Simple  Giemsa staining  was originally used for MN scoring. Later, the cytokinesis-block micronucleus (CBMN) method was established, where  Cyt-B , an inhibitor of the spindle assembly, was used to prevent  cytokinesis  occurring after nuclear division. The CBMN method is used for the assessment of chromosomal loss, breakage, and associated  apoptosis  and  necrosis  induced by different  mutagens . [ 3 ]


A  micronucleus  is the erratic (third) nucleus that is formed during the  anaphase  of  mitosis  or  meiosis .
Micronuclei (the name means 'small nucleus') are cytoplasmic bodies having a portion of acentric chromosome or whole chromosome which was not carried to the opposite poles during the anaphase. Their formation results in the daughter cell lacking a part or all of a chromosome. These chromosome fragments or whole chromosomes normally develop nuclear membranes and form as micronuclei as a third nucleus. After cytokinesis, one daughter cell ends up with one nucleus and the other ends up with one large and one small nucleus, i.e., micronuclei. There is a chance of more than one micronucleus forming when more genetic damage has happened.
The micronucleus test is used as a tool for genotoxicity assessment of various chemicals. It is easier to conduct than the chromosomal aberration test in terms of procedures and evaluation.
Using  fluorescent in situ hybridization  (FISH) with probes targeted to the  centromere  region, it can be determined if a whole chromosome, or only a fragment is lost.


Microsatellite instability  ( MSI ) is the condition of  genetic  hypermutability (predisposition to  mutation ) that results from impaired  DNA mismatch repair  (MMR). The presence of MSI represents  phenotypic  evidence that MMR is not functioning normally.


MMR corrects errors that spontaneously occur during  DNA replication , such as single base mismatches or short  insertions  and  deletions . The proteins involved in MMR correct  polymerase  errors by forming a complex that binds to the mismatched section of DNA, excises the error, and inserts the correct sequence in its place. [ 1 ]  Cells with abnormally functioning MMR are unable to correct errors that occur during DNA replication and consequently accumulate errors. This causes the creation of novel  microsatellite  fragments.  Polymerase chain reaction -based assays can reveal these novel  microsatellites  and provide evidence for the presence of MSI.


Microsatellites are repeated sequences of DNA. These sequences can be made of units of 1 to 6  base pairs  in length that are repeated and reside adjacent to each other in the genome. Although the length of microsatellites can vary from person to person and contributes to the individual DNA "fingerprint", each individual has microsatellites of a set length. The most common microsatellite in humans is a dinucleotide repeat of the  nucleotides   C  and  A , which occurs tens of thousands of times across the genome. Microsatellites are also known as simple sequence repeats (SSRs).


Microsatellite instability structure consists of repeated nucleotides, most often seen as GT/CA repeats. [ 2 ]


Researchers have yet to confirm the precise definition of the MSI structure. While all researchers agree that microsatellites are repeat sequences, the lengths of the sequences remain in question.  Some research suggests that MSIs are short tandem DNA repeat sequences of one to six base pairs throughout the genome, while other research suggests that the range may be two to five. [ 3 ]


Although researchers do not agree on a specific threshold for the number of tandem repeats that constitute a microsatellite, there is a consensus around their relative size. Longer sequences are called  minisatellite , and even longer sequences are called  satellite DNA  sites. Some scientists distinguish among the three categories by a minimum number of base pairs, and others use a minimum number of repeated units. The majority of repeats occur in untranslated regions, specifically  introns . However, microsatellites that occur in coding regions often inhibit the expansion of most downstream events. Microsatellites make up approximately three percent of the human genome, or more than one million fragments of DNA. Microsatellite density increases with genome size and is seen twice as much at the ends of chromosome arms than in the chromosome bodies. [ 4 ]


MSI was discovered in the 1970s and 1980s.


In a broad sense, MSI results from the inability of the mismatch repair (MMR) proteins to fix a DNA replication error. DNA replication occurs in the "S" phase of the  cell cycle ; the faulty event creating an MSI region occurs during the second replication event. The original strand is unharmed, but the daughter strand experiences a frame-shift mutation due to DNA polymerase slippage. Specifically, DNA polymerase slips, creating a temporary insertion-deletion loop, which is usually recognized by MMR proteins. However, when the MMR proteins do not function normally, as in the case of MSI, this loop results in frame-shift mutations, either through insertions or deletions, yielding non-functioning proteins. [ 5 ]


MSI is unique to DNA polymorphisms in that the replication errors vary in length instead of sequence. The rate and direction of the mutations yielding MSIs are the major components in determining genetic differences. To date, scientists agree that the mutation rates differ in loci position. The greater the length of the MSI, the greater the mutation rate. [ 4 ]


Although most mutations of MSI are the result of frame-shift mutations, occasionally the mutation events leading to MSI are derived from the hypermethylation of the h MLH1  (MMR protein) promoter. Hypermethylation occurs when a methyl group is added to a DNA nucleotide, resulting in gene silencing, thus yielding MSI. [ 6 ]


Researchers have shown that oxidative damage yields frame-shift mutations, thus yielding MSI, but they have yet to agree on a precise mechanism. It has been shown that the more oxidative stress is placed on the system, the more likely it is that mutations will occur. Additionally,  catalase  reduces mutations, whereas copper and nickel increase mutations by increasing reduction of peroxides. Some researchers believe that the oxidative stress on specific loci results in DNA polymerase pausing at those sites, creating an environment for DNA slippage to occur. [ 7 ]


Researchers first believed that MSI was random, but there is evidence suggesting that MSI targets include a growing list of genes. Examples include the  transforming growth factor Beta receptor  gene and the  BAX  gene. Each target leads to different phenotypes and pathologies. [ 8 ]


It is thought that microsatellites that form in intronic/non-coding regions leads to the formation of secondary DNA structures (e.g.  G-quadruplexes ) that can lead to DNA damage and cell death if not repaired. This is exemplified by the dependency of the  Werner syndrome helicase  in MSI-H cancers.  [ 9 ]


Microsatellite instability is associated with colon cancer, gastric cancer, endometrium cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer, and skin cancers. MSI is most prevalent in associations with colon cancers. Each year, there are over 500,000 colon cancer cases worldwide. Based on findings from over 7,000 patients stratified for MSI-High (MSI-H), MSI-Low (MSI-L), or Microsatellite Stable (MSS) colon cancers, those with MSI-H tumours had a more positive prognosis by 15% compared to MSI-L or MSS tumors. [ 10 ]


Colorectal tumors with MSI are found in the right colon, associated with poor differentiated tissue, high mucinogens, tumor infiltrating lymphocytes, and a presence of a Crohn's-like host response. [ 11 ] [ 12 ]  MSI-H tumors contributing to colorectal cancer exhibit less metastasis than other derived colorectal cancer. This is demonstrated by previous research showing that MSI-H tumors are more representative in Stage II rather than Stage III cancers. [ 3 ]


Scientists have explored the connection of  vacuolar protein sorting  (VPS) proteins to MSI. Like MSI, VPS is linked to gastric and colon cancers. One study reports that VPS proteins were linked to MSI-H cancers, but not MSI-L cancers, thus restricting VPS to MSI-H-specific cancers. [ 13 ]


MSI-H status raises the possibility of  Lynch syndrome , but MSI-H can also occur in patients without Lynch Syndrome and confirmation of Lynch Syndrome requires testing  germline  DNA. Lynch syndrome is associated with MSI and increases the risk for colon, endometrium, ovary, stomach, small intestine, hepatobiliary tract, urinary tract, brain, and skin cancers. [ 3 ]


One study conducted over 120 Lynch syndrome patients attributing Crohn's like reaction (CLR) associated with MSI to "tumor specific neopeptides generated during MSI-H carcinogenesis." This study further corroborated that the "presence of antimetastatic immune protection in MSI-H CRC patients may explain recent findings that adjuvant 5-FU chemotherapy has no beneficial or even adverse effects in this collective." The researchers assume that there is a protective role of lymphocytes against the MSI-H CRC that prevents tumor metastasis. [ 14 ]


MSI tumors in 15% of sporadic colorectal cancer result from the hypermethylation of the MLH 1 gene promoter, whereas MSI tumors in Lynch syndrome are caused by germline mutations in  MLH1 ,  MSH2 ,  MSH6 , and  PMS2 . [ 3 ]


MSI has been evident in the cause of sebaceous carcinomas. Sebaceous carcinomas are a subset of a larger pathology,  Muir-Torre syndrome . MSI is variably expressed in Muir-Torre syndrome, most often expressed with shared pathologies in patients with colon cancer. Furthermore, MMR proteins MLH 1, MSH 2, MSH6, and PMS2 are instrumental in periocular sebaceous carcinoma, which is seen on the eyelid in 40% of sebaceous carcinomas. [ 15 ]


In May 2017 the FDA approved an immunotherapeutic called Keytruda ( pembrolizumab ) ( PD-1 inhibitor ) for patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors that have progressed following prior treatment. This indication is independent of  PD-L1  expression assessment, tissue type and tumor location. [ 16 ]


MSI is a good marker for detecting Lynch syndrome and determining a prognosis for cancer treatments. In 1996, the National Cancer Institute (NCI) hosted an international workshop on Lynch Syndrome, which led to the development of the "Bethesda Guidelines" and loci for MSI testing.  During this first workshop the NCI has agreed on five microsatellite markers necessary to determine MSI presence: two mononucleotides, BAT25 and BAT26, and three dinucleotide repeats, D2S123, D5S346, and D17S250. MSI-H tumors result from MSI of greater than 30% of unstable MSI loci (>2 or more of the 5 loci). MSI-L tumors result from less than 30% of unstable MSI biomarkers. MSI-L tumors are classified as tumors of alternative etiologies. Several studies demonstrate that MSI-H patients respond best to surgery alone, rather than chemotherapy and surgery, thus preventing patients from needlessly experiencing chemotherapy. [ 3 ]


Six years later, during the second NCI hosted workshop to revisit Lynch Syndrome in 2002, the Bethesda Guidelines were revised (later published in 2004 [ 17 ] ) which recommended new criteria for MSI testing.  Specifically, they identified the five mononucleotide loci as being superior, over the mixture of mono and dinucleotide loci because the dinucleotide loci could appear shifted, when in fact they were not, thus increasing the possibility of a false positive MSI-H result.


The first commercially available kit was provided by  Promega Corporation , Madison, Wisconsin called the  Microsatellite Instability 1.2 Analysis System  (RUO).  Since then, the Promega MSI RUO has been widely adopted since 2004, with over 120 peer-reviewed publications citing its global standing as the gold standard in determining the MSI status of cancer tissue.


Recently, real-time PCR based MSI detection kits [ 18 ]  have been introduced in the market successfully. Single step closed-tube format, high accuracy and sensitivity of the real-time PCR based products without any additional analysis after the PCR amplification comparing to the traditional (PCR followed with fragment analysis) methods has been considered as critical advantages.


Artificial intelligence has also been used to predict MSI from the appearance of tumors under the microscope. [ 12 ] [ 19 ]  Digital  pathology  can be submitted to machine learning techniques and predictions about MSI can be made without any molecular testing. [ 12 ]  These methods have not yet shown results that are sufficient to incorporate in clinical care.


