“Silent” Mutation Linked to Poor Kidney Cancer Outcomes

F.For decades, researchers have also viewed mutations as unimportant quirks in the genome. Because of the way the genetic code is built – where multiple three base pair codons can encode the same amino acid – mutations can occur that do not alter the amino acid sequence of a protein. Scientists have largely dismissed these anomalies as harmless curiosities.

But as with other historically underestimated aspects of the genome, scientists recognize that many “silent” mutations may not be that silent after all. Research suggests that they are often subjected to selective pressures and could play a role in cancer, autism, and schizophrenia.

A study published online last week (February 12th) iScience adds to growing evidence that variations can have consequences too. The authors also describe a mutation in the gene. BAP1 This was associated with a worse-than-expected prognosis in a kidney cancer patient. Her subsequent experiments suggest that the mutation has this effect by disrupting the cells’ RNA splicing process, converting freshly transcribed messenger RNA (mRNA) into digestible fragments ready to be translated into protein. Since the cancer patient was missing a second healthy copy of the gene, the silent mutation may have led to a complete loss of function of BAP1.

“To the best of my knowledge, to bind a certain mutation [to] a clinical outcome [in cancer] is a novelty, ”notes Fran Supek, a cancer geneticist at the Institute for Research and Biomedicine in Barcelona, ​​who was not involved in the study. “I’m always happy to see that researchers think outside the box. . . and look at understaffed classes of genetic alterations that can help us resolve a certain number of patients with genetic diseases or cancer. “

[There is] little awareness of mutations and their role in cancer. Generally they are ignored in sequencing studies because they are believed to be very unlikely to be drivers.

– Thomas Mitchell, Wellcome Sanger Institute

Samuel Peña-Llopis and his colleagues searched the Cancer Genomatlas (TCGA) – a public database of genome samples from more than 11,000 patients around the world – and discovered an entry from a patient with an unusual course of disease. The 73-year-old Caucasian woman had clear cell renal cell carcinoma, the most common type of kidney cancer, with a mutation in PBRM1, a gene involved in chromatin remodeling.

Even though PBRM1 Mutations are usually associated with relatively good clinical outcomes in such patients – with a mean survival of 117 months according to TCGA data – the patient died just 56 months after diagnosis, says Peña-Llopis, a cancer geneticist who specializes in kidney cancer and uveal melanoma with the German Cancer consortium at the University Hospital Essen in Germany.

The team noticed that she also had a mutation in BAP1, which encodes an enzyme that is involved in regulating the breakdown of proteins. The mutation converts a thymine to a guanine, which still results in the same amino acid, glycine, encoded by both GGT and GGG. Oddly enough, the patient also had a very low frequency of BAP1 protein; in fact, it was comparable to renal cell carcinoma patients who experienced non-synonymous mutations with loss of function BAP1which tend to have serious consequences. The team suspected that the silence BAP1 A mutation could somehow affect the conversion of the gene into protein.

The path by which DNA becomes protein is long and winding. First, double-stranded DNA is pulled apart and the strands are individually transcribed into individual strings of pre-mRNA, a rough draft of the instructions needed to turn them into protein. Then it has to be spliced, whereby different proteins bind to different places in the pre-mRNA and cut out non-coding nucleotide sequences – introns – and the coding parts – exons – fuse together. Only then is the mRNA ready for other cellular machinists to convert into protein.

One way that mutations can interfere with this process is to change the specific binding sites of RNA splice proteins that are required to properly integrate different exons. If they cannot bind, or the altered codon binds the wrong proteins, they may skip important parts of the genetic code – known as “exon skipping” – which can result in a dysfunctional protein. Because the mutation is also in BAP1Exon 11 was located near a splice site that is critical to connecting this exon to the next. “We thought that the splice system might be affected,” recalls Peña-Llopis.

To find out, the team conducted a series of experiments with genetic constructs that contain BAP1‘s exon 11, into which they had inserted fluorescent proteins. They expressed the construct in a human cancer cell line. From the color that appeared under a microscope, they could tell if the exon was integrated or skipped. They observed almost 100 percent skip when the construct contained the synonymous mutation, significantly more than when using the construct based on the unmutated version of BAP1.

