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Researchers Link Mutation in SRC Oncogene to Familial Bleeding Disorder

NEW YORK (GenomeWeb) – Researchers have linked a gain-of-function mutation in the cancer-linked SRC gene to a bleeding disorder affecting multiple generations of one family.

As reported today in Science Translational Medicine, researchers led by the University of Cambridge's Willem Ouwehand sequenced two affected members of a family with an inherited bleeding disorder in order to narrow down a set of 67 candidate genes. After ranking those genes based on their overlap with Human Phenotype Ontology (HPO) terms uncovered in the affected people, SRC came out on top.

In addition, through in vitro and zebrafish studies, the researchers found that cells overexpressing the SRC mutation showed increased SRC kinase activity and tyrosine phosphorylation as well as reduced protoplatelet formation.

While SRC has been implicated in many cancers, the researchers noted that pathogenic germline mutations in the gene haven't before been described in humans.

"[W]e show that a germline gain-of-function SRC mutation leads to thrombocytopenia, myelofibrosis, bleeding, platelet dysfunction with abnormal α-granules, and bone pathologies," Ouwehand and his colleagues wrote in their paper.

A dominantly inherited bleeding disorder marked by reduced platelet levels and bone pathologies has affected three generations of a family, leading to death in one of the nine cases.  The affected members of the pedigree were diagnosed with inherited thrombocytopenia with early-onset myelofibrosis, though their platelet phenotype didn't match with any known inherited thrombocytopenias. Further, no known thrombocytopenia gene mutations were present in the cases.

Because of that, the researchers sequenced the genomes of two affected family members to find that they shared 67 plausible causal variants. They ranked these plausible causal variants based on their overlap with HPO coding and terms shared among three cases from the family. This approach placed SRC at the top of the list.

The SRC gene encodes the proto-oncogene tyrosine protein kinase SRC. The researchers further found through Sanger sequencing that the SRC variant c.1579G>A segregated with the disease phenotype in three additional cases and that that variant was absent from an unaffected relative.

In the SRC protein, the mutated E527 residue is three amino upstream of Y530 in the C-terminal tail of the SRC kinase domain, the researchers noted, adding that differential phosphorylation of Y530 and Y419 regulates the switching of SRC between its active and inactive states. Modeling indicated that the E527 mutation might affect the protein's configuration, leading it to adopt a constitutively active state, they added.

Through immunoblot assays, the researchers indeed found increased higher levels of active SRC in mutation carriers than controls. Additional antibody assays in a cell line further indicated that Y419 dephosphorylation is correlated with SRC activity level.

SRC is most strongly overexpressed in megakaryocytes (MKs) versus other blood cell progenitors, the researchers noted. Bone marrow samples obtained from one affected family member showed trilineage dysplasia in which a large portion of MKs showed dysplastic features and hypolobulated nuclei, which suggests that they blood cells are immature.

Further, E527-transduced MKs showed reduced proplatelet formation as well as altered actin organization and podosome structures.

Zebrafish injected with SRC mRNA with the equivalent of the human E527 mutation exhibit abnormal early hematopoiesis and reduced thrombocyte levels, though normal red blood cell formation as well as smaller bones, the researchers added, noting that it mimicked the disorder.

"In conclusion, our genome sequencing analysis approach … was used to discover a germline gain-of-function variant of SRC, linking the first ever discovered oncogene, which has been studied for decades, with a new syndrome," the researchers wrote.