The founder effect is a type of  genetic drift , occurring when a small group in a population splinters off from the original population and forms a new one. The new colony may have less genetic variation than the original population, and through the random sampling of  alleles  during reproduction of subsequent generations, continue rapidly towards  fixation . The homozygosity increase can be calculated as

 Δ

 f

 =
 1

 /

 2
 N

 {\displaystyle \Delta {f}=1/2N}

 , where

 f

 {\displaystyle f}

  equals inbreeding coefficient and

 N

 {\displaystyle N}

  equals population size. [ 13 ]  This consequence of inbreeding makes the colony more vulnerable to extinction. [ 14 ]


The per generation loss of heterozygosity can be calculated as

 Δ

 h

 =
 −
 1

 /

 2
 N

 {\displaystyle \Delta {h}=-1/2N}

 , where

 h

 {\displaystyle h}

  equals heterozygosity. [ 13 ]  The population of the founders of the colony can also be calculated if the loss of heterozygosity from the bottleneck is known using the same equation. [ 13 ]


When a newly formed colony is small, its founders can strongly affect the population's genetic makeup far into the future. In humans, who have a slow reproduction rate, the population will remain small for many generations, effectively amplifying the drift effect generation after generation until the population reaches a certain size. The post-bottleneck population growth rate can be calculated as

 N
 (
 t
 )
 =

 K

 1
 +
 b

 e

 r
 t

 {\displaystyle N(t)={K \over 1+be^{rt}}}

 , where

 t

 {\displaystyle t}

  equals the number of generations,

 r

 {\displaystyle r}

  is the growth rate,

 K

 {\displaystyle K}

  is the population equilibrium size,

 e

 {\displaystyle e}

  is the natural logarithm base, and

 b

 {\displaystyle b}

  is the constant

 (
 K
 −

 N

 0

 )

 /

 N

 0

 {\displaystyle (K-N_{0})/N_{0}}

 , where

 N

 0

 {\displaystyle N_{0}}

  is the original size of the founding colony. [ 13 ]


Alleles which were present but relatively rare in the original population can move to one of two extremes. The most common one is that the allele is soon lost altogether, but the other possibility is that the allele survives and within a few generations has become much more dispersed throughout the population. The new colony can experience an increase in the frequency of  recessive  alleles, as well, and as a result, an increased number who are  homozygous  for certain recessive traits. [ 13 ]  The equation to calculate reccessive allele frequencies is

 q
 ^

 =
 √

 N

 22

 /

 N

 {\displaystyle {\hat {q}}=\surd N_{22}/N}

  based on  Hardy-Wienberg  assumptions. [ 13 ]


The variation in gene frequency between the original population and colony may also trigger the two groups to  diverge  significantly over the course of many generations. As the variance, or  genetic distance , increases, the two separated populations may become distinctively different, both  genetically  and  phenotypically , although not only genetic drift, but also natural selection, gene flow and mutation all contribute to this divergence. This potential for relatively rapid changes in the colony's gene frequency led most scientists to consider the founder effect (and by extension, genetic drift) a significant driving force in the evolution of  new species .  Sewall Wright  was the first to attach this significance to random drift and small, newly isolated populations with his  shifting balance theory  of speciation. [ 16 ]  Following behind Wright,  Ernst Mayr  created many persuasive models to show that the decline in genetic variation and small population size accompanying the founder effect were critically important for new species to develop. [ 17 ]  However, much less support for this view is shown today, since the hypothesis has been tested repeatedly through experimental research, and the results have been equivocal at best. [ further explanation needed ]  Speciation by genetic drift is a specific case of  peripatric speciation  which in itself occurs in rare instances. [ 18 ]  It takes place when a random change in genetic frequency of population favours the survival of a few organisms of the species with rare genes which cause reproductive mutation. These surviving organisms then breed among themselves over a long period of time to create a whole new species whose reproductive systems or behaviors are no longer compatible with the original population.  [ further explanation needed ] [ 19 ]


Serial founder effects have occurred when populations migrate over long distances. Such long-distance migrations typically involve relatively rapid movements followed by periods of settlement. The populations in each migration carry only a subset of the genetic diversity carried from previous migrations. As a result, genetic differentiation tends to increase with geographic distance as described by the "isolation by distance" model. [ 20 ]  The migration of humans  out of Africa  is characterized by serial founder effects. [ 21 ]  Africa has the highest degree of human genetic diversity of any continent, which is consistent with an African origin of modern humans. [ 22 ]


Founder populations are essential to the study of  island biogeography  and  island ecology . A natural "blank slate" is not easily found, but a classic series of studies on founder population effects was done following the catastrophic  1883 eruption of Krakatoa , which erased all life on the island. [ 23 ] [ 24 ]  Another continuing study has been following the biocolonization of  Surtsey ,  Iceland , a new volcanic island that erupted offshore between 1963 and 1967. An earlier event, the  Toba eruption  in Sumatra about 73,000 years ago, covered some parts of India with 3–6 m (10–20 ft) of ash, and must have coated the  Nicobar Islands  and  Andaman Islands , much nearer in the ash fallout cone, with life-smothering layers, forcing the restart of their  biodiversity . [ 25 ]


However, not all founder effect studies are initiated after a natural disaster; some scientists study the reinstatement of a species that became locally extinct or hadn't existed there before. A study has been in place since 1958 studying the wolf/moose interaction on  Isle Royale  in Lake Superior after those animals naturally migrated there, perhaps on winter ice. Hajji and others, and Hundertmark & Van Daele, studied the current population statuses of past founder effects in  Corsican red deer  and Alaskan elk, respectively. Corsican red deer are still listed as an  endangered species , decades after a severe bottleneck. They inhabit the Tyrrhenian islands and surrounding mainlands currently, and before the bottleneck, but Hajji and others wanted to know how the deer originally got to the islands, and from what parent population or species they were derived. Through molecular analysis, they were able to determine a possible lineage, with red deer from the islands of  Corsica  and  Sardinia  being the most related to one another. These results are promising, as the island of Corsica was repopulated with red deer from the Sardinian island after the original Corsican red deer population became extinct, and the deer now inhabiting the island of Corsica are diverging from those inhabiting Sardinia. [ 26 ] [ 27 ]


Kolbe and others set up a pair of genetically sequenced and morphologically examined  lizards  on seven small islands to watch each new population's growth and adaptation to its new environment. Specifically, they were looking at the effects on limb length and perch width, both widely varying phenotypic ranges in the parent population. Unfortunately, immigration did occur, but the founder effect and adaptive differentiation, which could eventually lead to peripatric speciation, were statistically and biologically significant between the island populations after a few years. The authors also point out that although adaptive differentiation is significant, the differences between island populations best reflect the differences between founders and their genetic diversity that has been passed down through the generations. [ 28 ]


Founder effects can affect complex traits, such as song diversity. In the  Common Myna  ( Acridotheres tristis ), the percentage of unique songs within a repertoire and within‐song complexity were significantly lower in birds from founder populations. [ 29 ]


It was found by Tarr et al. (1998) that the loss of heterozygosity of the  Laysan finch  ( Telespiza cantans ) after founding events on small islands in the Pacific Ocean closely matched theoretical calculations upon examination of  microsatellite  loci. [ 30 ]


Genetic studies of founder effect have concentrated on discovering ancestral and novel genetic diseases caused by founder effect and, to a lesser degree, on ancestry-related founder effects on populations, races, and ancient migrations, as well other aspects. The founder population could be the common ancestry of race or ethnicity or the forced localizations caused by artificial countries inside the larger group of ancestry, hence causing an original founder effect. [ clarification needed ]  Race and specific founder effect mutation diseases are found in all races or ethnicities, [ citation needed ]  and country-specific mutation diseases are caused by increasing  homozygosity  (the existence of same gene on both chromosomes pairs, hence a recessive disease may increase in just few generations). The genetic abnormality will increase incrementally with the decrease of number of isolated populations making tribe-specific diseases (such as  Ashkenazi Jews ,  Amish , and  Bedouins ) and novel genetic defects. [ 31 ]
In recessive diseases, founder populations where underlying levels of genome-wide homozygosity are high due to shared common ancestry, but also for consanguineous populations that will have large genome-wide homozygous regions due to inbreeding. Having a catalog of disease-associated variation in these populations enables rapid, early, and accurate diagnoses that may improve patient outcomes due to informed clinical management and early interventions. Enclosed communities such as Amish communities, Ashkenazi communities, and relatively isolated islands allow scientists to better understand and further discover the mutated genes that cause these rare diseases and allow them to also discover protective genes as well. [ 32 ]


Due to various migrations throughout human history, founder effects are somewhat common among humans in different times and places. The  French Canadians  of  Quebec  are a classical example of founder population. Over 150 years of French colonization, between 1608 and 1760, an estimated 8,500 pioneers married and left at least one descendant on the territory. [ 33 ]  Following the takeover of the colony by the British crown in 1760, immigration from France effectively stopped, but descendants of French settlers continued to grow in number mainly due to their high fertility rate. Intermarriage occurred mostly with the deported  Acadians  and migrants coming from the British Isles. Since the 20th century, immigration in Quebec and mixing of French Canadians involve people from all over the world. While the French Canadians of Quebec today may be partly of other ancestries, the genetic contribution of the original French founders is predominant, explaining about 90% of regional gene pools, while Acadian (descended from other French settlers in eastern Canada) admixtures contributing 4% British and 2% Native American and other groups contributing less. [ 34 ]


In humans, founder effects can arise from cultural isolation, and inevitably,  endogamy . For example, the Amish populations in the United States exhibit founder effects because they have grown from a very few founders, have not recruited newcomers, and tend to marry within the community. Though still rare, phenomena such as  polydactyly  (extra fingers and toes, a symptom of a condition such as [ 35 ] [ 36 ]   Weyers acrodental dysostosis [ 35 ]  or  Ellis–Van Creveld syndrome [ 36 ] ) are more common in Amish communities than in the American population at large. [ 37 ]   Maple syrup urine disease  affects about one out of 180,000 infants in the general population. [ citation needed ]  Due in part to the founder effect, [ 38 ]  however, the disease has a much higher prevalence in children of Amish,  Mennonite , and  Jewish  descent. [ 39 ] [ 40 ]  Similarly, a high frequency of  fumarase deficiency  exists among the 10,000 members of the  Fundamentalist Church of Jesus Christ of Latter Day Saints , a community which practices both  endogamy  and  polygyny , where an estimated 75–80% of the community are blood relatives of just two men—founders  John Y. Barlow  and  Joseph Smith Jessop . [ 41 ]  In South Asia, castes like the  Gujjars , the  Baniyas  and the Pattapu Kapu have estimated founder effects about 10 times as strong as those of  Finns  and  Ashkenazi Jews . [ 42 ]


In Africa, many members of the  Vadoma  tribe inherit  ectrodactyly , giving them the nickname of the "two-toed tribe". [ 43 ] [ failed verification ]


The island of  Pingelap  also suffered a population bottleneck in 1775 following a typhoon that had reduced the population to only 20 people. As a result, complete  achromatopsia  has a current rate of occurrence of roughly 10%, with an additional 30% being carriers of this recessive condition.


