3.7.1. VSClinical ACMG

The ACMG Guidelines for variant interpretation provide a set of criteria for scoring germline variants, placing them into one of five classification tiers [1]:

  • Pathogenic

  • Likely Pathogenic

  • Benign

  • Likely Benign

  • Uncertain Significance

Several modifications of these guidelines have been proposed. These updates include new recommendations for interpreting the loss of function PVS1 criterion by Tayoun et al. [2] and the updated ACGS Guidelines for variant classification, which changes how Pathogenic and Likely Pathogenic classifications are reached [3]. While the updated PVS1 recommendations are always used, user can optionally select the ACGS classification system as an alternative to the traditional ACMG classification system.

This workflow requires the ACMG Guidelines product to be enabled as an add-on to your VarSeq license.

Project Options

When starting a VSClinical workflow, you will be greeted by the VSClinical Project Options dialog, which allows you to configure your workflow. These options are grouped into four tabs:

  • Options: The first tab allows you to select the database catalogs used by VSClinical to store interpretation data. The variant catalog stores interpretation information about classified variants, the CNV catalog stores information about classified CNVs, and the gene dosage sensitivity catalog stores evidence supporting or refuting each gene’s haploinsufficiency or triplosensitivity. See Assessment Catalog View for more information on assessment catalogs.

  • ACMG Consortium Sources: The second tab is used to specify consortium sources. These sources contain previously classified variants and are used to determine if a variant has been previously classified as pathogenic or benign.

  • ACMG Frequency Sources: The third tab is used to specify population frequency sources. These sources provide the variant-level allele frequencies used for recommending the criteria BA1, BS1, and PM2. If these sources include sub-population fields, then the sub-population with the largest allele frequency meeting the minimum allele count will be used for the recommendation of criteria.

  • ACMG Control Sources: The final tab is used to specify control sources. These sources are assumed to contain only healthy adult individuals and are used to recommend the BS2 criterion.

VSClinical Options

VSClinical Starting Page

From the VSClinical start page you can edit sample information, change project options, update source versions, start new evaluations, and manage existing evaluations.

VSClinical Home

The controls in the upper left corner of this screen allow you to update sample information, including the sample name, sex, and affection status. Once you have changed the sample information, you will need to click the Update button for your changes to take effect. This may require rerunning existing algorithms that depend on sample information. Instead of manually specifying the sample information, it can be imported from a text file. See Import Sample Information from Text File for more information on this functionality.

The bottom section of this screen is used to start new evaluations and manage existing evaluations. To start a new evaluations, simply click the Start New Evaluation button. To edit an existing evaluation, click the Open and Continue button in the card associated with the desired evaluation.

Evaluation Options

The Evaluation Options can be accessed from the top right corner of the VSClinical starting page. These options allow you to configure the settings used by each new evaluation. These settings are organized into four tabs.

Report Sections

The first tab allows you to specify the various variant, CNV, and coverage region record sets. There are four record set groups that can be specified from this tab:

  • Primary Findings: Used to define the primary findings section of the report.

  • Secondary Findings: Used to define the secondary findings section of the report.

  • Uncertain Significance: Used to define the uncertain significance section of the report.

  • Failed Target Regions: Defines regions to place in the report as failed targets.

These record sets can be created from this tab or can be selected from the list of existing record sets in the project.

VSClinical Options

General

The second tab allows you to specify the preferred HGVS representation for each variant’s protein description and the classification to be used by the recommendation engine. There are two currently supported classification systems:

  • ACMG (2015): ACMG Variant Classification Richards et al. 2015 [1]

  • ACGS (2020): ACGS Best Practice Guidelines for Variant Classification in Rare Disease (see Table 3) Ellard et al. 2020 [3]

GeneList/Target Panel

The third tab allows you to specify the name of the gene panel and a list of genes in the target panel used for filtering and reporting for the current evaluation. Using the dropdown, you can select a gene panel from the list of available panels in your customizable gene panel library.

