Identification of mutations related to Wilms’ tumor

Discovery published in the journal Nature Communications paves the way for developing therapies and new methods for earlier diagnosis of the renal neoplasia that most frequently affects children

By Karina Toledo

Agência FAPESP – In a study whose findings were published in the June issue of the journal Nature Communications, researchers from the A.C. Camargo Cancer Center identified a set of mutations related to development of Wilms’ tumor – the most common renal neoplasia found in children.

The discovery paves the way for developing new therapies and methods for an earlier diagnosis of the disease.

“Presently, the diagnosis is only possible when the tumor mass is palpable. However, if we can find a way to identify the neoplasia at an earlier stage, we could significantly reduce the intensity of the treatment as well as the side effects,” said Dirce Maria Carraro, head of the Genomics and Molecular Biology Laboratory at the A.C. Camargo Cancer Center and coordinator of the FAPESP-funded study.

According to Carraro, this type of cancer affects one out of every 10,000 children, most between the ages of 2 and 4. The tumor can appear anywhere in the kidney and is bilateral in 5% of all cases.

The treatment generally involves surgery to remove the affected organ in addition to chemotherapy and, in more advanced cases, radiation. The intensity of the chemotherapy treatment depends on the extent of the lesion.

“The prognosis is usually good, with cure rates of approximately 80%. However, many patients suffer adverse long-term effects from the treatment. The most frequent side effects include heart disorders, musculoskeletal disorders and the development of a second tumor. Therefore, many studies are searching for markers that will indicate whether it is in fact necessary to intensify the treatment,” Carraro explained.

MicroRNA biogenesis

Before the study that was recently published by the A.C. Camargo team, important genetic mutations had been identified in only 30% of Wilms’ tumors studied, mainly in genes WT1, WTX and β-catenin. “This means that in 70% of the cases, no genetic alteration had been found that justified the appearance of the tumor,” stated Carraro.

With the goal of finding new somatic mutations (those present only in the tumor and not in the patient’s germline) related to the disease, the group sequenced the entire exome (the part of the genome containing protein-coding genes) of a tumor from a patient undergoing hospital care.

The exome of blood leukocytes from the child and her parents were also sequenced. “The objective was to identify mutations found only in the tumor, in other words, the mutations acquired during the process of tumor formation,” Carraro said.

The researchers’ attention was caught by a mutation observed in a protein domain encoded by the DROSHA gene, which plays a crucial role in the biogenesis of microRNAs.

“MicroRNAs are small RNA molecules that, although they do not encode proteins, play an essential role in regulating the coding genes. The microRNAs therefore play an all-important role in controlling gene expression and thus ensuring the functioning of a particular cell. Alterations in a gene involved in the biogenesis of these small molecules could have a huge implication on the appearance of Wilms’ tumor,” Carraro emphasized.

According to the researcher, the mutation affects the magnesium-binding domain of the DROSHA protein, a domain that is essential for the enzyme to function properly in the cell. “This caught our attention, so we decided to expand our analysis to a large number of samples to determine whether it is a common mutation,” she said.

The next step involved the analysis of samples from more than 200 Wilms’ tumors, some of which were found in the A.C. Camargo Cancer Center tumor bank and some at the Children’s Oncology Group of the United States. The scientists detected this specific mutation in 10% of all tumors examined, which classified it as a recurrent mutation.

This finding spurred the important hypothesis that the disturbance of microRNA biogenesis is significant to the development of this tumor. To confirm this hypothesis, the scientists sequenced other important genes – in addition to the DROSHA gene – that play roles in the process of microRNA biogenesis.

The analyses revealed that 33% of the tumors presented mutations in these genes, including DGCR8, DICER1, XPO5 and TARBP2, and in other DROSHA gene domains, reiterating the crucial role that microRNA biogenesis plays in the formation of Wilms’ tumors.

In a subsequent phase, the researchers conducted functional tests on an embryonic cell line to understand precisely what impact the recurrent mutation in DROSHA has on microRNA synthesis.

“We induced the mutation in the cells and, upon evaluating the end of the process, noted that there was a preferential decrease in the expression level of mature microRNAs. Our experiments demonstrated that the recurrent mutation in the DROSHA gene prevented the generation of microRNA precursors that are capable of being recognized by another protein, called Dicer, which is also crucial to the process of microRNA biogenesis. In other words, the mutation prevented the appropriate processing of the microRNA precursor, thus affecting the generation of mature microRNAs,” Carraro said.

In parallel, the group confirmed that the phenomenon of preferential decreases in the expression level of mature microRNAs also occurred in Wilms’ tumors, with and without the DROSHA mutation, a fact that strongly suggests that this impediment to the maturation process is essential for tumor formation.

Impact on the disease

According to Carraro, the findings pave the way for the prospect of new studies that could lead to the development of new therapies, as an extremely important cell pathway that is frequently altered in Wilms’ tumors has now been identified.

“One of the strategies we plan to test in the future is the possibility of urine detection of the mutations in these and the other genes evaluated in this study, which, as a whole, comprised over 60% of the Wilms’ samples. This would offer promising prospects for low-cost and simple early diagnosis, prior to clinical manifestation,” related Carraro.

The article “Recurrent somatic mutation in DROSHA induces microRNA profile changes in Wilms’ tumor” (doi: 10.1038/ncomms5039) may be read at: www.nature.com/ncomms/2014/140609/ncomms5039/full/ncomms5039.html.