The 6–4 photoproduct (6–4 pyrimidine– pyrimidone , or 6–4 pyrimidine–pyrimidinone) is an alternate dimer configuration consisting of a single covalent bond linking the carbon at the 6 (C6) position of one pyrimidine ring and carbon at the 4 (C4) position of the adjoining base's ring. [ 11 ]  This type of conversion occurs at one third the frequency of CPDs and has a higher  mutagenic  risk. [ 12 ]


A third type of molecular lesion is a  Dewar  pyrimidinone, resulting from the reversible  isomerization  of a 6–4 photoproduct under further light exposure. [ 13 ]


Mutagenesis, the process of mutation formation, is significantly influenced by translesion  polymerases  which often introduce mutations at sites of pyrimidine dimers. This occurrence is noted both in  prokaryotes , through the  SOS response  to mutagenesis, and in  eukaryotes . Despite thymine-thymine CPDs being the most common lesions induced by UV, translesion polymerases show a tendency to incorporate  adenines , resulting in the accurate replication of thymine dimers more often than not. Conversely, cytosines that are part of CPDs are susceptible to  deamination , leading to a cytosine to thymine transition, thereby contributing to the mutation process. [ 14 ]


Pyrimidine dimers introduce local conformational changes in the  DNA structure , which allow recognition of the lesion by repair enzymes. [ 15 ]  In most organisms (excluding  placental mammals  such as humans) they can be repaired by photoreactivation. [ 16 ]  Photoreactivation is a repair process in which  photolyase  enzymes reverse CPDs using  photochemical  reactions. In addition, some photolyases can also repair 6-4 photoproducts of UV induced DNA damage. Photolyase enzymes utilize  flavin adenine dinucleotide (FAD)  as a cofactor in the repair process. [ 17 ]


The UV dose that reduces a population of wild-type yeast cells to 37% survival is equivalent (assuming a  Poisson distribution  of hits) to the UV dose that causes an average of one lethal hit to each of the cells of the population. [ 18 ]   The number of pyrimidine dimers induced per  haploid   genome  at this dose was measured as 27,000. [ 18 ]   A mutant yeast strain defective in the three pathways by which pyrimidine dimers were known to be  repaired  in yeast was also tested for UV sensitivity.  It was found in this case that only one or, at most, two unrepaired pyrimidine dimers per haploid genome are lethal to the cell. [ 18 ]   These findings thus indicate that the repair of thymine dimers in wild-type yeast is highly efficient. [ citation needed ]


Nucleotide excision repair , sometimes termed "dark reactivation", is a more general mechanism for repair of lesions and is the most common form of DNA repair for pyrimidine dimers in humans. This process works by using cellular machinery to locate the dimerized nucleotides and excise the lesion. Once the CPD is removed, there is a gap in the DNA strand that must be filled. DNA machinery uses the undamaged complementary strand to synthesize nucleotides off of and consequently fill in the gap on the previously damaged strand. [ 6 ]


Xeroderma pigmentosum  (XP) is a rare genetic disease in humans in which genes that encode for NER proteins are mutated and result in decreased ability to combat pyrimidine dimers that form as a result of UV damage. Individuals with XP are also at a much higher risk of cancer than others, with a greater than 5,000 fold increased risk of developing skin cancers. [ 7 ]  Some common features and symptoms of XP include skin discoloration, and the formation of multiple tumors proceeding UV exposure. [ citation needed ]


A few organisms have other ways to perform repairs:


Another type of repair mechanism that is conserved in humans and other non-mammals is  translesion synthesis.  Typically, the lesion associated with the pyrimidine dimer blocks cellular machinery from synthesizing past the damaged site. However, in translesion synthesis, the CPD is bypassed by translesion polymerases, and replication and or transcription machinery can continue past the lesion. One specific translesion DNA polymerase, DNA polymerase η, is deficient in individuals with XPD. [ 20 ]


Direct DNA damage is reduced by sunscreen, which also reduces the risk of developing a sunburn.  When the sunscreen is at the surface of the skin, it filters the UV rays, which attenuates the intensity. Even when the sunscreen molecules have penetrated into the skin, they protect against direct DNA damage, because the UV light is absorbed by the sunscreen and not by the DNA. [ 21 ]  Sunscreen primarily works by absorbing the UV light from the sun through the use of organic compounds, such as oxybenzone or avobenzone. These compounds are able to absorb UV energy from the sun and transition into higher-energy states. Eventually, these molecules return to lower energy states, and in doing so, the initial energy from the UV light can be transformed into heat. This process of absorption works to reduce the risk of DNA damage and the formation of pyrimidine dimers. UVA light makes up 95% of the UV light that reaches earth, whereas UVB light makes up only about 5%. UVB light is the form of UV light that is responsible for tanning and burning. Sunscreens work to protect from both UVA and UVB rays. Overall, sunburns exemplify DNA damage caused by UV rays, and this damage can come in the form of free radical species, as well as dimerization of adjacent nucleotides. [ 22 ]


Replication timing quantitative trait loci  (or  rtQTL ) are genetic variations that lead to differential use of replication origins, exhibiting allele-specific effects on  replication  timing. [ 1 ]  Originally, 16 rtQTL were identified in an analysis of human genomes. [ 2 ]


A  resistance mutation  is a  mutation  in a  virus   gene  that allows the virus to become resistant to treatment with a particular  antiviral drug .  The term was first used in the management of  HIV , the first virus in which genome sequencing was routinely used to look for drug resistance. At the time of infection, a virus will infect and begin to replicate within a preliminary  cell . As subsequent cells are infected, random mutations will occur in the viral genome. [ 1 ]  When these mutations begin to accumulate, antiviral methods will kill the  wild type  strain, but will not be able to kill one or many mutated forms of the original virus. At this point a resistance mutation has occurred because the new strain of virus is now resistant to the antiviral treatment that would have killed the original virus. [ 1 ]  Resistance mutations are evident and widely studied in HIV due to its high rate of mutation and prevalence in the general population. Resistance mutation is now studied in  bacteriology  and  parasitology .


Resistance mutations can occur through several mechanisms from single nucleotide substitutions to combinations of  amino acid  substitutions, deletions and insertions. [ 1 ]  Over time, these new genetic lines will persist if they become resistant to treatments being used against them. It has been shown that pathogens will favor and become more resistant to treatment in common host  genotypes  through  frequency-dependent selection . [ 2 ]  Further, strict adherence to a retroviral regimen correlates to a strong decrease in retroviral resistance mutations. [ 3 ]  There are five classes of drug that are used to treat HIV infection, and resistance mutations can effect the efficacy of these treatments as well.


Resistance mutations are found and become problematic in many viruses other than HIV. Notable examples of such viruses include the  herpes simplex  virus and  hepatitis B virus . [ 9 ]  In the herpes virus, drugs mainly target the viral DNA polymerase. As a result, mutations in the viral DNA polymerase that make it resistant to these drugs are selected for, which ultimately can cause complete resistance of the treatment. In hepatitis B, nucleoside and nucleotide analogs are used to cause early termination of viral transcription. Mutations in viral reverse transcriptase can cause the enzyme to not incorporate these nonfunctional analogs, in favor for their natural counterparts. If this mutation occurs, transcription will not be halted, and viral proteins can be created.


Rumpless chickens  are characterised by  caudal dysplasia , the absence of the  pygostyle  or caudal appendage – the "parson's nose". This is the result of inheritance of an  autosomal dominant  trait. The  mutation  which causes it is unknown; two candidates have been proposed. [ 1 ] : 10


Rumplessness in chickens has been documented for centuries. An early description is that of the  Bolognese  ornithologist  Ulisse Aldrovandi  in his  Ornithologiae Tomus Alter  of 1600. [ 2 ] : 127  [ 3 ] : 318


Rumpless breeds of chicken include: the  Araucana  or Rumpless Araucana of South America, both large fowl and  bantam ; [ 4 ] : 45   the Belgian  Barbu de Boitsfort ,  Barbu de Grubbe  and  Barbu d'Uccle  bantams; [ 4 ] : 75   the Japanese  Uzurao , a rumpless equivalent of the  Tosa-Kojidori  bantam, [ 5 ] : 95   and  Ingie  (large fowl) from  Kagoshima Prefecture ; [ 5 ] : 103   the  Rumpless Game  of the United Kingdom (both large fowl and bantam), sometimes called the Manx Rumpy or Persian Rumpless; [ 4 ] : 272  [ 6 ] : 65   and the German  Ruhlaer Zwerg-Kaulhühner  or Rumpless Booted Bantam. [ 4 ] : 92


This  poultry  article is a  stub . You can help Wikipedia by  expanding it .


In  genetics , a  selective sweep  is the process through which a new  beneficial mutation  that increases its frequency and becomes fixed (i.e., reaches a frequency of 1) in the population leads to the reduction or elimination of  genetic variation  among  nucleotide  sequences that are near the  mutation . In selective sweep,  positive selection  causes the new mutation to reach  fixation  so quickly that linked alleles can "hitchhike" and also become fixed.


A selective sweep can occur when a rare or previously non-existing  allele  that increases the  fitness  of the carrier (relative to other members of the  population ) increases rapidly in frequency due to  natural selection . As the prevalence of such a beneficial allele increases, genetic variants that happen to be present on the genomic background (the DNA neighborhood) of the beneficial allele will also become more prevalent. This is called  genetic hitchhiking . A selective sweep due to a strongly selected allele, which arose on a single genomic background, therefore results in a region of the genome with a large reduction of genetic variation in that  chromosome  region. The idea that strong positive selection could reduce nearby genetic variation due to hitchhiking was proposed by  John Maynard-Smith  and John Haigh in 1974. [ 1 ]


Not all sweeps reduce genetic variation in the same way. Sweeps can be placed into three main categories:


Sweeps do not occur when selection simultaneously causes very small shifts in allele frequencies at many loci each with standing variation ( polygenic adaptation ).


Whether or not a selective sweep has occurred can be investigated in various ways. One method is to measure  linkage disequilibrium , i.e., whether a given  haplotype  is overrepresented in the population. Under neutral evolution,  genetic recombination  will result in the reshuffling of the different alleles within a haplotype, and no single haplotype will dominate the population. However, during a selective sweep, selection for a positively selected gene variant will also result in selection of neighbouring alleles and less opportunity for recombination. Therefore, the presence of strong linkage disequilibrium might indicate that there has been a recent selective sweep, and can be used to identify sites recently under selection.