Direct and indirect mechanisms contribute to chemotherapy resistance. Direct mechanisms include pathways that metabolize the drug, while indirect mechanisms include pathways that respond to the chemotherapy treatment.  The NER DNA repair pathway plays a substantial role in reversing cell damage caused by chemotherapeutic agents such as 5-FU. [ 20 ]


It has been shown that MSI-H cancers are dependent on the  Werner syndrome helicase  (WRN) to repair the DNA secondary structures that are formed by expanded TA microsatellites.  [ 9 ]  Because of this targeted therapeutic hypothesis, inhibition of WRN has become an area of high interest for the treatment of MSI-H malignancies. [ 21 ]  Two first-in-class WRN inhibitors, HRO791 (an allosteric inhibitor,  Novartis ) and VVD-133214 (a covalent inhibitor, Vividion Therapeutics and  Roche ) are currently undergoing clinical trials.  [ 22 ] [ 23 ]  Both of these inhibitors induce the degradation of WRN through similar mechanisms. [ 24 ]


Researchers have found another MSI, called elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). However, EMAST is unique in that it is not derived from MMR, and it is commonly associated with  TP53  mutations. [ 5 ]


EMAST is seen in a variety of cancers including those of the lung, head and neck, colorectal, skin, urinary tract, and the reproductive organs. External organ sites have more potential for EMAST. Some researchers believe that EMAST maybe a consequence of mutagenesis. EMAST positive margins in otherwise negative cancer margins suggest disease relapse for patients. [ 25 ]


In  genetics , a  missense mutation  is a  point mutation  in which a single  nucleotide  change results in a  codon  that codes for a different  amino acid . [ 1 ]  It is a type of  nonsynonymous substitution .


Missense mutation refers to a change in one amino acid in a protein, arising from a  point mutation  in a single nucleotide. Missense mutation is a type of  nonsynonymous substitution  in a DNA sequence. Two other types of nonsynonymous substitution are the  nonsense mutations , in which a codon is changed to a premature  stop codon  that results in truncation of the resulting  protein , and the  nonstop mutations , in which a stop codon erasement results in a longer, nonfunctional protein.


Missense mutations can render the resulting protein nonfunctional, [ 2 ]  and such mutations are responsible for human diseases such as  Epidermolysis bullosa ,  sickle-cell disease ,  SOD1  mediated  ALS , and a substantial number of  cancers . [ 3 ] [ 4 ]


In the most common variant of sickle-cell disease, the 20th nucleotide of the gene for the  beta chain  of  hemoglobin  is altered from the  codon  GAG to GTG. Thus, the 6th amino acid  glutamic acid  is substituted by  valine —notated as an "E6V" mutation—and the protein is sufficiently altered to cause the sickle-cell disease. [ 5 ]


Not all missense mutations lead to appreciable protein changes. An amino acid may be replaced by an amino acid of very similar chemical properties, in which case, the protein may still function normally; this is termed a neutral, "quiet", "silent" or conservative mutation. Alternatively, the amino acid substitution could occur in a region of the protein which does not significantly affect the protein secondary structure or function.  When an amino acid may be encoded by more than one codon (so-called "degenerate coding") a mutation in a codon may not produce any change in translation; this would be a  synonymous substitution  and not a missense mutation.


LMNA  missense mutation (c.1580G>T) introduced at LMNA gene – position 1580 (nt) in the DNA sequence (CGT) causing the  guanine  to be replaced with the  thymine , yielding CTT in the DNA sequence. This results at the protein level in the replacement of the  arginine  by the  leucine  at the position 527. [ 6 ]  This leads to destruction of  salt bridge  and structure destabilization. At  phenotype  level this manifests with overlapping  mandibuloacral dysplasia  and  progeria syndrome .


The resulting transcript and protein product is:


Cancer associated missense mutations can lead to drastic destabilisation of the resulting protein. [ 7 ]  A  method to screen for such changes was proposed in 2012, namely  fast parallel proteolysis (FASTpp) . [ 8 ]


3RBN ,  4P7A


NM_001258273 NM_001258274


NM_026810 NM_001324522


NP_001245202 NP_001245203 NP_001341544 NP_001341546 NP_001341547 NP_001341548 NP_001341549 NP_001341550 NP_001341551 NP_001341552 NP_001341556 NP_001341557 NP_001341558 NP_001341559 NP_001341545 NP_001341553 NP_001341554 NP_001341555


NP_001311451 NP_081086


DNA mismatch repair protein Mlh1  or  MutL protein homolog 1  is a  protein  that in humans is encoded by the  MLH1   gene  located on  chromosome 3 .  The gene is commonly associated with  hereditary nonpolyposis colorectal cancer .  Orthologs  of human MLH1 have also been studied in other organisms including mouse and the budding yeast  Saccharomyces cerevisiae .


Variants in this gene can cause  hereditary nonpolyposis colon cancer  (Lynch syndrome). It is a human  homolog  of the  E. coli  DNA mismatch repair gene, mutL, which mediates protein-protein interactions during mismatch recognition, strand discrimination, and strand removal. Defects in MLH1 are associated with the  microsatellite instability  observed in hereditary nonpolyposis colon cancer. Alternatively spliced transcript variants encoding different isoforms have been described, but their full-length natures have not been determined. [ 4 ]


MLH1 protein is one component of a system of seven  DNA mismatch repair  proteins that work coordinately in sequential steps to initiate repair of  DNA mismatches  in humans. [ 5 ]   Defects in mismatch repair, found in about 13% of colorectal cancers, are much more frequently due to deficiency of MLH1 than deficiencies of other DNA mismatch repair proteins. [ 6 ]   The seven DNA mismatch repair proteins in humans are MLH1,  MLH3 ,  MSH2 ,  MSH3 ,  MSH6 ,  PMS1  and  PMS2 . [ 5 ]   In addition, there are  Exo1 -dependent and Exo1-independent DNA mismatch repair subpathways. [ 7 ]


DNA mismatches occur where one base is improperly paired with another base, or where there is a short addition or deletion in one strand of DNA that is not matched in the other strand.  Mismatches commonly occur as a result of DNA replication errors or during genetic recombination.  Recognizing those mismatches and repairing them is important for cells because failure to do so results in microsatellite instability] and an elevated spontaneous  mutation rate  (mutator phenotype).  Among 20 cancers evaluated,  microsatellite instable colon cancer (mismatch repair deficient)  had the second highest frequency of mutations (after melanoma).


A heterodimer between MSH2 and MSH6 first recognizes the mismatch, although a heterodimer between MSH2 and MSH3 also can start the process. The formation of the MSH2-MSH6 heterodimer accommodates a second heterodimer of MLH1 and PMS2, although a heterodimer between MLH1 and either PMS3 or MLH3 can substitute for PMS2. This protein complex formed between the 2 sets of heterodimers enables initiation of repair of the mismatch defect. [ 5 ]


Other gene products involved in mismatch repair (subsequent to initiation by DNA mismatch repair genes) include  DNA polymerase delta ,  PCNA ,  RPA ,  HMGB1 ,  RFC  and  DNA ligase I , plus  histone  and  chromatin  modifying factors. [ 8 ] [ 9 ]


Only a minority of sporadic cancers with a DNA repair deficiency have a mutation in a DNA repair gene.  However, a majority of sporadic cancers with a DNA repair deficiency do have one or more  epigenetic  alterations that reduce or silence DNA repair gene expression. [ 18 ]   In the table above, the majority of deficiencies of MLH1 were due to methylation of the promoter region of the  MLH1  gene.  Another epigenetic mechanism reducing MLH1 expression is over-expression of  miR-155 . [ 19 ]   MiR-155 targets MLH1 and MSH2 and an inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. [ 19 ]


A  field defect  is an area or "field" of epithelium that has been preconditioned by epigenetic changes and/or mutations so as to predispose it towards development of cancer.  As pointed out by Rubin, "The vast majority of studies in cancer research has been done on well-defined tumors  in vivo,  or on discrete neoplastic foci  in vitro. [ 20 ]   Yet there is evidence that more than 80% of the somatic mutations found in mutator phenotype human colorectal tumors occur before the onset of terminal clonal expansion." [ 21 ]  Similarly, Vogelstein et al. [ 22 ]  point out that more than half of somatic mutations identified in tumors occurred in a pre-neoplastic phase (in a field defect), during growth of apparently normal cells.


In the Table above, MLH1 deficiencies were noted in the field defects (histologically normal tissues) surrounding most of the cancers.  If MLH1 is epigenetically reduced or silenced, it would not likely confer a selective advantage upon a stem cell. However, reduced or absent expression of MLH1 would cause increased rates of mutation, and one or more of the mutated genes may provide the cell with a selective advantage. The expression-deficient  MLH1  gene could then be carried along as a selectively neutral or only slightly deleterious passenger (hitch-hiker) gene when the mutated stem cell generates an expanded clone. The continued presence of a clone with an epigenetically repressed  MLH1  would continue to generate further mutations, some of which could produce a tumor.


In a cancer, multiple DNA repair genes are often found to be simultaneously repressed. [ 18 ]   In one example, involving  MLH1 , Jiang et al. [ 23 ]  conducted a study where they evaluated the mRNA expression of 27 DNA repair genes in 40  astrocytomas  compared to normal brain tissues from non-astrocytoma individuals. Among the 27 DNA repair genes evaluated, 13 DNA repair genes,  MLH1 ,  MLH3 ,  MGMT ,   NTHL1 ,   OGG1 ,   SMUG1 ,   ERCC1 ,   ERCC2 ,  ERCC3 ,  ERCC4 ,   RAD50 ,  XRCC4  and  XRCC5  were all significantly down-regulated in all three grades (II, III and IV) of astrocytomas. The repression of these 13 genes in lower grade as well as in higher grade astrocytomas suggested that they may be important in early as well as in later stages of astrocytoma.  In another example, Kitajima et al. [ 24 ]  found that immunoreactivity for MLH1 and  MGMT  expression was closely correlated in 135 specimens of gastric cancer and loss of  MLH1 and MGMTappeared to be synchronously accelerated during tumor progression.


Deficient expression of multiple DNA repair genes are often found in cancers, [ 18 ]  and may contribute to the thousands of mutations usually found in cancers (see  Mutation frequencies in cancers ).


In addition to its role in DNA mismatch repair, MLH1 protein is also involved in  meiotic   crossing over . [ 25 ]   MLH1 forms a heterodimer with  MLH3  that appears to be necessary for  oocytes  to progress through metaphase II of  meiosis . [ 26 ]   Female and male  MLH1 (-/-) mutant mice are infertile, and sterility is associated with a reduced level of  chiasmata . [ 25 ] [ 27 ]   During  spermatogenesis  in  MLH1 (-/-) mutant mice chromosomes often separate prematurely and there is frequent arrest in the first division of meiosis. [ 25 ]   In humans, a common variant of the  MLH1  gene is associated with increased risk of sperm damage and male infertility. [ 28 ]


MLH1 protein appears to localize to sites of crossing over in meiotic chromosomes. [ 25 ]   Recombination  during meiosis is often initiated by a DNA double-strand break (DSB) as illustrated in the accompanying diagram. During recombination, sections of DNA at the 5' ends of the break are cut away in a process called  resection . In the  strand invasion  step that follows, an overhanging 3' end of the broken DNA molecule then "invades" the DNA of an homologous chromosome that is not broken forming a  displacement loop  ( D-loop ). After strand invasion, the further sequence of events may follow either of two main pathways leading to a crossover (CO) or a non-crossover (NCO) recombinant (see  Genetic recombination ).  The pathway leading to a CO involves a double  Holliday junction  (DHJ) intermediate. Holliday junctions need to be resolved for CO recombination to be completed.


