The Uproar of Silent Mutations
Introduction
According to the Encyclopedia of Genetics, “a silent mutation is a change in the nucleotide sequence of a gene that does not alter the amino acid sequence of the encoded protein.” (Hodgkin, 2001). It was previously believed that silent mutations did not influence human health due to their inability to change the amino acid sequences of polypeptides, and hence the overall proteins (Chamary and Hurst, 2009). Over the years however, discoveries were made that concluded that silent mutations do play a role in affecting human health (Chamary and Hurst, 2009). Indeed, Phenylketonuria, Marfan syndrome, Seckel syndrome, and some 47 other diseases are all prevalent in today’s world due to the simple yet complex concept that is silent mutations. While there is a thin line between silent mutations and the other types of mutations that can occur within our species, this essay will focus on the former type. The aim of this essay is to attempt to determine the extent to which these same silent mutations do indeed influence our health. The paper is divided into five main chapters, with each chapter discussing information relevant to understanding silent mutations and determining the extent of their effects. Following this introduction, the second chapter gives a general overview of the DNA processes of transcription and translation. The third chapter seeks to explore what silent mutations are and how they arise. Chapter four discusses mutations and health by investigating the role of silent mutations in Phenylketonuria (PKU) and Cystic Fibrosis, as well as the possibility of them being beneficial. Lastly, chapter five looks at the effects of silent mutations on one of the leading causes of death worldwide: cancer.
DNA processes and post-transcriptional modification
DNA, fully known as deoxyribonucleic acid, is a complex molecule that contains all the genetic information in humans and almost all other organisms (Miko and LeJeune, 2014). Every cell in a living organism’s body consists of identical DNA, and it serves as the primary unit of heredity in organisms of various species. The structure of DNA plays a vital role in its function and the processes it takes part in, such as transcription and translation, which will be discussed briefly. In the first stage of gene expression, DNA is transcribed into messenger RNA by the enzyme RNA polymerase. Proteins known as transcription factors control the rate of transcription by binding to specific promoter regions on the DNA molecule. They can either activate or repress the transcription of a gene (Cooper and Rogers, 2009). In transcription, only one of the two DNA strands is being transcribed. This strand is known as the template strand because it serves as a template for the RNA polymerase to add complementary nucleotides to the growing mRNA transcript in the 5’ to 3’ direction (Campbell et. al, 2017).
In eukaryotes, post-transcriptional modification occurs, which is the process by which primary transcript RNA is converted into mature mRNA. This process includes splicing, capping and polyadenylation. Splicing is a process whereby introns, non-coding regions in the mRNA, are removed and exons, protein-coding regions in the mRNA, are joined together. This process is catalysed by a complex called a spliceosome. Capping is a process in which a 5’ cap is attached to the mRNA molecule by a capping enzyme, preventing degradation and enabling ribosome binding during translation. Polyadenylation is the addition of a poly(A) tail, consisting of approximately 50-250 adenine bases, to the mRNA molecule. The poly (A) tail also prevents degradation of the final mRNA in the nucleus (Campbell et. al, 2017). Post-transcriptional mRNA modification is greatly affected by silent mutations, as we will see later.
Following post-transcriptional modification, a process occurs whereby polypeptide chains are formed using the triplet codons present on the mRNA sequence. This process is known as translation and can be divided into three main steps: initiation, elongation and termination. Ribosomes help coordinate the methodical linking of amino acids into polypeptide chains. A new form of RNA is introduced in translation, transfer RNA (tRNA). The function of tRNA is to transfer an amino acid from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome (Campbell et. al, 2017). A tRNA that binds to an mRNA codon specifying a particular amino acid must carry only that amino acid to the ribosome. The resulting aminoacyl tRNA, better known as charged tRNA, is released from the enzyme and can then deliver its amino acid to a growing polypeptide chain on a ribosome (Campbell et. Al, 2017). The tRNA anticodon must pair with the appropriate mRNA codon.
What are silent mutations & how do they arise?
As seen in Figure 1, a silent mutation is a change in the DNA that does not alter the final protein. Because of this, scientists assumed that these mutations were “silent”, and therefore innocuous. All genes are organized into codons, and each one of these codons corresponds to a specific amino acid. When a silent mutation occurs, one of the nucleotides in the codon is replaced with another. The amino acid corresponding to the codon, however remains the same. For example, GGC, GGA, GGU. GGG are all translated to the amino acid glycine (Chamary and Hurst, 2009). Silent mutations do not usually change a protein’s 3D conformation; however, they can affect protein expression and translation. This can be explained by a change in regulation, caused by the binding of another protein to the DNA or RNA at the site of the gene with the mutation. Furthermore, translation can be affected if the silent DNA mutation changes the secondary structure of the RNA molecule (Wheatley, 2016).
