Gene Fusion Detection
Overview
Gene fusions often result from large genomic rearrangements such as structural variants. While WGS secondary analysis pipelines typically contain alignment and variant calling stages, very few of them contain dedicated gene fusion callers. When they are included, they are usually associated with RNA-Seq pipelines where gene fusions can be readily observed.
Since gene fusions are frequently observed in cancer and since many sequencing experiments do not include paired RNA-Seq data, we have added gene fusion detection and annotation to Illumina Connected Annotations.
The rich diversity in gene fusion architectures and their likely mechanisms can be seen below:
Publication
Kumar-Sinha, C., Kalyana-Sundaram, S. & Chinnaiyan, A.M. Landscape of gene fusions in epithelial cancers: seq and ye shall find. Genome Med 7, 129 (2015)
Approach
Illumina Connected Annotations uses structural variant calls to evaluate if they form either putative intra-chromosomal or inter-chromosomal gene fusions. Let's consider two transcripts, NM_014206.3 (TMEM258) and NM_013402.4 (FADS1). Both of these genes are on the reverse strand in the genome. The vertical bar indicates the breakpoint where these transcripts are fused:
The above explains where the transcripts are fused together, but it doesn't explain in which orientation. By using the directionality encoded in the translocation breakend, we can rearrange these two transcripts in four ways:
Only two of the combinations yields a fusion containing both the transcription start site (TSS) and the stop codon. In one case, we can even detect an in-frame gene fusion.
If only unidirectional gene fusions are desired, only these two fusions can be detected. If enable-bidirectional-fusions is enabled, all four cases can be identified.
Interpreting translocation breakends
At first glance, translocation breakends are a bit daunting. However, once you understand how they work, they're actually quite simple. For more information, we recommend reading section 5.4 in the VCF 4.2 specification.
| REF | ALT | Meaning |
|---|---|---|
| s | t[p[ | piece extending to the right of p is joined after t |
| s | t]p] | reverse comp piece extending left of p is joined after t |
| s | ]p]t | piece extending to the left of p is joined before t |
| s | [p[t | reverse comp piece extending right of p is joined before t |
Variant Types
Specifically we can identify gene fusions from the following structural variant types:
- deletions (
<DEL>) - tandem_duplications (
<DUP:TANDEM>) - inversions (
<INV>) - translocation breakpoints (
AAAAAAAAAAAAAAAAAATTAGTCAGGCAC[chr3:153444911[)
Criteria
The following criteria must be met for Illumina Connected Annotations to identify a gene fusion:
- After accounting for gene orientation and genomic rearrangements, both transcripts must have the same orientation if
enable-bidirectional-fusionsis not enabled. They can have the same or different orientations ifenable-bidirectional-fusionsis set. - Both transcripts must be from the same transcript source (i.e. we won't mix and match between RefSeq and Ensembl transcripts)
- Both transcripts must belong to different genes
- Both transcripts cannot have a coding region that already overlaps without the variant (i.e. in cases where two genes naturally overlap, we don't want to call a gene fusion)
ETV6/RUNX1 Example
ETV6/RUNX1 is the most common gene fusion in childhood B-cell precursor acute lymphoblastic leukemia (ALL). Samples with this translocation are associated with a good prognosis and excellent response to treatment.