Skipping exon 11 will likely result in a loss of BAP1 for that copy of the gene, explains Peña-Llopis. Since this exon has 185 base pairs – which is not a multiple of three – its loss leads to a shift in the reading frame with three base pairs, which enzymes use for protein translation. This in turn would lead to a codon below being misunderstood as a stop codon, which signals that the protein translation machinery is ending. mRNA transcripts that contain premature stop codons are typically broken down by the cell. In this particular patient, this likely resulted in a complete loss of BAP1 as she lost her second copy due to the deletion of a small segment of chromosomes, which is common in this cancer subtype.

Synonymous mutations in kidney cancer patients

Back in the TCGA database, which includes nearly 500 patients with clear cell renal cell carcinoma, the team found another eight patients who also had mutations BAP1 Exons near sites essential for splicing, two of which were within the sites required to glue exons 10 and 11 together. When you consider that there are 32 splice sites in the 17 exons that make up BAP1Finding two in eight is a significant number, according to Peña-Llopis. “This suggests that this is a hotspot for inactivating BAP1. However, the two patients had very different clinical outcomes, suggesting that other genetic changes may also play a role in the prognosis.

“I think this is an important finding,” notes James Brugarolas, a physician-scientist and oncologist who directs the kidney cancer program at the University of Texas Medical Center. BAP1 is mutated in around 10–15 percent of all clear cell renal cell carcinomas, mostly due to non-synonymous changes. “The study provides relatively convincing evidence for this. . . Mutations that do not affect the protein sequence in BAP1 These could be pathogenic driver mutations that lead to the inactivation of the tumor suppressor protein, ”adds Brugarolas, who has worked with Peña-Llopis in the past but was not involved in the new research.

Brugarolas says the data would have been even more convincing if more data on RNA sequencing and immunohistochemistry were available from the patient’s tumor, which could provide clearer evidence of exon skipping. And of course, such results can always be better supported by larger samples and replication in independent data sets, adds Supek. That is, “I think the in vitro experiments they do [did] suggest that the mutation they identify can alter splicing. And of course, the escape of exon 11 would result in a non-functioning protein due to a premature stop codon. One could make a very convincing argument for this, ”says Brugarolas.

The clinical relevance of the finding is not yet apparent. Targeting loss of function mutations in tumor suppressor genes such as BAP1 has lagged behind targeting function gain mutations, such as enzymes that control cell growth and function, says Brugarolas; It is generally easier to inhibit mismatch proteins than to correct something that has already been removed by mutation. It is also unclear whether BAP1 Mutations could be used as biomarkers to predict patient response to therapies. “Like mutations in BAP1 [should] Its use for therapy is unknown, ”adds Brugarolas.

Overall, the results suggest researchers should also pay more attention to mutations, notes Thomas Mitchell, a clinician-scientist working on kidney cancer at the Wellcome Sanger Institute in the UK. “[There is] little awareness of mutations and their role in cancer. Generally they are ignored in sequencing studies because they are believed to be very unlikely to be drivers. “

However, larger studies in the past have predicted that mutations could also have pathogenic effects. In 2014, Supek and colleagues estimated that about 6 to 8 percent of pathogenic single nucleotide mutations in cancer genes are synonymous. Along with other studies, research seems to be converging on an estimate of 5 percent of all driver mutations that are synonymous – a “not negligible amount” that can be of great concern to some individual cancer patients, Supek says. Exon skipping is one mechanism by which such mutations could have deleterious effects, “but it will likely be different for each mutation as well.”

Understanding silent mutations, along with other overlooked genetic changes, could help uncover the underlying causes of disease for many individual patients for whom mutations do not clearly fall into the non-synonymous bucket, and open the door for treatment to be sought. “The human genome is a very complex thing, and there are many ways it can break down and lead to disease,” says Supek.

J. Niersch et al.ON BAP1 Synonym mutation leads to exon skipping, loss of function and poor patient prognosis. ” iScience, doi:10.1016 / j.isci.2021.102173.

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