The way in which silent mutations arise can be extracted from the definition of the mutation. Silent mutations occur when the change of a single DNA nucleotide within a protein-coding portion of a gene does not affect the sequence of amino acids that make up the protein coded for by that gene. This happens when a mutation alters a single nucleotide, but yields a synonymous codon (Chamary and Hurst, 2009). Mutations in which only a single DNA nucleotide is changed are known as point mutations. The protein-coding portions of a gene are known as exons, while the non-coding portions are known as introns. (Kimchi-Sarfaty et. al, 2006). A change in one nucleotide doesn't always change the triplet's meaning; the mutated triplet may still add the same amino acid. This result makes clear the degeneracy in the genetic code, which is also explained by the wobble hypothesis proposed by Francis Crick, explaining why multiple codons can code for the same amino acid. As a result, when the amino acids of a protein stay the same, researchers believed, so did its structure and function. (Vogel et. al, 2006). It was discovered that within various species, cells would preferentially employ certain codons because those choices optimized the rate or accuracy of protein synthesis (Chamary and Hurst, 2009). The same was found to be true for mammals; however, the mammalian pattern does not obviously suggest that the reason is to enhance protein synthesis (Chamary and Hurst, 2009). Silent mutations containing exonic splicing enhancer (ESE) motifs that function as splicing enhancers do not change an amino acid; however, they can have a major effect on a protein simply because they disrupt the proper removal of introns (Chamary and Hurst, 2009).
How do silent mutations contribute to human disease?
There are as many as 50 diseases afflicting most organ systems that have been associated with synonymous mutations (Sauna, 2011). Synonymous mutations can affect protein conformation and function by affecting post-transcriptional processing and regulation of RNA (Sauna, 2011). There are several prominent examples of silent mutations affecting human health in the form of disease. One of the most common hereditary diseases that arises as a result of silent mutations is phenylketonuria, often referred to as PKU. PKU is an inherited disorder that increases the level of the amino acid phenylalanine within the blood. Phenylalanine is an essential amino acid, meaning it has to be obtained through the individual’s diet as it cannot be made by the body. Within the body, phenylalanine is broken down by the enzyme phenylalanine hydroxylase. PKU arises when a silent mutation occurs in the PAH gene that provides instructions for producing phenylalanine hydroxylase within the body (Genetics Home Reference, 2018).
The mutation occurs when the adenine base is changed to a thymine base in complementary DNA (cDNA) nucleotide 1197 of the PAH gene. (Hastings, 2001). This is a silent mutation because the wild type GUA and mutant GUU alleles both code for the amino acid valine at codon 399. However, this substitution affects post-transcriptional PAH mRNA processing, resulting in an exon 11 deletion (Chao et. al, 2001). This in turn also results in the deletion of the codons coding for five amino acids needed for the proper functioning of phenylalanine hydroxylase. This can be clearly seen in the figure below, where the process from transcription to translation of the protein is shown in both the wild type allele and the mutant type. Ultimately, this leads to a significant reduction or in some extreme cases, completely elimination of the activity of phenylalanine hydroxylase, and hence there is not a proper breakdown of phenylalanine in the body. Buildup of phenylalanine in the body fluids can cause damage to the central nervous system (CNS), which can ultimately result in severe mental retardation and neurobehavioral abnormalities (Young-An et. al, 2012).
Aside from PKU, silent mutations are also prevalent in cystic fibrosis, which is one of the most prominent diseases in the world today. Like PKU, cystic fibrosis is a hereditary disease. It is characterized by the buildup of thick, sticky mucus that can accumulate within the body and is capable of damaging many of the body’s organs (Genetics Home Reference, 2018). Mucus is produced by every individual and is essential in lubricating and protecting the linings of the airways, digestive system, reproductive system and other organs (Genetics Home Reference, 2018). However, in individuals suffering from cystic fibrosis, the body produces abnormally thick and sticky mucus which can clog the airways. Ultimately, this can lead to severe breathing problems in addition to bacterial infections in the lungs. Cystic fibrosis results from the mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene (SDDC, 2017). The mutation on this gene causes decreased chloride transport across surface epithelia and dehydration of the airway surface liquid, which ultimately leads to the mucus production, as described earlier (SDDC, 2017).
Researchers have identified 270 synonymous single nucleotide polymorphisms (sSNPs), referred to here as synonymous mutations, in the CFTR gene. While these silent mutations do not by themselves cause cystic fibrosis, it is quite common in patients with the most severe cases of cystic fibrosis. Scientists at the University of Hamburg identified a silent mutation, known as T2562G, which modifies the local translation speed of the CFTR gene. This then results in harmful alterations in the protein folding and function (Kirchner et. al, 2017). T2652G is one of the most common synonymous mutations in the CFTR gene (Cuppens et. al, 1993). It is important to note, however, that this mutation does not cause cystic fibrosis, but when present in combination with cystic fibrosis-causing mutations, they can significantly alter the severity of the disease (SDDC, 2017).