Publication
Sun C., Chang L., Zhu X. Pathogenesis of ETV6/RUNX1-positive childhood acute lymphoblastic leukemia and mechanisms underlying its relapse. Oncotarget. 2017; 8: 35445-35459
VCF
Here's a simplified representation of the translocation breakends called by the Manta structural variant caller:
##fileformat=VCFv4.1
#CHROM POS ID REF ALT QUAL FILTER INFO
chr12 12026270 . C [chr21:36420865[C . PASS SVTYPE=BND
chr12 12026305 . A A]chr21:36420571] . PASS SVTYPE=BND
chr21 36420571 . C C]chr12:12026305] . PASS SVTYPE=BND
chr21 36420865 . C [chr12:12026270[C . PASS SVTYPE=BND
When you put these calls together, the resulting genomic rearrangement looks something like this:
JSON Output
The annotation for the first variant in the VCF looks like this:
{
"chromosome": "chr12",
"position": 12026270,
"refAllele": "C",
"altAlleles": [
"[chr21:36420865[C"
],
"filters": [
"PASS"
],
"cytogeneticBand": "12p13.2",
"clingen": [
{
"chromosome": "12",
"begin": 173786,
"end": 34835837,
"variantType": "copy_number_gain",
"id": "nsv995956",
"clinicalInterpretation": "pathogenic",
"phenotypes": [
"Decreased calvarial ossification",
"Delayed gross motor development",
"Feeding difficulties",
"Frontal bossing",
"Morphological abnormality of the central nervous system",
"Patchy alopecia"
],
"phenotypeIds": [
"HP:0002007",
"HP:0002011",
"HP:0002194",
"HP:0002232",
"HP:0005474",
"HP:0011968",
"MedGen:C0232466",
"MedGen:C1862862",
"MedGen:CN001816",
"MedGen:CN001820",
"MedGen:CN001989",
"MedGen:CN004852"
],
"observedGains": 1,
"validated": true
}
],
"variants": [
{
"vid": "12-12026270-C-[chr21:36420865[C",
"chromosome": "chr12",
"begin": 12026270,
"end": 12026270,
"isStructuralVariant": true,
"refAllele": "C",
"altAllele": "[chr21:36420865[C",
"variantType": "translocation_breakend",
"cosmicGeneFusions": [
{
"id": "COSF2245",
"numSamples": 249,
"geneSymbols": [
"ETV6",
"RUNX1"
],
"hgvsr": "ENST00000396373.4(ETV6):r.1_1283::ENST00000300305.3(RUNX1):r.504_6222",
"histologies": [
{
"name": "acute lymphoblastic B cell leukaemia",
"numSamples": 169
},
{
"name": "acute lymphoblastic leukaemia",
"numSamples": 80
}
],
"sites": [
{
"name": "haematopoietic and lymphoid tissue",
"numSamples": 249
}
],
"pubMedIds": [
7761424,
7780150,
8609706,
8751464,
8982044,
9067587,
9207408,
9226156,
9628428,
10463610,
10774753,
11091202,
12621238,
12661004,
12750722,
15104290,
15642392,
24557455,
26925663
]
}
],
"fusionCatcher": [
{
"genes": {
"first": {
"hgnc": "ETV6",
"isOncogene": true
},
"second": {
"hgnc": "RUNX1",
"isOncogene": true
}
},
"somaticSources": [
"DepMap CCLE",
"Cancer Genome Project",
"ChimerKB 4.0",
"ChimerPub 4.0",
"ChimerSeq 4.0",
"Known",
"Mitelman DB",
"OncoKB",
"TICdb"
]
}
],
"transcripts": [
{
"transcript": "ENST00000396373.4",
"source": "Ensembl",
"bioType": "protein_coding",
"introns": "5/7",
"geneId": "ENSG00000139083",
"hgnc": "ETV6",
"consequence": [
"transcript_variant",
"unidirectional_gene_fusion"
],
"geneFusions": [
{
"transcript": "ENST00000437180.1",
"bioType": "protein_coding",
"intron": 2,
"geneId": "ENSG00000159216",
"hgnc": "RUNX1",
"hgvsr": "ENST00000437180.1(RUNX1):r.?_58+274::ENST00000396373.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
},
{
"transcript": "ENST00000300305.3",
"bioType": "protein_coding",
"intron": 1,
"geneId": "ENSG00000159216",
"hgnc": "RUNX1",
"hgvsr": "ENST00000300305.3(RUNX1):r.?_58+274::ENST00000396373.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
},
{
"transcript": "ENST00000482318.1",
"bioType": "nonsense_mediated_decay",
"intron": 2,
"geneId": "ENSG00000159216",
"hgnc": "RUNX1",
"hgvsr": "ENST00000482318.1(RUNX1):r.?_58+274::ENST00000396373.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
},
{
"transcript": "ENST00000486278.2",