There have been many scans for selective sweeps in humans and other species, using a variety of statistical approaches and assumptions. [ 4 ]


In maize, a recent comparison of yellow and white corn genotypes surrounding  Y1 —the phytoene synthetase gene responsible for the yellow endosperm color, shows strong evidence for a selective sweep in yellow germplasm reducing  diversity  at this locus and linkage disequilibrium in surrounding regions.  White maize lines had increased diversity and no evidence of linkage disequilibrium associated with a selective sweep. [ 5 ]


Because selective sweeps allow for rapid adaptation, they have been cited as a key factor in the ability of  pathogenic  bacteria and viruses to attack their  hosts  and survive the medicines we use to treat them. [ 6 ]   In such systems, the competition between host and  parasite  is often characterized as an  evolutionary "arms race" , so the more rapidly one organism can change its method of attack or defense, the better.  This has elsewhere been described by the  Red Queen hypothesis .  Needless to say, a more effective pathogen or a more resistant host will have an adaptive advantage over its conspecifics, providing the fuel for a selective sweep.


One example comes from the human  influenza  virus, which has been involved in an adaptive contest with humans for hundreds of years.  While  antigenic drift  (the gradual change of surface antigens) is considered the traditional model for changes in the viral genotype, recent evidence [ 7 ]  suggests that selective sweeps play an important role as well.  In several flu populations, the time to the most recent common ancestor (TMRCA) of "sister" strains, an indication of relatedness, suggested that they had all evolved from a common progenitor within just a few years.  Periods of low genetic diversity, presumably resultant from genetic sweeps, gave way to increasing diversity as different strains adapted to their own locales.


A similar case can be found in  Toxoplasma gondii , a remarkably potent protozoan parasite capable of infecting warm-blooded animals.   T. gondii  was recently discovered to exist in only three clonal lineages in all of Europe and North America. [ 8 ]   In other words, there are only three genetically distinct strains of this parasite in all of the Old World and much of the New World.  These three strains are characterized by a single monomorphic version of the gene Chr1a, which emerged at approximately the same time as the three modern clones.  It appears then, that a novel genotype emerged containing this form of Chr1a and swept the entire European and North American population of  Toxoplasma gondii , bringing with it the rest of its genome via  genetic hitchhiking .  The South American strains of  T. gondii , of which there are far more than exist elsewhere, also carry this allele of Chr1a.


Rarely are genetic variability and its opposing forces, including adaptation, more relevant than in the generation of  domestic  and agricultural species.  Cultivated  crops , for example, have essentially been genetically modified for more than ten thousand years, [ 9 ]  subjected to artificial selective pressures, and forced to adapt rapidly to new environments.  Selective sweeps provide a baseline from which different varietals could have emerged. [ 10 ]


For example, recent study of the corn ( Zea mays ) genotype uncovered dozens of ancient selective sweeps uniting modern cultivars on the basis of shared genetic data possibly dating back as far as domestic corn's wild counterpart,  teosinte .  In other words, though artificial selection has shaped the genome of corn into a number of distinctly adapted cultivars, selective sweeps acting early in its development provide a unifying homoplasy of genetic sequence.  In a sense, the long-buried sweeps may give evidence of corn's, and teosinte's, ancestral state by elucidating a common genetic background between the two.


Another example of the role of selective sweeps in domestication comes from the chicken.  A Swedish research group recently used parallel sequencing techniques to examine eight cultivated varieties of chicken and their closest wild ancestor with the goal of uncovering genetic similarities resultant from selective sweeps. [ 11 ]   They managed to uncover evidence of several selective sweeps, most notably in the gene responsible for thyroid-stimulating hormone receptor ( TSHR ), which regulates the metabolic and  photoperiod -related elements of reproduction.  What this suggests is that, at some point in the domestication of the chicken, a selective sweep, probably driven by human intervention, subtly changed the reproductive machinery of the bird, presumably to the advantage of its human manipulators.


Examples of selective sweeps in humans are in variants affecting  lactase persistence , [ 12 ] [ 13 ]  and adaptation to high altitude. [ 14 ]


The  Seneca white deer  are a rare  herd  of  deer  living within the confines of the former  Seneca Army Depot  in  Seneca County, New York . When the 10,600-acre (43 km 2 ) depot was created in 1941, a 24-mile (39 km) fence was erected around its perimeter, isolating a small herd of  white-tailed deer  ( Odocoileus virginianus ), some of which had white coats.  These deer are not  albino , but instead have  leucism , which is an abnormal genetic condition that carries a set of  recessive genes  for all-white coats. [ 1 ]  While little is known about what caused leucism in the white-tailed deer, especially for this herd, [ 2 ]  researchers have noticed that its isolation causes high levels of inbreeding. [ 3 ]  With inbreeding, it leaves recessive  alleles  of the white-tailed population to be expressed. This recessive trait does not pose a danger to the animal. However, it is very rare.


In the 1950s, the depot commander forbade  GIs  from shooting any white deer. [ 4 ]  The deer population has since grown to about 700 head, approximately 300 of which are white, making it the largest herd of white deer in the world.  Seneca White Deer were created through years of selective breeding within the depot to create an environment where they can flourish. [ 5 ] [ 6 ] [ 7 ]


Since the depot's closure in 2000, the future of the deer has been uncertain. The visibility of white deer makes them easy prey for hunters and natural enemies like  coyotes , and such a herd would not survive in the wild  [ citation needed ] . The depot land is New York's largest block of land available for development, [ 8 ]  and some new uses have been found for it. The  Five Points   maximum security  state prison was built on the eastern rim, and the former  barracks  on the northern tip was converted into housing for troubled teenagers. Plans to buy 4,700 acres (1,900 ha) of land to build an  ethanol  and  biofuel   electricity production  center and to plant crops of  willow  trees and  switch grass  to be converted into fuel was proposed. [ 9 ]  In 2006, a business plan was submitted to the Seneca County Industrial Development Agency, owners of the depot property, to convert the land into a conservation park and  Cold War  museum, but the plan was denied. [ 3 ] [ 10 ]


In 2016, the 7,000 acres (2,800 ha) property was sold to local businessman Earl Martin of Seneca Iron Works for $900,000 and established as Deer Haven Park, LLC. [ 11 ] [ 12 ]  An agreement was later made between Martin and Dennis Money, founder of Seneca White Deer, Inc., to lease part of the land and operate it as a conservation park for the white deer. [ 12 ] [ 13 ]  Following renovations to the fencing and construction of a welcome center and museum, Seneca White Deer park opened for tours in November 2017, [ 13 ]  ending December 29, 2019. Tours reopened on Saturdays on June 27, 2020, as self-guided auto tours with the option to download an auto tour app. The current plans for the depot are to preserve the approximately 200 deer left through strategic investment and innovation. [ citation needed ]


The  Sequence Feature Variant Type  (SFVT) refers to the defined sequence variation (mutation) of a given sequence feature in a  protein . Consider a protein, its  amino acid sequence  determines its structure and function. Some regions of the sequence are primarily related to the  protein folding , thus the structure, while other regions are related to the  protein function , such as an enzyme catalytic site. Across many related  species , regions in a corresponding protein would remain conserved from species to species, while the exact amino acid at each location might, and often does, change. To clearly illustrate both the conserved nature of the functional regions and the precise change in the actual amino acid, the Sequence Feature Variant Type is defined.


A sequence feature is basically a collection of amino acids within a protein, where the collection may be a continuous range or some discrete residues. Most sequence features are from experimental results, but there are some that are from computational analysis.


For each sequence feature, the variations exist between many proteins from different related species. The variant types define the exact amino acid at each location for a feature. The larger the mutation at each location for a feature, the larger its number of variant types. For a given protein, once its amino acid sequence is determined, its sequence feature variant type is determined.


SFVTs can be used to study the protein functions across many related species. Such studies have been carried out on several human pathogens.


SFVTs are also available on  Influenza Research Database  for flu proteins and on  Virus Pathogen Database and Analysis Resource  for Dengue virus.


In agriculture,  shattering  is the  dispersal of a crop's seeds  upon their becoming ripe. From an agricultural perspective this is generally an undesirable process, and in the history of  crop domestication  several important advances have involved a  mutation  in a crop plant that reduced shattering—instead of the seeds being dispersed as soon as they were ripe, the mutant plants retained the seeds for longer, which made harvesting much more effective. Non-shattering  phenotype  is one of the prerequisites for  plant breeding  especially when  introgressing  valuable traits from wild varieties of domesticated crops. [ 1 ]


A particularly important mutation that was selected very early in the history of agriculture removed the "brittle rachis" problem from wheat. [ 2 ]  A ripe head ("ear") of wild-type wheat is easily shattered into dispersal units when touched, or blown by the wind, because during ripening a series of  abscission  layers forms that divides the  rachis  into short segments, each attached to a single  spikelet  (which contains 2–3 grains along with  chaff ).


A different class of shattering mechanisms involves  dehiscence  of the mature fruit, which releases the seeds.


Rapeseed  and the shattering types of  sesame  are harvested before the seed is fully mature, so that the pods do not split and drop the seeds. [ 3 ] [ 4 ] [ 5 ]  Harvesting shattering types of sesame involves wrapping the cut plants before processing. [ 5 ]


Current research priorities to understand the genetics of shattering include the following crops:


The  Sherman paradox  was a term used to describe the anomalous pattern of  inheritance  found in  fragile X syndrome . [ 1 ]  The phenomenon is also referred to as  anticipation  or  dynamic mutation .


The paradox was named in the late 1980s after American geneticist Stephanie Sherman, who studied the inheritance patterns of people with fragile X syndrome. Sherman observed that the effects of fragile X syndrome seemed to occur more frequently with each passing  generation . This observation became known as the Sherman paradox. [ 2 ]


The paradox was ultimately explained by insights into the mutation process that gives rise to the syndrome. Sherman theorized that the  gene  responsible for fragile X syndrome becomes mutated through a two-step process. The first mutation, called the 'premutation', doesn't cause any clinical symptoms. A second mutation was required to convert the 'premutation' into a 'full mutation' capable of causing the clinical symptoms associated with fragile X syndrome. Additionally, premutations must pass through females in order to transform into the full mutation. [ 2 ]


Fragile X syndrome is so named because of the appearance of the  X chromosome  in individuals with fragile X. Under an  electron microscope , a region on the long arm of the chromosome resembles a thin string. Investigation showed that this region consists of a  CGG repeat triplet  in both normal and diseased individuals. [ 1 ]  The difference between normal and diseased is the length of the repeat; the repeat is longer where fragile X syndrome is present. When the length of the repeat surpasses a critical threshold, symptoms of the disorder appear and they increase in likelihood and severity with further length. Even below this threshold there is a range where the repeat becomes unstable during  meiosis .


