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Biochemistry, Mutation

Editor: Josephine A. Orrick Updated: 10/24/2022 7:10:38 PM


Deoxyribonucleic acid (DNA) is a double helix composed of two strands of four types of nucleotides.[1] Each nucleotide consists of the sugar deoxyribose, a nitrogenous base, and a phosphate group.[2] These four nucleotides include adenine (A), thymine (T), cytosine (C), and guanine (G). They always pair together with the same partner; adenine pairs with thymine (A-T), and cytosine pairs with guanine (C-G). This allows base pairs to be replaced, repaired, or replicated and not ruin DNA’s backbone and subsequent information. This information is derived from the precise order of these base pairs, and as two strands compose every DNA molecule, each strand contains the same coding information. The two strands are joined together via hydrogen bonds between nucleotides, and they run antiparallel to one another. One strand goes from 5’ to 3’ and the other from 3’ to 5’, defined by numbering from deoxyribose’s carbon atoms.[3][4] 

Genes, in turn, are a sequence of nucleotides that serve as the basic unit of heredity and code for a specific product, either protein or RNA. The cell relies on genes to be accurately and fully copied, transcribed, and translated to create a functioning product, often a specific protein but occasionally RNA as well. However, genetic sequence mutation can occur, which refers to an unintended alteration in a gene’s coding.[5]

Mutations can reduce functionality or limit the expression of the gene product, potentially damaging or even killing the cell. Genetic mutations, whether acquired or inherited, form the basis for many disease states, cancer, and aging.[6][7]

Issues of Concern

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Issues of Concern

Genomes are unique entities that acquire changes over time and generations due to the cumulative results of both small- and large-scale changes. Small-scale changes are due to mutations, which occur due to several factors, either endogenous or exogenous. Endogenous mechanisms include DNA replication errors (insertion of the incorrect nucleotide), DNA repair errors (failure to remove the incorrect or damaged nucleotide), spontaneous base deamination (loss of the nucleotide’s amine group), base methylation, reactive oxygen species (byproducts of metabolism), and spontaneous hydrolysis of nitrogenous bases.[7][8] 

Exogenous causes of mutations include ionization radiation, ultraviolet radiation, alkylating agents (mustard gas), polycyclic aromatic hydrocarbons (found in tobacco smoke, charred food, combustion products), crosslinking agents (cyclophosphamide), insertional mutagenesis (integration of viral genome), and other toxins.[9][10] 

In contrast to small-scale changes, large-scale genetic changes are not due to mutations. They typically occur due to chromosomal abnormalities, such as translocation, duplication, inversion, or deletions of parts of chromosomes or entire chromosomes.[11][12]

If a mutation involves a change in a single nucleotide base pair, this is referred to as a point mutation. However, it is important to understand that proteins are coded for in groups of 3 nucleotides, termed codons. Each codon contains instructions to create exactly one amino acid, which is then added to the growing protein chain during translation. Point mutations have different consequences depending on which codon and what part of it they affect.[13]

Silent mutations can occur when a point mutation occurs in non-coding or non-regulatory regions of DNA, sparing genes and codons. Much of the human genome is neither used for genes or genetic regulation, with only about 8% used for biological activity. As a result, any mutation in the non-biologically active regions would be silent. Silent mutations may also occur even if a mutation targets a codon, and this is due to redundancy in the genetic code, also referred to as degeneracy.[14][15]

While there are 64 different codons based on varying combinations of the four nucleotides, this only codes for 20 amino acids and three stop codons—for example, CGU codes for the amino acid arginine. If a mutation were to occur in this stretch of DNA, changing CGU to CGC would still code for arginine! Therefore, silent mutations can occur when the codon mutates to another codon representing the same amino acid and this is because all but two amino acids are coded for by more than one codon. However, should a mutation within a codon result in it coding for a different amino acid, this is termed a missense or substitution mutation. In effect, one amino acid was replaced with another. For example, if the CGU that codes for arginine were changed to CAU, this would result in the placement of a histidine instead of arginine, thereby changing the amino acid composition of the protein.[16]

However, the consequences of a missense mutation vary depending on the exact amino acid that is replaced and exchanged. More specifically, their size, polarity, and presence/absence of hydrogen bonding with other nearby amino acids will determine the consequences of the genetic alteration.[17] A missense mutation that substitutes a chemically similar amino acid, such as isoleucine in place of leucine, is referred to as a synonymous substitution. The size and distribution of charge on each amino acid are similar and therefore would be less likely to cause drastic changes in protein function. In contrast, nonsynonymous substitutions involve chemically different amino acids and are more likely to cause significant changes in protein function. Sickle cell disease and certain forms of ALS are examples of pathology caused by nonsynonymous substitution missense mutations.[18]