In the budding yeast  Saccharomyces cerevisiae , as in the mouse, MLH1 forms a heterodimer with MLH3.  Meiotic CO requires resolution of  Holliday junctions  through actions of the MLH1-MLH3  heterodimer .  The MLH1-MLH3  heterodimer  is an  endonuclease  that makes single-strand breaks in  supercoiled  double-stranded DNA. [ 29 ] [ 30 ]   MLH1-MLH3 binds specifically to Holliday junctions and may act as part of a larger complex to process Holliday junctions during  meiosis . [ 29 ]   MLH1-MLH3 heterodimer (MutL gamma) together with  EXO1  and  Sgs1  (ortholog of  Bloom syndrome helicase ) define a joint molecule resolution pathway that produces the majority of crossovers in budding yeast and, by inference, in mammals. [ 31 ]


It can also be associated with  Turcot syndrome . [ 32 ]


MLH1 has been shown to  interact  with:


2O8B ,  2O8C ,  2O8D ,  2O8E ,  2O8F ,  3THW ,  3THX ,  3THY ,  3THZ


NM_000251 NM_001258281


NP_000242 NP_001245210


DNA mismatch repair protein Msh2  also known as  MutS homolog 2  or  MSH2  is a  protein  that in humans is encoded by the  MSH2   gene , which is located on  chromosome 2 . MSH2 is a  tumor suppressor gene  and more specifically a  caretaker gene  that codes for a  DNA mismatch repair  (MMR) protein, MSH2, which forms a  heterodimer  with  MSH6  to make the human MutSα mismatch repair complex. It also dimerizes with  MSH3  to form the MutSβ DNA repair complex.  MSH2 is involved in many different forms of  DNA repair , including  transcription-coupled repair , [ 5 ]   homologous recombination , [ 6 ]  and  base excision repair . [ 7 ]


Mutations in the MSH2 gene are associated with  microsatellite instability  and some cancers, especially with  hereditary nonpolyposis colorectal cancer  (HNPCC). At least 114 disease-causing mutations in this gene have been discovered. [ 8 ]


Hereditary nonpolyposis colorectal cancer  (HNPCC), sometimes referred to as Lynch syndrome, is inherited in an  autosomal dominant  fashion, where inheritance of only one copy of a mutated mismatch repair gene is enough to cause disease  phenotype . Mutations in the MSH2 gene account for 40% of genetic alterations associated with this disease and is the leading cause, together with MLH1 mutations. [ 9 ]  Mutations associated with HNPCC are broadly distributed in all domains of MSH2, and hypothetical functions of these mutations based on the crystal structure of the MutSα include  protein–protein interactions ,  stability ,  allosteric regulation , MSH2-MSH6 interface, and  DNA binding . [ 10 ]  Mutations in MSH2 and other mismatch repair genes cause DNA damage to go unrepaired, resulting in an increase in mutation frequency. These mutations build up over a person's life that otherwise would not have occurred had the DNA been repaired properly.


The viability of MMR genes including  MSH2  can be tracked via  microsatellite  instability, a biomarker test that analyzes short sequence repeats which are very difficult for cells to replicate without a functioning mismatch repair system. Because these sequences vary in the population, the actual number of copies of short sequence repeats does not matter, just that the number the patient does have is consistent from tissue to tissue and over time. This phenomenon occurs because these sequences are prone to mistakes by the DNA replication complex, which then need to be fixed by the mismatch repair genes. If these are not working, over time either duplications or deletions of these sequences will occur, leading to different numbers of repeats in the same patient.


71% of HNPCC patients show microsatellite instability. [ 11 ]  Detection methods for microsatellite instability include polymerase chain reaction (PCR) and immunohistochemical (IHC) methods, polymerase chain checking the DNA and immunohistochemical surveying mismatch repair protein levels. "Currently, there are evidences that universal testing for MSI starting with either IHC or PCR-based MSI testing is cost effective, sensitive, specific and is generally widely accepted." [ 12 ]


In eukaryotes from yeast to humans, MSH2 dimerizes with MSH6 to form the MutSα complex, [ 13 ]  which is involved in base mismatch repair and short insertion/deletion loops. [ 14 ]  MSH2 heterodimerization stabilizes MSH6, which is not stable because of its N-terminal disordered domain. Conversely, MSH2 does not have a nuclear localization sequence ( NLS ), so it is believed that MSH2 and MSH6 dimerize in the  cytoplasm  and then are imported into the  nucleus  together. [ 15 ]  In the MutSα dimer, MSH6 interacts with the DNA for mismatch recognition while MSH2 provides the stability that MSH6 requires. MSH2 can be imported into the nucleus without dimerizing to MSH6, in this case, MSH2 is probably dimerized to MSH3 to form MutSβ. [ 16 ]  MSH2 has two interacting domains with MSH6 in the MutSα heterodimer, a DNA interacting domain, and an ATPase domain. [ 17 ]


The MutSα dimer scans double stranded DNA in the nucleus, looking for mismatched bases. When the complex finds one, it repairs the mutation in an  ATP  dependent manner. The MSH2 domain of MutSα prefers  ADP  to ATP, with the MSH6 domain preferring the opposite. Studies have indicated that MutSα only scans DNA with the MSH2 domain harboring ADP, while the MSH6 domain can contain either ADP or ATP. [ 18 ]  MutSα then associates with MLH1 to repair the damaged DNA.


MutSβ is formed when MSH2 complexes with MSH3 instead of MSH6. This dimer repairs longer insertion/deletion loops than MutSα. [ 19 ]  Because of the nature of the mutations that this complex repairs, this is probably the state of MSH2 that causes the microsatellite instability phenotype. Large DNA insertions and deletions intrinsically bend the DNA double helix. The MSH2/MSH3 dimer can recognize this topology and initiate repair. The mechanism by which it recognizes mutations is different as well, because it separates the two DNA strands, which MutSα does not. [ 20 ]


Msh2 modulates accurate  homologous recombination , a prominent  DNA double-strand break repair  pathway in mammalian  chromosomes . [ 21 ]   Repair of DNA double-strand breaks by accurate homologous recombination predominates over the inaccurate double-strand break repair pathway of “non-homologous end joining” in hamster, mouse and human somatic cells. [ 21 ]


MSH2 has been shown to  interact  with:


DNA damage appears to be the primary underlying cause of cancer, [ 34 ]  and deficiencies in expression of DNA repair genes appear to underlie many forms of cancer. [ 35 ] [ 36 ]   If DNA repair is deficient, DNA damage tends to accumulate. Such excess DNA damage may increase  mutations  due to error-prone  translesion synthesis  and error prone repair (see e.g.  microhomology-mediated end joining ). Elevated DNA damage may also increase  epigenetic  alterations due to errors during DNA repair. [ 37 ] [ 38 ]  Such mutations and epigenetic alterations may give rise to  cancer .


Reductions in expression of DNA repair genes (usually caused by epigenetic alterations) are very common in cancers, and are ordinarily much more frequent than mutational defects in DNA repair genes in cancers. [ citation needed ]  (See  Frequencies of epimutations in DNA repair genes .)  In a study of  MSH2  in  non-small cell lung cancer  (NSCLC), no mutations were found while 29% of NSCLC had epigenetic reduction of  MSH2  expression. [ 39 ]   In  acute lymphoblastoid leukemia  (ALL), no MSH2 mutations were found [ 40 ]  while 43% of ALL patients showed MSH2 promoter methylation and 86% of relapsed ALL patients had MSH2 promoter methylation. [ 41 ]   There were, however, mutations in four other genes in ALL patients that destabilized the MSH2 protein, and these were defective in 11% of children with ALL and 16% of adults with this cancer. [ 40 ]


Methylation of the promoter region of the  MSH2  gene is correlated with the lack of expression of the MSH2 protein in esophageal cancer, [ 42 ]   in  non-small-cell lung cancer , [ 39 ] [ 43 ]   and in  colorectal cancer . [ 44 ]   These correlations suggest that methylation of the promoter region of the  MSH2  gene reduces expression of the MSH2 protein.  Such promoter methylation would reduce DNA repair in the four pathways in which MSH2 participates:  DNA mismatch repair ,  transcription-coupled repair [ 5 ]   homologous recombination , [ 6 ] [ 45 ] [ 46 ]  and  base excision repair . [ 7 ]    Such reductions in repair likely allow excess DNA damage to accumulate and contribute to  carcinogenesis .


The frequencies of  MSH2  promoter methylation in several different cancers are indicated in the Table.


In  genetics , a  mutagen  is a physical or chemical agent that permanently changes  genetic material , usually  DNA , in an  organism  and thus increases the frequency of  mutations  above the natural background level. As many mutations can cause  cancer  in animals, such mutagens can therefore be  carcinogens , although not all necessarily are. All mutagens have characteristic  mutational signatures  with some chemicals becoming mutagenic through cellular processes.


The process of DNA becoming modified is called  mutagenesis . Not all mutations are caused by mutagens: so-called "spontaneous mutations" occur due to spontaneous  hydrolysis ,  errors  in  DNA replication , repair and  recombination .


The first mutagens to be identified were  carcinogens , substances that were shown to be linked to  cancer .  Tumors  were described more than 2,000 years before the discovery of  chromosomes  and  DNA ; in 500 B.C., the Greek  physician   Hippocrates  named tumors resembling a crab  karkinos  (from which the word "cancer" is derived via Latin), meaning crab. [ 1 ]  In 1567, Swiss physician  Paracelsus  suggested that an unidentified substance in mined ore (identified as  radon  gas in modern times) caused a wasting disease in miners, [ 2 ]  and in England, in 1761,  John Hill  made the first direct link of cancer to chemical substances by noting that excessive use of  snuff  may cause nasal cancer. [ 3 ]  In 1775, Sir  Percivall Pott  wrote a paper on the high incidence of scrotal cancer in  chimney sweeps , and suggested chimney  soot  as the cause of scrotal cancer. [ 4 ]  In 1915, Yamagawa and Ichikawa showed that repeated application of coal tar to rabbit's ears produced malignant cancer. [ 5 ]  Subsequently, in the 1930s the carcinogen component in coal tar was identified as a  polyaromatic hydrocarbon  (PAH),  benzo[a]pyrene . [ 2 ] [ 6 ]  Polyaromatic hydrocarbons are also present in soot, which was suggested to be a causative agent of cancer over 150 years earlier.