Around 1814, a small group of British colonists founded a settlement on  Tristan da Cunha , a group of small islands in the Atlantic Ocean, midway between Africa and South America. One of the early colonists apparently carried a rare, recessive allele for  retinitis pigmentosa , a progressive form of blindness that afflicts  homozygous  individuals. As late as 1961, the majority of the genes in the gene pool on Tristan were still derived from 15 original ancestors; as a consequence of the inbreeding, of 232 people tested in 1961, four were suffering from retinitis pigmentosa. This represents a prevalence of 1 in 58, compared with a worldwide prevalence of around 1 in 4,000. [ 44 ]


The abnormally high rate of twin births in  Cândido Godói  could be explained by the founder effect. [ 45 ]


On 31 August 2023, researchers reported, based on genetic studies, that a  human ancestor   population bottleneck  (from a possible 100,000 to 1000 individuals) occurred "around 930,000 and 813,000 years ago ... lasted for about 117,000 years and brought human ancestors close to extinction." [ 46 ] [ 47 ]


A  frameshift mutation  (also called a  framing error  or a  reading frame shift ) is a  genetic mutation  caused by  indels  ( insertions  or  deletions ) of a number of  nucleotides  in a DNA sequence that is not divisible by three. Due to the triplet nature of  gene expression  by  codons , the insertion or deletion can change the  reading frame  (the grouping of the codons), resulting in a completely different  translation  from the original. The earlier in the sequence the deletion or insertion occurs, the more altered the protein. [ 1 ]  A frameshift mutation is not the same as a  single-nucleotide polymorphism  in which a nucleotide is replaced, rather than inserted or deleted. A  frameshift  mutation will in general cause the reading of the codons after the mutation to code for different amino acids. The frameshift mutation will also alter the first stop codon ("UAA", "UGA" or "UAG") encountered in the sequence. The polypeptide being created could be abnormally short or abnormally long, and will most likely not be functional. [ 2 ]


Frameshift mutations are apparent in severe genetic diseases such as  Tay–Sachs disease ; they increase susceptibility to certain cancers and classes of  familial hypercholesterolaemia ; in 1997, [ 3 ]  a frameshift mutation was linked to resistance to infection by the HIV retrovirus. Frameshift mutations have been proposed as a source of biological novelty, as with the alleged creation of  nylonase , however, this interpretation is controversial. A study by Negoro  et al.  (2006) [ 4 ]  found that a frameshift mutation was unlikely to have been the cause and that rather a two amino acid substitution in the  active site  of an ancestral  esterase  resulted in nylonase.


The information contained in DNA determines protein function in the cells of all organisms. Transcription and translation allow this information to be communicated into making proteins. However, an error in reading this communication can cause protein function to be incorrect and eventually cause disease even as the cell incorporates a variety of corrective measures.Genetic information is conveyed by DNA for protein synthesis within cells. Misinterpretation can lead to faulty function and disease, despite cellular correction mechanisms.


In 1956  Francis Crick  described the flow of genetic information from  DNA  to a specific amino acid arrangement for making a  protein  as the central dogma. [ 1 ]  For a cell to properly function, proteins are required to be produced accurately for structural and for  catalytic  activities. An incorrectly made protein can have detrimental effects on  cell  viability and in most cases cause the higher  organism  to become unhealthy by abnormal cellular functions. To ensure that the  genome  successfully passes the information on,  proofreading  mechanisms such as  exonucleases  and  mismatch repair  systems are incorporated in  DNA replication . [ 1 ]


After DNA replication, the reading of a selected section of genetic information is accomplished by  transcription . [ 1 ]
Nucleotides containing the genetic information are now on a single strand messenger template called  mRNA . The mRNA is incorporated with a subunit of the  ribosome  and interacts with an  rRNA . The genetic information carried in the codons of the mRNA are now read (decoded) by anticodons of the tRNA. As each codon (triplet) is read,  amino acids  are being joined until a  stop codon  (UAG, UGA or UAA) is reached. At this point the  polypeptide  (protein) has been synthesised and is released. [ 1 ]  For every 1000 amino acid incorporated into the protein, no more than one is incorrect. This fidelity of codon recognition, maintaining the importance of the proper reading frame, is accomplished by proper base pairing at the ribosome A site,  GTP  hydrolysis activity of  EF-Tu  a form of kinetic stability, and a proofreading mechanism as EF-Tu is released. [ 1 ]


Frameshifting may also occur during  prophase  translation, producing different proteins from overlapping open reading frames, such as the gag-pol-env  retroviral  proteins. This is fairly common in  viruses  and also occurs in  bacteria  and  yeast  (Farabaugh, 1996).  Reverse transcriptase , as opposed to  RNA Polymerase II , is thought to be a stronger cause of the occurrence of frameshift mutations. In experiments only 3–13% of all frameshift mutations occurred because of RNA Polymerase II. In  prokaryotes  the error rate inducing frameshift mutations is only somewhere in the range of .0001 and .00001. [ 5 ]


There are several biological processes that help to prevent frameshift mutations. Reverse mutations occur which change the mutated sequence back to the original  wild type  sequence. Another possibility for mutation correction is the use of a  suppressor mutation . This offsets the effect of the original mutation by creating a secondary mutation, shifting the sequence to allow for the correct amino acids to be read.  Guide RNA  can also be used to insert or delete Uridine into the mRNA after transcription, this allows for the correct reading frame. [ 1 ]


A  codon  is a set of three  nucleotides , a triplet that codes for a certain  amino acid . The first codon establishes the reading frame, whereby a new codon begins. A protein's amino acid backbone  sequence  is defined by contiguous triplets. [ 6 ]  Codons are key to translation of genetic information for the synthesis of proteins. The reading frame is set when translating the mRNA begins and is maintained as it reads one triplet to the next. The reading of the genetic code is subject to three rules the monitor codons in mRNA. First, codons are read in a 5' to 3' direction. Second, codons are nonoverlapping and the message has no gaps. The last rule, as stated above, that the message is translated in a fixed reading frame. [ 1 ]


Frameshift mutations can occur randomly or be caused by an external stimulus. The detection of frameshift mutations can occur via several different methods. Frameshifts are just one type of mutation that can lead to incomplete or incorrect proteins, but they account for a significant percentage of errors in DNA.In an unaltered gene, codons (triplets of nucleotides) are sequentially interpreted, with each codon encoding a specific amino acid. This is known as the standard reading frame. However, in cases of frame shift mutations, an extra nucleotide (or more) is inserted into the DNA sequence, disrupting the typical reading frame and causing a shift in the sequence.


This insertion prompts a shift in the reading frame due to the triplet nature of the genetic code. For instance, the addition of an extra "A" leads to a sequence shift, triggering the reading of an entirely different set of codons. This deviation in genetic information causes the ribosome, which reads the mRNA for protein synthesis, to misinterpret the genetic data. Consequently, an entirely different series of amino acids is generated, resulting in the generation of an altered protein sequence. In most instances, the new reading frame results in an early encounter with a stop codon, leading to the formation of a shortened and usually inactive protein. This form of mutation is termed an early stop codon or a nonsense mutation.


This is a genetic mutation at the level of nucleotide bases. Why and how frameshift mutations occur are continually being sought after. An environmental study, specifically the production of  UV -induced frameshift mutations by DNA polymerases deficient in 3′ → 5′ exonuclease activity was done. The normal sequence 5′ GTC GTT TTA CAA 3′ was changed to GTC GTT T TTA CAA (MIDT) of GTC GTT C TTA CAA (MIDC) to study frameshifts.  E. coli  pol I Kf and T7 DNA polymerase mutant  enzymes  devoid of 3′ → 5′ exonuclease activity produce UV-induced revertants at higher frequency than did their  exonuclease  proficient counterparts. The data indicates that loss of proofreading activity increases the frequency of UV-induced frameshifts. [ 7 ]


The effects of neighboring bases and secondary structure to detect the frequency of frameshift mutations has been investigated in depth using  fluorescence . Fluorescently tagged DNA, by means of base analogues, permits one to study the local changes of a DNA sequence. [ 8 ]  Studies on the effects of the length of the primer strand reveal that an equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. In contrast, a double-loop structure with an unusual unstacked DNA conformation at its downstream edge was observed when the extruded bases were positioned at the primer–template junction, showing that misalignments can be modified by neighboring DNA secondary structure. [ 9 ]


Sanger sequencing  and  pyrosequencing  are two methods that have been used to detect frameshift mutations, however, it is likely that data generated will not be of the highest quality. Even still, 1.96 million  indels  have been identified through Sanger sequencing that do not overlap with other databases. When a frameshift mutation is observed it is compared against the Human Genome Mutation Database (HGMD) to determine if the mutation has a damaging effect. This is done by looking at four features. First, the ratio between the affected and conserved DNA, second the location of the mutation relative to the transcript, third the ratio of conserved and affected amino acids and finally the distance of the indel to the end of the  exon . [ 10 ]


Massively Parallel Sequencing  is a newer method that can be used to detect mutations. Using this method, up to 17 gigabases can be sequenced at once, as opposed to limited ranges for  Sanger sequencing  of only about 1 kilobase. Several technologies are available to perform this test and it is being looked at to be used in clinical applications. [ 11 ]  When testing for different carcinomas, current methods only allow for looking at one gene at a time. Massively Parallel Sequencing can test for a variety of cancer causing mutations at once as opposed to several specific tests. [ 12 ]  An experiment to determine the accuracy of this newer sequencing method tested for 21 genes and had no false positive calls for frameshift mutations. [ 13 ]


A US  patent  (5,958,684) in 1999 by Leeuwen, details the methods and reagents for diagnosis of diseases caused by or associated with a gene having a somatic mutation giving rise to a frameshift mutation. The methods include providing a tissue or fluid sample and conducting gene analysis for frameshift mutation or a protein from this type of mutation. The nucleotide sequence of the suspected gene is provided from published gene sequences or from  cloning  and sequencing of the suspect gene. The amino acid sequence encoded by the gene is then predicted. [ 14 ]  NA Sequencing: Sanger sequencing or Next-Generation Sequencing (NGS) can be used to directly sequence the DNA and identify insertions or deletions.Polymerase Chain Reaction (PCR): PCR can be used to amplify the specific region containing the mutation for subsequent analysis.Multiplex Ligation-dependent Probe Amplification (MLPA): MLPA is a technique used to detect copy number variations and small insertions or deletions.Comparative Genomic Hybridization (CGH): CGH is used to detect chromosomal imbalances, which may include large insertions or deletions.