Relative to silent and missense mutations, nonsense and frameshift mutations are much less forgiving to the cell and organism and more likely to cause pathology. Nonsense mutations occur when a single nucleotide change results in a stop codon (UGA, UAA, UAG), resulting in premature termination of protein translation. The result of a nonsense mutation will vary, however, on how close the new stop codon is to the original and also how much of the protein’s functional sub-domains are still present. If the nonsense mutation resulted in a new stop codon just upstream of the original, this would likely disrupt protein function much less than a new stop codon placed immediately after the start codon.[19][20] The majority of cases of beta-thalassemia, for example, are caused by a variety of nonsense mutations with varying degrees of severity.[21] 

Frameshift mutations, in contrast, result from either the insertion of a new nucleotide into the DNA strand or the deletion of a pre-existing nucleotide. This can occur through transposons, toxins, mutagens, or viruses. The human papillomavirus (HPV), for example, relies on the insertion of its genetic material into the host’s genome for its life cycle to progress successfully.[22] 

The insertion of its genome alters the reading frame of nearby codons, and every codon downstream of insertion is changed. Consequently, whether viral or not in origin, the earlier a frameshift mutation occurs within a gene, the more deleterious its effects are. Interestingly, frameshift mutations can result in a premature stop codon if the downstream codon shift into one of the three stop codons. However, insertion or deletion of nucleotides will not cause a frameshift mutation in situations where the number of nucleotides is a multiple of 3. In this situation, a new amino acid would be inserted or deleted from the structure with varying effects on protein structure and function. Frameshift mutations are notable due to their implication in the pathogenesis of various cancers, particularly prostate and colorectal cancer.[23][24]

A unique type of small DNA mutation is the trinucleotide repeat expansion. This is a type of mutation where “slippage” occurs during DNA replication, resulting in repeated copying of the same codon. Slippage occurs due to repetitive sequences, typically G-C, located in the vicinity of one another. During DNA replication, when the double-stranded DNA (dsDNA) is unwound to single-stranded DNA (ssDNA), nucleotides located in the repetitive regions have the opportunity to base-pair to other nucleotides on the same strand resulting in the formation of a single strand loop. This single strand loop is then copied, which typically results in the expansion of the repetitive sequences.[25][26] The single-strand loop and subsequent expansion can also occur during DNA repair.[27]

If located within a gene or its regulatory components, this can result in either impaired production of a product or a dysfunctional product.[28] Examples of diseases that occur due to trinucleotide repeat expansion include Fragile X syndrome, Huntington disease, Friedrich Ataxia, and numerous other neurodegenerative disorders. Many of these diseases are hereditary and progressively worsen in each subsequent generation as the number of trinucleotide repeats increases.[28]

Cellular Level

Genetic mutations affect both prokaryotic and eukaryotic cells in both beneficial or harmful ways. Genetic mutations result in genetic variation, which is a prerequisite for natural selection. A genetic mutation may result in higher or lower fitness of the organism depending on the consequences of the mutation.[29]

Those organisms with higher fitness are more likely to reproduce and pass on their genetics, including any mutations, to subsequent generations. For example, bacteria can become resistant to antibiotics throughout thousands of generations due to, in part, the acquisition of new mutations.[30] 

Similarly, viruses such as HIV develop resistance to anti-viral medications, allowing their continued replication and spread. Bacteria, and particularly viruses, are prone to accumulating genetic mutations due to their very rapid generation times and decreased ability to repair their DNA. However, eukaryotic cells also are susceptible to acquiring mutations despite robust DNA damage repair mechanisms.[31]

Deleterious mutations in a somatic cell may render the cell less able to carry out its required roles within its organ. The rate of somatic mutations is much more rapid than DNA damage repair (DDR) pathways can function, allowing these mutated cells to continue proliferating. As organisms age, their cells accumulate mutations that are initially tolerated well but will gradually decline tissue and organ function over time. This occurs via the process of apoptosis, or programmed cell death. As cellular stress or damage crosses a certain threshold, an irreversible cascade of proteins and signaling molecules are activated within the cell, ultimately leading to its demise.[32][33]