The association of exposure to radiation and cancer had been observed as early as 1902, six years after the discovery of X-ray by  Wilhelm Röntgen  and radioactivity by  Henri Becquerel . [ 7 ]   Georgii Nadson  and German Filippov were the first who created fungi mutants under  ionizing radiation  in 1925. [ 8 ] [ 9 ]  The mutagenic property of mutagens was first demonstrated in 1927, when  Hermann Muller  discovered that  x-rays  can cause genetic mutations in  fruit flies , producing  phenotypic  mutants as well as observable changes to the chromosomes, [ 10 ] [ 11 ]  visible due to the presence of enlarged  "polytene" chromosomes  in fruit fly salivary glands. [ 12 ]  His collaborator  Edgar Altenburg  also demonstrated the mutational effect of UV radiation in 1928. [ 13 ]  Muller went on to use x-rays to create  Drosophila  mutants that he used in his studies of  genetics . [ 14 ]  He also found that X-rays not only mutate  genes  in fruit flies, [ 10 ]  but also have effects on the genetic makeup of humans. [ 15 ] [ better source needed ]  Similar work by  Lewis Stadler  also showed the mutational effect of X-rays on barley in 1928, [ 16 ]  and  ultraviolet  (UV) radiation on maize in 1936. [ 17 ]  The effect of sunlight had previously been noted in the nineteenth century where rural outdoor workers and sailors were found to be more prone to skin cancer. [ 18 ]


Chemical mutagens  were not demonstrated to cause mutation until the 1940s, when  Charlotte Auerbach  and  J. M. Robson  found that  mustard gas  can cause  mutations  in fruit flies. [ 19 ]  A large number of chemical mutagens have since been identified, especially after the development of the  Ames test  in the 1970s by  Bruce Ames  that screens for mutagens and allows for preliminary identification of carcinogens. [ 20 ] [ 21 ]  Early studies by Ames showed around 90% of known carcinogens can be identified in Ames test as mutagenic (later studies however gave lower figures), [ 22 ] [ 23 ] [ 24 ]  and ~80% of the mutagens identified through Ames test may also be carcinogens. [ 24 ] [ 25 ]


Mutagens are not necessarily carcinogens, and vice versa.  Sodium azide  for example may be mutagenic (and highly toxic), but it has not been shown to be carcinogenic. [ 26 ]  Meanwhile, compounds which are not directly mutagenic but stimulate cell growth which can reduce the effectiveness of DNA repair and indirectly increase the chance of mutations, and therefore that of cancer. [ 27 ]  One example of this would be  anabolic steroids , which stimulate growth of the prostate gland and increase the risk of  prostate cancer  among others. [ 28 ]  Other carcinogens may cause cancer through a variety of mechanisms without producing mutations, such as  tumour promotion ,  immunosuppression  that reduces the ability to fight cancer cells or pathogens that can cause cancer, disruption of the  endocrine system  (e.g. in breast cancer), tissue-specific toxicity, and  inflammation  (e.g. in colorectal cancer). [ 29 ]


A  DNA damaging agent  is an agent that causes a change in the structure of DNA that is not itself replicated when the  DNA is replicated . [ 30 ]   Examples of DNA damage include a chemical addition or disruption of a  nucleotide  base in DNA (generating an abnormal nucleotide or nucleotide fragment), or a break in one or both strands in DNA.  When duplex DNA containing a damaged base is replicated, an incorrect base may be inserted in the newly synthesized strand opposite the damaged base in the complementary template strand, and this can become a  mutation  in the next round of replication.  Also a DNA double-strand break may be repaired by an inaccurate process leading to an altered base pair, a mutation.  However, mutations and DNA damages differ in a fundamental way: mutations can, in principle, be replicated when DNA replicates, whereas DNA damages are not necessarily replicated.  Thus DNA damaging agents often cause mutations as a secondary consequence, but not all DNA damages lead to mutation and not all mutations arise from a DNA damage. [ 30 ]   The term genotoxic means toxic (damaging) to DNA.


Mutagens can cause changes to the DNA and are therefore  genotoxic . They can affect the transcription and replication of the DNA, which in severe cases can lead to cell death. The mutagen produces mutations in the DNA, and deleterious mutation can result in aberrant, impaired or loss of function for a particular gene, and accumulation of mutations may lead to cancer. Mutagens may therefore be also carcinogens. However, some mutagens exert their mutagenic effect through their metabolites, and therefore whether such mutagens actually become carcinogenic may be dependent on the metabolic processes of an organism, and a compound shown to be mutagenic in one organism may not necessarily be carcinogenic in another. [ 31 ]


Different mutagens act on DNA differently. Powerful mutagens may result in chromosomal instability, [ 32 ]  causing chromosomal breakages and rearrangement of the chromosomes such as  translocation ,  deletion , and  inversion . Such mutagens are called  clastogens .


Mutagens may also modify the DNA sequence; the changes in  nucleic acid  sequences by mutations include substitution of  nucleotide   base-pairs  and  insertions  and  deletions  of one or more nucleotides in DNA sequences. Although some of these mutations are lethal or cause serious disease, many have minor effects as they do not result in residue changes that have significant effect on the structure and function of the  proteins . Many mutations are  silent mutations , causing no visible effects at all, either because they occur in non-coding or non-functional sequences, or they do not change the  amino-acid  sequence due to the  redundancy  of  codons . [ 33 ]
Some mutagens can cause  aneuploidy  and change the number of chromosomes in the cell. They are known as aneuploidogens. [ 34 ]


In Ames test, where the varying concentrations of the chemical are used in the test, the dose response curve obtained is nearly always linear, suggesting that there may be no threshold for mutagenesis. Similar results are also obtained in studies with radiations, indicating that there may be  no safe threshold  for mutagens. However, the no-threshold model is disputed with some arguing for a  dose rate dependent threshold  for mutagenesis. [ 35 ] [ 10 ]  Some have proposed that low level of some mutagens may stimulate the  DNA repair  processes and therefore may not necessarily be harmful. More recent approaches with sensitive analytical methods have shown that there may be non-linear or bilinear dose-responses for genotoxic effects, and that the activation of DNA repair pathways can prevent the occurrence of mutation arising from a low dose of mutagen. [ 36 ]


Mutagens may be of physical, chemical or biological origin. They may act directly on the DNA, causing direct damage to the DNA, and most often result in replication error. Some however may act on the replication mechanism and chromosomal partition. Many mutagens are not mutagenic by themselves, but can form mutagenic metabolites through cellular processes, for example through the activity of the  cytochrome P450  system and other  oxygenases  such as  cyclooxygenase . [ 37 ]  Such mutagens are called  promutagens . [ 38 ]


Chemical mutagens either directly or indirectly damage DNA. On this basis, they are of 2 types:


They directly damage DNA, but may or may not undergo metabolism to produce promutagens (metabolites that can have higher mutagenic potential than their substrates).


They are not necessarily mutagenic by themselves, but they produce promutagens mutagenic compounds through metabolic processes in cells.


Some chemical mutagens additionally require  UV  or  visible light  activation for their mutagenic effect.  These are the  photomutagens , which include  furocoumarins  and  limettin . [ 46 ]


Many metals, such as  arsenic ,  cadmium ,  chromium ,  nickel  and their compounds may be mutagenic, but they may act, however, via a number of different mechanisms. [ 47 ]  Arsenic, chromium, iron, and nickel may be associated with the production of ROS, and some of these may also alter the fidelity of DNA replication. Nickel may also be linked to DNA hypermethylation and  histone  deacetylation, while some metals such as  cobalt , arsenic, nickel and cadmium may also affect DNA repair processes such as  DNA mismatch repair , and  base  and  nucleotide excision repair . [ 48 ]


Antioxidants  are an important group of  anticarcinogenic  compounds that may help remove  ROS  or potentially harmful chemicals. These may be found naturally in  fruits  and  vegetables . [ 51 ]  Examples of antioxidants are  vitamin A  and its  carotenoid  precursors,  vitamin C ,  vitamin E ,  polyphenols , and various other compounds.  β-Carotene  is the red-orange colored compounds found in vegetables like  carrots  and  tomatoes . Vitamin C may prevent some cancers by inhibiting the formation of mutagenic  N-nitroso  compounds (nitrosamine).  Flavonoids , such as  EGCG  in  green tea , have also been shown to be effective antioxidants and may have anti-cancer properties. Epidemiological studies indicate that a diet rich in fruits and vegetables is associated with lower incidence of some cancers and longer life expectancy, [ 52 ]  however, the effectiveness of antioxidant supplements in cancer prevention in general is still the subject of some debate. [ 52 ] [ 53 ]


Other chemicals may reduce mutagenesis or prevent cancer via other mechanisms, although for some the precise mechanism for their protective property may not be certain.  Selenium , which is present as a micronutrient in vegetables, is a component of important antioxidant enzymes such as gluthathione peroxidase. Many phytonutrients may counter the effect of mutagens; for example,  sulforaphane  in vegetables such as  broccoli  has been shown to be protective against  prostate cancer . [ 54 ]  Others that may be effective against cancer include  indole-3-carbinol  from  cruciferous vegetables  and  resveratrol  from red wine. [ 55 ]


An effective precautionary measure an individual can undertake to protect themselves is by limiting exposure to mutagens such as UV radiations and tobacco smoke. In Australia, where people with pale skin are often exposed to strong sunlight,  melanoma  is the most common cancer diagnosed in people aged 15–44 years. [ 56 ] [ 57 ]


In 1981, human epidemiological analysis by  Richard Doll  and  Richard Peto  indicated that smoking caused 30% of cancers in the US. [ 58 ]  Diet is also thought to cause a significant number of cancer, and it has been estimated that around 32% of cancer deaths may be avoidable by modification to the diet. [ 59 ]  Mutagens identified in food include  mycotoxins  from food contaminated with fungal growths, such as  aflatoxins  which may be present in contaminated peanuts and corn;  heterocyclic amines  generated in meat when cooked at high temperature; PAHs in charred meat and smoked fish, as well as in oils, fats, bread, and cereal; [ 60 ]  and nitrosamines generated from nitrites used as food preservatives in  cured meat  such as  bacon  ( ascorbate , which is added to cured meat, however, reduces nitrosamine formation). [ 51 ]  Overly-browned starchy food such as bread, biscuits and potatoes can generate  acrylamide , a chemical shown to cause cancer in animal studies. [ 61 ] [ 62 ]  Excessive  alcohol consumption  has also been linked to cancer; the possible mechanisms for its carcinogenicity include formation of the possible mutagen  acetaldehyde , and the induction of the  cytochrome P450  system which is known to produce mutagenic compounds from promutagens. [ 63 ]


For certain mutagens, such as dangerous chemicals and radioactive materials, as well as infectious agents known to cause cancer, government legislations and regulatory bodies are necessary for their control. [ 64 ]


Many different systems for detecting mutagen have been developed. [ 65 ] [ 66 ]  Animal systems may more accurately reflect the metabolism of human, however, they are expensive and time-consuming (may take around three years to complete), they are therefore not used as a first screen for mutagenicity or carcinogenicity.


Systems similar to Ames test have been developed in yeast.  Saccharomyces cerevisiae  is generally used. These systems can check for forward and reverse mutations, as well as recombinant events.


Sex-Linked  Recessive Lethal Test  – Males from a strain with yellow bodies are used in this test. The gene for the yellow body lies on the X-chromosome. The fruit flies are fed on a diet of test chemical, and progenies are separated by sex. The surviving males are crossed with the females of the same generation, and if no males with yellow bodies are detected in the second generation, it would indicate a lethal mutation on the X-chromosome has occurred.