Despite the rules that govern the genetic code and the various mechanisms present in a cell to ensure the correct transfer of genetic information during the process of DNA replication as well as during translation, mutations do occur; frameshift mutation is not the only type. There are at least two other types of recognized point mutations, specifically  missense mutation  and  nonsense mutation . [ 1 ]  A frameshift mutation can drastically change the coding capacity (genetic information) of the message. [ 1 ]  Small insertions or deletions (those less than 20 base pairs) make up 24% of mutations that manifest in currently recognized genetic disease. [ 10 ]


Frameshift mutations are found to be more common in repeat regions of DNA. A reason for this is because of slipping of the polymerase enzyme in repeat regions, allowing for mutations to enter the  sequence . [ 15 ]   Experiments  can be run to determine the frequency of the frameshift mutation by adding or removing a pre-set number of nucleotides. Experiments have been run by adding four basepairs, called the +4 experiments, but a team from  Emory University  looked at the difference in frequency of the mutation by both adding and deleting a base pair. It was shown that there was no difference in the frequency between the addition and deletion of a base pair. There is however, a difference in the result of the protein. [ 15 ]


Huntington's disease  is one of the nine codon reiteration disorders caused by polyglutamine expansion mutations that include spino-cerebellar ataxia (SCA) 1, 2, 6, 7 and 3, spinobulbar muscular atrophy and dentatorubal-pallidoluysianatrophy. There may be a link between diseases caused by polyglutamine and polyalanine expansion mutations, as frame shifting of the original SCA3 gene product encoding CAG/polyglutamines to GCA/polyalanines. Ribosomal slippage during translation of the SCA3 protein has been proposed as the mechanism resulting in shifting from the polyglutamine to the polyalanine-encoding frame. A dinucleotide deletion or single nucleotide insertion within the polyglutamine tract of huntingtin exon 1 would shift the CAG, polyglutamineen coding frame by +1 (+1 frame shift) to the GCA, polyalanine-encoding frame and introduce a novel epitope to the C terminus of Htt exon 1 (APAAAPAATRPGCG). [ 16 ]


Several diseases have frameshift mutations as at least part of the cause. Knowing prevalent mutations can also aid in the diagnosis of the disease. Currently there are attempts to use frameshift mutations beneficially in the treatment of diseases, changing the reading frame of the amino acids.


Frameshift mutations are known to be a factor in  colorectal  cancer as well as other  cancers  with  microsatellite instability . As stated previously, frameshift mutations are more likely to occur in a region of repeat sequence. When DNA mismatch repair does not fix the addition or deletion of bases, these mutations are more likely to be pathogenic. This may be in part because the tumor is not told to stop growing. Experiments in yeast and bacteria help to show characteristics of microsatellites that may contribute to defective DNA mismatch repair. These include the length of the  microsatellite , the makeup of the genetic material and how pure the repeats are. Based on experimental results longer microsatellites have a higher rate of frameshift mutations. The flanking DNA can also contribute to frameshift mutations. [ 17 ]  In prostate cancer a frameshift mutation changes the  open reading frame  (ORF) and prevents  apoptosis  from occurring. This leads to an unregulated growth of the  tumor . While there are environmental factors that contribute to the progression of  prostate cancer , there is also a genetic component. During testing of coding regions to identify mutations, 116 genetic variants were discovered, including 61 frameshift mutations. [ 18 ]  There are over 500 mutations on chromosome 17 that seem to play a role in the development of breast and ovarian cancer in the BRCA1 gene, many of which are frameshift. [ 19 ]


Crohn's disease  has an association with the NOD2 gene. The mutation is an insertion of a  Cytosine  at position 3020. This leads to a premature stop codon, shortening the protein that is supposed to be transcribed. When the protein is able to form normally, it responds to bacterial liposaccharides, where the 3020insC mutation prevents the protein from being responsive. [ 20 ]


Cystic fibrosis  (CF) is a disease based on mutations in the CF  transmembrane  conductance regulator (CFTR) gene. There are over 1500 mutations identified, but not all cause the disease. [ 21 ]  Most cases of cystic fibrosis are a result of the ∆F508 mutation, which deletes the entire amino acid. Two frameshift mutations are of interest in diagnosing CF, CF1213delT and CF1154-insTC. Both of these mutations commonly occur in tandem with at least one other mutation. They both lead to a small decrease in the function of the  lungs  and occur in about 1% of patients tested. These mutations were identified through Sanger sequencing. [ 22 ]


CCR5  is one of the cell entry co-factors associated with HIV, most frequently involved with nonsyncytium-inducing strains, is most apparent in HIV patients as opposed to AIDS patients. A 32 base pair deletion in CCR5 has been identified as a mutation that negates the likelihood of an HIV infection. This region on the open reading frame  ORF  contains a frameshift mutation leading to a premature stop codon. This leads to the loss of the HIV-coreceptor function in vitro. CCR5-1 is considered the wild type and CCR5-2 is considered to be the mutant allele. Those with a heterozygous mutation for the CCR5 were less susceptible to the development of HIV. In a study, despite high exposure to the HIV virus, there was no one homozygous for the CCR5 mutation that tested positive for HIV. [ 3 ]


Tay–Sachs disease  is a fatal disease affecting the central nervous system. It is most frequently found in infants and small children. Disease progression begins in the  womb  but symptoms do not appear until approximately 6 months of age. There is no cure for the disease. [ 23 ]  Mutations in the β-hexosaminidase A (Hex A) gene are known to affect the onset of Tay-Sachs, with 78 mutations of different types being described, 67 of which are known to cause disease. Most of the mutations observed (65/78) are single base substitutions or SNPs, 11 deletions, 1 large and 10 small, and 2 insertions. 8 of the observed mutations are frameshift, 6 deletions and 2 insertions. A 4 base pair insertion in exon 11 is observed in 80% of Tay-Sachs disease presence in the  Ashkenazi  Jewish population. The frameshift mutations lead to an early stop codon which is known to play a role in the disease in infants. Delayed onset disease appears to be caused by 4 different mutations, one being a 3 base pair deletion. [ 24 ]


Smith–Magenis syndrome  (SMS) is a complex  syndrome  involving intellectual disabilities, sleep disturbance, behavioural problems, and a variety of craniofacial, skeletal, and visceral anomalies. The majority of SMS cases harbor an ~3.5 Mb common deletion that encompasses the retinoic acid induced-1 ( RAI1 ) gene. Other cases illustrate variability in the SMS  phenotype  not previously shown for RAI1 mutation, including hearing loss, self-abusive behaviours, and mild global delays. Sequencing of RAI1 revealed mutation of a heptamericC-tract (CCCCCCC) in exon 3 resulting in frameshift mutations. Of the seven reported frameshift mutations occurring in poly C-tracts in RAI1, four cases (~57%) occur at this heptameric C-tract. The results indicate that this heptameric C-tract is a preferential  recombination hotspot  insertion/deletions (SNindels) and therefore a primary target for analysis in patients suspected for mutations in RAI1. [ 25 ]


Hypertrophic cardiomyopathy  is the most common cause of  sudden death  in young people, including trained athletes, and is caused by mutations in genes encoding proteins of the cardiac sarcomere. Mutations in the Troponin C gene ( TNNC1 ) are a rare genetic cause of hypertrophic cardiomyopathy. A recent study has indicated that a frameshift mutation (c.363dupG or p.Gln122AlafsX30) in Troponin C was the cause of hypertrophic cardiomyopathy (and sudden cardiac death) in a 19-year-old male. [ 26 ]


Finding a cure for the diseases caused by frameshift mutations is rare. Research into this is ongoing. One example is a  primary immunodeficiency  (PID), an inherited condition which can lead to an increase in infections. There are 120 genes and 150 mutations that play a role in primary immunodeficiencies. The standard treatment is currently  gene therapy , but this is a highly risky treatment and can often lead to other diseases, such as leukemia. Gene therapy procedures include modifying the zinc fringer nuclease fusion protein, cleaving both ends of the mutation, which in turn removes it from the sequence. Antisense-oligonucleotide mediated  exon skipping  is another possibility for Duchenne  muscular dystrophy . This process allows for passing over the mutation so that the rest of the sequence remains in frame and the function of the protein stays intact. This, however, does not cure the disease, just treats symptoms, and is only practical in structural proteins or other repetitive genes. A third form of repair is  revertant mosaicism , which is naturally occurring by creating a reverse mutation or a mutation at a second site that corrects the reading frame. This reversion may happen by intragenic  recombination ,  mitotic  gene conversion, second site DNA slipping or site-specific reversion. This is possible in several diseases, such as  X-linked severe combined immunodeficiency  (SCID),  Wiskott–Aldrich syndrome , and  Bloom syndrome . There are no drugs or other pharmacogenomic methods that help with PIDs. [ 27 ]


A European patent (EP1369126A1) in 2003 by Bork records a method used for prevention of cancers and for the curative treatment of cancers and precancers such as DNA-mismatch repair deficient (MMR) sporadic tumours and HNPCC associated tumours. The idea is to use  immunotherapy  with combinatorial mixtures of tumour-specific frameshift mutation-derived peptides to elicit a cytotoxic T-cell response specifically directed against tumour cells. [ 28 ]


The  Functional Element  SNPs  Database  ( FESD ) is a  biological database  of  single nucleotide polymorphisms  in  molecular biology . [ 1 ]  The database is a tool designed to organize functional elements into categories in human gene regions and to output their sequences needed for genotyping [ 2 ]  experiments as well as provide a set of SNPs that lie within each region. The database defines functional elements into ten types: promoter regions,  CpG islands ,  5' untranslated regions  (5'-UTRs), translation start sites,  splice sites , coding exons,  introns , translation stop sites, polyadenylation signals, and 3' UTRs. [ 3 ]  People may reference this database for haplotype information or obtain a flanking sequence for genotyping. [ 4 ]  This may help in finding mutations that contribute to common and polygenic diseases. Researchers can manually choose a group of SNPs of special interest for certain functional elements along with their corresponding sequences. [ 5 ]  The database combines information from sources such as HapMap, UCSC GoldenPath, dbSNP, OMIM, and TRANSFAC. Users can obtain information about tag SNPs and simulate LD blocks for each gene. FESD is still a developing database and is not widely known so was unable to find projects that used the database. Research was found using similar databases or databases that are combined in FESD's information pool. [ 6 ]


The database is a reference of all known SNPs in functional units that may affect a given phenotype in which some cases may be a disease. SNPs are selected based on disease, gene, or factor. The database has a link on the HapMap Project website. [ 7 ]  The database may have been used in one of the following research examples. The Korean HapMap Project website is quoted saying "We have developed a series of software programs for association studies as well as the comparison and analysis of Korean HapMap data with other populations, such as European, Chinese, Japanese, and African populations. The developed software includes HapMapSNPAnalyzer, SNPflank, HWE Test, FESD, D2GSNP, SNP@Domain, KMSD, KFOD, KFRG, and SNP@WEB." [ 8 ]  It is unknown how many species are included; however, there is an alphabetical list of all the genes included in case the gene name is known but not the disease it is associated with or factor it is located in. Some of the genes were from humans, mice, and some were from D. Melanogaster. Information cannot be submitted to the database publicly. The database is updated regularly and the information is pooled from the sites listed on the page. There is also a user guide tab that displays what each process should look like.