VSClinical Options

By default the current Project Gene List is selected. This list can be modified by clicking on the blue Edit button on the right. This will allow you to enter a list of genes. When finished click the validate button to look up each gene in the latest gene track for the assembly of the current project. If a gene can not be found, an error is displayed below the gene entry box.

Gene Thresholds

The final tab is used to specify allele count and frequency thresholds. There are four sets of thresholds that can be specified from this tab:

  • Minimum Allele Count for Sub-Populations: Minimum allele count required for a sub-population to be considered when applying population frequency criteria.

  • Common: A variant’s allele frequency exceeding this threshold is considered stand-alone evidence for a benign classification and results in the application of the BA1 criterion.

  • High for Disorder: This threshold is used to determine if a variant’s allele frequency is greater than expected for the disorder and is used to recommend the BS1 criterion.

  • Extremely Rare: Variants with an allele frequency falling below this threshold are considered extremely rare, resulting in the application of the PM2 criterion.

Source Versions

The annotation source versions used by VSClinical can be configured by clicking Source Versions in the upper right corner of the VSClinical starting page. This dialog allows you to select the currently used version of each annotation source by clicking on the source version in the rightmost column. Sources can be locked to a specific version by clicking on the lock icon next to the source version. You can also specify whether to use the locally downloaded version of a source or the cloud-hosted version on the Golden Helix Annotation Server. This dialog will also indicate when a new version of an annotation source is available. Annotation source updates can be downloaded by clicking the blue Download button at the bottom of the dialog.

Source Version

Evaluation Tab

The Evaluation Tab is the first tab you will see when opening a new ACMG Guideline evaluation. This tab is broken into six sections. The first section is the Evaluation Summary. This section shows sample information, report information, data sources, and a summary of variants and CNVs. At the bottom of this section are three buttons which can be used to finalize, delete, or close the current evaluation.

Evaluation Summary

Sample and Patient

The second section is used to edit sample and patient information. This includes sample details such as the accession number and collection method, patient details such as the patient name and sex, information such as the ordering physician and facility, and family information such as the names of the father and mother.

Variants to Evaluate

The third section is used to manage variants within the current evaluation. This section displays the variants to be evaluated in a table, which includes summary information about each variant. The order in which variants appear in the report matches the variant order in the table. The variant order can be modified by clicking Edit Variant Order in the lower left hand corner of the table. Variants can be added to the evaluation by clicking Manually Enter Variants or Add Variants From Project

Variants to Evaluate

The Add Variants From Project dialog is used to add variants from the current VarSeq project to the evaluation. A list of filtered variants is shown on the left of this dialog. A dropdown above the list of filtered variants can be used to change which variants are shown. Using the checkboxes on the left, you can select the variants you wish to add. After clicking Prepare to Add, a preview of the variant details will be shown in a table on the right. Once you have finished entering variants, you may click the Add Variants button in the bottom right corner to add the variants to the evaluation.

Add Variants From Project

The Manually Enter Variants dialog can be used to add variants that are not in the current VarSeq project. To add a variant using this dialog, simply enter a query into the search box in the upper left-hand corner of the window. Several different formats are supported for the query string:

  • Genomic Coordinates with Ref/Alt (7: 140,453,136 A/T)

  • HGVS Coding Notation (NM_004333.6:c.1799T>A)

  • HGVS Amino Acid Notation (NP_004324.2:p.V600E)

  • Gene with Amino Acid Change (BRAF V600E)

Variants matching the entered query will be shown below the search box. Once a variant has been selected, you may set the variant’s allele counts and zygosity before clicking Prepare to Add. As with the Add Variants From Project dialog, you can click the Add Variants button to add the variants to the evaluation.

Variants to Evaluate

CNVs to Evaluate

The fourth section is used to manage CNVs within the current evaluation. As with the Variants to Evaluate section, CNVs can be either added manually or added from the current VarSeq project.