In normal individuals, an insertion of extra CGGs is unlikely. However, as the length of the repeat increases, the  probability  of additional triplet insertions increases. When the expansion reaches the danger range, the carrier is still unaffected, but the risk of further mutation becomes significant. This is called the premutation range. Once the fragile X syndrome emerges, symptoms worsen from generation to generation because of the self-promoting aspect of the mutation. [ 2 ]


A similar mechanism, involving triplet repeats, underlies  myotonic dystrophy ,  spinocerebellar ataxia  and  Huntington's disease . [ 2 ]  In Huntington's disease, in contrast to fragile X, somatic as well as  germline mutations  occur.  Autopsies  of affected individuals reveal an accumulation of long repeats of CAG in DNA in the  striatum . In both conditions, effects are less severe if the long repeat is interspersed with other triplets.


Silent mutations , also called synonymous or samesense mutations, are  mutations  in  DNA  that do not have an observable effect on the organism's phenotype. The phrase  silent mutation  is often used interchangeably with the phrase  synonymous mutation ; however, synonymous mutations are not always silent, nor vice versa. [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ]  Synonymous mutations can affect  transcription ,  splicing ,  mRNA  transport, and  translation , any of which could alter phenotype, rendering the synonymous mutation non-silent. [ 3 ]  The substrate specificity of the  tRNA  to the rare  codon  can affect the timing of translation, and in turn the co-translational folding of the protein. [ 1 ]  This is reflected in the  codon usage bias  that is observed in many species. Mutations that cause the altered codon to produce an  amino acid  with similar functionality ( e.g.  a mutation producing  leucine  instead of  isoleucine ) are often classified as silent; if the properties of the amino acid are conserved, this mutation does not usually significantly affect protein function. [ 6 ]


The genetic code translates mRNA nucleotide sequences to amino acid sequences. Genetic information is coded using this process with groups of three nucleotides along the mRNA which are commonly known as codons. [ 7 ]  The set of three nucleotides almost always produce the same amino acid with a few exceptions like UGA which typically serves as the  stop codon  but can also encode  tryptophan  in mammalian  mitochondria . [ 7 ]  Most amino acids are specified by multiple codons demonstrating that the  genetic code  is  degenerate –different codons result in the same amino acid. [ 7 ]  Codons that code for the same amino acid are termed synonyms. Silent mutations are base substitutions that result in no change of the amino acid or amino acid functionality when the altered  messenger RNA  (mRNA) is translated. For example, if the codon AAA is altered to become AAG, the same amino acid –  lysine  – will be incorporated into the  peptide  chain.


Mutations are often linked to diseases or negative impacts but silent mutations can be extremely beneficial in creating genetic diversity among species in a population.  Germ-line mutations  are passed from the parent to the offspring. [ 8 ] [ additional citation(s) needed ]  Scientists have predicted that people have approximately 5 to 10 deadly mutations in their genomes but this is essentially harmless because there is usually only one copy of a particular bad gene so diseases are unlikely. [ 8 ]  Silent mutations can also be produced by  insertions  or  deletions , which cause a shift in the  reading frame . [ 9 ]


Because silent mutations do not alter protein function they are often treated as though they are  evolutionarily neutral . Many organisms are known to exhibit  codon usage biases , suggesting that there is  selection  for the use of particular codons due to the need for translational stability.  Transfer RNA  (tRNA) availability is one of the reasons that silent mutations might not be as silent as conventionally believed. [ 10 ]


There is a different tRNA molecule for each codon. For example, there is a specific tRNA molecule for the codon UCU and another specific for the codon UCC, both of which code for the amino acid  serine . In this instance, if there was a thousand times less UCC tRNA than UCU tRNA, then the incorporation of  serine  into a polypeptide chain would happen a thousand times more slowly when a mutation causes the codon to change from UCU to UCC. If amino acid transport to the  ribosome  is delayed,  translation  will be carried out at a much slower rate. This can result in lower expression of a particular gene containing that silent mutation if the mutation occurs within an exon. Additionally, if the ribosome has to wait too long to receive the amino acid, the ribosome could terminate translation prematurely. [ 6 ]


A nonsynonymous mutation that occurs at the genomic or transcriptional levels is one that results in an alteration to the amino acid sequence in the protein product.  A protein's  primary structure  refers to its amino acid sequence.  A substitution of one amino acid for another can impair protein function and tertiary structure, however its effects may be minimal or tolerated depending on how closely the properties of the amino acids involved in the swap correlate. [ 11 ]   The premature insertion of a  stop codon , a  nonsense mutation , can alter the primary structure of a protein. [ 12 ]   In this case, a truncated protein is produced.  Protein function and folding is dependent on the position in which the stop codon was inserted and the amount and composition of the sequence lost.


Conversely, silent mutations are mutations in which the amino acid sequence is not altered. [ 12 ]  Silent mutations lead to a change of one of the letters in the triplet code that represents a  codon , but despite the single base change, the amino acid that is coded for remains unchanged or similar in biochemical properties.  This is permitted by the  degeneracy of the genetic code .


Historically, silent mutations were thought to be of little to no significance.  However, recent research suggests that such alterations to the triplet code do affect protein translation efficiency and protein folding and function. [ 13 ] [ 14 ]


Furthermore, a change in primary structure is critical because the fully folded tertiary structure of a protein is dependent upon the primary structure.  The discovery was made throughout a series of experiments in the 1960s that discovered that reduced and denatured RNase in its unfolded form could refold into the native tertiary form.  The tertiary structure of a protein is a fully folded polypeptide chain with all hydrophobic R-groups folded into the interior of the protein to maximize entropy with interactions between secondary structures such as beta sheets and alpha helixes.  Since the structure of proteins determines its function, it is critical that a protein be folded correctly into its tertiary form so that the protein will function properly.  However, it is important to note that polypeptide chains may differ vastly in primary structure, but be very similar in tertiary structure and protein function. [ 15 ]


Silent mutations alter the  secondary structure  of  mRNA .


Secondary structure of proteins consists of interactions between the atoms of the backbone of a polypeptide chain, excluding the R-groups.  One common type of secondary structures is the alpha helix, which is a right-handed helix that results from hydrogen bonds between the  nth  amino acid residue and the  n+4th  amino acid residue.  The other common type of secondary structure is the beta sheet, which displays a right-handed twist, can be parallel or anti-parallel depending on the direction of the direction of the bonded polypeptides, and consists of hydrogen bonds between the carbonyl and amino groups of the backbone of two polypeptide chains. [ 16 ]


mRNA  has a secondary structure that is not necessarily linear like that of DNA, thus the shape that accompanies complementary bonding in the structure can have significant effects. For example, if the mRNA molecule is relatively unstable, then it can be rapidly degraded by enzymes in the  cytoplasm . If the RNA molecule is highly stable, and the complementary bonds are strong and resistant to unpacking prior to translation, then the gene may be under expressed.  Codon usage influences mRNA stability. [ 10 ]


Furthermore, since all organisms contain a slightly different genetic code, their mRNA structures differ slightly as well, however, multiple studies have been conducted that show that all properly folded mRNA structures are dependent on the primary sequence of the polypeptide chain and that the structure is maintained by dinucleotide relative abundances in the cell matrix.  It has also been discovered that mRNA secondary structure is important for cell processes such as transcript stability and translation. The general idea is that the functional domains of mRNA fold upon each other, while the start and stop codon regions generally are more relaxed, which could aid in the signaling of initiation and termination in translation. [ 17 ]


If the oncoming ribosome pauses because of a knot in the RNA, then the polypeptide could potentially have enough time to fold into a non-native structure before the  tRNA  molecule can add another  amino acid .  Silent mutations may also affect  splicing , or  transcriptional control .


Silent mutations affect protein folding and function. [ 1 ]   Normally a misfolded protein can be refolded with the help of molecular chaperones. RNA typically produces two common misfolded proteins by tending to fold together and become stuck in different conformations and it has a difficulty singling in on the favored specific tertiary structure because of other competing structures.  RNA-binding proteins can assist RNA folding problems, however, when a silent mutation occurs in the mRNA chain, these chaperones do not bind properly to the molecule and are unable to redirect the mRNA into the correct fold. [ 18 ]


Recent research suggests that silent mutations can have an effect on subsequent protein structure and activity. [ 19 ] [ 20 ]  The timing and rate of protein folding can be altered, which can lead to functional impairments. [ 21 ]


Silent mutations have been employed as an experimental strategy and can have clinical implications.


Steffen Mueller  at the Stony Brook University designed a  live vaccine  for  polio  in which the virus was engineered to have synonymous codons replace naturally occurring ones in the genome. As a result, the virus was still able to infect and reproduce, albeit more slowly. Mice that were vaccinated with this vaccine and exhibited resistance against the natural polio strain.


In molecular  cloning  experiments, it can be useful to introduce silent mutations into a gene of interest in order to create or remove recognition sites for  restriction enzymes .


Mental disorders can be caused by silent mutations. One silent mutation causes the  dopamine receptor D2  gene to be less stable and degrade faster, underexpressing the gene.


A silent mutation in the multidrug resistance gene 1 ( MDR1 ), which codes for a cellular membrane pump that expels drugs from the cell, can slow down translation in a specific location to allow the peptide chain to bend into an unusual conformation. Thus, the mutant pump is less functional.