The study involved the investigation of ITIH3 (inter alpha trypsin inhibitor heavy chain 3). Researchers used functional analysis, linkage disequilibrium mapping, SNP markers, and the HapMap database. FESD Version II may have been used since it has the information from HapMap as well as other databases. The researchers discovered that the gene was on chromosome 3. In vitro functional analyses showed that this SNP enhanced the transcriptional level of the ITIH3 gene. Furthermore, expression of the ITIH3 protein in the vascular smooth muscle cells and macrophages in the human atherosclerotic lesions was found, suggesting ITIH3 SNP to be a novel genetic risk factor of MI. The gene has been shown to be related to the proinflammatory process of immune response. ITIH3 makes a complex with the locally synthesized  hyaluronan  (HA) and interacts with inflammatory cells. The ITIH3-HA complex has been reported to be involved in inflammatory diseases, including rheumatoid arthritis and inflammatory bowel diseases. The most statistically-significant SNP did not substitute an amino acid of ITIH3 protein. The researchers hypothesized that this synonymous SNP affected the transcriptional regulation, because several papers reported that some transcriptional factors bound to the exonic coding sequences of some genes and regulated their transcriptional level. There was expression of ITIH3 protein in the atherosclerotic lesion. ITIH3 could play a critical role in the pathogenesis of atherosclerosis and subsequent myocardial infarction. [ 9 ]


These researchers used SNP selecting web tools and could have used FESD Version II to select their SNPs. They hypothesized that the polymorphisms in circadian genes may be associated with prognosis of hepatocellular carcinoma. The circadian negative feedback regulation genes- CRY1, CRY2, PER1, PER2 and PER3 were identified based on a comprehensive literature review and the potentially functional SNPs were selected through web tools. Finally, a total of twelve potentially functional SNPs in five genes were selected, including CRY1: rs1056560, rs3809236; CRY2: rs6798, rs2292910; PER1: rs2585405, rs3027178; PER2: rs934945, rs2304669; PER3: rs2640908, rs172933, rs2859390, rs228669. When the dominant genetic model was tested, their data showed that only SNP rs2640908 in PER3 gene was significantly associated with overall survival of hepatocellular carcinoma patients. Patients carrying at least one variant allele of rs2640908 had a significantly decreased risk of death when compared with those carrying homozygous wild-type alleles. [ 10 ]


A polymorphism interaction analysis (PIA) algorithm was used to obtain cooperating single nucleotide polymorphisms (SNPs) that contribute to RA disease. The acquired SNP pairs were used to construct a SNP-SNP network. Sub-networks defined by hub SNPs were then extracted and turned into gene modules by mapping SNPs to genes using dbSNP database. The FESD Version II would also be a good choice in database because it also covers the dbSNP database. They ran the Haseman Elston test, then a random forest algorithm. The shared SNPs detected were then run through a polymorphism interaction analysis (PIA) algorithm. They performed gene ontology analysis of the 5 modules that were created. It was suggested that Ets-1 may be an important transcription factor in the cytokine-mediated inflammatory pathway and destructive cascade characteristic of RA. The ATP-binding cassette (ABC) transporter, ABCA1, was induced during differentiation of human monocytes into macrophages, and there was a dual regulatory function for ABCA1 in macrophage lipid metabolism and inflammation. Extracellular signals are transduced intracellularly via multiple pathways, resulting in alterations in the transcription and translation of specific proteins. Some of these signaling pathways result in the production of proteins, including cytokines and matrix metalloproteinases, which are implicated in the pathogenesis of RA. The zinc-finger protein 238 (ZNF238) is attached to zinc-finger proteins that can regulate the human immunodeficiency virus type 1. Hub gene CD160 is a potential RA association gene. The CD160 receptor represents a unique triggering surface molecule that is expressed by cytotoxic NK cells, it participates in the inflammatory response and determines the type of subsequent specific immunity. [ 11 ]


In  genetics , a  fusion gene  is a hybrid  gene  formed from two previously independent genes. It can occur as a result of  translocation ,  interstitial deletion , or  chromosomal inversion . Fusion genes have been found to be prevalent in all main types of human  neoplasia . [ 1 ]  The identification of these fusion genes play a prominent role in being a diagnostic and prognostic  marker . [ 2 ]


The first fusion gene [ 1 ]  was described in cancer cells in the early 1980s. The finding was based on the discovery in 1960 by  Peter Nowell  and  David Hungerford  in Philadelphia of a small abnormal marker  chromosome  in patients with  chronic myeloid leukemia —the first consistent chromosome abnormality detected in a human malignancy, later designated the  Philadelphia chromosome . [ 3 ]  In 1973,  Janet Rowley  in Chicago showed that the Philadelphia chromosome had originated through a translocation between chromosomes  9  and  22 , and not through a simple deletion of chromosome 22 as was previously thought. Several investigators in the early 1980s showed that the Philadelphia  chromosome translocation  led to the formation of a new BCR::ABL1 fusion gene, composed of the 3' part of the  ABL1  gene in the breakpoint on chromosome 9 and the 5' part of a gene called  BCR  in the breakpoint in chromosome 22. In 1985 it was clearly established that the fusion gene on chromosome 22 produced an abnormal  chimeric  BCR::ABL1 protein with the capacity to induce chronic myeloid leukemia.


It has been known for 30 years that the corresponding gene fusion plays an important role in tumorigenesis. [ 4 ]   Fusion genes can contribute to tumor formation because fusion genes can produce much more active abnormal protein than non-fusion genes. Often, fusion genes are  oncogenes  that cause  cancer ; these include  BCR-ABL , [ 5 ]  TEL-AML1 ( ALL  with t(12 ; 21)), AML1-ETO ( M2 AML  with t(8 ; 21)), and  TMPRSS2 - ERG  with an interstitial deletion on  chromosome 21 , often occurring in prostate cancer. [ 6 ]
In the case of TMPRSS2-ERG, by disrupting androgen receptor (AR) signaling and inhibiting AR expression by oncogenic ETS transcription factor, the fusion product regulates the prostate cancer. [ 7 ]
Most fusion genes are found from  hematological cancers ,  sarcomas , and  prostate cancer . [ 1 ] [ 8 ]
 BCAM-AKT2  is a fusion gene that is specific and unique to high-grade  serous ovarian cancer . [ 9 ]


Oncogenic fusion genes may lead to a gene product with a new or different function from the two fusion partners. Alternatively, a proto-oncogene is fused to a strong  promoter , and thereby the oncogenic function is set to function by an  upregulation  caused by the strong promoter of the upstream fusion partner. The latter is common in  lymphomas , where oncogenes are juxtaposed to the promoters of the  immunoglobulin  genes. [ 10 ]  Oncogenic  fusion transcripts  may also be caused by  trans-splicing  or  read-through  events. [ 11 ]


Since chromosomal translocations play such a significant role in neoplasia, a specialized database of chromosomal aberrations and gene fusions in cancer has been created.  This database is called Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer. [ 12 ]


Presence of certain chromosomal aberrations and their resulting fusion genes is commonly used within cancer diagnostics in order to set a precise diagnosis. Chromosome  banding analysis ,  fluorescence  in situ  hybridization  (FISH), and  reverse transcription polymerase chain reaction  (RT-PCR) are common methods employed at diagnostic laboratories. These methods all have their distinct shortcomings due to the very complex nature of cancer  genomes . Recent developments such as  high-throughput sequencing [ 13 ]  and custom  DNA microarrays  bear promise of introduction of more efficient methods. [ 14 ]


Gene fusion plays a key role in the evolution of gene architecture. We can observe its effect if gene fusion occurs in coding sequences. [ 15 ]  Duplication, sequence divergence, and recombination are the major contributors at work in gene evolution. [ 16 ]  These events can probably produce new genes from already existing parts. When gene fusion happens in non-coding sequence region, it can lead to the misregulation of the expression of a gene now under the control of the  cis-regulatory  sequence of another gene. If it happens in coding sequences, gene fusion cause the assembly of a new gene, then it allows the appearance of new functions by adding peptide modules into a multi-domain protein. [ 15 ]  The detecting methods to inventory gene fusion events on a large biological scale can provide insights about the multi modular architecture of proteins. [ 17 ] [ 18 ] [ 19 ]


The  purines   adenine  and  guanine  are two of the four information encoding bases of the universal  genetic code .  Biosynthesis of these purines occurs by similar, but not identical, pathways in different species of the three domains of life, the  Archaea ,  Bacteria  and  Eukaryotes . A major distinctive feature of the purine biosynthetic pathways in Bacteria is the prevalence of gene fusions where two or more purine biosynthetic enzymes are encoded by a single gene. [ 20 ]   Such gene fusions are almost exclusively between genes that encode enzymes that perform sequential steps in the biosynthetic pathway.  Eukaryotic species generally exhibit the most common gene fusions seen in the Bacteria, but in addition have new fusions that potentially increase metabolic flux.


In recent years, next generation sequencing technology has already become available to screen known and novel gene fusion events on a genome wide scale. However, the precondition for large scale detection is a paired-end sequencing of the cell's  transcriptome . The direction of fusion gene detection is mainly towards data analysis and visualization. Some researchers already developed a new tool called Transcriptome Viewer (TViewer) to directly visualize detected gene fusions on the transcript level. [ 21 ]


Biologists may also deliberately create fusion genes for research purposes. The fusion of  reporter genes  to the regulatory elements of genes of interest allows researches to study gene expression. Reporter gene fusions can be used to measure activity levels of gene regulators, identify the regulatory sites of genes (including the signals required), identify various genes that are regulated in response to the same stimulus, and artificially control the expression of desired genes in particular cells. [ 22 ]  For example, by creating a fusion gene of a protein of interest and  green fluorescent protein , the protein of interest may be observed in  cells  or  tissue  using fluorescence  microscopy . [ 23 ]  The protein synthesized when a fusion gene is  expressed  is called a  fusion protein .