Plants such as  Zea mays ,  Arabidopsis thaliana  and  Tradescantia  have been used in various test assays for mutagenecity of chemicals.


Mammalian cell lines such as Chinese hamster V79 cells,  Chinese hamster ovary  (CHO) cells or mouse lymphoma cells may be used to test for mutagenesis. Such systems include the  HPRT  assay  for resistance to 8-azaguanine or  6-thioguanine , and  ouabain -resistance (OUA) assay .


Rat primary hepatocytes may also be used to measure DNA repair following DNA damage. Mutagens may stimulate unscheduled DNA synthesis that results in more stained nuclear material in cells following exposure to mutagens.


These systems check for large scale changes to the chromosomes and may be used with cell culture or in animal test. The chromosomes are stained and observed for any changes.  Sister chromatid exchange  is a symmetrical exchange of chromosome material between sister chromatids and may be correlated to the mutagenic or carcinogenic potential of a chemical. In  micronucleus Test , cells are examined for micronuclei, which are fragments or chromosomes left behind at anaphase, and is therefore a test for clastogenic agents that cause chromosome breakages. Other tests may check for various chromosomal aberrations such as chromatid and chromosomal gaps and deletions, translocations, and ploidy.


Rodents  are usually used in  animal test . The chemicals under
test are usually administered in the food and in the drinking water, but sometimes by dermal application, by  gavage , or by inhalation, and carried out over the major part of the life span for rodents. In tests that check for carcinogens, maximum tolerated dosage is first determined, then a range of doses are given to around 50 animals throughout the notional lifespan of the animal of two years. After death the animals are examined for sign of tumours. Differences in metabolism between rat and human however means that human may not respond in exactly the same way to mutagen, and dosages that produce tumours on the animal test may also be unreasonably high for a human, i.e. the equivalent amount required to produce tumours in human may far exceed what a person might encounter in real life.


Mice with recessive mutations for a visible phenotype may also be used to check for mutagens. Females with recessive mutation crossed with wild-type males would yield the same phenotype as the wild-type, and any observable change to the phenotype would indicate that a mutation induced by the mutagen has occurred.


Mice may also be used for  dominant lethal assays  where early embryonic deaths are monitored. Male mice are treated with chemicals under test, mated with females, and the females are then sacrificed before parturition and early fetal deaths are counted in the  uterine horns .


Transgenic mouse  assay  using a mouse strain infected with a viral  shuttle vector  is another method for testing mutagens. Animals are first treated with suspected mutagen, the mouse DNA is then isolated and the phage segment recovered and used to infect  E. coli . Using similar method as the  blue-white screen , the plaque formed with DNA containing mutation are white, while those without are blue.


Many mutagens are highly toxic to proliferating cells, and they are often used to destroy cancer cells. Alkylating agents such as  cyclophosphamide  and  cisplatin , as well as intercalating agent such as  daunorubicin  and  doxorubicin  may be used in  chemotherapy . However, due to their effect on other cells which are also rapidly dividing, they may have side effects such as hair loss and nausea. Research on better targeted therapies may reduce such side-effects. Ionizing radiations are used in  radiation therapy .


In  science fiction , mutagens are often represented as substances that are capable of completely changing the form of the recipient or granting them superpowers. Powerful radiations are the agents of mutation for the  superheroes  in  Marvel Comics 's  Fantastic Four ,  Daredevil , and  Hulk , while in the  Ninja Turtles  franchise the MUTAGEN  "ooze" for  Inhumans  the mutagen is the  Terrigen Mist . Mutagens are also featured in video games such as  Cyberia ,   System Shock ,  The Witcher ,  Metroid Prime: Trilogy ,  Resistance: Fall of Man ,  Resident Evil ,  Infamous ,   Freedom Force ,  Command & Conquer ,  Gears of War 3 ,  StarCraft ,  BioShock ,  Fallout ,  Underrail , and  Maneater .
In the "nuclear monster" films of the 1950s, nuclear radiation mutates humans and common insects often to enormous size and aggression; these films include  Godzilla ,  Them! ,  Attack of the 50 Foot Woman ,  Tarantula! , and  The Amazing Colossal Man .


In  biology , and especially in  genetics , a  mutant  is an organism or a new genetic character arising or resulting from an instance of  mutation , which is generally an alteration of the  DNA  sequence of the  genome  or  chromosome  of an  organism . It is a characteristic that would not be observed naturally in a specimen. The term mutant is also applied to a  virus  with an alteration in its  nucleotide sequence  whose  genome  is in the  nuclear genome . The natural occurrence of genetic mutations is integral to the process of  evolution . The study of mutants is an integral part of biology; by understanding the effect that a mutation in a gene has, it is possible to establish the normal function of that gene. [ 2 ]


Mutants arise by  mutations  occurring in pre-existing  genomes  as a result of errors of  DNA replication  or errors of  DNA repair .  Errors of replication often involve  translesion synthesis  by a  DNA polymerase  when it encounters and bypasses a damaged base in the template strand. [ 3 ]   A  DNA damage  is an abnormal chemical structure in DNA, such as a strand break or an oxidized base, whereas a mutation, by contrast, is a change in the sequence of standard base pairs. Errors of repair occur when repair processes inaccurately replace a damaged DNA sequence.  The DNA repair process  microhomology-mediated end joining  is particularly error-prone. [ 4 ] [ 5 ]


Although not all mutations have a noticeable  phenotypic  effect, the common usage of the word "mutant" is generally a  pejorative  term, only used for genetically or phenotypically noticeable mutations. [ 6 ]  Previously, people used the word " sport " (related to  spurt ) to refer to abnormal specimens. The scientific usage is broader, referring to any organism differing from the  wild type . The word finds its origin in the Latin term mūtant- (stem of mūtāns), which means "to change". [ 6 ]


Mutants should not be confused with organisms born with  developmental abnormalities , which are caused by errors during  morphogenesis . In a developmental abnormality, the DNA of the organism is unchanged and the abnormality cannot be passed on to progeny.  Conjoined twins  are the result of developmental abnormalities.


Chemicals that cause developmental abnormalities are called  teratogens ; these may also cause mutations, but their effect on development is not related to mutations.  Chemicals that induce mutations are called  mutagens . Most mutagens are also considered to be  carcinogens .


Mutations  are distinctly different from  epigenetic  alterations, although they share some common features. Both arise as a  chromosomal  alteration that can be replicated and passed on to subsequent cell generations. Both, when occurring within a gene, may silence expression of the gene.  Whereas mutant cell lineages arise as a change in the sequence of standard bases, epigenetically altered cell lineages retain the sequence of standard bases but have gene sequences with changed levels of expression that can be passed down to subsequent cell generations.  Epigenetic alterations include  methylation  of  CpG islands  of a gene  promoter  as well as specific chromatin histone modifications.  Faulty repair of chromosomes at sites of DNA damage can give rise both to mutant cell lineages [ 4 ]  and/or  epigenetically altered  cell lineages. [ 7 ]


A  mutant protein  is the  protein  product encoded by a  gene  with  mutation . [ 1 ]  Mutated protein can have single amino acid change (minor, but still in many cases significant change leading to disease) or wide-range amino acid changes by e.g. truncation of C-terminus after introducing premature stop codon.


Mutation bias  refers to a predictable or systematic difference in rates for different types of  mutation . The types are most often defined by the molecular nature of the mutational change, but sometimes they are based on downstream effects, e.g., Ostrow, et al.  [ 1 ]  refer to "mutational bias for body size".


The concept of mutation bias appears in several scientific contexts, most commonly in molecular studies of evolution, where mutation biases may be invoked to account for such phenomena as  systematic differences in codon usage  or genome composition between species. [ 2 ]   The  short tandem repeat  (STR) loci used in forensic identification may show biased patterns of gain and loss of repeats. [ 3 ]   In cancer research, some types of tumors have distinctive  mutational signatures  that reflect differences in the contributions of mutational pathways.  Mutational signatures have proved useful in both detection and treatment.


Recent studies of the emergence of resistance to anti-microbials and anti-cancer drugs show that mutation biases are an important determinant of the prevalence for different types of resistant strains or tumors. [ 4 ] [ 5 ]   Thus, a knowledge of mutation bias can be used to design more evolution-resistant therapies. [ 4 ]


When mutation bias is invoked as a possible cause of some pattern in evolution, this is generally an application of the  theory of arrival biases , and the alternative hypotheses may include selection, biased gene conversion, and demographic factors.  Evidence for an evolutionary impact of mutation biases on changes involved in adaptation is summarized in the   Arrival Bias  article (note that  [ 6 ]  argued in 2019 that this line of argument is flawed and that apparently mutation-biased patterns of change are better explained by selection).


In the past, due to the technical difficulty of detecting rare mutations, most attempts to characterize the mutation spectrum were based on reporter gene systems, or based on patterns of presumptively neutral change in pseudogenes.  More recently, there has been an effort to use the MA (mutation accumulation) method and high-throughput sequencing (e.g., [ 7 ]  ).


The canonical DNA nucleotides include 2  purines  (A and G) and 2  pyrimidines  (T and C).  In the  molecular evolution  literature, the term  transition  is used for nucleotide changes within a chemical class, and  transversion  for changes from one chemical class to the other.  Each nucleotide is subject to one transition (e.g., T to C) and 2 transversions (e.g., T to A or T to G).


Because a site (or a sequence) is subject to twice as many transversions as transitions, the total rate of transversions for a sequence may be higher even when the rate of transitions is higher on a per-path basis.  In the molecular evolution literature, the per-path rate bias is typically denoted by  κ  (kappa), so that, if the rate of each transversion is  u , the rate of each transition is  κu .  Then, the aggregate rate ratio (transitions to transversions) is  R = (1 * κu) / (2 * u) = κ / 2 .  For instance, in yeast,  κ ~ 1.2 , [ 8 ]  therefore the aggregate bias is  R = 1.2 / 2 = 0.6 , whereas in E. coli,  κ ~ 4  so that  R ~ 2 .