Deviations from average pain sensitivity are caused by both an ATG to GTG mutation ( nonsynonymous ), and a CAT to CAC mutation ( synonymous ). These two mutations are both shared by the low pain sensitivity and high pain sensitivity gene. Low pain sensitivity has an additional CTC to CTG silent mutation, while high pain sensitivity does not and shares the CTC sequence at this location with average pain sensitivity. [ 22 ]


Around 99.8% of genes that undergo mutations are deemed silent because the nucleotide change does not change the amino acid being translated. [ 23 ]  Although silent mutations are not supposed to have an effect on the phenotypic outcome, some mutations prove otherwise like the Multi-Drug Resistance Gene 1. MDR1 codes for the P-glycoprotein which helps get rid of drugs in the body. It is located in the intestines, liver, pancreas, and brain. MDR 1 is located in the same places that CYP3A4 is located in, which is an enzyme that helps get rid of toxins or drugs from the liver and intestines. Silent mutations like MDR 1 do express a change in phenotypic response. A study done on mice showed when they did not have enough of the MDR 1 gene, their body did not recognize the ivermectin or cyclosporine drug, leading to the creation of toxins in their bodies. [ 23 ]


MRD1 has over fifty single nucleotide polymorphisms (SNP's) which are changes in the nucleotide base sequence. [ 24 ] [ 23 ]  In MDR1 the gene exon 26 which represents 3535C can mutate to 3535T which then changes the transfer RNA into one that is not often as seen, leading to changes in the outcome during translation. This is an example of how some silent mutations are not always silent. [ 25 ]  The multi-drug resistance genes at Exon 26 C3435T, exon 21 G2677T/A, and exon 12 C1236T have been studied to have SNP's that occur at the same time, therefore making the phenotypic "function "change. This suggests a haplotype dependency between exon 26 and other exon that have polymorphisms. For example, efavirenz and nelfinavir are two types of drugs that help decrease the HIV infection in a person's body. When the SNP from exon 26 is coupled with other SNP exons, the drugs have a lower chance of maintaining the HIV infection. Although, when the TT nucleotides in exon 26 are expressed the patient has a lower concentration of the virus but when the genotype morphs into CC or CT the infection is able to spread like normal leaving the MDR 1 gene almost defenseless. These changes in bases of exon 26 for MDR 1 show a correlation between the MDR 1 gene mutations and the ability of the antiretroviral drugs to suppress the HIV infection. [ 23 ]


Exon 26 has also been studied as to whether it is haplotype dependent or not. The presence of the SNP of exon 26 changes phenotypic functions when it is paired with the presence of mutations from exons 12 and 21. But when acting alone, it does not affect the phenotypic outcome as strongly. An example of exon 26’s haplotype dependency is seen when looking at chemotherapy. Since MDR 1 removes drugs from our cells, inhibitors have been used to block MRD 1's ability to remove drugs, thus letting beneficial drugs like chemotherapy and immunosuppressants aid the body in recovery more efficiently. MDR1 has different proteins that help exile these specific drugs from cancer cells. [ 26 ]  Verapamil and cyclosporine A are common inhibitors for MDR 1. [ 23 ]  Unfortunately, when C3435T is mutated with a mutation from either exon 12 or exon 21 (or if all three mutations occur at the same time creating a haplotype), the inhibitors are less likely to weaken the function of MDR1. Multiple silent mutated genes tend to be more resistant against these inhibitors. [ 26 ]


Looking at the molecular level, the reason why C3435T in exon 26 of MDR 1 gene is not silent is because of the pace at which the amino acids are being translated to proteins. [ 25 ]  mRNA’s secondary structures can fold which means different codons correspond to different folding's of the mRNA. For example, when exon 26 changes ATC to ATT both codons produce the same amino acid but ATC is seen more often than the mutation codon. As a consequence, the amount of time it takes for the ribosome to produce its protein confirmation is changed. This leads to a protein structure different from the usual shape of the protein which leads to different functions of the protein. [ 27 ]


Other reasons behind MDR1’s “silent mutation” occurs in messenger RNA. In mRNA, codons also work as exon splicing enhancers. Codons decide when to cut out introns based on the codon it is reading in mRNA. [ 24 ]  The mutated codons have a higher risk of making a mistake when splicing introns out of the mRNA sequence leading to the wrong exons being produced. Therefore, making a change to the mature messenger RNA. [ 27 ]  Mutations in the Multi-Drug Resistance Gene 1 show how silent mutations can have an effect on the outcome of the phenotype.


Slipped strand mispairing  ( SSM , also known as  replication slippage ) is a mutation process which occurs during  DNA replication . It involves  denaturation  and displacement of the  DNA  strands, resulting in mispairing of the complementary bases. Slipped strand mispairing is one explanation for the origin and evolution of  repetitive DNA sequences . [ 1 ]


It is a form of  mutation  that leads to either a trinucleotide or dinucleotide expansion, or sometimes contraction, during  DNA replication . [ 2 ]   A slippage event normally occurs when a sequence of repetitive nucleotides ( tandem repeats ) are found at the site of replication. Tandem repeats are unstable regions of the genome where frequent insertions and deletions of nucleotides can take place, resulting in genome rearrangements. [ 3 ]   DNA polymerase , the main enzyme to catalyze the polymerization of free  deoxyribonucleotides  into a newly forming DNA strand, plays a significant role in the occurrence of this mutation. When DNA polymerase encounters a direct repeat, it can undergo a replication slippage. [ 4 ]


Strand slippage may also occur during the DNA synthesis step of  DNA repair  processes.  Within DNA trinucleotide repeat sequences, the repair of  DNA damage  by the processes of  homologous recombination ,  non-homologous end joining ,  DNA mismatch repair  or  base excision repair  may involve strand slippage mispairing leading to  trinucleotide repeat expansion  when the repair is completed. [ 5 ]


Slipped strand mispairing has also been shown to function as a  phase variation  mechanism in certain bacteria. [ 6 ]


Slippage occurs through five main stages:


Tandem repeats (the main influence for slippage replication) can be found in coding and non-coding regions. If these repeats are found in coding regions then the variations to the polynucleotide sequence can result in the formation of abnormal proteins in eukaryotes. Many human diseases have been reported to be associated with trinucleotide repeat expansions including  Huntington's disease . [ 7 ]  The HD gene [ 8 ]  is found in all human genomes. In the event that a slippage event occurs there can be a large expansion in the tandem repeats of the HD gene. [ 8 ]  An individual who is not affected by Huntington's disease will have 6-35 tandem repeats at the HD locus. However, an affected individual will have 36- 121 repeats present. [ 7 ]  The expansion of the HD locus results in a dysfunctional protein leading to Huntington's disease.


Huntington disease is normally progressive and results in movement, cognitive and psychiatric disorders. These disorders can lead to a severe impact on an individual's daily activities, making it hard for proper communication and independent actions to take place. [ 9 ]  Replication slippage can also lead to other neurodegenerative diseases in humans. These include  spinal and bulbar muscular atrophy  ( trinucleotide  expansion in the AR gene),   dentatorubral–pallidoluysian atrophy  ( trinucleotide expansion in the DRPLA gene),   spinocerebellar ataxia type 1  ( trinucleotide expansion in the SCA1gene),  Machado-Joseph disease  ( trinucleotide expansion in the SCA3 gene),  myotonic dystrophy  ( trinucleotide expansion in the DMPK gene), and  Friedreich's ataxia  ( a trinuncleotide expansion in the X25 gene). [ 7 ]  Therefore, replication slippage leads to a form of trinucleotide expansion which results in serious changes to protein structure.


SSM events can result in either insertions or deletions. Insertions are thought to be self-accelerating: as repeats grow longer, the probability of subsequent mispairing events increases. Insertions can expand simple tandem repeats by one or more units. In long repeats, expansions may involve two or more units. For example, insertion of a single repeat unit in GAGAGA expands the sequence to GAGAGAGA, while insertion of two repeat units in [GA] 6  would produce [GA] 8 . Genomic regions with a high proportion of repeated DNA sequences ( tandem repeats ,  microsatellites ) are prone to strand slippage during  DNA replication  and  DNA repair .


Trinucleotide repeat expansion  is a cause of a number of human diseases including  fragile X syndrome ,  Huntington's disease , several  spinocerebellar ataxias ,  myotonic dystrophy  and  Friedrich ataxia . [ 5 ]


The combination of SSM events with  point mutation  is thought to account for the evolution of more complex repeat units. Mutations followed by expansion would result in the formation of new types of adjacent  short tandem repeat  units. For example, a  transversion  could change the simple two- base repeat [GA] 10  to [GA] 4 GATA[GA] 2 . This could then be expanded to[GA] 4 [GATA] 3 [GA] 2  by two subsequent SSM events. Simple  repetitive DNA sequences  containing a variety of adjacent short tandem repeats are commonly observed in non-protein coding regions of  eukaryotic   genomes .


A  somatic mutation  is a change in the  DNA sequence  of a  somatic cell  of a  multicellular organism  with dedicated  reproductive cells ; that is, any  mutation  that occurs in a cell other than a  gamete ,  germ cell , or  gametocyte . Unlike  germline mutations , which can be passed on to the descendants of an organism, somatic mutations are not usually transmitted to descendants. This distinction is blurred in plants, which lack a dedicated  germline , and in those animals that can  reproduce asexually  through mechanisms such as  budding , as in members of the cnidarian  genus  Hydra .


While somatic mutations are not passed down to an organism's offspring, somatic mutations will be present in all descendants of a cell within the same organism. Many  cancers  are the result of accumulated somatic mutations.


The term somatic generally refers to the cells of the body, in contrast to the reproductive ( germline ) cells, which give rise to the  egg  or  sperm .  For example, in  mammals , somatic cells make up the internal organs, skin, bones, blood, and connective tissue. [ 1 ]


In most animals, separation of germ cells from somatic cells ( germline development ) occurs during early stages of  development . Once this segregation has occurred in the embryo, any mutation outside of the germline cells can not be passed down to an organism's offspring.


However, somatic mutations are passed down to all the progeny of a mutated cell within the same organism. A major section of an organism therefore might carry the same mutation, especially if that mutation occurs at earlier stages of development. [ 2 ]  Somatic mutations that occur later in an organism's life can be hard to detect, as they may affect only a single cell—for instance, a post- mitotic  neuron; [ 3 ] [ 4 ]  improvements in  single cell sequencing  are therefore an important tool for the study of somatic mutation. [ 5 ]  Both the  nuclear DNA  and  mitochondrial DNA  of a cell can accumulate mutations; somatic mitochondrial mutations have been implicated in development of some neurodegenerative diseases. [ 6 ]


There are many exceptions to the rule that somatic mutations cannot be inherited by offspring. Many organisms simply do not dedicate a separate germline during early development. Plants and  basal animals  such as  sponges  and  corals  instead  generate gametes from  pluripotent  stem cells in adult somatic tissues. [ 7 ] [ 8 ]  In flowering plants, for example, germ cells can arise from adult somatic cells in the floral  meristem . Other animals without a designated germ line include  tunicates  and  flatworms . [ 9 ]


Somatic mutations can also be passed down to offspring in organisms that can  reproduce asexually , without production of gametes. For instance, animals in the  cnidarian  genus  Hydra  can reproduce asexually through the mechanism of  budding  (they can also reproduce sexually). In  hydra , a new bud develops directly from somatic cells of the parent hydra. [ 10 ]  A mutation present in the tissue that gives rise to the daughter organism would be passed down to that offspring.


Many plants naturally reproduce through  vegetative reproduction —growth of a new plant from a fragment of the parent plant—propagating somatic mutations without the step of seed production. Humans artificially induce vegetative reproduction via  grafting  and stem cuttings.


As with germline mutations, mutations in somatic cells may arise due to endogenous factors, including errors during  DNA replication  and repair, and exposure to  reactive oxygen species  produced by normal cellular processes. Mutations can also be induced by contact with  mutagens , which can increase the rate of mutation.