Fusion transcript  is a chimeric  RNA  encoded by a  fusion gene [ 1 ]  or by two different genes by subsequent  trans-splicing . [ 2 ] [ 3 ]  Certain fusion transcripts are commonly produced by  cancer  cells, and detection of fusion transcripts is part of routine  diagnostics  of certain cancer types.


Gene duplication  (or  chromosomal duplication  or       gene amplification ) is a major mechanism through which new genetic material is generated during  molecular evolution . It can be defined as any duplication of a region of  DNA  that contains a  gene .  Gene duplications can arise as products of several types of errors in  DNA replication  and  repair  machinery as well as through fortuitous capture by selfish genetic elements.  Common sources of gene duplications include  ectopic recombination ,   retrotransposition  event,  aneuploidy ,  polyploidy , and  replication slippage . [ 1 ]


Duplications arise from an event termed  unequal crossing-over  that occurs during meiosis between misaligned homologous chromosomes. The chance of it happening is a function of the degree of sharing of repetitive elements between two chromosomes. The products of this recombination are a duplication at the site of the exchange and a reciprocal deletion. Ectopic recombination is typically mediated by sequence similarity at the duplicate breakpoints, which form direct repeats.  Repetitive genetic elements such as  transposable  elements offer one source of repetitive DNA that can facilitate recombination, and they are often found at duplication breakpoints in plants and mammals. [ 2 ]


Replication slippage  is an error in DNA replication that can produce duplications of short genetic sequences.  During replication  DNA polymerase  begins to copy the DNA.  At some point during the replication process, the polymerase dissociates from the DNA and replication stalls.  When the polymerase reattaches to the DNA strand, it aligns the replicating strand to an incorrect position and incidentally copies the same section more than once.  Replication slippage is also often facilitated by repetitive sequences, but requires only a few bases of similarity. [ citation needed ]


Retrotransposons , mainly  L1 , can occasionally act on cellular mRNA. Transcripts are reverse transcribed to DNA and inserted into random place in the genome, creating retrogenes.  Resulting sequence usually lack introns and often contain poly(A) sequences that are also integrated into the genome. Many retrogenes display changes in gene regulation in comparison to their parental gene sequences, which sometimes results in novel functions. Retrogenes can move between different chromosomes to shape chromosomal evolution. [ 3 ]


Aneuploidy  occurs when nondisjunction at a single chromosome results in an abnormal number of chromosomes. Aneuploidy is often harmful and in mammals regularly leads to spontaneous abortions (miscarriages).  Some aneuploid individuals are viable, for example trisomy 21 in humans, which leads to  Down syndrome .  Aneuploidy often alters gene dosage in ways that are detrimental to the organism; therefore, it is unlikely to spread through populations.


Polyploidy , or  whole genome duplication  is a product of  nondisjunction  during meiosis which results in additional copies of the entire genome.  Polyploidy is common in plants, but it has also occurred in animals, with two rounds of whole genome duplication ( 2R event ) in the vertebrate lineage leading to humans. [ 4 ]  It has also occurred in the hemiascomycete yeasts ~100 mya. [ 5 ] [ 6 ]


After a whole genome duplication, there is a relatively short period of genome instability, extensive gene loss, elevated levels of nucleotide substitution and regulatory network rewiring. [ 7 ] [ 8 ]   In addition, gene dosage effects play a significant role. [ 9 ]  Thus, most duplicates are lost within a short period, however, a considerable fraction of duplicates survive. [ 10 ]   Interestingly, genes involved in regulation are preferentially retained. [ 11 ] [ 12 ]  Furthermore, retention of regulatory genes, most notably the  Hox genes , has led to adaptive innovation.


Rapid evolution and functional divergence have been observed at the level of the transcription of duplicated genes, usually by point mutations in short transcription factor binding motifs. [ 13 ] [ 14 ]  Furthermore, rapid evolution of protein phosphorylation motifs, usually embedded within rapidly evolving intrinsically disordered regions is another contributing factor for survival and rapid adaptation/neofunctionalization of duplicate genes. [ 15 ]  Thus, a link seems to exist between gene regulation (at least at the post-translational level) and genome evolution. [ 15 ]


Polyploidy is also a well known source of speciation, as offspring, which have different numbers of chromosomes compared to parent species, are often unable to interbreed with non-polyploid organisms.  Whole genome duplications are thought to be less detrimental than aneuploidy as the relative dosage of individual genes should be the same.


Comparisons of genomes demonstrate that gene duplications are common in most species investigated. This is indicated by variable copy numbers ( copy number variation ) in the genome of humans [ 16 ] [ 17 ]  or fruit flies. [ 18 ]  However, it has been difficult to measure the rate at which such duplications occur. Recent studies yielded a first direct estimate of the genome-wide rate of gene duplication in  C. elegans , the first multicellular eukaryote for which such as estimate became available. The gene duplication rate in  C. elegans  is on the order of 10 −7  duplications/gene/generation, that is, in a population of 10 million worms, one will have a gene duplication per generation. This rate is two orders of magnitude greater than the spontaneous rate of point mutation per nucleotide site in this species. [ 19 ]  Older (indirect) studies reported locus-specific duplication rates in bacteria,  Drosophila , and humans ranging from 10 −3  to 10 −7 /gene/generation. [ 20 ] [ 21 ] [ 22 ]


Gene duplications are an essential source of genetic novelty that can lead to evolutionary innovation. Duplication creates genetic redundancy, where the second copy of the gene is often free from  selective pressure —that is,  mutations  of it have no deleterious effects to its host organism.  If one copy of a gene experiences a mutation that affects its original function, the second copy can serve as a 'spare part' and continue to function correctly.  Thus, duplicate genes accumulate mutations faster than a functional single-copy gene, over generations of organisms, and it is possible for one of the two copies to develop a new and different function. Some examples of such neofunctionalization is the apparent mutation of a duplicated digestive gene in a family of  ice fish  into an antifreeze gene and duplication leading to a novel snake venom gene [ 23 ]  and the synthesis of 1 beta-hydroxytestosterone in pigs. [ 24 ]


Gene duplication is believed to play a major role in  evolution ; this stance has been held by members of the scientific community for over 100 years. [ 25 ]   Susumu Ohno  was one of the most famous developers of this theory in his classic book  Evolution by gene duplication  (1970). [ 26 ]   Ohno argued that gene duplication is the most important evolutionary force since the emergence of the  universal common ancestor . [ 27 ]
Major  genome duplication  events can be quite common. It is believed that the entire  yeast   genome  underwent duplication about 100 million years ago. [ 28 ]   Plants  are the most prolific genome duplicators.  For example,  wheat  is hexaploid (a kind of  polyploid ), meaning that it has six copies of its genome.


Another possible fate for duplicate genes is that both copies are equally free to accumulate degenerative mutations, so long as any defects are complemented by the other copy. This leads to a neutral " subfunctionalization " (a process of  constructive neutral evolution ) or DDC (duplication-degeneration-complementation) model, [ 29 ] [ 30 ]  in which the functionality of the original gene is distributed among the two copies.  Neither gene can be lost, as both now perform important non-redundant functions, but ultimately neither is able to achieve novel functionality.


Subfunctionalization can occur through neutral processes in which mutations accumulate with no detrimental or beneficial effects.  However, in some cases subfunctionalization can occur with clear adaptive benefits.  If an ancestral gene is  pleiotropic  and performs two functions, often neither one of these two functions can be changed without affecting the other function.  In this way, partitioning the ancestral functions into two separate genes can allow for adaptive specialization of subfunctions, thereby providing an adaptive benefit. [ 31 ]


Often the resulting genomic variation leads to gene dosage dependent neurological disorders such as  Rett-like syndrome  and  Pelizaeus–Merzbacher disease . [ 32 ]   Such detrimental mutations are likely to be lost from the population and will not be preserved or develop novel functions. However, many duplications are, in fact, not detrimental or beneficial, and these neutral sequences may be lost or may spread through the population through random fluctuations via  genetic drift .


The two genes that exist after a gene duplication event are called  paralogs  and usually code for  proteins  with a similar function and/or structure.  By contrast,  orthologous  genes present in different species which are each originally derived from the same ancestral sequence.  (See  Homology of sequences in genetics ).


It is important (but often difficult) to differentiate between paralogs and orthologs in biological research. Experiments on human gene function can often be carried out on other  species  if a homolog to a human gene can be found in the genome of that species, but only if the homolog is orthologous. If they are paralogs and resulted from a gene duplication event, their functions are likely to be too different. One or more copies of duplicated genes that constitute a gene family may be affected by insertion of  transposable elements  that causes significant variation between them in their sequence and finally may become responsible for  divergent evolution . This may also render the chances and the rate of  gene conversion  between the homologs of gene duplicates due to less or no similarity in their sequences.


Paralogs can be identified in single genomes through a sequence comparison of all annotated gene models to one another.  Such a comparison can be performed on translated amino acid sequences (e.g. BLASTp, tBLASTx) to identify ancient duplications or on DNA nucleotide sequences (e.g. BLASTn, megablast) to identify more recent duplications.  Most studies to identify gene duplications require reciprocal-best-hits or fuzzy reciprocal-best-hits, where each paralog must be the other's single best match in a sequence comparison. [ 33 ]


Most gene duplications exist as  low copy repeats  (LCRs), rather highly repetitive sequences like transposable elements. They are mostly found in  pericentronomic ,  subtelomeric  and  interstitial  regions of a chromosome. Many LCRs, due to their size (>1Kb), similarity, and orientation, are highly susceptible to duplications and deletions.


Technologies such as genomic  microarrays , also called array comparative  genomic  hybridization (array CGH), are used to detect chromosomal abnormalities, such as microduplications, in a high throughput fashion from genomic DNA samples. In particular, DNA  microarray  technology can simultaneously monitor the  expression  levels of thousands of genes across many  treatments or experimental conditions, greatly facilitating the evolutionary studies of  gene regulation  after gene duplication or  speciation . [ 34 ] [ 35 ]


Gene duplications can also be identified through the use of next-generation sequencing platforms.  The simplest means to identify duplications in genomic resequencing data is through the use of paired-end sequencing reads.  Tandem duplications are indicated by sequencing read pairs which map in abnormal orientations.  Through a combination of increased sequence coverage and abnormal mapping orientation, it is possible to identify duplications in genomic sequencing data.