These cells are replaced by the division of other neighboring cells so that loss in function in a tissue is not noticeable to the organism. In other situations, cells do not undergo apoptosis but instead are restricted from further progression through the cell cycle and enter an irreversible state termed senescence. They are still able to perform many normal cellular activities but can no longer replicate. Gradually, either through apoptosis or senescence, tissues and organs have decreased functioning and slower healing rates, and the organism experiences the process of aging. However, not all cells enter a state of senescence or undergo apoptosis even if they have acquired a significant mutational burden. Either process and its regulatory pathways can experience genetic mutations, thereby becoming defective and resulting in continued cell survival and replication.[34]

This can lead to the development of cancerous cells should these cells continue to replicate and acquire new mutations. For example, the mutation of a key regulatory gene, TP53, and its product p53, can affect numerous apoptotic and DNA repair pathways and is implicated in the development of various cancers. Normally, p53 can activate DNA repair enzymes in the presence of DNA damage, it can arrest cell growth at the G1/S restriction point, and it can also initiate apoptosis or cellular senescence. TP53 mutation can interrupt any of these crucial functions. This mutation is often found alongside mutations in genes that enhance growth, limit inhibitory signals, and reduce dependence on signaling from neighboring cells.[35]

Other disruptions in TP53 can occur due to HPV, as it encodes a protein called E6, which binds to and inactivates p53. This is part of the pathogenesis of HPV and how certain HPV types, such as HPV 16, have a higher probability of causing pre-cancerous and cancerous lesions. Typically, such cancer-causing mutations are spontaneous and only affect somatic cells, thereby sparing the offspring of any individual with such mutations. However, genetic mutations can also affect the gametes, in which case such acquired mutations would be spread to offspring.


The acquisition of genetic mutations, particularly those that impact protein structure, function, or expression, is a prerequisite for natural selection, particularly if they significantly impact the organism’s phenotype. Mutations can result in either a positive or negative impact on an organism’s fitness. However, most tend to be slightly deleterious and selected against. However, it is important to note that traits which offer high fitness in one environment may be deleterious in others. A mutation’s variable impact on fitness in different environments can lead to speciation. It results in different populations with varying fitness levels in a given context, thereby causing a barrier to reproduction.[36][37]

Barriers to reproduction between populations are crucial in forming new species, and genetic mutations are ultimately necessary for this to occur. Furthermore, there are vast differences between species in their baseline rate of genetic mutation, which also contributes to natural selection and evolution. For example, RNA viruses have a rate of 10^-3 per base per generation, while humans have a rate of 10^-8 per base per generation.[38][39] 

This evolutionary process is one explanatory mechanism for the tendency of viruses to become rapidly resistant to anti-viral medications. Although mutations serve as the driver of natural selection and evolution, they also contribute to many disease states, cancer formation, and aging.[40]


The mechanisms of genetic mutations can be divided into four categories: mutations induced by mutagens, molecular decay, errors during DNA repair, and errors during DNA replication.[41]

Mutagen-induced: Mutagens alter the structure of DNA after direct contact and cause induced mutations either through sources of radiation or chemicals. The most common sources of radiation that act as mutagens include ultraviolet (UV) and ionizing radiation.[42][43] Most skin cancers are caused due to UV light exposure, either from sunlight or artificial sources. UV light most frequently causes pyrimidine dimers and pyrimidine pyrimidone photoproducts, although other minor products are also formed, such as pyrimidine hydrate. These products, particularly the dimers, require TLS for replication to proceed, further adding to their potential for mutation. Furthermore, UV light can cause DNA adducts, DNA-protein crosslinks, and DNA strand breakages.[44][45]

Ionizing radiation, such as x-rays or gamma rays, causes DNA damage either directly through single-strand or double-strand breaks or indirectly through the production of ROS or other free radicals.[46][47] The autosomal recessive condition xeroderma pigmentosum is associated with dysfunctional TLS, typically due to a deletion mutation. Affected individuals are at increased sensitivity to UV and ionizing radiation-induced genetic mutations with a significantly elevated risk of most forms of pre-malignant skin lesions as well as skin cancer.[48]