Most mutagens act by causing DNA damage—alterations in DNA structure such as  pyrimidine dimers , or breakage of one or both DNA strands.  DNA repair  processes can remove DNA damages that would, otherwise, upon DNA replication, cause mutation. Mutation results from damage when mistakes in the mechanism of  DNA repair  cause changes in the nucleotide sequence, or if replication occurs before repair is complete.


Mutagens can be physical, such as radiation from  UV rays  and  X-rays , or chemical—molecules that interact directly with DNA—such as  metabolites  of  benzo[ a ]pyrene , a potent  carcinogen  found in  tobacco smoke . [ 11 ]  Mutagens associated with cancers are often studied to learn about cancer and its prevention.


Research suggests that the  frequency of mutations  is generally higher in somatic cells than in cells of the germline; [ 12 ]  furthermore, there are differences in the types of mutation seen in the germ and in the soma. [ 13 ]  There is variation in mutation frequency between different somatic tissues within the same organism [ 13 ]  and between species. [ 2 ]


Milholland et al. (2017) examined the mutation rate of  dermal fibroblasts  (a type of somatic cell) and germline cells in humans and in mice. They measured the rate of  single nucleotide variants  (SNVs), most of which are a consequence of replication error. Both in terms of mutational load (total mutations present in a cell) and mutation rate per  cell division  (new mutations with each  mitosis ), somatic mutation rates were more than ten times that of the germline, in humans and in mice.


In humans, mutation load in fibroblasts was over twenty times greater than germline (2.8 × 10 −7  compared with 1.2 × 10 −8  mutations per base pair). Adjusted for differences in the estimated number of cell divisions, the fibroblast mutation rate was about 80 times greater than the germ (respectively, 2.66 × 10 −9   vs. 3.3 × 10 −11  mutations per base pair per mitosis). [ 2 ]


The disparity in mutation rate between the germline and somatic tissues likely reflects the greater importance of genetic integrity in the germline than in the soma. [ 12 ]  Variation in mutation frequency may be due to differences in rates of DNA damage or to differences in the DNA repair process as a result of elevated levels of DNA repair enzymes. [ 13 ]


In April 2022 it has been reported that most mammals have about the same number of mutations by the time they reach the end of their lifespan, so those that have similar lifespan will have similar somatic  mutation rates  and those who live less/more will have a higher/lower rate of somatic mutations respectively. [ 14 ] [ 15 ]


Post-mitotic  neurons  accumulate somatic mutations at a constant rate throughout life, and this rate is roughly similar to the mutation rates of  mitotically  active tissues. [ 16 ]   The mutations in neurons may arise as a consequence of  endogenous DNA damage  and the somewhat inaccurate  repair  of such damage that occurs all the time in cells. [ 16 ]


As a part of the  adaptive immune response , antibody-producing  B cells  experience a mutation rate many times higher than the normal rate of mutation. The mutation rate in  antigen-binding coding sequences of the immunoglobulin genes is up to 1,000,000 times higher than in cell lines outside the lymphoid system. A major step in  affinity maturation , somatic hypermutation helps B cells produce antibodies with greater  antigen  affinity. [ 17 ]


Somatic mutations accumulate within an organism's cells as it ages and with each round of cell division; the role of somatic mutations in the development of cancer is well established, and the accumulation of somatic mutations is implicated in the biology of aging. [ 4 ]


Mutations in neuronal  stem cells  (especially during  neurogenesis ) [ 18 ]  and in post-mitotic  neurons  lead to genomic heterogeneity of neurons—referred to as "somatic brain mosaicism". [ 3 ]  The accumulation of age-related mutations in neurons may be linked to  neurodegenerative diseases , including  Alzheimer's disease , but the association is unproven. The majority of central-nervous system cells in the adult are post-mitotic, and adult mutations might affect only a single neuron. Unlike in cancer, where mutations result in clonal proliferation, detrimental somatic mutations might contribute to neurodegenerative disease by cell death. [ 19 ]  Accurate assessment of somatic mutation burden in neurons therefore remains difficult to assess.


If a mutation occurs in a cell of an organism, that mutation will be present in all the descendants of this cell within the same organism. The accumulation of certain mutations over generations of somatic cells is part of the process of  malignant transformation , from normal cell to cancer cell.


Cells with heterozygous loss-of-function mutations (one good copy of a gene and one mutated copy) may function normally with the unmutated copy until the good copy has been spontaneously somatically mutated. This kind of mutation happens often in living organisms, but it is difficult to measure the rate. Measuring this rate is important in predicting the rate at which people may develop cancer.


The  somatic mutation and recombination tests (SMARTs)  are  in vivo   genotoxicity   tests  performed in  Drosophila melanogaster  (Fruit fly). [ 1 ]  These fruit fly tests are a short-term test and a non-mammalian approach for  in vivo  testing of putative  genotoxins  found in the environment. D. melanogaster has a short lifespan, which allows for fast reproductive cycles and high-throughput genotoxicity testing. D. melanogaster also has around 75% functional orthologs of human disease-related genes, making it an attractive in vivo model for human research. [ 2 ]  The tests identify loss of  heterozygosity  for the specified  genetic markers  in heterozygous or trans-heterozygous adults using phenotypically observable genetic markers in adult tissues. [ 3 ] [ 4 ]  Although diverse events like  point mutations / deletions ,  nondisjunction , and  homologous mitotic recombination  might theoretically cause this loss of heterozygosity, nondisjunction processes are generally not relevant for most of the examined chemicals. [ 5 ] [ 6 ]  SMARTs are two different tests that use the same genetic foundation, but target different adult tissues and are named accordingly: the wing-spot test [ 5 ]  and the eye-spot test. [ 6 ]


In the developmental phase, larval structures and  imaginal discs  - clusters of diploid cells of undifferentiated epithelium- are formed in the embryo. [ 7 ]  The pupa emerges following the completion of the larval stages, and  metamorphosis  occurs as a result of systemic hormonal regulation, with histolysis of the larval organs and differentiation of the imaginal discs into adult components. [ 7 ] [ 8 ]  When these imaginal discs are exposed to genotoxic substances genetic mutations occur due to possible  DNA damage  that can be inherited by the progeny cells during  mitosis . The phenotypic forms of these genetic mutations can be observed in adult body forms, like the wings and the eyes, and thus can be examined using the wing-spot test and the eye-spot test, respectively. The loss of heterozygosity (LOH) for specific genetic markers in heterozygous individuals enables for visual scoring of DNA damage/genotoxicity in adult tissues [ 9 ]


The wing-spot test in D. melanogaster was first described by Graf and Würgler. [ 3 ]  The wing-spot test determines for the induction of  mutant  spots that represent the loss of heterozygozity due to point mutation, deletion, nondisjunction, or mitotic recombination using the recessive genetic markers multiple wing hair (mwh) and flare-3 (flr3), located on chromosome number 3.


The eye w/w+ SMART assay uses the  X-chromosome  white (w) gene as a recessive marker to monitor the presence of white clones in wild-type eyes, which indicate the occurrence of loss of heterozygosity at the white locus due to point mutations and/or deletions, as well as nondisjunction and  homologous  mitotic recombination in w/w+ somatic cells of Drosophila in vivo [ 6 ]


These tests are particularly effective instruments for analyzing (in vivo) the potential genotoxicity of chemicals in the somatic cells of a higher  eukaryotic  organism as they detect primarily the production of  gene mutation  and homologous recombination. It's worth noting that measuring mitotic recombination in somatic cells is important for genotoxicity screening since abnormal recombination activity is frequently linked to  cancer . [ 10 ]  Indeed, these tests have been used to determine the genotoxicity of a variety of agents, including radiation, [ 11 ] [ 12 ] [ 13 ]  metals, [ 14 ] [ 15 ] [ 16 ] [ 17 ]  chemicals, [ 18 ]  plant extracts/products, [ 19 ] [ 20 ]  therapeutic drugs, [ 21 ] [ 22 ]  food products [ 13 ] [ 23 ]  and various types of pollutants [ 24 ] [ 25 ]  and  nanoparticles .They've also been employed to look for potential  antimutagens .


A  splice site mutation  is a  genetic   mutation  that  inserts ,  deletes  or changes a number of nucleotides in the specific site at which  splicing  takes place during the processing of  precursor messenger RNA  into  mature messenger RNA . Splice site consensus sequences that drive  exon  recognition are located at the very termini of  introns . [ 1 ]  The deletion of the splicing site results in one or more introns remaining in  mature mRNA  and may lead to the production of abnormal  proteins . When a splice site mutation occurs, the mRNA transcript possesses information from these introns that normally should not be included. Introns are supposed to be removed, while the exons are expressed.


The mutation must occur at the specific site at which intron splicing occurs: within non-coding sites in a gene, directly next to the location of the exon. The mutation can be an insertion, deletion,  frameshift , etc. The splicing process itself is controlled by the given sequences, known as splice-donor and splice-acceptor sequences, which surround each exon. Mutations in these sequences may lead to retention of large segments of intronic DNA by the mRNA, or to entire exons being spliced out of the mRNA. These changes could result in production of a nonfunctional protein. [ 2 ]  An intron is separated from its exon by means of the splice site. Acceptor-site and donor-site relating to the splice sites signal to the  spliceosome  where the actual cut should be made. These donor sites, or recognition sites, are essential in the processing of mRNA. The average vertebrate gene consists of multiple small exons (average size, 137 nucleotides) separated by introns that are considerably larger. [ 1 ]


In 1993,  Richard J. Roberts  and  Phillip Allen Sharp  received the  Nobel Prize in Physiology or Medicine  for their discovery of " split genes ". [ 4 ]  Using the model  adenovirus  in their research, they were able to discover splicing—the fact that pre-mRNA is processed into mRNA once introns were removed from the RNA segment. These two scientists discovered the existence of splice sites, thereby changing the face of genomics research. They also discovered that the splicing of the messenger RNA can occur in different ways, opening up the possibility for a mutation to occur.


Today, many different types of technologies exist in which splice sites can be located and analyzed for more information.  The  Human Splicing Finder  is an  online database  stemming from the  Human Genome Project  data. The genome database identifies thousands of mutations related to medical and health fields, as well as providing critical research information regarding splice site mutations. The tool specifically searches for pre-mRNA splicing errors, the calculation of potential splice sites using complex algorithms, and correlation with several other online genomic databases, such as the  Ensembl  genome browser. [ 5 ]


Due to the sensitive location of splice sites, mutations in the acceptor or donor areas of splice sites can become detrimental to a human individual. In fact, many different types of diseases stem from anomalies within the splice sites.


A study researching the role of splice site mutations in cancer supported that a splice site mutation was common in a set of women who were positive for breast and ovarian cancer. These women had the same mutation, according to the findings. An intronic single base-pair substitution destroys an acceptor site, thus activating a cryptic splice site, leading to a 59 base-pair insertion and chain termination. The four families with both breast and ovarian cancer had chain termination mutations in the N-terminal half of the protein. [ 6 ]  The mutation in this research example was located within the splice-site.


