Germline mutations can occur before fertilization and during various stages of zygote development. [ 3 ]  When the mutation arises will determine the effect it has on offspring. If the mutation arises in either the sperm or the oocyte before development, then the mutation will be present in every cell in the individual's body. [ 4 ]  A mutation that arises soon after fertilization, but before germline and somatic cells are determined, then the mutation will be present in a large proportion of the individual's cell with no bias towards germline or somatic cells, this is also called a gonosomal mutation. [ 4 ]  A mutation that arises later in zygote development will be present in a small subset of either somatic or germline cells, but not both. [ 3 ] [ 4 ]


A germline mutation often arises due to  endogenous  factors, like errors in cellular replication and oxidative damage. [ 5 ]  This damage is rarely repaired imperfectly, but due to the high rate of germ cell division, can occur frequently. [ 5 ]


Endogenous mutations are more prominent in sperm than in ova. [ 6 ]  This is because  spermatocytes  go through a larger number of cell divisions throughout a male's life, resulting in more replication cycles that could result in a DNA mutation. [ 5 ]  Errors in maternal ovum also occur, but at a lower rate than in paternal sperm. [ 5 ]  The types of mutations that occur also tend to vary between the sexes. [ 7 ]  A mother's eggs, after production, remain in stasis until each is utilized in ovulation. This long stasis period has been shown to result in a higher number of chromosomal and large sequence deletions, duplications, insertions, and transversions. [ 7 ]  The father's sperm, on the other hand, undergoes continuous replication throughout his lifetime, resulting in many small point mutations that result from errors in replication. These mutations commonly include single base pair substitutions, deletions, and insertions. [ 6 ]


Oxidative damage is another endogenous factor that can cause germline mutations. This type of damage is caused by  reactive oxygen species  that build up in the cell as a by-product of  cellular respiration . [ 8 ]  These reactive oxygen species are missing an electron, and because they are highly  electronegative  (have a strong electron pull) they will rip an electron away from another molecule. [ 8 ]  This can initiate DNA damage because it causes the nucleic acid guanine to shift to 8-oxoguanine (8-oxoG). This 8-oxoG molecule is then mistaken for a thymine by  DNA polymerase  during replication, causing a G>T  transversion  on one DNA strand, and a C>A transversion on the other. [ 9 ]


In mice and humans the spontaneous  mutation rate  in the male germ line is significantly lower than in  somatic cells . [ 10 ]   Furthermore, although the spontaneous mutation rate in the male germ line increases with age, the rate of increase is lower than in somatic tissues.  Within the testicular  spermatogonial  stem cell population the integrity of  DNA  appears to be maintained by highly effective  DNA damage  surveillance and protective  DNA repair  processes. [ 10 ]   The progressive increase in the mutation rate with age in the male germ line may be a result of a decline in the accuracy of the repair of DNA damages, or of an increase in  DNA replication  errors. Once  spermatogenesis  is complete, the differentiated spermatozoa that are formed no longer have the capability for DNA repair, and are thus vulnerable to attack by prevalent oxidative free radicals that cause oxidative DNA damage. Such damaged  spermatozoa  may undergo programmed cell death ( apoptosis ). [ 10 ]


A germline mutation can also occur due to  exogenous  factors. Similar to somatic mutations, germline mutations can be caused by exposure to harmful substances, which damage the DNA of germ cells. This damage can then either be repaired perfectly, and no mutations will be present, or repaired imperfectly, resulting in a variety of mutations. [ 11 ]  Exogenous  mutagens  include harmful chemicals and  ionizing radiation ; the major difference between germline mutations and somatic mutations is that germ cells are not exposed to  UV radiation , and thus not often directly mutated in this manner. [ 12 ] [ 13 ]


Different germline mutations can affect an individual differently depending on the rest of their genome. A  dominant mutation  only requires a single mutated gene to produce the disease  phenotype , while a recessive mutation requires both  alleles  to be mutated to produce the disease phenotype. [ 14 ]  For example, if the embryo inherits an already mutated allele from the father, and the same allele from the mother underwent an endogenous mutation, then the child will display the disease related to that mutated gene, even though only one parent carries the mutant allele. [ 14 ]  This is only one example of how a child can display a recessive disease while a mutant gene is only carried by one parent. [ 14 ]  Detection of chromosomal abnormalities can be found in utero for certain diseases by means of blood samples or ultrasound, as well as invasive procedures such as an  amniocentesis . Later detection can be found by genome screening.