In a variety of organisms, transition mutations occur several-fold more frequently than expected under uniformity. [ 9 ]   The bias in animal viruses is sometimes much more extreme, e.g., 31 of 34 nucleotide mutations in a recent study in HIV were transitions. [ 10 ]   As noted above, the bias toward transitions is weak in yeast, and appear to be absent in the grasshopper  Podisma pedestris . [ 11 ]


Male mutation bias is also called "Male-Driven Evolution". The rate of male germline mutations is generally higher than in females. [ 12 ]  The phenomenon of Male mutation bias have been observed in many species. [ 13 ]


In 1935, the British-Indian scientist J.B.S. Haldane found that in hemophilia, the blood clotting disorder originated on the X chromosomes is due to fathers' germline mutation. [ 14 ]  Then he proposed the hypothesis that the male germline contributes inordinately more mutations to succeeding generations than that in the female  germline mutation . [ 15 ]


In 1987, Takashi Miyata at al. designed an approach to test Haldane’s hypothesis. [ 16 ]  If α is the ratio of the male mutation rate to the female mutation rate, Y and X are denoted as Y and X-linked sequence mutation rate, he include that the ratio of Y-linked sequence mutation rate to X-linked sequence mutation rate is:


Y

 /

 X
 =

 3
 α

 2
 +
 α

 {\displaystyle Y/X={\frac {3\alpha }{2+\alpha }}}


The mean Y/X ratio is 2.25 in higher primates. [ 17 ]  By using the equation, we could estimate the ratio of male to female mutation rates α ≈ 6. In some organisms with a shorter generation time than humans, the mutation rate in males is also larger than those in females. Because their cell divisions in males are usually not that large. The ratio of the number of germ cell divisions from one generation to the next in males to females is less than that in human. [ 18 ] [ 19 ] [ 20 ]


There are also other hypotheses that want to explain the male mutation bias. They think it may be caused by the mutation rate in the Y-linked sequence higher than the X-linked sequence mutation rate. The male germline genome is heavily methylated and more inclined to mutate than females. X chromosomes experience more purifying selection mutations on hemizygous chromosomes. [ 21 ]  To test this hypothesis, people use birds to study their mutation rate. [ 22 ] [ 23 ]  Contrary to humans, bird males are homogametes (WW), and females are heterogametes (WZ). They found that the bird male-to-female ratio in mutation rates ranges from 4 to 7. [ 24 ]  It also proved that the mutation bias is mostly resulted from more male germline mutation than the female.


A  mutation  is a heritable variation in the genetic information of a short region of DNA sequences. Mutations can be categorized into replication-dependent mutations and replication-independent mutations. Therefore, there are two kinds of mutation mechanisms to explain the phenomenon of male mutation bias.


The number of  germ cell  divisions in females are constant and are much less than that in males. In females, most primary oocytes are formed at birth. The number of cell divisions occurred in the production of a mature ovum is constant. In males, more cell divisions are required during the process of  spermatogenesis . Not only that, the cycle of spermatogenesis is never-ending. Spermatogonia will continue to divide throughout the whole productive life of the male. The number of male  germline  cell divisions at production is not only higher than female germline cell divisions but also mounting as the age of the male increases. [ 25 ]


One might expect the male mutation rate would be similar to the rate of male germline cell divisions. But only few species conform to the estimation of the male mutation rate. [ 20 ]  Even in these species, the ratio of male-to-female mutation rate is lower than the ratio of male-to-female in the number of germline cell divisions. [ 26 ]


The skew estimates of the male-to-female mutation rate ratio introduce the other important mechanism that highly influences male mutation bias. Mutations at  CpG sites  result in a C-to-T transition. [ 27 ]  These C-to-T nucleotide substitutions occur 10-50 times faster than that at rest sites in DNA sequences, especially likely appeared in the male and female germlines. [ 28 ]  The CpG mutation barely expresses any sex biases because of the independence of replication, and effectively lower the ratio of male-to-female mutation rate. [ 29 ]  Besides, neighbor-dependent mutations can also cause biases in mutation rate, and may have no relevance to DNA replication. For example, if mutations originated by the effect of mutagens show weak male mutation bias, such as exposure to the UV light. [ 30 ]


A GC-AT bias is a bias with a net effect on GC content.  For instance, if G and C sites are simply more mutable than A and T sites, other things being equal, this would result in a net downward pressure on GC content.  Mutation-accumulation studies indicate a strong many-fold bias toward AT in mitochondria of  D. melanogaster , [ 31 ]  and a more modest 2-fold bias toward AT in yeast. [ 8 ]


A common idea in the literature of molecular evolution is that codon usage and genome composition reflect the effects of mutation bias, e.g., codon usage has been treated with a mutation-selection-drift model combining mutation biases, selection for translationally preferred codons, and drift. To the extent that mutation bias prevails under this model, mutation bias toward GC is responsible for genomes with high GC content, and likewise the opposite bias is responsible for genomes with low GC content. [ 32 ]


Starting in the 1990s, it became clear that GC-biased  gene conversion  was a major factor—previously unanticipated—in affecting GC content in diploid organisms such as mammals. [ 33 ]


Similarly, although it may be the case that bacterial genome composition strongly reflects GC and AT biases, the proposed mutational biases have not been demonstrated to exist.  Indeed, Hershberg and Petrov suggest that mutation in most bacterial genomes is biased toward AT, even when the genome is not AT-rich. [ 2 ]


Mutation biases are not constant, but vary taxonomically, as shown in the table below from, [ 39 ]  and by conditions such as nutritional state. [ 40 ]


The concept of mutation bias, as defined above, does not imply foresight, design, or even a specially evolved tendency, e.g., the bias may emerge simply as a side-effect of DNA repair processes.  Currently there is no established terminology for mutation-generating systems that tend to produce useful mutations.  The term "directed mutation" or  adaptive mutation  is sometimes used with the implication of a process of mutation that senses and responds to conditions directly.  When the sense is simply that the mutation system is tuned to enhance the production of helpful mutations under certain conditions, the terminology of "mutation strategies"  [ 41 ]  or " natural genetic engineering " [ 42 ]   has been suggested, but these terms are not widely used. As argued in Ch. 5 of Stoltzfus 2021, [ 43 ]  various mechanisms of mutation in pathogenic microbes, e.g., mechanisms for  phase variation  and  antigenic variation , appear to have evolved so as to enhance lineage survival, and these mechanisms are routinely described as strategies or adaptations in the microbial genetics literature, such as by Foley 2015. [ 44 ]


Mutation Frequency Decline  ( mfd ) is the gene which encodes the protein Mfd (also known as Transcription Repair Coupling Factor, TRCF). Mfd functions in  transcription-coupled repair  to remove a stalled  RNA polymerase  that has encountered DNA damage and is unable to continue translocating.


The protein is named after the fact that it reduces the chances of suppressor mutations in UV-irradiated cells (or rather, knockout cells show higher rates of such mutations). It does not reduce the chance of every kind of mutation. [ 1 ]  In fact, it seems to increase the chances of mutation in general, helping to evolve new  traits such as antimicrobial resistance. [ 2 ]


Mfd utilizes  ATP  to translocate along DNA, most likely forcing RNA polymerase forward and ultimately dissociating it from the DNA template. [ 3 ]   Mfd also contains binding domains which recruit  UvrA  and trigger the associated  nucleotide excision repair  pathway and was initially discovered when its mutation led to a decrease in mutation rates after irradiation by UV light. Structural studies of  E. coli  Mfd by  X-ray crystallography  have revealed that this molecule is autoinhibited for UvrA-binding in its apo form due to a "clamp" interaction between the  N-terminal  UvrB-homology module and the  C-terminal  domain. [ 4 ] [ 5 ]


In 2002, it was shown that Mfd may also re-initiate transcription at backtracked RNAP by forcing the polymerase forward and out of its backtracked state. [ 6 ]


In 2015, Merrikh Lab at  University of Washington  discovered that Mfd quickens the bacterial mutation process. [ 2 ]  This work researches ways to slow the rate of bacterial mutations and to block their evolution, in order to fight against  antibiotic resistance . [ 7 ]


In 2022, a small molecule inhibitor of Mfd was identified by the Merrikh lab. As expected, it slowed down the evolution of antibiotic resistance. [ 8 ]


Animal immune systems try to kill bacteria in a number of ways, one being the release of nitrogen monoxide (NO). NO damages bacterial DNA, but some species can survive this attack by expressing  Mfd . [ 9 ]


In  genetics , the  mutation rate  is the frequency of new  mutations  in a single  gene ,  nucleotide sequence , or organism over time. [ 2 ]  Mutation rates are not constant and are not limited to a single type of mutation; there are many different types of mutations. Mutation rates are given for specific classes of mutations.  Point mutations  are a class of mutations which are changes to a single base.  Missense ,  nonsense , and  synonymous mutations  are three subtypes of point mutations. The rate of these types of substitutions can be further subdivided into a mutation spectrum which describes the influence of the genetic context on the mutation rate. [ 3 ]


There are several natural units of time for each of these rates, with rates being characterized either as mutations per base pair per cell division, per gene per generation, or per genome per generation. The mutation rate of an organism is an evolved characteristic and is strongly influenced by the genetics of each organism, in addition to strong influence from the environment. The upper and lower limits to which mutation rates can evolve is the subject of ongoing investigation. However, the mutation rate does vary over the  genome . [ 4 ]


When the mutation rate in humans increases certain health risks can occur, for example,  cancer  and other hereditary diseases. Having knowledge of mutation rates is vital to understanding the future of cancers and many hereditary diseases. [ 5 ]


Different genetic variants within a species are referred to as alleles, therefore a new mutation can create a new allele. In  population genetics , each allele is characterized by a selection coefficient, which measures the expected change in an allele's frequency over time. The selection coefficient can either be negative, corresponding to an expected decrease, positive, corresponding to an expected increase, or zero, corresponding to no expected change. The distribution of fitness effects of new mutations is an important parameter in population genetics and has been the subject of extensive investigation. [ 6 ]  Although measurements of this distribution have been inconsistent in the past, it is now generally thought that the majority of mutations are mildly deleterious, that many have little effect on an organism's fitness, and that a few can be favorable.


Because of  natural selection , unfavorable mutations will typically be eliminated from a population while favorable changes are generally kept for the next generation, and neutral changes accumulate at the rate they are created by mutations. This process happens by reproduction. In a particular generation the 'best fit' survive with higher probability, passing their genes to their offspring. The sign of the change in this probability defines mutations to be beneficial, neutral or harmful to organisms. [ 7 ]


An organism's mutation rates can be measured by a number of techniques.


One way to measure the mutation rate is by the fluctuation test, also known as the  Luria–Delbrück experiment . This experiment demonstrated that bacteria mutations occur in the absence of selection instead of the presence of selection. [ 8 ]


This is very important to mutation rates because it proves experimentally mutations can occur without selection being a component—in fact, mutation and selection are completely distinct  evolutionary forces . Different DNA sequences can have different propensities to mutation (see below) and may not occur randomly. [ 9 ]


The most commonly measured class of mutations are substitutions, because they are relatively easy to measure with standard analyses of DNA sequence data. However substitutions have a substantially different rate of mutation (10 −8  to 10 −9  per generation for most cellular organisms) than other classes of mutation, which are frequently much higher (~10 −3  per generation for satellite DNA expansion/contraction [ 10 ] ).


Many sites in an organism's genome may admit mutations with small fitness effects. These sites are typically called neutral sites. Theoretically mutations under no selection become  fixed  between organisms at precisely the mutation rate. Fixed synonymous mutations, i.e.  synonymous substitutions , are changes to the sequence of a gene that do not change the protein produced by that gene. They are often used as estimates of that mutation rate, despite the fact that some synonymous mutations have fitness effects. As an example, mutation rates have been directly inferred from the whole genome sequences of experimentally evolved replicate lines of  Escherichia coli  B. [ 11 ]


A particularly labor-intensive way of characterizing the mutation rate is the mutation accumulation line.