Splice-site mutations are recurrently found in key lymphoma genes [ 7 ]  like  BCL7A [ 8 ]  or  CD79B [ 7 ]  due to aberrant somatic hypermutation as the sequence targeted by  AID  overlaps with the sequences of the splice-sites. [ 9 ]


According to a research study conducted Hutton, M et al, a missense mutation occurring on the 5' region of the RNA associated with the tau protein was found to be correlated with inherited dementia (known as FTDP-17). The splice-site mutations all destabilize a potential stem–loop structure which is most likely involved in regulating the alternative splicing of exon10 in chromosome 17. Consequently, more usage occurs on the 5' splice site and an increased proportion of tau transcripts that include exon 10 are created. Such drastic increase in mRNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17, a type inherited dementia. [ 10 ]


Some types of  epilepsy  may be brought on due to a splice site mutation.
In addition to a mutation in a  stop codon ,  a splice site mutation on the 3' strand was found in a gene coding for  cystatin B  in Progressive Myoclonus Epilepsy [ 11 ]  patients. This combination of mutations was not found in unaffected individuals. By comparing sequences with and without the splice site mutation, investigators were able to determine that a G-to-C nucleotide  transversion  occurs at the last position of the first intron. This transversion occurs in the region that codes for the cystatin B gene. Individuals suffering from Progressive Myoclonus Epilepsy possess a mutated form of this gene, which results in decreased output of mature mRNA, and subsequently decreases in protein expression.


A study has also shown that a type of Childhood Absence Epilepsy (CAE) causing  febrile seizures  may be linked to a splice site mutation in the sixth intron of the  GABRG2 gene .  This splice site mutation was found to cause a nonfunctional GABRG2 subunit in affected individuals. [ 12 ]  According to this study, a  point mutation  was the culprit for the splice-donor site mutation, which occurred in intron 6. A nonfunctional protein product is produced, leading to the also nonfunctional subunit.


Several  genetic diseases  may be the result of splice site mutations. For example, mutations that cause the incorrect splicing of  β-globin  mRNA are responsible of some cases of  β-thalassemia . Another Example is  TTP  (thrombotic thrombocytopenic purpura). TTP is caused by deficiency of  ADAMTS-13 . A splice site mutation of ADAMTS-13 gene can therefore cause TTP. It is estimated that 15% of all point mutations causing human genetic diseases occur within a splice site. [ 13 ]


When a splice site mutation occurs in intron 2 of the gene that produces the  parathyroid hormone , a parathyroid deficiency can prevail. In one particular study, a G to C substitution in the splice site of intron 2 produces a skipping effect in the messenger RNA transcript. The exon that is skipped possesses the initiation start codon to produce parathyroid hormone. [ 14 ]  Such failure in initiation causes the deficiency.


Using the model organism  Drosophila melanogaster , data has been compiled regarding the genomic information and sequencing of this organism. A prediction model exists in which a researcher can upload his or her genomic information and use a splice site prediction database to gather information about where the splice sites could be located.  The Berkeley Drosophila Project  can be used to incorporate this research, as well as annotate high quality euchromatic data. The splice site predictor can be a great tool for researchers studying human disease in this  model organism .


Splice site mutations can be analyzed using  information theory . [ 15 ]


In  botany , a  sport  or  bud sport , traditionally called  lusus , [ 2 ]  is a part of a plant that shows morphological differences from the rest of the plant. Sports may differ by foliage shape or color,  flowers , fruit, or  branch  structure. The cause is generally thought to be a chance  genetic mutation . [ 3 ]


Sports with desirable characteristics are often propagated  vegetatively  to form new  cultivars  that retain the characteristics of the new morphology. [ 4 ]  Such selections are often prone to "reversion", meaning that part or all of the plant reverts to its original form. An example of a bud sport is the  nectarine , at least some of which developed as a bud sport from  peaches . Other common fruits resulting from a sport mutation are the  red Anjou  pear, the  Ruby Red grapefruit , and the  variegated pink lemon , which is a sport of the  "Eureka" lemon . [ 5 ]


This  plant morphology  article is a  stub . You can help Wikipedia by  expanding it .


Surveyor nuclease assay  is an enzyme mismatch cleavage assay used to detect  single base mismatches  or small insertions or deletions ( indels ).


Surveyor nuclease is part of a family of mismatch-specific  endonucleases  that were discovered in celery (CEL nucleases). [ 1 ]  The enzyme recognizes all base substitutions and insertions/deletions, and cleaves the 3′ side of mismatched sites in both DNA strands with high specificity [ 2 ]


This assay has been used to identify and analyze mutations in a variety of organisms and cell types, as well as to confirm genome modifications following genome editing (using  CRISPR / TALENs / zinc fingers ).


The ability to discover and detect known and unknown mutations is of great importance in biomedical research and genetic diagnosis (see Applications). Therefore, multiple methods have been developed to enable research-based and clinical diagnostic detection of such mutations.


The most direct manner to identify the sequence changes/differences is through reading the DNA sequence with traditional and high throughput DNA sequencing methods (see  Sanger sequencing  and  DNA sequencing ). However, these methods provide large amounts of unnecessary data and are costly to use. [ 3 ]  In addition, traditional sequencing can be useful for detection of germline mutations, but may be less successful in detecting somatic minor alleles at low frequencies ( mosaicism ). Therefore, other non-sequencing based approaches to detect the mutation or polymorphisms are required.


Other widely used methods depend on physical properties of DNA, for example melting temperature-based systems such as  Single-stranded conformational polymorphism  analysis (SSCP) and  Denaturing high-performance liquid chromatography  (DHPLC). These techniques are generally limited to the analysis of short DNA fragments (< 1000 bp) and are only able to indicate the presence of polymorphism(s), but do not easily yield the location of a mutation within a DNA sequence. Therefore, this must be followed with additional techniques in order to pinpoint the mutation or map multiple mutations in the same fragment. [ 3 ]


Enzymatic mismatch cleavage assays exploit the properties of mismatch-specific endonucleases to detect and cleave mismatches. These methods are simple to run using standard laboratory techniques and equipment, and can detect polymorphisms, single base pair mismatches, and insertions and deletions at low frequencies. [ 4 ]  Several such enzymes have been discovered (including CEL I, T4 endonuclease VII,  Endonuclease V , T7 endonuclease I). [ 3 ]


One of the commonly used enzymes is Surveyor nuclease (CEL II), which cleaves the 3′ side of both DNA strands with high specificity at sites of base substitution or insertion/deletion. This enzyme is capable of cleaving at multiple mutations in large DNA fragments, and produces detectable cleavage products from mismatch DNA representing only a small proportion of the DNA in a population, thus making it suitable for use in enzyme mismatch cleavage assays. [ 2 ]


In 1998, Olekowski et al. [ 5 ]  identified a new mismatch specific endonuclease. The enzyme was purified from celery and was given the name CEL I.


CEL I was shown to cut DNA with high specificity on both strands at the 3′ side of base-substitution mismatches, and can therefore be used in enzyme mutation detection methods to identify mutations and polymorphisms. Olekowski and colleagues demonstrated this technique by using this enzyme to detect a variety of mutations and polymorphisms in the human BRCA1 gene. [ 1 ] [ 5 ] [ 6 ]


While monitoring the purification of CEL I using  polyacrylamide gel electrophoresis , Yang et al. [ 1 ]  noticed that there were two nuclease bands that stayed together during all the purification steps. The major nuclease activity was designated CEL I, while the minor activity on SDS-PAGE was named CEL II. They concluded that CEL I and CEL II are similar, and that both are able to cleave a DNA mismatch.


In 2004, Qiu et al. developed a mutation detection technology based on CEL II, also known as Surveyor Nuclease. [ 2 ]  Since then the method has been used to detect mutations and polymorphisms in many different organisms and cell types (see applications).


Surveyor nuclease was licensed from the  Fox Chase Cancer Center  by  Transgenomic, Inc.  and was subsequently sold to  IDT , which currently distributes it.


Initially, the DNA of interest (nuclear or  mitochondrial DNA ) is extracted from tissues or cell culture. This can be done by standard extraction methods such as Proteinase K digestion followed by ethanol precipitation or by other commercially available methods.


If the DNA is predicted to be heterogeneous, e.g. from a pool of differentially modified cells or from heterozygous mutation carriers, there is no need to add control DNA.


The region of interest in both mutant and wild-type reference DNA is amplified by  polymerase chain reaction  (PCR). The PCR reaction should be carried out using a high-fidelity proofreading polymerase to avoid introducing PCR errors that will be interpreted by the nuclease. The PCR reaction should be optimized to create a single, strong PCR band, as non-specific bands will increase the  background noise  of the assay.


If the allele of interest is expected to be present in low frequency, such as in the case of somatic  mosaicism  or heteroplasmic mitochondrial DNA, a modified PCR protocol that enriches variant alleles from a mixture of wild-type and mutation-containing DNA might be considered (e.g.  COLD-PCR ).


The DNA of interest is denatured and annealed in order to form heteroduplexes containing a mismatch at the point of the mutation, which can then be identified by the Surveyor nuclease. If the DNA is predicted to be homogenous (e.g. homoplasmic mitochondrial DNA or identical alleles on both chromosomes of genomic DNA) then DNA from a control sample is needed in order to form a heteroduplex which is then recognizable by the nuclease. If the DNA sample is heterogeneous, no additional control DNA is needed; however, the PCR products should still be denatured and annealed in order to create heteroduplexes.


The annealed DNA is treated with Surveyor nuclease to cleave the heteroduplexes.  All types of mismatches are identifiable by Surveyor nuclease, although the mismatch cutting preferences fall into four groups from most to least preferred: CT, AC, and CC are preferred equally over TT, followed by AA and GG, and finally followed by the least preferred, AG and GT. Sequence context also influences the Surveyor nuclease digestion rate. [ 2 ]


Digested DNA products can be analyzed using conventional gel electrophoresis or high-resolution capillary electrophoresis. The detection of cleaved products indicates the presence of a heteroduplex formed by a mismatch. The location of the mutation/polymorphism can be inferred by observing the fragment length after cleavage. If fluorescent labelled primers are used to mark the 5’ and 3’ end of the PCR products, different colored bands will be observed in the analysis. The size of each band independently confirms the position of the mutation/polymorphism. Multiple mutations can be detected by the presence of several fragments. [ 1 ] [ 5 ]


One of the main advantages of detecting mutations and polymorphisms using mismatch nuclease methods is that no previous knowledge is required regarding the nature of the mutation, as opposed to other methods, such as  restriction fragment length polymorphisms  (RFLP) that has been used for SNP analysis in the past.


