;

   • ClinGen dosage sensitivity scores:

     • TS - ClinGen triplosensitivity score,

     • HI - ClinGen haploinsufficiency score;

   • HI predictors:

     • gnomAD pLI score (colored in red if pLI > 0.9),

     • DECIPHER HI index (colored in red if HI < 10);

   • Canonical transcript:

     • RefSeq ID,

     • 5’ UTR - affected or not,

     • CDS:

       • exons involved out of total,

       • NMD flag if the CNV is predicted to undergo nonsense mediated decay.

       • ClinVar flag if there are Clinvar Path SNV in the last exon
2. b. Clicking on a section box reveals the active criterion, its score, and notes box. Here you can: i. add notes; ii. change the criterion's score where applicable*.

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    ![](https://downloads.intercomcdn.com/i/o/766802351/b444b5fa721acd910d4e425d/cnv_acmg_see_description.gif)

   c. With the Edit tag option, users can modify a particular criterion: 1. select a different criterion within a section, 2. add notes, 3. change the criterion's score where applicable*.

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    ![](https://downloads.intercomcdn.com/i/o/765176533/2fc6bebf8813f5e81c84b602/cnv_acmg_edit_tag%2Bnotes.gif)

   d. You may choose to review and adjust evidence section-by-section using the Reclassify option.

*Criteria with variable score:

• 2F, 2I;

• 4A, 4B, 4C, 4D, 4E, 4I, 4J, 4K, 4L, 4M, 4N, 4O;

• 5A, 5B, 5C, 5E, 5G, 5H.

Where can I review and edit the ACMG Classification of a SNV?

The ACMG SNV Classification wizard is located in the Evidence section of the Variant page. It facilitates classification of variant pathogenicity through the automation of 23 out of 28 ACMG criteria and enabling manual review and editing of the tags presented as interactive buttons.

Starting from version 32.0, the ACMG SNV Classification wizard includes a pathogenicity bar that visually represents the pathogenicity score.

The wizard is available for tagged sequence variants in disease-associated genes. The results of the classification are also highlighted in the Clinical Significance section of the Variant page. Unlike the wizard, automatically assigned criteria and resulting variant class are shown in the Clinical Significance section for all variants in disease-associated genes, regardless of their tagging status.

How do I review and edit the ACMG Classification of a SNV?

Each ACMG tag is represented by an interactive button including checkbox (1), name (2) and evidence strength indicator (3).

Pathogenic criteria are represented by red boxes, while benign criteria boxes are colored green. Each ACMG criterion has three possible states:

• Neutral (1) - represented by an empty checkbox. Criterion requires further investigation.

• Negative (2) - represented by a cross. Criterion is not applicable.

• Positive (3) - represented by a tick and dark color. Criterion is applicable.

Each ACMG tag can be manually checked, unchecked, or set to an undefined state by clicking the interactive button's checkbox element.

To examine in detail or modify the underlying evidence for the particular ACMG tag, select it by clicking on the tag name. The button becomes flood-filled (b), as opposed to it's original, non-selected, state (a).

Upon selection, a description of the criterion and its underlying evidence emerges below. Yes and No radio buttons accompany each piece of evidence. The tag can be assigned if Yes has been selected for all the underlying conditions.

You may modify evidence strength in the Strength dropdown (Stand Alone, Very Strong, Strong, Moderate, Supporting), which will impact both the pathogenicity class and score calculations.

On versions 32.0+, you have the capability to add a note alongside a tag.

After you've modified ACMG classification, you can either save manual changes by pressing the Save button or reset via Revert manual changes. Keep in mind that after saving your edits, Revert manual changes will become unavailable.

ACMG classification of mtDNA variants

The ACMG SNV Classification wizard is available for ACMG classification of tagged mtDNA variants. To classify an mtDNA variant, please manually assign the relevant criteria; the resulting ACMG classification will be calculated automatically.

Seven criteria have been removed in compliance with Specifications of the ACMG/AMP standards and guidelines for mitochondrial DNA variant interpretation (2020): PM1, PM3, PP2, PP5, BP1, BP3, BP6.

Any variant can be tagged by the AI Shortlist or a user as:

• Most Likely Candidate - most promising for solving the case;

• Candidate - worth considering;

• - found in one of the medically actionable genes defined by the ACMG;

• Carrier - identified by the Carrier analysis pipeline;

• In Report - manually selected to be reported;

• Not relevant - automatically tagged variant that has been disregarded after manual review;

• Any custom tag used in your organization (e.g., Submitted for Sanger confirmation).