"bioType": "protein_coding",
"intron": 2,
"geneId": "ENSG00000159216",
"hgnc": "RUNX1",
"hgvsr": "ENST00000486278.2(RUNX1):r.?_-15+274::ENST00000396373.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
},
{
"transcript": "ENST00000455571.1",
"bioType": "protein_coding",
"intron": 2,
"geneId": "ENSG00000159216",
"hgnc": "RUNX1",
"hgvsr": "ENST00000455571.1(RUNX1):r.?_58+274::ENST00000396373.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
},
{
"transcript": "ENST00000475045.2",
"bioType": "protein_coding",
"intron": 11,
"geneId": "ENSG00000159216",
"hgnc": "RUNX1",
"hgvsr": "ENST00000475045.2(RUNX1):r.?_58+274::ENST00000396373.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
},
{
"transcript": "ENST00000416754.1",
"bioType": "protein_coding",
"intron": 2,
"geneId": "ENSG00000159216",
"hgnc": "RUNX1",
"hgvsr": "ENST00000416754.1(RUNX1):r.?_58+274::ENST00000396373.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
}
],
"isCanonical": true,
"proteinId": "ENSP00000379658.3"
},
{
"transcript": "NM_001987.4",
"source": "RefSeq",
"bioType": "protein_coding",
"introns": "5/7",
"geneId": "2120",
"hgnc": "ETV6",
"consequence": [
"transcript_variant",
"unidirectional_gene_fusion"
],
"geneFusions": [
{
"transcript": "NM_001754.4",
"bioType": "protein_coding",
"intron": 2,
"geneId": "861",
"hgnc": "RUNX1",
"hgvsr": "NM_001754.4(RUNX1):r.?_58+274::NM_001987.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
}
],
"isCanonical": true,
"proteinId": "NP_001978.1"
}
]
}
]
}
| Field | Type | Notes |
|---|---|---|
| transcript | string | transcript ID |
| bioType | string | descriptions of the biotypes from Ensembl |
| exon | int | exon that contained fusion breakpoint |
| intron | int | intron that contained fusion breakpoint |
| geneId | string | gene ID. e.g. ENSG00000116062 |
| hgnc | string | gene symbol. e.g. MSH6 |
| hgvsr | string | HGVS RNA nomenclature |
Gene Fusion Data Sources
To provide more context to our gene fusions, we provide the following gene fusion data sources:
Consequences
When a gene fusion is identified, we add the following Sequence Ontology consequence:
"consequence": [
"transcript_variant",
"gene_fusion"
],
- If both transcripts have the same orientation, we label it as
unidirectional_gene_fusion, if they have different orientations, we label it asbidirectional_gene_fusion - If both unidirectional and bidirectional ones are detected, we label it as
gene_fusion.
Gene Fusions Section
The geneFusions section is contained within the object of the originating transcript. It will contain all the pairwise gene fusions that obey the criteria outline above. In the case of ENST00000396373.4, there 7 other Ensembl transcripts that would produce a gene fusion. For NM_001987.4, there was only one transcript (NM_001754.4) that produce a gene fusion.
For each originating transcript, we report the following for each partner transcript:
- transcript ID
- gene ID
- HGNC gene symbol
- transcript bio type (e.g. protein_coding)
- intron or exon number containing the breakpoint
- HGVS RNA notation
- gene fusion directionality
tip
Before Illumina Connected Annotations 3.15, we provided HGVS coding notation. However, HGVS r. notation is more appropriate for these types fusion splicing events (see HGVS SVD-WG007).
"geneFusions": [
{
"transcript": "NM_001754.4",
"bioType": "protein_coding",
"intron": 2,
"geneId": "861",
"hgnc": "RUNX1",
"hgvsr": "NM_001754.4(RUNX1):r.?_58+274::NM_001987.4(ETV6):r.1009+3367_?",
"directionality":"uniDirectional"
}
],
The HGVS RNA notation above indicates that the gene fusion starts with NM_001754.4 (RUNX1) until CDS position 58 and continues with NM_001987.4 (ETV6). 1009+3367 indicates that the fusion occurred 3367 bp within intron 2.