CNVs to Evaluate

The Add CNVs From Project dialog behaves much the same way as the Add Variants From Project dialog. A list of filtered CNVs are shown on the left hand side of the dialog, each of which can be added to a table of selected CNVs which displays summary information about each CNV.

The Manually Enter CNVs dialog is used to add CNVs that are not in the current VarSeq project. CNVs are selected by entering a query into the search box in the upper left-hand corner of the window. Two different formats are supported for the query string:

  • Genomic Coordinates (7: 1,000,000-2,000,000)

  • Gene Name and Exon Numbers (BRCA2 3-5)

CNVs matching the entered query will be shown below the search box. Once a CNV has been selected, you can set the CNV type (Het Del, Deletion, or Duplication) and may include the CNV’s Ratio and Z-Score values before clicking Prepare to Add. Once the CNVs have been added to the staging table, you can click the Add CNVs button to add the CNVs to the evaluation. By default, both dialogs will expand the CNV coordinates to match the boundaries of the first and last exons overlapping the event. This functionality can be disabled by clicking the checkbox labeled Expand CNVs to Exon Boundaries.

NGS Sequencing Summary

The fifth section is the NGS Sequencing Summary, which displays sample-level summary information about the variants in the current VarSeq project. This includes a variant count broken down by zygosity and type. In order to view this section, you must run the Sample Statistics algorithm by clicking Add -> Computed Data and selecting Sample Statistics from the algorithm list.

NGS Sequencing Summary

The final section is the Changelog History, which shows a table listing all changes made to the evaluation.

Genes Tab

The Genes Tab displays information about target-level coverage and reported genes. This tab requires the Coverage Statistics algorithm, which can be added to a VarSeq project by clicking Add -> Secondary Tables -> Coverage Region Annotation, provided that the samples in your project have associated BAM files. The first section of this tab is the NGS Coverage Summary. This section displays sample-level coverage information, including the mean depth, number of targets, and percent of bases covered at various levels of coverage.

NGS Coverage Summary

The second section is the Reported Genes section, which lists the genes to be included in the report. There are four options for selecting the list of reported genes:

  • Don’t Report Gene Coverage: Gene coverage will be excluded from the report.

  • Coverage Regions: Gene coverage will be reported for all genes overlapping targets in the Coverage Statistics table.

  • Evaluation Options: Coverage will be reported for all genes specified in the Evaluation Options gene panel. This gene list can be edited from this section by clicking the blue edit button on the bottom right.

  • Sample Table Gene List: Coverage will be reported for all genes in the sample table gene list or panel for this sample.

Reported Genes

The final section is the Reported Failed Targets section, which displays a gene level coverage chart, highlighting in red any genes with targets which fail to meet minimum depth requirements.

Reported Failed Targets (Gene View)

Clicking on an individual gene in this chart will plot of the coverage for each individual target within the gene.

Reported Failed Targets (Target View)

Each bar in this chart shows the minimum, maximum, and average depth for the corresponding gene or target. This chart can also be used to display mapping quality by toggling the Depth/MQ button in the upper right hand corner of the chart. Below this chart is a table showing summary information for genes and targets that fail to meet coverage requirements.

Failed Targets Table

Variants Tab

The Variants Tab is used to evaluate individual variants in accordance with the ACMG Guidelines. Each variant is automatically evaluated by the ACMG recommendation engine, with many of the individual criteria receiving recommendations on whether they should be answered Yes or No along with detailed reasons for these recommendations.

Evidence Summary

In the Evidence Summary section, criteria that are recommended will be listed under Recommended to Score Pathogenic or Recommended to Score Benign. The codes for the recommended criteria are shown in colored boxes with arrow links to navigate to the section containing the criteria questions and supporting evidence.

Evidence Summary

Each ACMG criteria has an associated card in it’s corresponding section. These criteria cards display the recommended answer for each associated question, reasons supporting the recommended answer, and caveats associated with the given criteria. These cards allow you to select answers for each criteria question, change the selected evidence strength of the criteria, and enter any criteria-specific comments.