In comparison to methods based on melting temperature, mismatch-specific endonuclease methods are not only faster, but can also detect multiple mutations in large DNA fragments.


The method is feasible to use in high-throughput systems using automated injection, making it suitable for screening. [ 7 ]


Surveyor nuclease is a reasonably sensitive enzyme, producing detectable cleavage products from sequences representing only a small proportion of DNA in the population. It can detect a ratio of 1:32 heteroduplex to homoduplex for smaller PCR products (~0.6 kb), and 1:16 heteroduplex to homoduplex for longer PCR products (~2.3 kb). This property makes it possible to  pool  clinical samples in order to increase the heteroduplex formation and hence the sensitivity. [ 3 ]  This is also useful for detection of minor variants in a heterogeneous population (such as heterogeneous tumor). In the case of genome editing by CRISPR or other methods, this property can enhance detection of rare editing events in a population of cells prior to creation and testing of individual edited clones.


Surveyor nuclease cleaves all types of mismatches, even if some are more preferred then others: CT, AC, and CC are preferred equally over TT, followed by AA and GG, and finally followed by the least preferred, AG and GT. It also detects  Indels  up to at least 12 bp. [ 2 ]


Surveyor nuclease assay can also detect multiple mutations in the same fragment. However, this requires several additional processing steps that may also increase the background of the assay (see Limitations).


One of the main limitations of this assay is that it relies on PCR amplification, and is therefore influenced by the quality of the amplified product. PCR artifacts (e.g. primer-dimers or truncated products) can increase the background noise and obscure the signal. Primer-dimers can also inhibit the activity of surveyor nuclease, reducing the signal.


As the PCR method has the potential to introduce its own mutations during the amplification, these errors can also increase background noise. Therefore, it is best to use a high-fidelity polymerase to minimize the amplification errors.


Mitochondrial DNA analysis might be susceptible to contamination by nuclear mitochondrial DNA sequences co-amplified during the PCR reaction, thus confounding the analysis of homoplasmic versus heteroplasmic mitochondrial DNA. [ 8 ]


In order to detect multiple mutations in the same fragment, post-PCR clean-up must be done before Surveyor nuclease digestion. Detection of multiple mismatches can also be improved by increasing time and amount of surveyor nuclease in the reaction, but this also increases the background due to non-specific cleavage.


Surveyor nuclease also has a 5′ exonuclease activity that attacks the ends of double‐stranded DNA increasing background signal during extended incubation. [ 2 ]  This can be reduced by shortening the digestion time and by adding DNA polymerase.


A number of genome editing technologies have emerged in recent years, including  zinc-finger nucleases  (ZFNs), transcription activator-like effector nucleases ( TALENs ) and the RNA-guided  CRISPR/Cas9  nuclease system. These methods promote genome editing by introduction of a double strand DNA break, followed by repair through the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. While HDR is expected to introduce a consistent modification to the genome, NHEJ can introduce a heterogeneous mix of mutations (usually small indels), which will be difficult to identify using Sanger sequencing. Point mutations and indels can be detected by surveyor nuclease assay, making it useful to detect genome editing in a pool of cells without the need for clonal expansion prior to analysis, and it can also provide an estimate of the targeting efficiency achieved. [ 9 ] [ 10 ] [ 11 ]  Even after clonal expansion, detection of mutations using Sanger sequence may be difficult as each allele can undergo a different editing event. In this case, the Surveyor nuclease assay will actually use this effect to create the required heteroduplexes for detection by the mismatch endonuclease.


Surveyor nuclease assay has been used to detect  germline mutations  in human genes. For example, ATRX for X-linked mental retardation, [ 12 ]  and the HBB gene linked to β-thalassemia. [ 7 ]  The assay has also been used to detect mitochondrial and nuclear DNA mutations associated with respiratory chain defects, [ 13 ]  and mutations associated with kidney disease. [ 14 ] [ 15 ]


Surveyor nuclease assay has been used to detect  somatic mutations  in various  cancer-related genes , and as stated above, can be used even when the sample is heterogeneous and the mutant allele only comprises 1%–5% of the total alleles. [ 16 ]  The method has been used to detect mutations in  epidermal-growth-factor-receptor  (EGFR), [ 16 ] [ 17 ] [ 18 ] [ 19 ]   Janus Kinase 2  (JAK2), [ 20 ] [ 21 ]   p53 [ 22 ]  and others.


The method has been used to detect mutations that cause drug resistance in  Mycobacterium tuberculosis . [ 23 ]


A  synonymous substitution  (often called a  silent  substitution  though they are not always silent) is the  evolutionary   substitution  of one  base  for another in an  exon  of a  gene  coding for a  protein , such that the produced  amino acid sequence  is not modified. This is possible because the  genetic code  is " degenerate ", meaning that some amino acids are coded for by more than one three-base-pair  codon ; since some of the codons for a given amino acid differ by just one base pair from others coding for the same amino acid, a mutation that replaces the "normal" base by one of the alternatives will result in incorporation of the same amino acid into the growing  polypeptide chain  when the gene is translated. Synonymous substitutions and mutations affecting  noncoding DNA  are often considered  silent mutations ; however, it is not always the case that the mutation is silent. [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ]


Since there are 22 codes for 64 codons, roughly we should expect a random substitution to be synonymous with probability about 22/64 = 34%. The actual value is around 20%. [ 6 ]


A synonymous mutation can affect  transcription ,  splicing ,  mRNA  transport, and  translation , any of which could alter the resulting  phenotype , rendering the synonymous mutation non-silent. [ 3 ]  The  substrate specificity  of the  tRNA  to the rare codon can affect the timing of translation, and in turn the co-translational  folding of the protein . [ 1 ]  This is reflected in the  codon usage bias  that is observed in many  species .  A  nonsynonymous substitution  results in a change in amino acid that may be arbitrarily further classified as conservative (a change to an amino acid with similar  physiochemical properties ), semi-conservative (e.g.  negatively  to  positively charged  amino acid), or radical (vastly different amino acid).


Protein  translation  involves a set of twenty  amino acids . Each of these amino acids is coded for by a sequence of three  DNA  base pairs called a  codon . Because there are 64 possible codons, but only 20-22  encoded amino acids  (in nature) and a stop signal (i.e. up to three codons that do not code for any amino acid and are known as  stop codons , indicating that translation should stop), some amino acids are coded for by 2, 3, 4, or 6 different codons. For example, the codons TTT and TTC both code for the amino acid  phenylalanine . This is often referred to as  redundancy  of the genetic code . There are two mechanisms for redundancy: several different transfer RNAs can deliver the same amino acid, or one  tRNA  can have a non-standard  wobble base   in position three of the anti-codon, which recognises more than one base in the codon.


In the above phenylalanine example, suppose that the base in position 3 of a TTT codon got substituted to a C, leaving the codon TTC. The amino acid at that position in the protein will remain a phenylalanine. Hence, the substitution is a synonymous one.


When a synonymous or  silent mutation  occurs, the change is often assumed to be  neutral , meaning that it does not affect the fitness of the individual carrying the new gene to survive and reproduce.


Synonymous changes may not be neutral because certain codons are translated more efficiently (faster and/or more accurately) than others.  For example, when a handful of synonymous changes in the fruit fly alcohol dehydrogenase gene were introduced, changing several codons to sub-optimal synonyms, production of the encoded enzyme was reduced [ 7 ]  and the adult flies showed lower ethanol tolerance. [ 8 ]
Many organisms, from bacteria through animals, display biased use of certain synonymous codons.  Such  codon usage bias  may arise for different reasons, some selective, and some neutral. In  Saccharomyces cerevisiae  synonymous codon usage has been shown to influence  mRNA  folding stability, with mRNA encoding different protein  secondary structure  preferring different codons. [ 9 ]


Another reason why synonymous changes are not always neutral is the fact that exon sequences close to exon-intron borders function as  RNA splicing  signals. When the splicing signal is destroyed by a synonymous mutation, the exon does not appear in the final protein. This results in a truncated protein. One study found that about a quarter of synonymous variations affecting exon 12 of the  cystic fibrosis transmembrane conductance regulator  gene result in that exon being skipped. [ 10 ]


Transition , in  genetics  and  molecular biology , refers to a  point mutation  that changes a  purine   nucleotide  to another purine ( A  ↔  G ), or a  pyrimidine  nucleotide to another pyrimidine ( C  ↔  T ). Approximately two out of three  single nucleotide polymorphisms  (SNPs) are transitions. [ 1 ]


Transitions can be caused by  oxidative deamination  and  tautomerization . Although there are twice as many possible  transversions , transitions appear more often in  genomes , [ 2 ]  possibly due to the molecular mechanisms that generate them.  [ 3 ]  Transitions are more likely to be  synonymous substitutions  than transversions, as one observes in the  codon table .


5-Methylcytosine  is more prone to transition than unmethylated  cytosine , due to spontaneous  deamination . This mechanism is important because it dictates the rarity of  CpG islands .


Transversion , in  molecular biology , refers to a  point mutation  in  DNA  in which a single (two ring)  purine  ( A  or  G ) is changed for a (one ring)  pyrimidine  ( T  or  C ), or vice versa. [ 1 ]  A transversion can be spontaneous, or it can be caused by  ionizing radiation  or  alkylating agents . It can only be reversed by a spontaneous  reversion .


Although there are two possible transversions but only one possible  transition  per base, transition mutations are more likely than transversions because substituting a single ring structure for another single ring structure is more likely than substituting a double ring for a single ring. Also, transitions are less likely to result in amino acid substitutions (due to  wobble base pair ), and are therefore more likely to persist as "silent substitutions" in populations as  single nucleotide polymorphisms  (SNPs). [ 2 ]  A transversion usually has a more pronounced effect than a transition  because the second and third nucleotide  codon  position of the  DNA , which to a large extent is responsible for the  degeneracy  of the  code , is more tolerant of transition than a transversion: transitions are more likely to be  synonymous substitutions  than transversions, as one observes in the  codon table .


8-oxo-2'-deoxyguanosine  (8-oxodG) is an oxidized derivative of  deoxyguanosine , and is one of the major products of  DNA oxidation . During  DNA replication  in the germ line of mice, the oxidized base  8-oxoguanine  (8-oxoG) causes spontaneous and heritable G to T transversion mutations. [ 3 ]   These mutations occur in different stages of the  germ cell  lineage and are distributed throughout the  chromosomes .