Mutation accumulation lines have been used to characterize mutation rates with the  Bateman-Mukai Method  and direct sequencing of well-studied experimental organisms ranging from intestinal bacteria ( E. coli ), roundworms ( C. elegans ), yeast ( S. cerevisiae ), fruit flies ( D. melanogaster ), and small ephemeral plants ( A. thaliana ). [ 12 ]


Mutation rates differ between species and even between different regions of the genome of a single species. Mutation rates can also differ even between genotypes of the same species; for example, bacteria have been observed to evolve hypermutability as they adapt to new selective conditions. [ 13 ]  These different rates of nucleotide substitution are measured in substitutions ( fixed mutations ) per base pair per generation. For example, mutations in intergenic, or non-coding, DNA tend to accumulate at a faster rate than mutations in DNA that is actively in use in the organism ( gene expression ). That is not necessarily due to a higher mutation rate, but to lower levels of  purifying selection . A region which mutates at predictable rate is a candidate for use as a  molecular clock .


If the rate of neutral mutations in a sequence is assumed to be constant (clock-like), and if most differences between species are neutral rather than adaptive, then the number of differences between two different species can be used to estimate how long ago two species diverged (see  molecular clock ). In fact, the mutation rate of an organism may change in response to environmental stress. For example, UV light damages DNA, which may result in error prone attempts by the cell to perform  DNA repair .


The human mutation rate is higher in the male germ line (sperm) than the female (egg cells), but estimates of the exact rate have varied by an order of magnitude or more. This means that a human genome accumulates around 64 new mutations per generation because each full generation involves a number of cell divisions to generate gametes. [ 14 ]  Human mitochondrial DNA has been estimated to have mutation rates of ~3× or ~2.7×10 −5  per base per 20 year generation (depending on the method of estimation); [ 15 ]  these rates are considered to be significantly higher than rates of human genomic mutation at ~2.5×10 −8  per base per generation. [ 16 ]  Using data available from whole genome sequencing, the human genome mutation rate is similarly estimated to be ~1.1×10 −8  per site per generation. [ 17 ]


The rate for other forms of mutation also differs greatly from  point mutations . An individual  microsatellite  locus often has a mutation rate on the order of 10 −4 , though this can differ greatly with length. [ 18 ]


Some sequences of DNA may be more susceptible to mutation. For example, stretches of DNA in human sperm which lack methylation are more prone to mutation. [ 19 ]


In general, the mutation rate in unicellular  eukaryotes  (and  bacteria ) is roughly 0.003 mutations per genome per  cell  generation. [ 14 ]  However, some species, especially the  ciliate  of the genus  Paramecium  have an unusually low mutation rate. For instance,  Paramecium tetraurelia  has a base-substitution mutation rate of ~2 × 10 −11  per site per cell division. This is the lowest mutation rate observed in nature so far, being about 75× lower than in other eukaryotes with a similar genome size, and even 10× lower than in most prokaryotes. The low mutation rate in  Paramecium  has been explained by its transcriptionally silent  germ-line   nucleus , consistent with the hypothesis that replication fidelity is higher at lower  gene expression  levels. [ 20 ]


The highest per base pair per generation mutation rates are found in viruses, which can have either RNA or DNA genomes. DNA viruses have mutation rates between 10 −6  to 10 −8  mutations per base per generation, and RNA viruses have mutation rates between 10 −3  to 10 −5  per base per generation. [ 14 ]


A mutation spectrum is a distribution of rates or frequencies for the mutations relevant in some context, based on the recognition that rates of occurrence are not all the same. In any context, the mutation spectrum reflects the details of mutagenesis and is affected by conditions such as the presence of chemical mutagens or genetic backgrounds with mutator alleles or damaged DNA repair systems. The most fundamental and expansive concept of a mutation spectrum is the distribution of rates for all individual mutations that might happen in a genome (e.g.,  [ 21 ] ).  From this full de novo spectrum, for instance, one may calculate the relative rate of mutation in  coding  vs  non-coding  regions.  Typically the concept of a spectrum of mutation rates is simplified to cover broad classes such as  transitions  and  transversions  (figure), i.e., different mutational conversions across the genome are aggregated into classes, and there is an aggregate rate for each class.


In many contexts, a mutation spectrum is defined as the observed frequencies of mutations identified by some selection criterion, e.g., the distribution of mutations associated clinically with a particular type of cancer, [ 22 ]  or the distribution of adaptive changes in a particular context such as antibiotic resistance (e.g.,
 [ 23 ]
).
Whereas the spectrum of de novo mutation rates reflects mutagenesis alone, this kind of spectrum may also reflect effects of selection and ascertainment biases (e.g., both kinds of spectrum are used in  [ 24 ] ).


The theory on the evolution of mutation rates identifies three principal forces involved: the generation of more deleterious mutations with higher mutation, the generation of more advantageous mutations with higher mutation, and the metabolic costs and reduced replication rates that are required to prevent mutations. Different conclusions are reached based on the relative importance attributed to each force. The optimal mutation rate of organisms may be determined by a trade-off between costs of a high mutation rate, [ 25 ]  such as deleterious mutations, and the  metabolic  costs of maintaining systems to reduce the mutation rate (such as increasing the expression of DNA repair enzymes. [ 26 ]  or, as reviewed by Bernstein et al. [ 27 ]  having increased energy use for repair, coding for additional gene products and/or having slower replication). Secondly, higher mutation rates increase the rate of beneficial mutations, and evolution may prevent a lowering of the mutation rate in order to maintain optimal rates of adaptation. [ 28 ] [ 29 ]  As such, hypermutation enables some cells to rapidly adapt to changing conditions in order to avoid the entire population from becoming extinct. [ 30 ]  Finally, natural selection may fail to optimize the mutation rate because of the relatively minor benefits of lowering the mutation rate, and thus the observed mutation rate is the product of neutral processes. [ 31 ] [ 32 ]


Studies have shown that treating  RNA viruses  such as  poliovirus  with  ribavirin  produce results consistent with the idea that the viruses mutated too frequently to maintain the integrity of the information in their genomes. [ 33 ]  This is termed  error catastrophe .


The characteristically high mutation rate of  HIV  (Human Immunodeficiency Virus) of 3 x 10 −5  per base and generation, coupled with its short replication cycle leads to a high  antigen  variability, allowing it to evade the immune system. [ 34 ]


Mutation–selection balance  is an equilibrium in the number of deleterious  alleles  in a population that occurs when the rate at which deleterious alleles are created by  mutation  equals the rate at which deleterious alleles are eliminated by  selection . [ 1 ] [ 2 ] [ 3 ] [ 4 ]  The majority of genetic mutations are neutral or deleterious; beneficial mutations are relatively rare. The resulting influx of deleterious mutations into a population over time is counteracted by  negative selection , which acts to purge deleterious mutations. Setting aside other factors (e.g.,  balancing selection , and  genetic drift ), the equilibrium number of deleterious alleles is then determined by a balance between the deleterious mutation rate and the rate at which selection purges those mutations.


Mutation–selection balance was originally proposed to explain how  genetic variation  is maintained in populations, although several other ways for deleterious mutations to persist are now recognized, notably  balancing selection . [ 3 ]  Nevertheless, the concept is still widely used in  evolutionary genetics , e.g. to explain the persistence of deleterious alleles as in the case of  spinal muscular atrophy , [ 5 ] [ 4 ]  or, in theoretical models, mutation-selection balance can appear in a variety of ways and has even been applied to beneficial mutations (i.e. balance between selective loss of variation and creation of variation by beneficial mutations). [ 6 ]


As a simple example of mutation-selection balance, consider a single  locus  in a  haploid  population with two possible alleles: a normal allele  A  with  frequency

 p

 {\displaystyle p}

 , and a mutated deleterious allele  B  with frequency

 q

 {\displaystyle q}

 , which has a small relative  fitness  disadvantage of

 s

 {\displaystyle s}

 . Suppose that deleterious mutations from  A  to  B  occur at rate

 μ

 {\displaystyle \mu }

 , and the reverse beneficial mutation from  B  to  A  occurs rarely enough to be negligible (e.g. because the mutation rate is so low that

 q

 {\displaystyle q}

  is small). Then, each generation selection eliminates deleterious mutants reducing

 q

 {\displaystyle q}

  by an amount

 s
 p
 q

 {\displaystyle spq}

 , while mutation creates more deleterious alleles increasing

 q

 {\displaystyle q}

  by an amount

 μ
 p

 {\displaystyle \mu p}

 . Mutation–selection balance occurs when these forces cancel and

 q

 {\displaystyle q}

  is constant from generation to generation, implying

 q
 =
 μ

 /

 s

 {\displaystyle q=\mu /s}

 . [ 3 ]  Thus, provided that the mutant allele is not weakly deleterious (very small

 s

 {\displaystyle s}

 ) and the mutation rate is not very high, the equilibrium frequency of the deleterious allele will be small.


In a  diploid  population, a deleterious allele  B  may have different effects on individual fitness in heterozygotes  AB  and homozygotes  BB  depending on the degree of dominance of the normal allele  A . To represent this mathematically, let the relative fitness of deleterious  homozygotes  and  heterozygotes  be smaller than that of normal homozygotes  AA  by factors of

 1
 −
 h
 s

 {\displaystyle 1-hs}

  and

 1
 −
 s

 {\displaystyle 1-s}

  respectively, where

 h

 {\displaystyle h}

  is a number between

 0

 {\displaystyle 0}

  and

 1

 {\displaystyle 1}

  measuring the degree of dominance (

 h
 =
 0

 {\displaystyle h=0}

  indicates that  A  is completely dominant while

 h
 =
 1

 /

 2

 {\displaystyle h=1/2}

  indicates no dominance). For simplicity, suppose that  mating is random .


The degree of dominance affects the relative importance of selection on heterozygotes versus homozygotes. If  A  is not completely dominant (i.e.

 h

 {\displaystyle h}

  is not close to zero), then deleterious mutations are primarily removed by selection on heterozygotes because heterozygotes contain the vast majority of deleterious  B  alleles (assuming that the deleterious mutation rate

 μ

 {\displaystyle \mu }

  is not very large). This case is approximately equivalent to the preceding haploid case, where mutation converts normal homozygotes to heterozygotes at rate

 μ

 {\displaystyle \mu }

  and selection acts on heterozygotes with selection coefficient

 h
 s

 {\displaystyle hs}

 ; thus

 q
 ≈
 μ

 /

 h
 s

 {\displaystyle q\approx \mu /hs}

 . [ 1 ]


In the case of complete dominance (

 h
 =
 0

 {\displaystyle h=0}

 ), deleterious alleles are only removed by selection on  BB  homozygotes. Let

 p

 A
 A

 {\displaystyle p_{AA}}

 ,

 2

 p

 A
 B

 {\displaystyle 2p_{AB}}

  and

 p

 B
 B

 {\displaystyle p_{BB}}

  be the frequencies of the corresponding  genotypes . The frequency

 p
 =

 p

 A
 A

 +

 p

 A
 B

 {\displaystyle p=p_{AA}+p_{AB}}

  of normal alleles  A  increases at rate

 1

 /

 (
 1
 −
 s

 p

 B
 B

 )

 {\displaystyle 1/(1-sp_{BB})}

  due to the selective elimination of recessive homozygotes, while mutation causes

 p

 {\displaystyle p}

  to decrease at rate

 1
 −
 μ

 {\displaystyle 1-\mu }

  (ignoring  back mutations ). Mutation–selection balance then gives

 p

 B
 B

 =
 μ

 /

 s

 {\displaystyle p_{BB}=\mu /s}

 , and so the frequency of deleterious alleles is

 q
 =

 μ

 /

 s

 {\displaystyle q={\sqrt {\mu /s}}}

 . [ 1 ]  This equilibrium frequency is potentially substantially larger than for the case of partial dominance, because a large number of mutant alleles are carried in heterozygotes and are shielded from selection.


