Fusion genes originate from two genes that normally existed separately from each other. They are characteristic of different types of cancer, as the new combinations of DNA – the genetic rearrangement – in these fusion genes lead to altered properties of the gene, which can lead to cancer. As such, the detection of fusion genes in tumor cells is important for diagnosis, prognosis, treatment options and the ability to measure the result. Fusion genes are known to play a role particularly in forms of cancer that are relatively common in children, such as acute lymphatic leukemia (ALL) and chronic myeloid leukemia (CML).
Princess Máxima Center
Researcher Ronald Stam from the Princess Máxima Center is involved in this research. 'Because I do research with my research group into high-risk forms of childhood leukemia, in which fusion genes play an important role. If implemented in our diagnostics, the new FUDGE technique could make it possible to detect fusion genes much more quickly, or in some cases to detect fusion genes for which it is often not possible or time-consuming with today’s techniques to unravel the exact composition of the fusion genes. This is important because the occurrence of fusion genes and often the composition of the fusion genes are related to the patient’s prognosis.'
Fusion genes are hard to detect because genes can fuse different partner genes. The fusion can also take place at different places on the genes. Common detection methods focus on already known rearrangements of genetic material. 'However, these methods do not detect new fusions,' says researcher Glen Monroe (department of Genetics). 'Reliable techniques that offer greater clarity, such as Next Generation Sequencing which examines the entire RNA or DNA, on the other hand, are time-consuming and still expensive. For quick use in the diagnosis of cancer patients, that’s a drawback.'
DNA cutting with Cas9
The UMC Utrecht has now developed a new technique to detect fusion genes quickly and precisely. 'We read the DNA from one side to find the fusion gene,' says researcher Christina Stangl, who developed the technique. 'This technique involves the use of CRISPR-Cas9. The Cas9 protein works like scissors with which you can cut the DNA in precise places. We have developed guides that direct Cas9 in a highly targeted fashion to specific places in the genome, where it does its work as scissors.'
The cut DNA is then read from one side using nanopore sequencing. To this end, strands of DNA are each pulled through a 'gate' and carefully analyzed. In this way, the unknown fusion partner is found, as well as the exact place where the two genes fused. This knowledge is important for diagnostics, choosing cancer treatment and monitoring its effects. The investigators call it the FUDGE technique, which stands for FUsion Detection from Gene Enrichment.
Within 48 hours
In this research the UMC Utrecht collaborated with the Princess Máxima Center for Pediatric Oncology. The new FUDGE technique was used to quickly identify gene fusion partners in samples of eight solid tumors and ten patients’ leukemia. The researchers found 22 unique, differently composed fusion genes. 'In one case, the new technique enabled us to find the fusion partner within two days, which we were unable to find with the usual diagnostic options,' says researcher Gijs van Haaften. 'The FUDGE technique makes it possible to accurately determine the fusion of genes within 48 hours of reading the sample.'
Children with leukemia
The researchers at the UMC Utrecht are currently working together with colleagues from the Princess Máxima Center to enhance the FUDGE technique for detecting fusion genes and other rearrangements of the genetic material in tumor cells of children with leukemia. 'This helps accelerate the diagnosis in these children,' says Gijs. 'The technique can also be used to determine very precisely for each patient whether and to what extent the treatment of their specific form of cancer is effective. To this end, we examine the blood or bone marrow for the presence of fusion genes as DNA traces of cancer.' The aim is to be able to introduce the FUDGE technique in the diagnosis and treatment of childhood cancer within a few years. This time is needed to test the technique and to check that it really is precise enough in practice.
The researchers’ article on this technique was published in Nature Communications on Friday June 5 under the title Partner independent fusion gene detection by multiplexed CRISPR-Cas9 enrichment and long read nanopore sequencing.