The location of a transversion mutation on a gene coding for a protein correlates with the extent of the mutation. If the mutation occurs at a site that is not involved with the shape of a protein or the structure of an enzyme or its active site, the mutation will not have a significant effect on the cell or the enzymatic activity of its proteins. If the mutation occurs at a site that changes the structure or function of a protein, therefore changing its enzymatic activity, the mutation can have significant effects on the survival of the cell. [ 4 ]


Of the natural nitrogenous bases of DNA, guanine is most prone to oxidation. Oxidation of guanine, also known as oxidative guanine damage, results in the formation of many products. These products trigger mutations, leading to DNA damage, and can pair with adenine and guanine through hydrogen bonding causing G-T transversions and G-C transversions, respectively. [ 5 ]


The mutation of the  P53  gene is the most common gene mutation found in cancer cells. A study has shown that p53 mutations are common in tobacco-related cancers, with a variation in the amount of G-T transversions in lung cancer from smokers and non-smokers. In smokers’ lung cancer, the prevalence of G-T transversions is 30% compared to that of 12% in non-smokers. At many p53 mutational hotspots, a large number of the mutations are G-T events in lung cancers but almost exclusively G-A transitions in non-tobacco-related cancers. [ 6 ]


Tumour mutational burden  (abbreviated as  TMB ) is a genetic characteristic of tumorous tissue that can be informative to  cancer research  and treatment. It is defined as the number of  non-inherited mutations  per million  bases  (Mb) of investigated genomic sequence, [ 1 ]  and its measurement has been enabled by  next generation sequencing . High TMB and DNA damage repair mutations were discovered to be associated with superior clinical benefit from immune checkpoint blockade therapy by Timothy Chan and colleagues at the Memorial Sloan Kettering Cancer Center. [ 2 ]


TMB has been validated as a predictive  biomarker  with several applications, including associations reported between different TMB levels and patient response to immune checkpoint inhibitor (ICI) therapy in a variety of cancers. [ 3 ] [ 4 ]  TMB is also strongly predictive of overall as well as disease-specific survival, independently of cancer type, stage or grade. Patients with both low and high TMB fare notably better than those with intermediate burden. [ 5 ]


While both TMB and  mutational signatures  provide critical information about cancer behaviour, they have different definitions. TMB is defined as the number of somatic mutations/megabase whereas mutational signatures are distinct mutational patterns of single base substitutions, double base substitutions, or small insertions and deletions in tumors. [ 6 ]  For instance,  COSMIC  single base substitution signature 1 is characterized by the enzymatic deamination of cytosine to thymine and has been associated with age of an individual. [ 6 ]


Scientists postulate that high TMB is associated with an increased amount of neoantigens, which are tumour specific markers displayed by cells. [ 2 ] [ 7 ]  An increase in these  antigens  may then lead to increased detection of cancer cells by the immune system and more robust activation of cytotoxic  T-lymphocytes . Activation of T-cells is further regulated by  immune checkpoints  that can be displayed by cancer cells, thus treatment with ICIs can lead to improved patient  survival . [ 8 ]


On June 16, 2020 the  U.S. Food and Drug Administration  expanded the approval of the immunotherapy drug  pembrolizumab  to treat any advanced solid-tumor cancers with a TMB greater than 10 mutations per Mb and continued growth following prior treatments. [ 9 ]  This marks the first time that the FDA has approved a drug with its use based on TMB measurements. [ 10 ]


One survival mechanism in tumors is to increase the expression of  immune checkpoint  molecules that can bind to tumor-specific T-cells and inactivate them, so that the tumor cells cannot be detected and killed. [ 11 ]  ICIs have been shown to improve patients' response and the  survival rates  as they help the immune system to target tumor cells. [ 1 ] [ 10 ]  However, there is a variation in response to ICIs among patients and it is crucial to know which patients can benefit from ICI therapy. [ 1 ]  The expression of  PD-L1  (programmed death-ligand 1; one of the  immune checkpoints ) has been demonstrated to be a good  biomarker  of  PD-L1  blockade therapy in some cancers. [ 10 ]  However, there is a need for better biomarkers as there are some predictive errors with  PD-L1  expression. [ 10 ]  Studies on TMB have illustrated that there is an association between patients' outcome (of ICI therapy) and the TMB value. [ 1 ]  It has been proposed that TMB can be used as a predictive marker of response in ICI therapy across many cancer types. [ 10 ]  Also, TMB can be helpful to identify individuals that can benefit from ICI therapy with cancers that generally have low TMB values. [ 10 ]  Furthermore, it has been shown that tumors with higher TMB values usually result in a higher number of neoantigens, the  antigens  that are presented on the tumor cells surface that are usually a result of missense mutations. [ 10 ]  So, TMB can be a good estimator of neoantigen load and can help find the patients who can benefit from ICI therapy by increasing the chance of detecting the neoantigens. [ 10 ]  However, it is important to note that different sequencing platforms and bioinformatics pipelines have been used to estimate TMB and it is important to harmonize TMB quantification protocols and procedures before it can be used as a reliable  biomarker . [ 1 ] [ 12 ]  There have been some efforts to standardize these methods. [ 1 ]


TMB has been found to correlate with patient response to therapies such as immune checkpoint inhibitors (ICIs). An analysis of a large cohort of patients receiving ICI therapy revealed that higher TMB levels (≥ 20 mutations/Mb) corresponded to a 58%  response rate  to ICIs while lower TMB levels (<20 mutations/Mb) reduced response to 20%. [ 13 ]  Researchers could also show a significant correlation between treatment response rate and TMB level in patients treated with anti-PD-1 or anit-PD-L1 (types of ICIs). [ 14 ]  Additionally, it has been reported that when ICIs were the only treatments used by patients, 55% of the differences in the objective response rate across cancer types were explained by TMB. [ 14 ]


Associations have been reported between TMB and patient outcome in a variety of cancers. In one study, scientists observed differences in  survival rates , with high TMB individuals having a median progression-free survival of 12.8 months and a median overall survival not reached by the time of publication, compared to 3.3 months and 16.3 months respectively for individuals with lower TMB. [ 13 ]  Another study examining patients who had not received ICI therapy found that intermediate levels of TMB (>5 and <20 mutations/Mb) correlate with significantly decreased  survival , likely as a result of the accumulation of mutations in  oncogenes . [ 7 ]  This relationship does not appear to be significantly disparate across different tissues types and is only modestly affected by corrections for confounders such as smoking, sex, age, and ethnicity. [ 7 ]  This suggests that TMB is both an independent and reliable indicator of poor patient outcomes in the absence of ICI therapy. [ 7 ]  Interestingly, very high levels of TMB (≥ 50 mutations/Mb) were reported to correlate with increased  survival , giving an overall parabolic shape to the trend. [ 7 ]  While this association is still under investigation, it has been hypothesized that the decreased risk of death under very high TMB could result from reduced cell viability due to  genetic instability  or increased production of neoantigens recognized by the immune system. [ 7 ]


There is a large variation in TMB values across different cancer types as the number of  somatic mutations  can span from 0.01 to 400 mutations per megabase of genome. [ 1 ] [ 10 ] [ 11 ]  It has been shown that  melanoma ,  NSCLC  and other  squamous carcinomas  have the highest levels of TMB in this order, while  leukemias  and  pediatric  tumors have the lowest levels of TMB and other cancers like  breast ,  kidney , and  ovary  have intermediate TMB values. [ 10 ]  There is also variation in TMB across different subtypes of different cancers. [ 10 ]  Due to high variability in TMB across different cancer types and subtypes, it is important to define different cut-offs to have an improved survival prediction and a better treatment decision. [ 1 ] [ 10 ] [ 11 ]  For example, Fernandez et al. showed that TMB can range from 0.03 to 14.13 mutations per megabase (mean=1.23) in  TCGA  prostate cancer cohort while this range is from 0.04-99.68 mutations per megabase (mean=6.92) in  TCGA  bladder cancer cohort. [ 15 ]  A recent study illustrated that different cut-offs are needed for different cancer types to find the patients who can benefit from ICI therapy. [ 1 ]  In addition, it is crucial to understand that usually there are different clusters of cells in a tumor, known as  tumor heterogeneity , that can affect TMB and consequently the response to ICIs. [ 10 ]  Another factor that can affect TMB is whether the source of the sample is primary or  metastatic  tissue. [ 16 ]  Most  metastatic  samples have been shown to be monoclonal (i.e. there is only one cluster of cells in the tumor), while primary tumors usually consist of a higher number of clusters and have higher overall genetic diversity (more heterogeneous). [ 16 ]  Scientists have shown that  metastatic  tumors usually have a higher TMB level compared to primary tumors and this can be due to monoclonal nature of  metastatic  lesions. [ 16 ]


There are disparities between how TMB is calculated in clinical and research settings. [ 17 ]  Broadly,  whole genome sequencing ,  whole exome sequencing , and panel based approaches can be used to help to calculate TMB. [ 17 ]  Studies of TMB from research perspectives typically incorporate  whole exome sequencing , and occasionally  whole genome sequencing  within their workflows while clinical applications use panel sequencing to estimate TMB primarily for their comparatively quicker speed and low cost. [ 17 ]  Within panel based approaches, different strategies to calculate TMB have been adopted. [ 17 ]  For instance, consider MSK-IMPACT developed by the  Memorial Sloan Kettering Cancer Center  and F1CDx developed by  Foundation Medicine . [ 18 ] [ 19 ]  F1CDx utilizes tumor-only sequencing strategy while MSK-IMPACT requires sequencing of both the tumor and its matched normal sample. Additionally, F1CDx counts synonymous mutations while excluding hotspot driver mutations. [ 18 ]  MSK-IMPACT calculates TMB with similar filtering criteria to those used in  whole exome sequencing , considering both  synonymous mutations  and  hotspot driver mutations . [ 19 ]  Ensembles of targeted panels and  whole exome sequencing  panels have been recommended for optimal results. [ 20 ]  As an approach that is potentially more expedient and cost effective than sequencing, TMB can be calculated directly from  H&E stained  pathology images using  deep learning . [ 21 ]


Overall, 5 primary factors have been identified to influence TMB calculations. [ 22 ]


Greater tumor cell content and sequencing coverage play a key role in the quality of TMB data. [ 22 ]  For instance, targeted panels may enable deeper sequencing compared to  whole exome sequencing , enabling higher sensitivity, that have been shown to perform well even when tumor cell content is low (defined as <10%). [ 22 ]  Targeted panels have shown to enable much greater coverage than in  whole exome sequencing . [ 22 ]  For example, one recent study reached a mean sequencing  coverage  across all tumor samples of 744× when using the MSK-IMPACT panel, while the WES led to a mean target coverage of 232× in tumor sequences. [ 23 ]