The  International System for Human Cytogenomic Nomenclature  (ISCN) is an international standard for  human chromosome   nomenclature , which includes band names, symbols and abbreviated terms used in the description of human chromosome and chromosome abnormalities. Abbreviations include  dup  for duplications of parts of a chromosome. [ 36 ]  For example, dup(17p12) causes  Charcot–Marie–Tooth disease  type 1A. [ 37 ]


Gene duplication does not necessarily constitute a lasting change in a species' genome.  In fact, such changes often don't last past the initial host organism.  From the perspective of  molecular genetics ,  gene amplification  is one of many ways in which a  gene  can be  overexpressed .  Genetic amplification can occur artificially, as with the use of the  polymerase chain reaction  technique to amplify short strands of  DNA   in vitro  using  enzymes , or it can occur naturally, as described above.  If it's a natural duplication, it can still take place in a  somatic cell , rather than a  germline  cell (which would be necessary for a lasting evolutionary change).


Duplications of  oncogenes  are a common cause of many types of  cancer . In such cases the genetic duplication occurs in a somatic cell and affects only the genome of the cancer cells themselves, not the entire organism, much less any subsequent offspring. Recent comprehensive patient-level classification and quantification of driver events in  TCGA  cohorts revealed that there are on average 12 driver events per tumor, of which 1.5 are amplifications of oncogenes. [ 38 ]


Whole-genome duplications are also frequent in cancers, detected in 30% to 36% of tumors from the most common cancer types. [ 40 ] [ 41 ]  Their exact role in carcinogenesis is unclear, but they in some cases lead to loss of chromatin segregation leading to chromatin conformation changes that in turn lead to oncogenic epigenetic and transcriptional modifications. [ 42 ]


Genome instability  (also  genetic instability  or  genomic instability ) refers to a high frequency of  mutations  within the genome of a  cellular lineage . These mutations can include changes in  nucleic acid sequences ,  chromosomal rearrangements  or  aneuploidy . Genome instability does occur in bacteria. [ 1 ]  In multicellular organisms genome instability is central to carcinogenesis, [ 2 ]  and in humans it is also a factor in some  neurodegenerative  diseases such as  amyotrophic lateral sclerosis  or the neuromuscular disease  myotonic dystrophy .


The sources of genome instability have only recently begun to be elucidated. A high frequency of externally caused DNA damage [ 3 ]  can be one source of genome instability since DNA damage can cause inaccurate  translesion  DNA synthesis past the damage or errors in repair, leading to  mutation . Another source of genome instability may be  epigenetic  or  mutational  reductions in expression of DNA repair genes. Because  endogenous (metabolically-caused) DNA damage  is very frequent, occurring on average more than 60,000 times a day in the genomes of human cells, any reduced DNA repair is likely an important source of genome instability.


Usually, all cells in an individual in a given species (plant or animal) show a constant number of  chromosomes , which constitute what is known as the  karyotype  defining this species (see also  List of number of chromosomes of various organisms ), although some species present a very high karyotypic variability. In humans, mutations that would change an amino acid within the protein coding region of the genome occur at an average of only 0.35 per generation (less than one mutated protein per generation). [ 4 ]


Sometimes, in a species with a stable karyotype, random variations that modify the normal number of chromosomes may be observed. In other cases, there are structural alterations (e.g.,  chromosomal translocations ,  deletions ) that modify the standard chromosomal complement. In these cases, it is indicated that the affected organism presents genome instability (also  genetic instability , or even  chromosomic instability ). The process of genome instability often leads to a situation of  aneuploidy , in which the cells present a chromosomic number that is either higher or lower than the normal complement for the species.


In the cell cycle, DNA is usually most vulnerable during replication. The  replisome  must be able to navigate obstacles such as tightly wound chromatin with bound proteins, single and double stranded breaks which can lead to the stalling of the replication fork. Each protein or enzyme in the  replisome  must perform its function well to result in a perfect copy of DNA. Mutations of proteins such as DNA polymerase or DNA ligase can lead to impairment of replication and lead to spontaneous chromosomal exchanges. [ 5 ]  Proteins such as Tel1 and Mec1 (ATR, ATM in humans) can detect single and double-stranded breaks and recruit factors such as Rmr3  helicase  to stabilize the replication fork in order to prevent its collapse. Mutations in Tel1, Mec1, and Rmr3  helicase  result in a significant increase of chromosomal recombination. ATR responds specifically to stalled replication forks and single-stranded breaks resulting from UV damage while ATM responds directly to double-stranded breaks. These proteins also prevent progression into mitosis by inhibiting the firing of late replication origins until the DNA breaks are fixed by phosphorylating CHK1 and CHK2, which results in a signaling cascade arresting the cell in S-phase. [ 6 ]  For single stranded breaks, replication occurs until the location of the break, then the other strand is nicked to form a double stranded break, which can then be repaired by Break Induced Replication or homologous recombination using the sister  chromatid  as an error-free template. [ 7 ]  In addition to S-phase checkpoints, G1 and G2 checkpoints exist to check for transient DNA damage which could be caused by mutagens such as UV damage. An example is the Saccharomyces pombe gene rad9 which arrests the cells in late S/G2 phase in the presence of DNA damage caused by radiation. The yeast cells with defective rad9 failed to arrest following irradiation, continued cell division, and died rapidly; the cells with wild-type rad9 successfully arrested in late S/G2 phase and remained viable. The cells that arrested were able to survive due to the increased time in S/G2 phase allowing for DNA repair enzymes to function fully. [ 8 ]


There are hotspots in the genome where DNA sequences are prone to gaps and breaks after inhibition of DNA synthesis such as in the aforementioned checkpoint arrest. These sites are called fragile sites, and can occur commonly as naturally present in most mammalian genomes or occur rarely as a result of mutations, such as DNA-repeat expansion. Rare fragile sites can lead to genetic disease such as fragile X mental retardation syndrome, myotonic dystrophy, Friedrich's ataxia, and Huntington's disease, most of which are caused by expansion of repeats at the DNA, RNA, or protein level. [ 9 ]  Although, seemingly harmful, these common fragile sites are conserved all the way to yeast and bacteria. These ubiquitous sites are characterized by trinucleotide repeats, most commonly CGG, CAG, GAA, and GCN. These trinucleotide repeats can form into hairpins, leading to difficulty of replication. Under  replication stress , such as defective machinery or further DNA damage, DNA breaks and gaps can form at these fragile sites. Using a sister chromatid as repair is not a fool-proof backup as the surrounding DNA information of the n and n+1 repeat is virtually the same, leading to copy number variation. For example, the 16th copy of CGG might be mapped to the 13th copy of CGG in the sister chromatid since the surrounding DNA is both CGGCGGCGG..., leading to 3 extra copies of CGG in the final DNA sequence. [ citation needed ]


In both E. coli and Saccharomyces pombe, transcription sites tend to have higher recombination and mutation rates. The coding or non-transcribed strand accumulates more mutations than the template strand. This is due to the fact that the coding strand is single-stranded during transcription, which is chemically more unstable than double-stranded DNA. During elongation of transcription, supercoiling can occur behind an elongating RNA polymerase, leading to single-stranded breaks. When the coding strand is single-stranded, it can also hybridize with itself, creating DNA secondary structures that can compromise replication. In E. coli, when attempting to transcribe GAA triplets such as those found in Friedrich's ataxia, the resulting RNA and template strand can form mismatched loops between different repeats, leaving the complementary segment in the coding strand available to form its own loops which impede replication. [ 10 ]  Furthermore, replication of DNA and transcription of DNA are not temporally independent; they can occur at the same time and lead to collisions between the replication fork and RNA polymerase complex. In S. cerevisiae, Rrm3 helicase is found at highly transcribed genes in the yeast genome, which is recruited to stabilize a stalling replication fork as described above. This suggests that transcription is an obstacle to replication, which can lead to increased stress in the chromatin spanning the short distance between the unwound replication fork and transcription start site, potentially causing single-stranded DNA breaks. In yeast, proteins act as barriers at the 3' of the transcription unit to prevent further travel of the DNA replication fork. [ 11 ]


In some portions of the genome, variability is essential to survival. One such locale is the Ig genes. In a pre-B cell, the region consists of all V, D, and J segments. During development of the B cell, a specific V, D, and J segment is chosen to be spliced together to form the final gene, which is catalyzed by RAG1 and RAG2 recombinases.  Activation-Induced Cytidine Deaminase (AID) then converts cytidine into uracil. Uracil normally does not exist in DNA, and thus the base is excised and the nick is converted into a double-stranded break which is repaired by non-homologous end joining (NHEJ). This procedure is very error-prone and leads to somatic hypermutation. This genomic instability is crucial in ensuring mammalian survival against infection.  V, D, J recombination  can ensure millions of unique B-cell receptors; however, random repair by NHEJ introduces variation which can create a receptor that can bind with higher affinity to antigens. [ 12 ]


Of about 200 neurological and neuromuscular disorders, 15 have a clear link to an inherited or acquired defect in one of the DNA repair pathways or excessive genotoxic oxidative stress. [ 13 ] [ 14 ]   Five of them ( xeroderma pigmentosum ,  Cockayne's syndrome ,  trichothiodystrophy ,  Down's syndrome , and  triple-A syndrome ) have a defect in the DNA nucleotide excision repair pathway.  Six ( spinocerebellar ataxia  with axonal neuropathy-1,  Huntington's disease ,  Alzheimer's disease ,  Parkinson's disease ,  Down's syndrome  and  amyotrophic lateral sclerosis ) seem to result from increased oxidative stress, and the inability of the base excision repair pathway to handle the damage to DNA that this causes.  Four of them (Huntington's disease, various  spinocerebellar ataxias ,  Friedreich's ataxia  and  myotonic dystrophy  types 1 and 2) often have an unusual expansion of repeat sequences in DNA, likely attributable to genome instability.  Four ( ataxia-telangiectasia , ataxia-telangiectasia-like disorder,  Nijmegen breakage syndrome  and Alzheimer's disease) are defective in genes involved in repairing DNA double-strand breaks. Overall, it seems that oxidative stress is a major cause of genomic instability in the brain.  A particular neurological disease arises when a pathway that normally prevents oxidative stress is deficient, or a DNA repair pathway that normally repairs damage caused by oxidative stress is deficient. [ citation needed ]


In  cancer , genome instability can occur prior to or as a consequence of transformation. [ 15 ]  Genome instability can refer to the accumulation of extra copies of  DNA  or  chromosomes ,  chromosomal translocations ,  chromosomal inversions , chromosome  deletions , single-strand breaks in DNA,  double-strand breaks  in DNA, the intercalation of foreign substances into the DNA double helix, or any abnormal changes in DNA tertiary structure that can cause either the loss of DNA, or the misexpression of genes. Situations of genome instability (as well as aneuploidy) are common in cancer cells, and they are considered a "hallmark" for these cells. The unpredictable nature of these events are also a main contributor to the  heterogeneity  observed among tumour cells. [ citation needed ]