PM1 Criteria Card

The right sidebar can be used to switch the evaluation to other variants as well as see summary information about the current variant. Each variant is represented with a box with the variant names available on hover. The boxes are filled in when the variant is evaluated and saved. The color of the outline and the fill-in color correlate to the ACMG classification of each variant. The color scheme is as follows:

Classification Color Scheme

As you answer criteria questions, the right bar will update to show the currently scored criteria and resulting ACMG Classification.

ACMG Classification

The ACMG Classification section displays a list of the currently applied criteria along with the current classification based on these criteria. On the right side of this section is a plot showing the probabilities for each classification, based on our probabilistic model.

ACMG Classification

The ACMG Criteria Summary, which is located at the bottom of this section, can be expanded to display the recommendations for all relevant criteria. A switch at the top of the summary can be toggled to show all criteria. Comments for each criteria can also be entered in the ACMG Criteria Summary.

Criteria Summary

Variant Interpretation for Sample

The Variant Interpretation for Sample section is used to enter the variant interpretation along with other relevant details including:

  • Report Section (Primary Findings, Secondary Findings, or Uncertain Significance)

  • Variant Set

  • Classification (Pathogenic, Likely Pathogenic, Uncertain Significance, Benign, or Likely Benign)

  • Disorder Associated with Classification

  • Inheritance Type

  • Internal Notes

  • Flags or Comments

When entering an interpretation, references for PubMed publications and clinical trials will be automatically added to the set of inline references provided that these references take the following form: PMID: 12345678 or NCT01234567. Once the interpretation has been finalized, it can be saved by clicking Review & Save Now.

Variant Interpretation

The right side of this section displays a variant details card which contains five tabs:

  • Scoring: This tab includes the variant auto-interpretation, classification, and scored criteria.

  • Annotations: This tab displays transcript annotations, computational evidence, and splice predictions.

  • Gene: This tab contains gene information from NCBI, the Clinical Genomic Database, OMIM, and Genetics Home Reference.

  • Literature: This section provides tools for searching relevant literature and displays in-line references from ClinVar and dbSNP.

  • Assessments: This section displays ClinVar assessments.

Variant Interpretation Card

The auto-interpretation text generated by VSClinical can be added to the variant interpretation by clicking the Add to Interpretation button at the bottom of this card.

Tolerated Frequency

This section is used to select the Gene/Disorder Inheritance Model and view the associated variant frequency thresholds used to recommend the population-frequency-based ACMG criteria BA1, BS1, and PM2. The thresholds associated with each inheritance model can be changed in the Evaluation Options.

Tolerated Frequency

Population Frequency

This section displays evidence related to the population-frequency-based criteria. This includes a plot of the frequency and homozygous count for the highest frequency sub-population, along with a plot of the Allele Count and Frequencies for every sub-population. These plots can be displayed for both gnomAD Exomes and 1kG Variant Frequencies. The currently selected catalog can be changed by clicking on the desired catalog name in the tabs above these plots. In addition to the population frequency plots, this section includes the ACMG criteria cards for BA1, BS1, BS2, and PM2.

Population Frequency

Gene and Transcript

The Gene and Transcript section displays details about the gene, including the NCBI gene description and the variant’s predicted effect on the protein. The Gene Impact card on the right side of this section presents a summary of the variant’s impact on the gene. This includes the overlapping exon number and the predicted effect along with the HGVS protein and coding change notation. Through this card, you can change the preferred amino acid representation by clicking on the gear icon in the upper right-hand corner. The selected transcript can also be changed by clicking on the blue Transcripts button.

Gene Impact

For details on the heuristics used to select clinically relevant transcripts see Gene and Transcript Preferences.

Gene Region and Mutation Profile

This section displays gene-level counts of classified variants along with a table of all classified variants in the gene. This table can be filtered based on variant type (Missense or LoF), genomic region (Same Gene, Exon, or Codon), classification (Benign or Pathogenic), and star rating. This section also includes the ACMG criteria card for PM1.