Many chemical compounds can induce DNA damage and mutation through several different mechanisms. Alkylating agents, such as tobacco smoke, chemotherapeutics, and nitrogen gas, function through their high affinity to the nitrogen atoms of the nucleotide base, particularly N7 of guanine and N3 of adenine.[49][50] They react strongly with these nitrogens and produce N7-methyl guanine and N3-methyladenine, respectively, which are more susceptible to N-glycosidic bond cleavage and subsequent apurinic site formation. Alkylating agents induce DNA adducts, intra-strand crosslinks, interstrand crosslinks, and DNA-protein crosslinks.[51][52] This is the primary mechanism of action of cyclophosphamide, which is the most common chemotherapeutic alkylating agent, often used in combination with other drugs to treat multiple myeloma, leukemias, breast cancer, and small cell lung cancers. Any type of crosslink results in DNA replication blockage, requiring the use of the translesion synthesis (TLS) pathway or other repair pathways.[53][54]

Other classes of compounds, such as polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and N-nitrosamines, often require activation by the cytochrome P450 system, after which they attain their mutagenic properties.[55][56] Aromatic amines are, interestingly, converted to alkylating agents after activation by P450 enzymes. PAHs, while also requiring P450 activation, will typically function to generate reactive chemical intermediates. Nitrosamines, in contrast, do not require P450 activation and function through the formation of DNA adducts and strand breaks.[57]

Molecular decay: DNA is not entirely stable as a molecule, and various spontaneous chemical reactions can occur. DNA can undergo depurination, in which the N-glycosyl bond between deoxyribose (the sugar-phosphate backbone) and adenine or guanine (nitrogenous base) is cleaved. Although less common, this can also occur with cytosine and thymine, termed apyrimidation. Both apurinic and apyrimidinic sites are commonly referred to as abasic sites. In the average human cell, 10,000 abasic sites are formed per day. Due to their instability and reactivity, apurinic sites alter DNA structure, converting into single-strand DNA through either beta-elimination or delta-elimination.[58][59] 

In addition to apurination/apyrimidation, nucleotides can undergo deamination in which they lose an amine group, or tautomerism, in which the nucleotide is changed by hydrogen atom repositioning.[60] Both of these processes ultimately result in incorrect base pairing during DNA replication, thereby propagating this change.[61] Alongside the spontaneous molecular reactions, DNA can also undergo molecular decay and modification via normal cellular processes and metabolism. Reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals, normally form byproducts of aerobic cellular respiration, anabolic reactions, and peroxisomal enzymes.[62][63][64] Normally, cells limit the damaging effects of ROS through anti-oxidant enzymes or restricting aerobic metabolism to the mitochondria. However, it is impossible to entirely mitigate their harmful effects.[65]

Over 100 different ROS-induced base lesions have been noted, resulting in incorrect base-pairing. Furthermore, ROS can react with the DNA backbone and cause single-strand breaks (SSB). Although these can be repaired with the SSB repair pathway, this is error-prone and may result in the placement of the incorrect nucleotide. Other normal cellular agents apart from ROS can also result in DNA mutation. This includes s-adenosylmethionine, which acts as a methyl donor in several metabolic pathways, along with other methylating agents such as bile salts, choline, and betaine. These methylating agents can either cause transition mutations (e.g., G:C to A:T) or react further to generate apurinic sites.[66][67]

DNA replication-induced: There are numerous polymerases and various other proteins involved in the copying of DNA as a human cell replicates. These polymerases have a very low error rate due to their proofreading capabilities, further supported by the mismatch repair (MMR) pathway, capable of correcting incorrectly paired nucleotides.[68][69] Despite this, base substitutions, insertions, and deletions occur at the rate of 10 to 10 per cell per generation. Furthermore, uracil can be incorrectly incorporated by polymerases, or polymerases may have reduced fidelity based on concentrations of deoxyribonucleotides and ribonucleotides relative to one another.[70]

Other enzymes involved in the DNA replication process, such as topoisomerase, can result in mutation. Normally, they remove superhelical tension during replication and transcription as DNA unwinds by nicking and re-ligating the DNA. However, they can misalign the nicked DNA ends, resulting in the formation of lesions such as DNA adducts, abasic sites, or mismatches.[71] DNA repair-induced: DNA can undergo double-strand breaks (DSBs) through endogenous or exogenous means, which can contribute to cancer formation if unresolved. Human cells can undergo both homology-directed repair (HDR) and non-homologous end joining (NHEJ) as a mechanism to address the DSD.[72][73] In contrast to HDR, which requires a homologous sequence for repair to occur, NHEJ involves ligating the ends of the two DNA molecules directly. Although often accurate, an imprecise repair can result in a loss of nucleotides or even cause translocations.[74]

