My thoughts and ideas
My thoughts and ideas
ClinVar aggregates information about genomic variation and its relationship to human health. To date, thousands of variants and associated phenotypes have been deposited in ClinVar. The main use case for ClinVar is when you have one or more variants observed in a human sample and you would like to know if these variants have established clinical relevance. The resource is fairly heavily biased towards germline inherited variants that predispose an individual for disease. Many diseases and phenotypes have been reported. Some of the variant observations in ClinVar come from sources that mine published literature (e.g. DoCM, OMiM, etc.). However, the majority of submissions to ClinVar come from clinical sequencing labs that identify variants in patients and report that variant along with the phenotype of the patient(see Data Sources for more details). Several types of variants are supported in ClinVar. Many are single nucleotide variants (SNVs) or small insertions and deletions, but large structural variants are also supported. In general, all variants in ClinVar are designated using the HGVS nomenclature. ClinVar strongly recommends following the ACMG guidelines in deciding on the clinical significance (pathogenicity) of variants. The following tutorial explores these concepts in greater detail.
ClinVar offers a few video tutorials to get you started: Intro to ClinVar and Find All Variants with ClinVar. The ClinVar website is quite simple. The home page offers various links to frequently used sections. The tool bar at the top of each page includes the simple search bar and various menus to navigate to additional information.
ClinVar has several key entities used to organize variant data. (1) Each submission of an interpretation for a variant is assigned a submission accession of the format SCV000000000.0. Multiple labs could report independent interpretations for the same variant. Each would be entered as a distinct record. If a lab wishes to update that submission, they can do so, and the version number will be updated to reflect this change. (2) When there are multiple submissions about the same variation/condition relationship, they are aggregated under a reference accession of the format RCV000000000.0. If the same variant is associated with multiple conditions, there will be multiple reference accessions. (3) All information about a single distinct variant are organized under one Variation ID and a corresponding Allele ID. Both are simple integers. Each allele corresponds to a distinct genomic variation that must be described using one or more HGVS expressions.
Consider the following example: NM_007294.3(BRCA1):c.5558dupA (p.Tyr1853Terfs)
A more detailed explanation of these identifiers can be found in the Identifiers section of the ClinVar documentation.
ClinVar several search modes. (1) you can simply type free form text in the search box near the top of every page, (2) if you know the neccessary field codes, you can construct complex queries in this same search box, (3) you can use the Advanced Search Builder.
Search for variants within a narrow window on a single chromosome: “5[chr] AND 1264532:1264552[chrpos]”. What gene does this region correspond to? What is the significance of this gene?
Use the Advanced Search page to construct the same query. Note that you can use the show index list option to see valid search options/examples for each field.
If you just want the complete list of high quality variants that you might consider returning results for in a clinical setting. You might decide to peform a query that requires the variant to have been reviewed by an expert panel, belong to a practice guideline, or have been submitted by multiple entities that agree on the interpretation. This list could be further limited to those that are Pathogenic and where the variant origin is stated as “germline”. If you put all of this together the query looks like this:
((("reviewed by expert panel"[Review status]) OR "criteria provided, multiple submitters, no conflicts"[Review status]) OR "practice guideline"[Review status]) AND "clinsig pathogenic"[Properties] AND "germline"[Origin]
In completing the exercises above in ClinVar you will have noticed that variants have rather complex looking names. This is a deliberate choice on the part of ClinVar. It is common in the literature and in day to day conversations to refer to variants with shorthand names. For example, someone refers to BRAF V600E you may have a good idea what they are referring to. However, such names can be ambigiguous. There are often multiple correct variations in the genome that could lead to a V600E effect in the protein. Furthermore, if you want to design an assay to detect this variant in RNA, cDNA, or genomic DNA, then V600E is not very useful. For this reason, the community has adopted a set of standards for describing variants in a much more precise way that is unambiguous and leads to less confusion. The Human Genome Variation Society (HGVS) acts as a steward for these standards. They have also released detailed guidelines (the rulebook) on the correct way to create HGVS expressions for almost any variant. These guidelines are maintained, updated, and versioned. They are available at: varnomen.hgvs.org. ClinVar also provides a simple overview of the HGVS types they use. Here is an example of the same variant (AKT G17K) represented as HGVS expressions describing it at the:
ClinVar variant name: NM_005163.2(AKT1):c.49G>A (p.Glu17Lys) HGVS for protein: NP_005154.2:p.Glu17Lys HGVS for cDNA: NM_005163.2:c.49G>A HGVS for genome: NC_000014.9:g.104780214C>T (GRCh38)
Each HGVS has several required elements. First you have a sequence accession with a version number (e.g. NM_005163.2). This tells you unambiguously what the reference sequence is (a published protein, cDNA, or genome sequence). Then you have a delimiter “:”. Next you have a letter that indicates the general type of HGVS expression to follow: “p” for protein, “c” for cDNA, “g” for genomic DNA. Finally, you have the expression that describes the sequence variation itself. The rules for these expressions depend on the type of variation (substitution, duplication, deletion, etc.) and the level of HGVS (protein, cDNA, gDNA, RNA, …).