Gene Region Table

Tolerant Loss of Function Mutations

This section is used to evaluate the impact of loss of function variants on the gene. It displays a plot of the variant’s position relative to the penultimate exon junction along with information from gnomAD Gene Constraints including:

  • Obs/Exp LoF: The observed/expected score for loss-of-function variants

  • pLi: The probability of gene being loss-of-function intolerant

  • Z-Score: Distance in standard deviations from the average for loss-of-fucntion variants

This section also includes the ACMG criteria card for PVS1.

Tolerant Loss of Function Mutations

Missense as Mechanism of Disease

This section is used to determine whether missense mutations are a common mechanism of disease for the gene of interest. It displays a scale representation of the missense Z-score computed on the gnomAD population frequencies for this transcript with supporting counts, along with plots for the missense badness and missense deleteriousness scores. This section also includes the ACMG criteria card for PP2 and BP1.

Missense as Mechanism of Disease

Coding Change and Repeats

This section provides a visualization of the surrounding region in both the coding sequence and the amino acid sequence, to help the user determine if the variant is in a repeat region. This includes a plot of nearby Low Complexity Regions, Genomic Super Dups, and Repeat Regions. This section also includes the ACMG criteria card for PM4/BP3.

Coding Change and Repeats

Computational Evidence

This section displays all computational evidence associated with the variant. This includes the functional prediction algorithms SIFT and PolyPhen2, along with the results for the following splice prediction algorithms:

  • GeneSplicer

  • MaxEntScan

  • PWM

  • NNSplice

Computational Evidence

By clicking Open Splice Site Analysis on Region you can open the splice region window. This window displays the splice predictions for all potential splice sites in the surrounding region for both the reference and alternate sequence. Regions where there is potential splicing disruption are highlighted in orange.

Splice Region

This section also displays the multi-sequence alignment for 100 vertebrates. The multi-sequence alignment can be switched between the DNA view and the Amino Acid view using a toggle in the right hand corner of the display. This section also includes the ACMG criteria card for PP3/BP4.

Multi-Sequence Alignment

Clinical and Functional Studies

The Clinical and Functional Studies section is used to explore relevant literature and identify functional studies supportive of a damaging effect on the gene. This section provides fields for entering information on clinical and functional studies, including the reported amino acid change, inheritance state, disorder, and study summary.

Clinical and Functional Studies

This section also includes buttons for searching Google, Google Scholar, and PubMed for relevant literature, along with a list of ClinVar assessments and their associated citations. This section also includes the ACMG criteria cards for PS1, PM5, and PS3/BS3.

ClinVar Assessments

Inheritance and Allelic State

This section includes information on the variant’s inheritance and allelic state. This section also allows you to specify if the variant has been observed in trans or in cis with another pathogenic variant. The last component of this section includes the ACMG criteria cards for PP4, PS4, BP5, PP1/BS4, and PS2/PM6.

Inheritance and Allelic State

CNVs Tab

The CNVs Tab is used to evaluate individual copy number variants in accordance with the ACMG Guidelines and capture the interpretation for reporting CNVs. The gene impact of each CNV is automatically evaluated and, when a CNV overlaps an established dosage sensitive gene or region, it is often possible to achieve a pathogenic or benign classification without the need for further evaluation. However, many genes have insufficient evidence to establish haploinsufficiency or triplosensitivity. For CNVs overlapping such genes, a detailed evaluation of the relevant clinical literature is required, unless the CNV overlaps a large number of genes. The dosage sensitivity scoring tool helps the user perform the necessary literature review to establish a gene as either haploinsufficient or triplosensitive.

CNV Summary

The CNV Summary section provides a short description of the CNV under evaluation, including its predicted effect on the relevant genes, along with a description of the clinically relevant gene’s dosage sensitivity.

CNV Summary

The CNV Evidence card provides an overview of important information about the CNV, including the genomic position, type, cytogenetic location, and relevant metrics.