Translesion synthesis (TLS) is another repair mechanism that allows the cell to continue DNA replication past certain lesions such as apurinic sites or thymine dimers.[75][76] However, in doing so, it requires swapping the high fidelity polymerases for specialized translesion polymerases with lower fidelity.[77] Notably, more than 60% of single base-pair mutations occur in certain species such as yeast due to translesion synthesis.[77]


In the last two decades, rapid advances in genomic and biologic technologies have allowed genetic testing and analysis to become essential components of clinical practice and academic research. Furthermore, testing methods have become widely accessible and have substantially decreased in cost, allowing for the participation of more laboratories.[78] In addition, the availability of a variety of public online genomic databases allows the researcher, clinician, and even patient to better understand and compare the significance of a variety of genetic mutations.

Significant genomic alterations, such as chromosomal translocations, deletions, or duplications, could be detected by conventional methods such as karyotyping. However, single nucleotide changes were difficult to detect.[79] However, the discovery of polymerase chain reaction (PCR) allowed for the rapid development of numerous molecular genetic technologies.[80]

PCR was used in techniques such as restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) testing. However, these methods were not able to detect every mutation, particularly if the sequence of a gene was not known. The development and subsequent automatization of Sanger sequencing overcome this limitation, thereby allowing the human genome to be sequenced rapidly and accurately. Sanger sequencing allowed for the Human Genome Project to be launched and completed ahead of time. However, despite advances in Sanger sequencing, newer techniques such as massively parallel sequencing (MPS) allowed for even more rapid and cost-effective technologies.[81] Newer techniques and technologies have evolved since then, allowing for sequencing costs of an entire genome to approach $1000.[82]

Currently, targeted sequencing, whole-exome sequencing (WES), and whole-genome sequencing (WGS) are the three main sequencing approaches used in the clinical setting to evaluate for the presence of a rare or novel genetic variant. These techniques are often collectively referred to as next-generation sequencing (NGS). The first of these, gene panels, evaluate a certain, specific number of genes or regions within genes that have been reported to contain mutations contributory to a specific disease or group of diseases.[83]

Gene panels are economical, rapid, and flexible but may vary between laboratories regarding the included genes and their reasoning for inclusion. This makes a comparison between different panels difficult. Furthermore, as the literature expands on certain diseases, panels must be updated routinely to capture newly described mutations. Additionally, many of the current panels are predominantly based on genetic analysis of individuals with European ancestry, limiting generalizability to other groups. In contrast, WES and WGS are both more extensive regarding the extent to which they sequence the genome. WES involves the sequencing of protein-coding regions of the genome, which can identify genetic mutations not yet associated with specific pathologies.

Furthermore, WES can be used to evaluate a pre-selected list of genes or evaluate all potential genetic mutations. In contrast, WGS is the most extensive and can reveal the entire genome. It has the most potential to discover new genetic mutations, genes, and other novel findings.[84][85] However, it also has the most potential to uncover incidental findings, in which mutations are uncovered, which indicate a patient is at risk for a certain disease that is not currently present.[86] Additionally, variants of unknown significance (VUS) can be uncovered.[87] VUS refers to the presence of genetic mutations or genetic variants. There is not enough information in the reported literature to determine whether the mutation is pathologic, benign, or beneficial.[88]

Clinical Significance

Genetic mutations underlie much of human disease and evolution. Although rare in themselves, approximately 1 in 15 people are affected by a genetic disorder. Furthermore, various diseases have a multifactorial etiology, of which genetic mutations are a contributory factor, including conditions such as asthma, diabetes, hypertension, obesity, and infertility. Additionally, the role of acquired genetic mutations cannot be understated in their role in carcinogenesis, particularly if the affected genes are critical in cell cycle progression and growth regulation, such as TP53.

Furthermore, the acquisition of genetic mutations throughout an individual’s lifetime leads to aging, as cells either undergo apoptosis or enter senescence. All healthcare practitioners encounter genetic mutations in their work, even if they may be unaware, that is the case. Cancer, chronic conditions, and the diseases of aging all share the common foundation of genetic mutations.

As NGS techniques continue to evolve and become more cost-effective, healthcare practitioners will more readily have access to genetic testing for their patients and families. Though there may be pitfalls, such as VUS, more genetic variants will be accounted for and described in the literature over time. The combination of lower barriers to genetic testing and robust literature will provide practitioners the opportunity for improved decision-making in many of their patients.



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