Here are some excellent resources for working with HGVS:
You may have noticed in the exercises above various references to the clinical significance or pathogenicity of variants. In some queries of ClinVar we limited variants to only those that are thought to be Pathogenic (i.e. if inherited, the predispose an individual to a particular disease). How does one establish whether a variant is Pathogenic, or Benign? To help establish how this should be done, the ACMG has released detailed Standards and guidelines for the interpretation of sequence variants. For the full details, one should definitely read that paper and refer back to it extensively as you try to interepret specific variants. Briefly, the ACMG established a variant classification approach that is meant to be applicable to variants in all Mendelian genes. They categorize the types of evidence that one should use to support the pathogenicity of a variant. Each type of evidence is indicated by an evidence code. Evidence codes are broken down into two main categories, those that support a pathogenic interpretation, and those that support a benign interpretation. Within these two broad groups, sub-categories are defined for different types of evidence (e.g. functional data, population frequency, inheritance patterns, algorithmic predictions, etc.). The guidelines describe how to document this evidence and how to use that evidence to make a final conclusion for the pathogenicity of each variant for each disease/phenotype. The ACMG defines five tiers for the pathogenicity of a variant: “pathogenic,” “likely pathogenic,” “uncertain significance,” “likely benign,” and “benign”.
We strongly encourage you to read the ACMG guidelines carefully. The critical summaries are provided in several tables of this paper.
Final comment: If used properly, the ACMG guidelines require a lot of evidence to define a variant as pathogenic or even likely pathgenic. Similarly strong evidence is needed to conclude a variant is benign or even likely benign. Many variants are thus initially classified as “uncertain significance”. Note that this does not mean unknown it means uncertain. i.e. lacking certainty. Since variant classifications are meant to be used in a clinical setting the goal is to avoid misinforming patients that may take life changing actions upon hearing these results.
How many ClinVar variants are there for BRCA2 that are Germline, Pathogenic and have Expert Panel review status?
Get a hint!
Simply enter BRCA2 in the ClinVar search box, then apply the three filters requested.
At the time of writing this post, there were 2069 germline, pathogenic, expert panel reviewed BRCA2 variants.
Use ClinVar to find valid HGVS expressions at the genome, cDNA, and protein level for the following variant: PIK3CA H1047R.
Get a hint!
Simply enter PIK3CA H1047R in the ClinVar search box, then review the HGVS section.
NG_012113.2:g.90775A>G (genome), NM_006218.3:c.3140A>G (cDNA), NP_006209.2:p.His1047Arg (protein).
Find all of the variants in ClinVar that correspond to exon 2 of VHL. How many are there? Export these to a spreadsheet.
Get a hint!
Use a genome browser such as IGV to obtain the build38 coordinates for exon 2 of VHL. Then use the advanced ClinVar search box to search for variants in VHL and within the exon 2 coordinates.
To get VHL exon 2 variants use this query. (VHL[Gene Name]) AND 10146514:10146636[Base Position]. At the time this question was created there were 517 variants in VHL and 63 of these were within exon 2.