CNV Evidence Card

As with the variant tab, the right sidebar can be used to switch the evaluation to other CNVs as well as see summary information about the current CNV. Each CNV is represented with a box labeled with the clinically relevant gene. The color of the outline and the fill-in color indicate the classification of each CNV.

CNV Interpretation for Sample

This section is used to enter the CNV interpretation along with other relevant details including:

  • Report Section (Primary Findings, Secondary Findings, or Uncertain Significance)

  • CNV Sets

  • CNV State

  • CNV Origin (Do novo, Maternal, Paternal, etc)

  • Classification (Pathogenic, Likely Pathogenic, Uncertain Significance, Benign, or Likely Benign)

  • Internal Notes

  • Flags or Comments

As with the variant interpretation, references for PubMed publications and clinical trials will be automatically added to the set of inline references.

CNV Interpretation

The right side of this section displays the CNV scoring card which contains four tabs:

  • Scoring: This tab includes the CNV Summary, classification, and scored criteria.

  • Genes: This tab contains information on overlapping genes including the clinically relevant transcript, effected exons, and gene impact.

  • Previous CNVs: This tab lists relevant previously scored CNVs.

  • Annotations: This section displays annotation information, including ClinGen, gnomAD, ClinVar, DECIPHER, and DGV.

CNV Scoring Card

Genomic Region

This section displays a plot of the genomic region surrounding the CNV. This plot includes several annotation sources:

  • ClinGen Gene/Region Dosage Sensitivity

  • GnomAD High Frequency CNV Regions

  • DECIPHER Population CNV

  • DGV CNVs - Gold Standard Variants

  • 1kG Phase3 CNVs and Large Variants

  • ClinVar CNVs and Large Variants

  • ClinVar Variants

  • Previous CNV Interpretations

  • RefSeq Genes

Each annotation plot’s visibility can be changed by selecting the Tracks arrow in the upper right hand corner and clicking the checkbox associated with the track. Details for each annotation feature can be viewed by clicking on the feature in the plot.

CNV Genomic Region

Below the region plot are the controls for criteria 1A-B, 3A-C, and 4O. VSClinical will automatically select the appropriate criteria in this section, but the selected criteria can be changed using the checkbox or radio button in each criteria card.

Overlapping Genes

This section is used to select the clinically relevant gene, evaluate criteria for sections 2, 4, and 5, and enter gene-level report information. This information includes:

  • Relevance to Patient

  • Disorder

  • Mode of Inheritance

  • Notes on Relevance to Patient

  • Notes on Gene

For CNVs overlapping multiple genes, VSClinical will automatically select a clinically relevant gene, but you may select a different gene by clicking on a row of the overlapping genes table. Genes are sorted based on dosage sensitivity and relevant annotations, such as OMIM, ClinGen, and MONDO.

CNV Genes Table

The CNV Gene Scoring card on the right of this section displays information on CNV scoring and gene details. This card is broken into the following tabs:

  • Scoring: This tab contains information on applied scoring criteria and the CNV’s impact on the gene.

  • Gene: This tab includes gene summary information from NCBI RefSeq, the Clinical Genomic Database, OMIM, and Genetics Home Reference.

  • Annotations: This tab contains gene-level annotations from ClinGen, gnomAD, DGV, OMIM, Orphanet, and DECIPHER.

  • Inheritance: This tab displays information on the gene’s inheritance model and associated conditions.

CNV Gene Scoring Card

From the scoring tab you can selected which criteria sections to apply. If the gene is a known dosage sensitive gene, Section 2 will be automatically applied. However, if the gene lacks sufficient evidence for dosage sensitivity, Section 4 will be selected instead. Each criteria section can be viewed and edited by clicking the button on the right side of that section’s row.