Variant tags are shown in the Variant tagging widget of the . If the variant has been tagged manually (besides or instead of automatic tagging by the AI Shortlist), the user-selected tag will be shown in the Variant tag field. Otherwise, you'll see an automatically chosen tag or N/A for no tag.

Variant tagging widget how-to

Add or change a tag

Click on the dropdown icon in the Variant tag field and select a suitable tag. The Variant tag field showcases the most recently assigned tag.

Review the tagging history

• 25.0.0+: click on the Variant tag field and scroll to the Assigned tags section.

• 23.0.0 and older: click on the View all tags.

Remove an automatic tag

Click on the dropdown array in the Variant tag field and select Not relevant.

Clear your manual tag:

Click on the dropdown array in the Variant tag field and select Clear.

1) A Pathogenicity box

where you and other users in your group can manually assign variant pathogenicity by selecting an option from the dropdown (Pathogenic, Likely Pathogenic, VUS, Likely Benign, Benign).

If the variant is already in your database, the previously selected pathogenicity will be marked with a Curate logo.

2) Variant Interpretation notes

that include basic variant details added by the AI Shortlist algorithm.

The notes can be manually edited (Edit text link). In editing mode, Paste icon becomes available. You can select actions from the dropdown menu, including:

1. Import data from Curate:

   1. Gene - import Interpretation from )

   2. Variant - import Interpretation from

2. Choose from related cases - connect summary notes available for the variant if it was classified in one of your organization's previous cases.

3. Choose from template - generate variant interpretation using the Variant interpretation template.

3) A See evidence button

to review and modify the automatically assigned genomic variant pathogenicity class. Mostly automated.

5) Sanger Confirmation toggle button

to indicate if the variant should or has been submitted for validation through Sanger sequencing.

Note: Keep in mind that the Evidence section is active only for variants that have been automatically or manually tagged. To enable the Evidence section, you need to assign any tag to the variant under consideration.

The Related Cases section highlights variants that appear in previously analyzed cases, both within your organization and among organizations in your .

Note: data is automatically lifted over between genome references on the fly.

The section features:

data View mode switch (32.0+);

1. Simple mode allows you to show or hide Network data altogether.

1. Advanced mode gives you the flexibility to select specific networks to display.

1. Green - Benign,

2. Blue - Likely Benign,

3. Light grey - VUS,

4. Orange - Likely Pathogenic,

5. Red - Pathogenic,

6. Dark grey - N/A;

You can filter cases by Pathogenicity simply by clicking on the respective section of the percent bar. To clear filters, click on See all.

CNV variants: Dynamic CNV overlap percentage filter;

A table that summarizes details of previous variant interpretation.

Variant and case specifics are available with a single click on the corresponding row of the table (31.0+):

• Proband ID,

• proband phenotypes,

• proband age,

• proband sex,

• maternal and paternal ethnicity,

• case type.

The table comprises the following information:

1. Collaborator (32.0+)

1. Case status icon (32.0+)

1. Lock icon(32.0+)

1. Case ID (32.0+) or Case name (before 32.0)

1. Variant Details (2.29+, CNV variants)

Displays variant coordinates in GRCh37 and GRCh38 genome references, along with the CNV length.

1. Overlap (2.29+, CNV variants)

1. Pathogenicity

1. Date

Date of case creation.

1. Tag

1. Zygosity (30.0+) or Variant Inheritance (before 30.0)

1. Link icon (32.0+)

1. Letter icon (32.0+)

ACMG score

Since version 32.0, the software calculates the variant score based on a 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.

​

ACMG classification

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: “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.

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.

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.

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.

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.

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.

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.

The collapsible Variant page sidebar allows you to access key case information, connect with Alamut and IGV, and view activity log, all within the Variant page. To expand the sidebar, click the arrow icon on the top right, and click again to collapse it.

The Variant page sidebar consists of three tabs:

1. Case Info tab displays relevant details about the case: sample names, the location of the BAM files connected to the case (if any), affected vs healthy sample status, and proband phenotypes.

2. Desktop App tab allows users to manage integration with the IGV and Alamut desktop applications.

3. Activity tab records variant-level user activities such as tagging a variant, adding comments, drafting variant interpretation notes, and editing the evidence graph. This aids collaboration and ensures a traceable record of variant interpretation.