Over the years, scientists observed and recorded patterns of changes in the DNA of various species that indicated that many silent mutations were preserved over time. This suggested that these mutations were useful to the organisms possessing them. It seemed as if that these synonymous mutations helped the cells of every other species except humans to make proteins more efficiently (Chamary and Hurst, 2009). Recently, it was discovered that silent mutations provide evolutionary advantages. However, this evolutionary advantage has only been found to occur in bacteria so far. Studies have shown that single highly beneficial silent mutations can provide organisms with the ability to rapidly evolve and adapt to their surrounding environment.
Within the experiments, researchers created multiple variants of a gene, fae, which is a metabolic enzyme necessary for survival and growth of the bacterium Methylobacterium extorquens, as they live in environments where the only source of carbon comes from methanol or methylamine. In these conditions, bacteria tend to retain the fae gene function. When these bacteria were grown in environments where only methanol was present, all bacterial populations with the synonymous fae gene variants performed poorly when compared to the controlled bacterial population that carried the normal gene. These populations were grown over a long period of time in the same conditions, and it was later discovered that within 100-200 generations, the experimental bacterial populations began to recover through additional mutations to the gene variants. Several of these additional mutations were also classified as silent mutations. When this experiment was repeated, the same mutations recurred, giving rise to the same results and proving that synonymous mutations can have evolutionary advantages in bacterial strains. Although benefits of silent mutations were discovered in bacterial populations, there have not been any recorded findings of silent mutations having the same, or similar effects on multicellular organisms.
Silent mutations in cancer
Different categories of genomic alterations, such as somatic point mutations, loss or gain of chromosome material, and structural genome rearrangements, have been observed in the DNA of tumor cells (Zheng et. al, 2013). According to Zheng et. al, synonymous mutations account for approximately 20 to 40% of somatic mutations detected in cancer exomes (2013). Synonymous mutations are able to alter oncogene activity through modulation of the post-transcriptional mRNA splicing machinery. Selection on synonymous mutations in oncogenes is cancer-type specific and these silent mutations repeatedly alter exonic motifs that regulate splicing and are associated with changes in oncogene splicing in tumors (Supek et. al, 2014). In a study carried out by Supek et. al, it was found that silent mutations are associated with increased exon expression variability. This indicated to the researchers the presence of tumor-cell-specific transcripts. In similar cancer related studies carried out by Tabernero et. al, it was discovered that small interfering RNAs (siRNAs) play a major role in that they are capable of mediating target mRNA cleavage and degrading these mRNAs with a high specificity. This in turn, holds promise for effective cancer-cell-specific gene silencing (2013). In particular, more than 40% of exon splicing enhancer-altering synonymous mutations were found to be associated with the known binding site of a proto-oncogene splicing factor. In addition to this, other silent mutations were found to cluster near the binding site of an exon splicing silencer (ESS) factor that has been found to play a role in drug resistance (Stark et. al, 2011). In addition, a study conducted by Ke et. al depicted that cancer-associated silent mutations close to exon boundaries within genes preferentially gain ESE motifs and lose ESS motifs. However, the opposite occurs for silent mutations that occur in tumor suppressor genes, such as p53. In these genes, silent mutations do not create ESEs (2011). It is important to note that apart from containing cancer-causing “driver” mutations, cancer genomes also contain accumulated additional “passenger” mutations that do not directly contribute to the tumor phenotype. A challenge in cancer research is to distinguish between these two types of mutations.
Conclusion
In conclusion, silent mutations are changes in the nucleotide sequence of a gene that do not alter the amino acid sequence of the encoded protein. Silent mutations primarily affect the post-transcriptional modification of mRNA in eukaryotic cells, specifically they can disrupt the proper splicing of introns from the eukaryotic mRNA. This in turn can result in lethal consequences. As exemplified by PKU, cystic fibrosis and cancer, silent mutations can have major negative influences on our health. Although there is more research to be done, the positive impacts of silent mutations have been proven in certain bacterial populations. This study can act as a starting point for future research on the possible beneficial silent mutations in multicellular organisms. Altogether, the information that can be derived from studies on the effects of synonymous mutations on our health can aid in progression of medical advancements. This can then possibly lead researchers to more effective treatments or even cures for such diseases. Finally, we can conclude that silent mutations have significant effects on human health, as seen from the variety of diseases that can arise from them. Further supporting this conclusion is the lack of evidence in favor of the notion that silent mutations can be beneficial to human health.