Section 2

Clicking on the Edit button next to the Section 2 row of the Scoring tab will open a new window containing the Section 2 wizard. Section 2 is applied if the CNV overlaps an established haploinsufficient, triplosensitive, or benign gene/region. A gene is considered haploinsufficient or triplosensitive if it has been classified as having “sufficient evidence for dosage pathogenicity” in either the ClinGen Dosage Sensitivity Map or in the clinical lab’s internal database. Established benign genes are those that have been classified as “dosage sensitivity unlikely” by ClinGen.

The Section 2 wizard determines the most applicable Section 2 criterion by answering a series of simple questions about the CNV. While the Section 2 criteria are applied automatically for genes with established dosage sensitivity, the Section 2 wizard allows the user to review and change the answers to each question used to determine the applicable criterion. In the example below, criterion 2C-1 has been automatically recommended by VSClinical, as this deletion overlaps the 5’ end of the gene and the coding sequence is involved. The user can navigate to previous questions by clicking the back button in the upper left hand corner of the window or by clicking the breadcrumb links shown below the selected criteria.

CNV Section2 Wizard

Section 4

When a CNV overlaps an established dosage sensitive gene or region, it may be possible to achieve a pathogenic or benign classification without the need for further evaluation. However, many genes have insufficient evidence to establish haploinsufficiency or triplosensitivity. For CNVs overlapping such genes, a detailed evaluation of the relevant clinical literature is usually required.

If the gene under evaluation has not been previously classified as either haploinsufficient or triplosensitive, the Section 4 checkbox will be selected in the CNV Gene Scoring card. Clicking on the Section 4 Edit button will open the Section 4 dialog. This dialog allows you to enter evidence obtained from relevant peer-reviewed medical literature. At the top of this dialog is a table listing all Section 4 evidence records associated with the gene.

Create Evidence Record

Evidence associated with an individual case or case-control study can be added by clicking the Create New Evidence Record button below the table. This will launch a new window where you can enter the evidence source, reported mutation, disorder, and mode of inheritance, along with an evidence summary.

Create Evidence Record

Once you have entered all of the relevant information, you can click Start Wizard to launch the Section 4 wizard, which will ask a series of questions about the current evidence record and determine the most applicable Section 4 criteria.

Section 4 Wizard

Once created, each evidence record will be displayed in the Evidence Record table at the top of the Section 4 dialog. Below the Evidence Record table is a plot showing overlapping gene annotations, alongside the card for criteria 4O. This plot aids the user in determining whether the gene is fully contained by a common CNV.

Section 4 Plot

At the bottom of the dialog is a form used to enter the gene’s evidence strength, associated disorder, and evidence for dosage sensitivity.

Haploinsufficiency Evidence Curation

To the right of this form, is the Section 4 Evidence Card, which includes four tabs:

  • Scoring: Displays all applied Section 4 criteria, the total Section 4 score, and the evidence for dosage sensitivity.

  • Variants: Lists assessments for previously classified CNVs in this gene.

  • Gene: Includes gene summary information from NCBI RefSeq, the Clinical Genomic Database, OMIM, and Genetics Home Reference.

  • Annotations: Contains gene-level annotations from ClinGen, gnomAD, DGV, OMIM, Orphanet, and DECIPHER.

  • Inheritance: Displays information on the gene’s inheritance model and associated conditions.

Haploinsufficiency Evidence Card

To finalize the Section 4 literature review, click the Save button in the upper right hand corner of the screen. After completing the Section 4 analysis, VSClinical will tabulate each of the individual pieces of literature evidence in accordance with the guidelines rules and apply the correct code and point level.

Section 5

The Section 5 wizard is used to evaluate the inheritance information and family history for the current patient. While this information can provide evidence supporting dosage sensitivity, it should be noted that it is difficult to determine dosage sensitivity based on the inheritance pattern of a CNV in a single family. Much of the criteria described in Section 5 require the application of the Section 4 criteria to the current proband, essentially treating the current patient as a single piece of individual case-level evidence. Because of this, the Section 5 wizard in VSClinical asks many of the same questions that the Section 4 wizard asks in the context of individual case evidence.