It is currently accepted that sporadic tumors (non-familial ones) are originated due to the accumulation of several genetic errors. [ 16 ]   An average cancer of the breast or colon can have about 60 to 70 protein altering mutations, of which about 3 or 4 may be "driver" mutations, and the remaining ones may be "passenger" mutations [ 17 ]  Any genetic or epigenetic lesion increasing the  mutation  rate will have as a consequence an increase in the acquisition of new mutations, increasing then the probability to develop a tumor. [ 18 ]  During the process of  tumorogenesis , it is known that  diploid  cells acquire mutations in genes responsible for maintaining genome integrity  ( caretaker genes ), as well as in genes that are directly controlling cellular proliferation  ( gatekeeper genes ). [ 19 ]  Genetic instability can originate due to deficiencies in DNA repair, or due to loss or gain of chromosomes, or due to large scale chromosomal reorganizations. Losing genetic stability will favour tumor development, because it favours the generation of mutants that can be selected by the environment. [ 20 ]


The  tumor microenvironment  has an inhibitory effect on  DNA repair  pathways contributing to genomic instability, which promotes tumor survival, proliferation, and malignant transformation. [ 21 ]


The protein coding regions of the human genome, collectively called the  exome , constitutes only 1.5% of the total genome. [ 22 ]   As pointed out above, ordinarily there are only an average of 0.35 mutations in the exome per generation (parent to child) in humans.  In the entire genome (including non-protein coding regions) there are only about 70 new mutations per generation in humans. [ 23 ] [ 24 ]


The likely major underlying cause of mutations in cancer is DNA damage. [ citation needed ]   For example, in the case of lung cancer,  DNA damage is caused by agents in  exogenous   genotoxic  tobacco smoke (e.g.  acrolein , formaldehyde,  acrylonitrile , 1,3-butadiene, acetaldehyde, ethylene oxide and isoprene). [ 25 ]   Endogenous (metabolically-caused) DNA damage  is also very frequent, occurring on average more than 60,000 times a day in the genomes of human cells (see  DNA damage (naturally occurring) ).  Externally and endogenously caused damages may be converted into mutations by inaccurate  translesion synthesis  or inaccurate DNA repair (e.g. by  non-homologous end joining ).  In addition, DNA damages can also give rise to  epigenetic  alterations during DNA repair. [ 26 ] [ 27 ] [ 28 ]  Both mutations and epigenetic alterations (epimutations) can contribute to progression to  cancer .


As noted above, about 3 or 4 driver mutations and 60 passenger mutations occur in the exome (protein coding region) of a cancer. [ 17 ]   However, a much larger number of mutations occur in the  non-protein-coding regions  of DNA.  The average number of DNA sequence mutations in the entire genome of a breast cancer tissue sample is about 20,000. [ 29 ]   In an average melanoma tissue sample (where melanomas have a higher  exome  mutation frequency [ 17 ] ) the total number of DNA sequence mutations is about 80,000. [ 30 ]


The high frequency of mutations in the total genome within cancers suggests that, often, an early carcinogenic alteration may be a deficiency in DNA repair.  Mutation rates substantially increase (sometimes by 100-fold) in cells defective in  DNA mismatch repair [ 31 ] [ 32 ]  or in  homologous recombinational   DNA repair . [ 33 ]   Also, chromosomal rearrangements and aneuploidy increase in humans defective in DNA repair gene  BLM . [ 34 ]


A deficiency in DNA repair itself can allow DNA damages to accumulate, and error-prone  translesion synthesis  past some of those damages may give rise to mutations. In addition, faulty repair of these accumulated DNA damages may give rise to  epigenetic alterations or epimutations .  While a mutation or epimutation in a DNA repair gene itself would not confer a selective advantage, such a repair defect may be carried along as a passenger in a cell when the cell acquires an additional mutation/epimutation that does provide a proliferative advantage.  Such cells, with both proliferative advantages and one or more DNA repair defects (causing a very high mutation rate), likely give rise to the 20,000 to 80,000 total genome mutations frequently seen in cancers. [ citation needed ]


In somatic cells, deficiencies in DNA repair sometimes arise by mutations in DNA repair genes, but much more often are due to epigenetic reductions in expression of DNA repair genes.  Thus, in a sequence of 113 colorectal cancers, only four had somatic missense mutations in the DNA repair gene MGMT, while the majority of these cancers had reduced MGMT expression due to methylation of the MGMT promoter region. [ 35 ]   Five reports, listed in the article  Epigenetics  (see section "DNA repair epigenetics in cancer") presented evidence that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region.


Similarly, for 119 cases of colorectal cancers classified as mismatch repair deficient and lacking DNA repair gene PMS2 expression, Pms2 was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1). [ 36 ]   The other 10 cases of loss of PMS2 expression were likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1. [ 37 ]


In  cancer epigenetics  (see section  Frequencies of epimutations in DNA repair genes ), there is a partial listing of epigenetic deficiencies found in DNA repair genes in sporadic cancers.  These include frequencies of between 13–100% of epigenetic defects in genes  BRCA1 ,  WRN ,  FANCB ,  FANCF ,  MGMT ,  MLH1 ,  MSH2 ,  MSH4 ,  ERCC1 , XPF,  NEIL1  and  ATM  located in cancers including breast, ovarian, colorectal and head and neck.  Two or three epigenetic deficiencies in expression of ERCC1, XPF and/or PMS2 were found to occur simultaneously in the majority of the 49 colon cancers evaluated. [ 38 ]   Some of these DNA repair deficiencies can be caused by epimutations in  microRNAs  as summarized in the  MicroRNA  article section titled  miRNA, DNA repair and cancer .


Cancers usually result from disruption of a tumor repressor or dysregulation of an oncogene. Knowing that B-cells experience DNA breaks during development can give insight to the genome of lymphomas. Many types of lymphoma are caused by chromosomal translocation, which can arise from breaks in DNA, leading to incorrect joining. In Burkitt's lymphoma,  c-myc , an oncogene encoding a transcription factor, is translocated to a position after the promoter of the immunoglobulin gene, leading to dysregulation of c-myc transcription. Since immunoglobulins are essential to a lymphocyte and highly expressed to increase detection of antigens, c-myc is then also highly expressed, leading to transcription of its  targets , which are involved in cell proliferation.  Mantle cell lymphoma  is characterized by fusion of  cyclin D1  to the immunoglobulin locus. Cyclin D1 inhibits Rb, a tumor suppressor, leading to tumorigenesis.  Follicular lymphoma  results from the translocation of the immunoglobulin promoter to the Bcl-2 gene, giving rise to high levels of Bcl-2 protein, which inhibits apoptosis. DNA-damaged B-cells no longer undergo apoptosis, leading to further mutations which could affect driver genes, leading to tumorigenesis. [ 39 ]  The location of translocation in the oncogene shares structural properties of the target regions of  AID , suggesting that the oncogene was a potential target of AID, leading to a double-stranded break that was translocated to the immunoglobulin gene locus through  NHEJ  repair. [ 40 ]


Genotoxicity  is the  property  of chemical agents that damage the genetic information within a cell causing  mutations , which may lead to  cancer . While genotoxicity is often confused with  mutagenicity , all mutagens are genotoxic, but some genotoxic substances are not mutagenic. The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. The permanent, heritable changes can affect either  somatic cells  of the organism or  germ cells  to be passed on to future generations. [ 1 ]  Cells prevent expression of the genotoxic mutation by either  DNA repair  or  apoptosis ; however, the damage may not always be fixed leading to  mutagenesis .


To  assay  for genotoxic molecules, researchers assay for DNA damage in cells exposed to the toxic substrates. This DNA damage can be in the form of single- and double-strand breaks, loss of excision repair, cross-linking, alkali-labile sites, point mutations, and structural and numerical chromosomal aberrations. [ 2 ]  The compromised integrity of the genetic material has been known to cause cancer. As a consequence, many sophisticated techniques including Ames Assay,  in vitro  and  in vivo  Toxicology Tests, and Comet Assay have been developed to assess the chemicals' potential to cause DNA damage that may lead to cancer.


Genotoxic substances induce damage to the genetic material in the cells through interactions with the DNA sequence and structure.  For example, the transition metal  chromium  interacts with DNA in its high-valent oxidation state, incurring DNA lesions which lead to  carcinogenesis .  The metastable oxidation state Cr(V) is achieved through reductive activation.  Researchers performed an experiment to study the interaction between DNA with the carcinogenic chromium by using a Cr(V)-Salen complex at the specific oxidation state. [ 3 ]   The interaction was specific to the  guanine  nucleotide in the genetic sequence.  In order to narrow the interaction between the Cr(V)-Salen complex with the guanine base, the researchers modified the bases to 8-oxo-G so to have site specific oxidation.  The reaction between the two molecules caused DNA lesions; the two lesions observed at the modified base site were guanidinohydantoin and spiroiminodihydantoin.  To further analyze the site of lesion, it was observed that polymerase stopped at the site and  adenine  was inappropriately incorporated into the DNA sequence opposite of the 8-oxo-G base.  Therefore, these lesions predominately contain G→T  transversions .  High-valent chromium is seen to act as a carcinogen as researchers found that "the mechanism of damage and base oxidation products for the interaction between high-valent chromium and DNA...  are relevant to  in vivo  formation of DNA damage leading to cancer in chromate-exposed human populations". [ 3 ]   Consequently, it shows how high-valent chromium can act as a carcinogen with 8-oxo-G forming  xenobiotics . [ 3 ]


Another example of a genotoxic substance causing DNA damage are  pyrrolizidine alkaloids  (PAs).  These substances are found mainly in plant species and are poisonous to animals, including humans; about half of them have been identified as genotoxic and many as tumorigenic.  The researchers concluded from testing that when metabolically activated, "PAs produce DNA adducts, DNA cross-linking, DNA breaks, sister chromatid exchange, micronuclei, chromosomal aberrations, gene mutations, and chromosome mutations  in vivo  and  in vitro ." [ 4 ]   The most common mutation within the genes are G:C → T:A tranversions and tandem base substitution. The pyrrolizidine alkaloids are mutagenic  in vivo  and  in vitro  and, therefore, responsible for the carcinogenesis prominently in the liver. [ 4 ]   Comfrey  is an example of a plant species that contains fourteen different PAs.  The active metabolites interact with DNA to cause DNA damage, mutation induction, and cancer development in liver  endothelial cells  and  hepatocytes .  The researchers discovered in the end that the "comfrey is mutagenic in liver, and PA contained in comfrey appear to be responsible for comfrey-induced toxicity and tumor induction,". [ 5 ]