Many properties of a non random mating population can be explained by a random mating population whose  effective population size  is adjusted. However, in non-steady state population dynamics there can be a lower prevalence for recessive disorders in a random mating population during and after a growth phase. [ 7 ] [ 8 ]


The first paper on the subject was ( Haldane , 1935), which used the prevalence and fertility ratio of  haemophilia  in males to estimate mutation rate in human genes. [ 9 ] [ 10 ]


The prevalence of hemophilia among males is

 p
 ∈
 [
 4
 ,
 17
 ]
 ×

 10

 −
 5

 {\displaystyle p\in [4,17]\times 10^{-5}}

 . The fertility ratio of males with hemophilia to males without hemophilia is

 f
 ∈
 [
 0.1
 ,
 0.25
 ]

 {\displaystyle f\in [0.1,0.25]}

 , where

 f
 =

 #
 (

 offsprings of abnormal allele)

 #
 (

 offsprings of normal allele)

 {\displaystyle f={\frac {\#({\text{offsprings of abnormal allele)}}}{\#({\text{offsprings of normal allele)}}}}}

 .


Assuming hemophilia is purely due to mutations on the X chromosome, the mutation rate can be estimated as follows.


At mutation-selection balance, the rate of new hemophilia cases due to mutations should be equal to the rate of hemophilia cases lost due to the lower fitness of hemophilia patients. Since every male has one X chromosome, the rate of new hemophilia cases due to mutations is

 μ

 {\displaystyle \mu }

 . On the other hand, the relative fitness of hemophilia patients is

 f

 {\displaystyle f}

 , so

 (
 1
 −
 f
 )

 {\displaystyle (1-f)}

  times the existing hemophilia cases are lost every generation due to selection. The mutation-selection balance thus gives

 μ
 =
 (
 1
 −
 f
 )
 p
 .

 {\displaystyle \mu =(1-f)p.}

However, since females have two X chromosomes, only about 1/3 of the new mutations would appear in males (assuming an equal sex ratio at birth). Thus, the equation

 μ
 ≈
 (
 1
 −
 f
 )
 p

 /

 3
 ∈
 [
 1
 ,
 5
 ]
 ×

 10

 −
 5

 ,

 {\displaystyle \mu \approx (1-f)p/3\in [1,5]\times 10^{-5},}

is obtained, where the numerical range was obtained by plugging in the ranges for

 p

 {\displaystyle p}

  and

 f

 {\displaystyle f}

 . Subsequent research using different methods showed that the mutation rate in many genes is indeed on the order of

 10

 −
 5

 {\displaystyle 10^{-5}}

  per generation.


Mutationism  is one of several  alternatives to evolution by natural selection  that have existed both before and after the publication of  Charles Darwin 's 1859 book  On the Origin of Species . In the theory, mutation was the source of novelty, creating new forms and new species, potentially instantaneously, [ 1 ]  in sudden jumps. [ 2 ]  This was envisaged as driving evolution, which was thought to be limited by the supply of mutations.


Before Darwin, biologists commonly believed in  saltationism , the possibility of large evolutionary jumps, including immediate  speciation . For example, in 1822  Étienne Geoffroy Saint-Hilaire  argued that species could be formed by sudden transformations, or what would later be called macromutation. Darwin opposed saltation, insisting on  gradualism  in evolution as  geology's uniformitarianism . In 1864,  Albert von Kölliker  revived Geoffroy's theory. In 1901 the  geneticist   Hugo de Vries  gave the name "mutation" to seemingly new forms that suddenly arose in his experiments on the evening primrose  Oenothera lamarckiana . In the first decade of the 20th century, mutationism, or as de Vries named it  mutationstheorie , became a rival to Darwinism supported for a while by geneticists including  William Bateson ,  Thomas Hunt Morgan , and  Reginald Punnett .


Understanding of mutationism is clouded by the mid-20th century portrayal of the early mutationists by supporters of the  modern synthesis  as opponents of Darwinian evolution and rivals of the biometrics school who argued that selection operated on continuous variation. In this portrayal, mutationism was defeated by a synthesis of genetics and natural selection that supposedly started later, around 1918, with work by the mathematician  Ronald Fisher . However, the alignment of Mendelian genetics and natural selection began as early as 1902 with a paper by  Udny Yule , and built up with theoretical and experimental work in Europe and America. Despite the controversy, the early mutationists had by 1918 already accepted natural selection and explained continuous variation as the result of multiple genes acting on the same characteristic, such as height.


Mutationism, along with other alternatives to Darwinism like  Lamarckism  and  orthogenesis , was discarded by most biologists as they came to see that  Mendelian genetics and natural selection could readily work together ; mutation took its place as a source of the genetic variation essential for natural selection to work on.  However, mutationism did not entirely vanish. In 1940,  Richard Goldschmidt  again argued for single-step speciation by macromutation, describing the organisms thus produced as "hopeful monsters", earning widespread ridicule. In 1987,  Masatoshi Nei  argued controversially that evolution was often mutation-limited. Modern biologists such as  Douglas J. Futuyma  conclude that essentially all claims of evolution driven by large mutations can be explained by Darwinian evolution.


Prior to  Charles Darwin , most naturalists were  saltationists , [ a ]  believing that species evolved and that  speciation  took place in sudden jumps. [ 4 ]   Jean-Baptiste Lamarck  was a gradualist but similar to other scientists of the period had written that saltational evolution was possible. [ 5 ]


In 1822, in the second volume of his  Philosophie anatomique ,  Étienne Geoffroy Saint-Hilaire  endorsed a theory of saltational evolution that "monstrosities could become the founding fathers (or mothers) of new species by instantaneous transition from one form to the next." [ 6 ]  Geoffroy wrote that environmental pressures could produce sudden transformations to establish new  species  instantaneously. [ 7 ]


In his 1859 book  On the Origin of Species ,  Charles Darwin  denied saltational evolution. He argued that evolutionary transformation always proceeds gradually, never in jumps: " natural selection  acts solely by accumulating slight successive favourable variations, it can produce no great or sudden modification; it can act only by very short steps". Darwin continued in this belief throughout his life. [ 8 ]


Thomas Henry Huxley  warned Darwin that he had taken on "an unnecessary difficulty in adopting  Natura non facit saltum  ["Nature does not take leaps"] so unreservedly." [ 9 ]  Huxley feared this assumption could discourage naturalists ( catastrophists ) who believed that major leaps and cataclysms played a significant role in the history of life. [ 10 ]


In 1864  Albert von Kölliker  revived Geoffroy's theory that evolution proceeds by large steps, under the name of  heterogenesis , but this time assuming the influence of a nonmaterial force [ b ]  to  direct the course of evolution . [ 11 ] [ 12 ]


Darwin's cousin,  Francis Galton , considered Darwin's evidence for evolution, and came to an opposite conclusion about the type of variation on which natural selection must act. He carried out his own experiments and published a series of papers and books setting out his views. Already by 1869 when he published  Hereditary Genius , he believed in evolution by saltation. In his 1889 book  Natural Inheritance  he argued that natural selection would benefit from accepting that the steps need not, as Darwin had stated, be minute. In his 1892 book  Finger Prints , he stated directly that "The progress of evolution is not a smooth and uniform progression, but one that proceeds by jerks, through successive 'sports' (as they are called), some of them implying considerable organic changes; and each in its turn being favoured by Natural Selection". [ 13 ]


From 1860 to 1880 saltation had been a minority viewpoint, to the extent that Galton felt his writings were being universally ignored. By 1890 it became a widely held theory, and his views helped to launch a major controversy. [ 14 ] [ 15 ]


William Bateson 's 1894 book  Materials for the Study of Variation, Treated with Especial Regard to Discontinuity in the Origin of Species  marked the arrival of mutationist thinking, before the rediscovery of Mendel's laws. [ 16 ]  He examined discontinuous variation (implying a form of saltation [ 17 ] ) where it occurred naturally, following  William Keith Brooks , Galton,  Thomas Henry Huxley  and  St. George Jackson Mivart . [ 17 ]


The main principle of the mutation theory is that species and varieties have originated by mutation, but are, at present, not known to have originated in any other way. — Hugo de Vries [ 18 ]


Hugo de Vries 's careful 1901 studies of wild variants of the evening primrose  Oenothera lamarckiana  showed that distinct new forms could arise suddenly in nature, apparently at random, and could be propagated for many generations without dissipation or blending. He gave such changes the name " mutation ". [ c ] [ 20 ] [ 21 ]  By this, de Vries meant that a new form of the plant was created in a single step (not the same as a mutation in the modern sense); no long period of natural selection was required for speciation, and nor was  reproductive isolation . [ 22 ]
In the view of the historian of science  Peter J. Bowler , De Vries used the term to mean [ 1 ]


large-scale genetic changes capable of producing a new subspecies, or even species, instantaneously. [ 1 ]


The historian of science Betty Smocovitis described mutationism as: [ 2 ]


the case of purported  saltatory evolution  that Hugo de Vries had mistakenly interpreted for the evening primrose,  Oenothera . [ 2 ]


De Vries set out his position, known as  Mutationstheorie  (mutation theory) on the creative nature of mutation in his 1905 book  Species and Varieties: their Origin by Mutation . [ 23 ]   In the view of the historian of science Edward Larson, de Vries was the person largely responsible for transforming Victorian era saltationism into early 20th century mutation theory, "and in doing so pushed Darwinism near the verge of extinction as a viable scientific theory". [ 24 ]


In the early 1900s, Darwin's mechanism of natural selection was understood by believers in continuous variation, principally the  biometricians   Walter Weldon  and  Karl Pearson , to be able to work on a continuously varying characteristic, whereas de Vries argued that selection on such characteristics would be ineffective.  Wilhelm Johannsen 's "pure line" experiments on  Phaseolus vulgaris  beans appeared to refute this mechanism. Using the true-breeding Princess variety of bean, carefully inbred within weight classes, Johannsen's work appeared to support de Vries. The offspring had a smooth random distribution. Johanssen believed that his results showed that continuous variability was not inherited, so evolution must rely on discontinuous mutations, as de Vries had argued. [ 25 ] [ 26 ] [ 27 ] [ 28 ]  Johanssen published his work in Danish in a 1903 paper  Om arvelighed i samfund og i rene linier  (On inheritance in populations and in pure lines), [ 29 ]  and in his 1905 book  Arvelighedslærens Elementer  (The Elements of Heredity). [ 30 ]


In 1915,  Reginald Punnett  argued in his book  Mimicry in Butterflies  that the 3 morphs (forms) of the butterfly  Papilio polytes , which  mimic  different host species of butterfly, demonstrated discontinuous evolution in action. The different forms existed in a stable  polymorphism  controlled by 2 Mendelian factors ( genes ). The alleles of these genes were certainly discontinuous, so Punnett supposed that they must have evolved in discontinuous leaps. [ 31 ]