Often it will be informative to annotate variants with additional information in order to get a sense of a variants impact on a phenotype. One tool that makes this process quick and straightforward is the ensembl Variant Effect Predictor (VEP). This program is available both as a stand alone software program based in perl and as a web based GUI. In this module we will learn how to use VEP in both forms.
In order to use stand alone VEP we will first need to download and install perl, a high level scripting language. First let’s check if you have perl ≥ 5.10 already installed, open a command prompt “terminal” on your local machine and run the code below.
# check perl version perl -v
If you see a message to the effect of “This is perl 5” then you can ignore the next bit, otherwise you will need to download and install perl. To do this navigate to the perl downloads page at https://www.perl.org/get.html and select get started for your specific operating system.
Then select activeState perl, once the installer is downloaded follow the on screen instructions and check your install with
perl -v from a terminal window.
In order to use VEP on widnows we will first need to download and install a special flavor of perl called DWIMperl. Navigate to http://dwimperl.com/windows.html and download the “Dwimperl-22.214.171.124-v7-32.exe” executable at the bottom of the page. Then run the executable and follow the on screen instructions. Once finished search and open Command Prompt from the “Start Menu” and type
perl -v to check that the installation was successful.
With perl now installed we can go ahead and download and install VEP itself. The recommended way to do this is by cloning the github repo available here. We won’t actually be cloning the repo, though you certainly could. Instead select the green Clone or download button to the right and then select Download Zip. Once the file is downloaded go ahead and unzip it.
Once that is complete you will need to navigate to the directory where you unziped the VEP repo and run the code below.
# change to directory where vep is cd ./ensembl-vep-release-90 # initate VEP installations perl INSTALL.pl --NO_HTSLIB --NO_TEST
This will start the VEP installation process. You will be asked if you want to “download local cache files”, “download fasta files”, and “install plugins”. Because of their size we will answer “no” to all these however you can always change this by re-running
As we have mentioned previously VEP can either be run via the command line or through a web GUI. For the remainder of this section we will be doing both side by side however we should note that using VEP from the command line is more flexible and there is a greater range of features.
To start let’s go ahead and annotate the germline variant rs1799966 using the default ensembl input format. Essentially we need to create a file with 5 columns corresponding to “chromosome”, “start” (1-based), “stop” (1-based), “reference/variant”, and “strand” (corresponding to reference/variant). You can optionally have a sixth column to add a unique identifier for this row. After creating the file
variant.txt we run VEP with the following options.
-iinput file, the file format is automatically detected
-ooutput file to write results
--databasemake queries to public ensembl databases instead of looking for local copies
--speciesspecies for which annotations should be obtained
--everythingflag to output additional annotations
# make a file with a single variant in echo "17 43071077 43071077 A/G - variant_1" > variant.txt # run VEP ./vep -i variant.txt -o variant.anno.txt --database --species "human" --everything
We can do the same thing through the web interface, navigate to the vep homepage at http://www.ensembl.org/info/docs/tools/vep/index.html, and click on Launch VEP.
Then input your variant and click on Run.
This will submit the job to ensembl servers, the page will refresh every few seconds. When the job completes click on view results.
Doing so will take you to a web page where you will be able to view summary statistics, the results, and options to filter or export a file.
You have probably noticed from your exploration of the results that even though only one variant was supplied multiple rows were output. By default VEP returns annotations for each “transcript” and each “variant consequence”. While this is informative it is often desireable to only have one annotation per gene. We can achieve this on our command line by adding the
--per_gene parameter. The same thing can be achieved through the web interface by expanding the filtering options tab and setting Restrict results to Show one selected consequence per gene. Go ahead and do that now through either the command line or web GUI.
There are many features through both forms of VEP, to many to cover in it’s entirety for this course. However extensive documentation for the web based version is available here and the stand alone perl script available here.
Now that you have an inital VEP result try and answer a few questions about your data. If you need help in understanding a certain column hover over it with your cursor or look at the VEP documentation available here.
What is the maximum allele frequency observed for this vairant in the 1000 genomes european population?
In what gene is this variant located?
What is the amino acid change for this variant?