Section 5 Wizard

It is only through the accumulation of evidence across many different families that a true measure of clinical significance can be obtained. For this reason, VSClinical stores all criteria applied in Section 5, so that it can be leveraged in future interpretations as Section 4 case-level evidence. As the clinical lab accumulates evidence across different individuals and families, a clearer picture of the gene’s dosage sensitivity can emerge, allowing for the classification of variants that were previously of uncertain significance.

Phenotypes Tab

The Phenotypes Tab is used to manage the patient’s associated disorders and phenotypes. Disorder and phenotype terms can be extracted from clinical notes by entering the notes into the form in the top left of the tab and clicking the Extract Terms button.

Clinical Notes

The extracted disorders and phenotypes will be shown in a list to the right of the clinical notes. Each disorder and phenotype in this list has an associated card which displays its ID, name, synonyms, and description. You can add the complete list of extracted disorders and phenotypes to the evaluation by clicking the Add All button in the upper right corner of the list. Alternatively, individual disorders and phenotypes from this list can be added by clicking the Add Disorder/Phenotype button in the associated card.

Extracted Phenotypes

Disorders and phenotypes can also be added using the Phenotype and Disorder search interface. To manually search for a specific phenotype, scroll down to the Selected Phenotypes and Disorders section, and enter the phenotype into the search bar. A list of matching phenotypes will then be shown. The details for the currently selected phenotype are shown to the right of the search results. The Disorders tab contains a similar search interface for manually adding disorders. This section also includes tabs listing disorders and genes associated with the list of selected phenotypes.

Phenotype Search

The added phenotypes and disorders are listed in a panel on the right side of the Phenotypes tab. The details of each added phenotype and disorder can be viewed by clicking on the phenotype/disorder name in the panel.

Phenotypes Side Panel

Report Tab

The report tab is used to edit report details, generate reports, and finalize the evaluation. It shows all reportable fields and is organized into the following sections:

  • Report: Includes the result summary, along with patient and sample information.

  • Variants: Includes information on reported variants.

  • CNVs: Includes information on reported CNVs.

  • Uncertain Significance: Includes information on variants and CNVs of uncertain significance.

  • NGS Report: Includes summary statistics along with coverage information.

  • Evaluation Options: Includes information on the classification system and population frequency thresholds.

  • Annotation Sources: Includes all annotation sources used for the evaluation along with the version number and type.

  • Inline References: Includes all inline PubMed references with hyperlinks to the source data.

When you click on a group of fields in the report data display, a form will be shown on the right side of the tab, allowing you to edit any of the fields in the selected group.

Edit Report Fields

Once you have finished editing the report fields, you can click Back to Report Data to begin the export and sign out process. From this screen, you will see an option to set up and generate a clinical report as a Word file, and subsequently as a PDF. The first time you select the Word file item, you will need to create a new Report Template. For more details on which report template to choose and how to configure them to match your needs, see Customizing VSClinical Reports.

Render Word Report

After selecting a template, you can click the Render button to generate the final report from the full set of reviewed data. The Word file will automatically be opened after it is rendered. A PDF can be generated from the Microsoft Word report by clicking on the PDF icon in the right bar. PDF reports are stored in the same location and can be previewed by clicking on the blue Preview button.

New exports can be added to the report output by hovering over the blue + button in the right bar. Three export options are available:

  • New Word Export

  • New PDF Export

  • New Custom Export

Custom report exports can be used to generate HTML, XML, JSON, and more. The JSON outputs generated by these custom report exports can then be used as inputs into other report templates. Before generating a custom report export, you must specify the custom template file in the menu under the Custom Script Report Inputs section. The outputs of custom report exports are determined by the associated javascript file, which can be edited by selecting Open from the Custom Template File menu. For more details on custom report exports see Customizing VSClinical Reports.

Custom Export

Below is an example of a rendered report.

Example Report

Select the green Sign Out & Finalize button when you are ready to complete the evaluation.

This will finalize the evaluation content and will not allow any changes to be made without first revoking the report finalization (available in the Report History list).