Fragment Modification

From ReactomeWiki

FragmentModification Use cases

If the protein you want to annotate is a disease variant containing a large insertion or a deletion, you will represent this large scale rearrangement by using FragmentModification attribute.

Annotation of Large Deletions

To annotate an EWAS with a large deletion, for example deletion of amino acid residues 30 to 297 in the glioblastoma variant of EGFR, EGFRvIII, create a new EGFR EWAS instance. Set the referenceEntity - UniProt: P00533 EGFR in this example and specify startCoordinate, endCoordinate and compartment as previously described. Specify the name(s) of the disease variant. One name can be a commonly used name – EGFRvIII in this example. Another name should specify the position of the deletion in accordance with the accepted nomenclature. In this example, the alternative name is EGFR V30_R297del, a deletion of amino acid residues in EGFR starting at Val30 and ending at Arg297. Right click on the hasModifiedResidue field and select Add. In the new window that opens, under AbstractModifiedResidue, select FragmentDeletionModification and click on New Instance. Set the referenceSequence (UniProt: P00533 EGFR in this example). Specify the startPositionInReferenceSequence – the first amino acid residue deleted (30 in this example). Specify the endPositionInReferenceSequence – the last amino acid residue deleted (297 in this example). Press OK. When the FragmentDeletionModification window closes, press OK again.

To specify the Disease in which the mutant protein is present
Browse the EBI disease ontology at http://www.ebi.ac.uk/ontology-lookup/browse.do?ontName=DOID. The DOID for adult glioblastoma multiforme, the cancer expressing EGFRvIII, is 3075. After browsing the gk_central and confirming that the DOID of interest is not in the central repository, right click on disease field in the EGFRvIII EWAS window and select Add. In the new window that opens, click on New Instance. Set the Identifier to 3075 and press Enter. You will be asked if you want the tool to fetch the information from the EBI ontology for the specified disease instance. Select “Yes”. Values for the specified identifier will populate automatically. Press OK. When the Disease window closes, press OK again. The Disease slot is multivalued, so if you are curating a disease process that involves a variant protein for which the right EWAS already exists (i.e., the same referenceGeneProduct, same compartment, exactly the same modifications), add the identifier for your disease to the disease slot on the existing EWAS and re-use it in your annotation – do not create a new EWAS identical to the old one except for its “disease” attribute. A disease attribute should also be assigned to events involving a disease variant EWAS.

Annotation of Large Insertions

To annotate a large insertion, for example duplication of the kinase domain of EGFR in glioblastoma (amino acid residues 664 to 1030 duplicated and inserted at the position 1031), create a new EWAS for the mutant protein. Set the referenceEntity - UniProt: P00533 EGFR in this example - and specify startCoordinate, endCoordinate and compartment as previously described. Specify the name of the disease variant. In this example, one name will be TKD-EGFR, a commonly used abbreviation for tandem kinase domain mutant of EGFR. An alternative name will be EGFR 1031insI664_S1030. Right click on hasModifiedResidue field and select Add. Under AbstractModifiedResidue, select FragmentInsertionModification and press New Instance. In the FragmentInsertionModification window that opens, set the referenceSequence (UniProt: P00533 EGFR in this example). Specify the startPositionInReferenceSequence – the first amino acid residue of the inserted fragment (664 in this example). Specify the endPositionInReferenceSequence – the last amino acid residue of the inserted fragment (1030 in this example). Specify the Coordinate - the amino acid residue in the referenceEntity at which the inserted fragment starts (1031 in this example). Press OK. When the FragmentDeletionModification window closes, press OK again.

To specify the Disease in which the mutant protein is present
Rght click on disease field and select Add. In the window that opens, select Disease and “adult glioblastoma mutliforme”, which was annotated in the EGFRvIII example for large deletions. Press OK.

Annotation of Fusion Proteins

To annotate a fusion protein, follow the example of BCR-ABL1, resulting from the translocation between BCR locus on chromosome 9 and ABL1 locus on chromosome 22 in chronic myeloid leukemia. The fusion protein contains BCR amino acid residues 1 to 927 at the N-terminus, and ABL1 amino acid residues 27 to 1130 at the C-terminus. Create a new EWAS and set the referenceEntity to UniProt P11274 BCR. Specify the startCoordinate as 1 and endCoordinate as 927. Specify the name of the disease variant as “BCR-ABL 1 fusion protein”. Right click on hasModifiedResidue field and select Add. Under AbstractModifiedResidue, select FragmentDeletionModification and press New Instance. In the FragmentInsertionModification window that opens, set the referenceSequence to UniProt P00519 ABL1. Specify the startPositionInReferenceSequence as 27 and the endPositionInReferenceSequence as 1130. Specify the Coordinate as 928 - the amino acid residue in the referenceEntity at which the fused ABL1 fragment starts. Press OK. Once the FragmentInsertionModification window closes, click OK again.

To specify the Disease in which the mutant protein is present
The DOID for chronic myeloid leukemia, the cancer expressing BCR/ABL fusion protein is 8552. Add disease attribute to BCR-ABL1 EWAS as described above.