The purpose of genotoxicity testing is to determine if a substrate will influence genetic material or may cause cancer.  They can be performed in bacterial, yeast, and mammalian cells. [ 2 ]   With the knowledge from the tests, one can control early development of vulnerable organisms to genotoxic substances. [ 1 ]


The Bacterial Reverse Mutation Assay, also known as the  Ames Assay , is used in laboratories to test for gene mutation.  The technique uses many different bacterial strains in order to compare the different changes in the genetic material.  The result of the test detects the majority of genotoxic  carcinogens  and genetic changes; the types of mutations detected are  frame shifts  and base substitutions. [ 6 ]


The purpose of  in vitro  testing is to determine whether a substrate, product, or environmental factor induces genetic damage.  One technique entails cytogenetic assays using different mammalian cells. [ 6 ]   The types of  aberrations  detected in cells affected by a genotoxic substance are chromatid and chromosome gaps, chromosome breaks, chromatid deletions, fragmentation, translocation, complex rearrangements, and many more. The  clastogenic  or  aneugenic  effects from the genotoxic damage will cause an increase in frequency of structural or numerical aberrations of the genetic material. [ 6 ]   This is similar to the  micronucleus test  and chromosome aberration assay, which detect structural and numerical chromosomal aberrations in mammalian cells. [ 1 ]


In a specific mammalian tissue, one can perform a mouse  lymphoma  TK+/- assay to test for changes in the genetic material. [ 6 ]   Gene mutations are commonly point mutations, altering only one base within the genetic sequence to alter the ensuing transcript and amino acid sequence; these point mutations include base substitutions, deletions, frame-shifts, and rearrangements.  Also,  chromosomes'  integrity may be altered through chromosome loss and clastogenic  lesions  causing multiple gene and multilocus deletions.  The specific type of damage is determined by the size of the colonies, distinguishing between genetic mutations (mutagens) and chromosomal aberrations (clastogens). [ 6 ]


The  SOS/umu assay test  evaluates the ability of a substance to induce DNA damage; it is based on the alterations in the induction of the SOS response due to DNA damage.  The benefits of this technique are that it is a fast and simple method and convenient for numerous substances.  These techniques are performed on water and wastewater in the environment. [ 7 ]


The purpose for  in vivo  testing is to determine the potential of DNA damage that can affect chromosomal structure or disturb the  mitotic apparatus  that changes chromosome number; the factors that could influence the genotoxicity are ADME and DNA repair.  It can also detect genotoxic agents missed in  in vitro  tests.  The positive result of induced chromosomal damage is an increase in frequency of micronucelated PCEs. [ 6 ]   A  micronucleus  is a small structure separate from the nucleus containing nuclear DNA arisen from DNA fragments or whole chromosomes that were not incorporated in the daughter cell during mitosis.  Causes for this structure are mitotic loss of acentric chromosomal fragments (clastogenicity), mechanical problems from chromosomal breakage and exchange, mitotic loss of chromosomes (aneugenicity), and apoptosis.  The micronucleus test  in vivo  is similar to the  in vitro  one because it tests for structural and numerical chromosomal aberrations in mammalian cells, especially in rats' blood cells. [ 6 ]


Comet assays  are one of the most common tests for genotoxicity. The technique involves lysing cells using detergents and salts. The DNA released from the lysed cell is electrophoresed in an agarose gel under  neutral pH  conditions. Cells containing DNA with an increased number of double-strand breaks will migrate more quickly to the anode. This technique is advantageous in that it detects low levels of DNA damage, requires only a very small number of cells, is cheaper than many techniques, is easy to execute, and quickly displays results. However, it does not identify the mechanism underlying the genotoxic effect or the exact chemical or chemical component causing the breaks. [ 8 ]


Genotoxic effects such as deletions, breaks and/or rearrangements can lead to cancer if the damage does not immediately lead to cell death. Regions sensitive to breakage, called  fragile sites , may result from genotoxic agents (such as pesticides). Some chemicals have the ability to induce fragile sites in regions of the chromosome where  oncogenes  are present, which could lead to carcinogenic effects. In keeping with this finding, occupational exposure to some mixtures of pesticides are positively correlated with increased genotoxic damage in the exposed individuals. DNA damage is not uniform in its severity across populations because Individuals vary in their ability to activate or detoxify genotoxic substances, which leads to variability in the incidence of cancer among individuals. The difference in ability to detoxify certain compounds is due to individuals' inherited  polymorphisms  of genes involved in the metabolism of the chemical. Differences may also be attributed to individual variation in efficiency of DNA repair mechanisms [ 9 ]


The  metabolism  of some chemicals results in the production of  reactive oxygen species  (ROS), which is a possible mechanism of genotoxicity. This is seen in the metabolism of  arsenic , which produces  hydroxyl radicals , which are known to cause genotoxic effects. [ 10 ]   Similarly, ROS have been implicated in genotoxicity caused by particles and fibers. Genotoxicity of nonfibrous and fibrous particles is characterized by high production of ROS from  inflammatory cells . [ 11 ]


Major genotoxic agents responsible for the four most common cancers worldwide (lung, breast, colon and stomach) have been identified.


Lung cancer  is the most frequent cancer in the world, both in terms of yearly cases (1.61 million cases; 12.7% of all cancer cases) and deaths (1.38 million deaths; 18.2% of all cancer deaths). [ 12 ]   Tobacco smoke  is the main cause of lung cancer. Risk estimates for lung cancer indicate that tobacco smoke is responsible for 90% of lung cancers in the United States.  Tobacco smoke contains more than 5,300 identified chemicals. The most significant carcinogens in tobacco smoke have been determined by a "Margin of Exposure" approach. [ 13 ]  By this approach, the tumorigenic compounds in tobacco smoke were, in order of importance,  acrolein ,  formaldehyde ,  acrylonitrile ,  1,3-butadiene ,  cadmium ,  acetaldehyde ,  ethylene oxide , and  isoprene . In general, these compounds are genotoxic and cause DNA damage. As examples, DNA damaging effects have been reported for acrolein, [ 14 ]  formaldehyde, [ 15 ]  and acrylonitrile. [ 16 ]


Breast cancer  is the second most frequent cancer worldwide on a yearly basis [(1.38 million cases, 10.9% of all cancer cases), and ranks 5th as cause of death (458,000, 6.1% of all cancer deaths)]. [ 12 ]  Breast cancer risk is associated with persistently high blood levels of  estrogen . [ 17 ]   Estrogen likely contributes to breast  carcinogenesis  by the following three processes; (1) metabolic conversion of estrogen to genotoxic, mutagenic carcinogens, (2) stimulation of growth of tissues, and (3) repression of phase II  detoxification  enzymes that metabolize genotoxic  reactive oxygen species , thus resulting in increased oxidative DNA damage. [ 18 ] [ 19 ] [ 20 ]  The principal human estrogen,  estradiol , can be metabolized to  quinone  derivatives that form  DNA adducts . [ 21 ]   These derivatives can cause the removal of bases from the phosphodiester backbone of DNA (e.g.  depurination ).  This removal may be followed by inaccurate repair or replication of the apurinic site leading to  mutation  and eventually cancer.


Colorectal cancer  is the third most frequent cancer worldwide [1.23 million cases (9.7% of all cancer cases), 608,000 deaths (8.0% of all cancer deaths)]. [ 12 ]  In the United States, tobacco smoke may be responsible for up to 20% of colorectal cancers. [ 22 ]  In addition,  bile acids  are implicated by substantial evidence as an important genotoxic factor in colon cancer. [ 23 ]  In particular, the bile acid  deoxycholic acid  acid causes the production of DNA-damaging reactive oxygen species in human and rodent colon epithelial cells. [ 23 ]


Stomach cancer  is the fourth most common cancer worldwide [990,000 cases (7.8% of all cancer cases), 738,000 deaths (9.7% of all cancer deaths )]. [ 12 ]  Infection by  Helicobacter pylori   is the main causative factor in stomach cancer. Chronic inflammation due to  H. pylori , if untreated, is often long-standing.  H. pylori  infection of gastric epithelial cells causes increased production of genotoxic  reactive oxygen species  (ROS). [ 24 ] [ 25 ]   ROS cause oxidative damage to DNA that includes the major base alteration  8-Oxo-2'-deoxyguanosine .  In a recent retrospective study it was found that use of a bile acid sequestrant was associated with a significant reduction in gastric cancer risk, suggesting that bile acids may be a contributory factor in stomach cancer. [ 26 ]


Genotoxic chemotherapy is the treatment of cancer with the use of one or more genotoxic drugs. The treatment is traditionally part of  standardized regime . By utilizing the destructive properties of genotoxins treatments aims to induce DNA damage into cancer cells. Any damage done to a cancer is passed on to descendent cancer cells as  proliferation  continues. If this damage is severe enough, it will induce cells to undergo  apoptosis . [ 27 ]


A drawback of treatment is that many genotoxic drugs are effective on cancerous cells and normal cells alike. Selectivity of a particular drug's action is based on the sensitivity of the cells themselves.  So, while rapidly dividing cancer cells are particularly sensitive to many drug treatments, often normal functioning cells are affected. [ 27 ]


Another risk of treatment is that, in addition to being genotoxic, many of the drugs are also  mutagenic  and  cytotoxic . So the effects of these drugs are not limited to just DNA damage. In addition, some of these drugs that are meant to treat cancers are also  carcinogens  themselves, raising the risk of secondary cancers, such as  leukemia . [ 27 ]


This table depicts different genotoxic-based cancer treatments along with examples. [ 27 ]


Genotoxic  DNA damage  in the  kidney  has been implicated in both acute and  chronic kidney injury , as well as in  renal cell carcinoma . [ 28 ]    Studies of humans with genetic deficiencies in  DNA repair  pathways have revealed that their kidneys are particularly vulnerable to DNA damages such as DNA crosslinks, DNA breaks and transcription-blocking damage. [ 28 ]


A  germline mutation , or  germinal mutation , is any detectable variation within  germ cells  (cells that, when fully developed, become  sperm  and  ova ). [ 1 ]  Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated  sperm  or  oocyte  come together to form a  zygote . [ 2 ]  After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every  somatic  and germline cell in the offspring; this is also known as a constitutional mutation. [ 2 ]  Germline mutation is distinct from  somatic mutation .


Germline mutations can be caused by a variety of endogenous (internal) and exogenous (external) factors, and can occur throughout zygote development. [ 3 ]  A mutation that arises only in germ cells can result in offspring with a genetic condition that is not present in either parent; this is because the mutation is not present in the rest of the parents' body, only the germline. [ 3 ]