Mutations in  tumour suppressor genes  or  proto-oncogenes  can predispose an individual to developing tumors. [ 15 ]  It is estimated that inherited genetic mutations are involved in 5-10% of cancers. [ 16 ]  These mutations make a person susceptible to tumor development if the other copy of the  oncogene  is randomly mutated. These mutations can occur in germ cells, allowing them to be  heritable . [ 15 ]  Individuals who inherit germline mutations in  TP53  are predisposed to certain cancer variants because the protein produced by this gene suppresses tumors. Patients with this mutation are also at a risk for  Li–Fraumeni syndrome . [ 16 ]  Other examples include mutations in the  BRCA1  and  BRCA2  genes which predispose to breast and ovarian cancer, or mutations in  MLH1  which predispose to  hereditary non-polyposis colorectal cancer .


Huntington's disease  is an  autosomal dominant  mutation in the HTT gene. The disorder causes degradation in the brain, resulting in uncontrollable movements and behavior. [ 17 ]  The mutation involves an expansion of repeats in the Huntington protein, causing it to increase in size. Patients who have more than 40 repeats will most likely be affected. The onset of the disease is determined by the amount of repeats present in the mutation; the greater the number of repeats, the earlier symptoms of the disease will appear. [ 17 ] [ 18 ]  Because of the dominant nature of the mutation, only one mutated allele is needed for the disease to be in effect. This means that if one parent is affected, the child will have a 50% chance of inheriting the disease. [ 19 ]  This disease does not have carriers because if a patient has one mutation, they will (most likely) be affected. The disease typically has a late onset, so many parents have children before they know they have the mutation. The HTT mutation can be detected through  genome screening .


Trisomy 21 (also known as  Down syndrome ) results from a child having three copies of chromosome 21. [ 20 ]  This chromosome duplication occurs during germ cell formation, when both copies of chromosome 21 end up in the same  daughter cell  in either the mother or father, and this mutant germ cell participates in fertilization of the zygote. [ 20 ]  Another, more common way this can occur is during the first cell division event after the formation of the zygote. [ 20 ]  The risk of Trisomy 21 increases with maternal age with the risk being 1/2000 (0.05%) at age 20 increasing to 1/100 (1%) at age 40. [ 21 ]  This disease can be detected by non-invasive as well as invasive procedures prenatally. Non-invasive procedures include scanning for  fetal DNA  through maternal plasma via a blood sample. [ 22 ]


Cystic fibrosis is an  autosomal recessive  disorder that causes a variety of symptoms and complications, the most common of which is a thick mucous lining in lung  epithelial  tissue due to improper salt exchange, but can also affect the  pancreas ,  intestines ,  liver , and  kidneys . [ 23 ] [ 24 ]  Many bodily processes can be affected due to the hereditary nature of this disease; if the disease is present in the DNA of both the sperm and the egg, then it will be present in essentially every cell and organ in the body; these mutations can occur initially in the germline cells, or be present in all parental cells. [ 23 ]  The most common mutation seen in this disease is ΔF508, which means a deletion of the amino acid at the 508 position. [ 25 ]  If both parents have a mutated  CFTR  (cystic fibrosis transmembrane conductance regulator) protein, then their children have a 25% of inheriting the disease. [ 23 ]  If a child has one mutated copy of CFTR, they will not develop the disease, but will become a carrier of the disease. [ 23 ]  The mutation can be detected before birth through amniocentesis, or after birth via prenatal genetic screening. [ 26 ]


Many Mendelian disorders stem from  dominant  point mutations within genes, including  cystic fibrosis ,  beta-thalassemia ,  sickle-cell anemia , and  Tay–Sachs disease . [ 14 ]  By inducing a double stranded break in sequences surrounding the disease-causing point mutation, a dividing cell can use the non-mutated strand as a template to repair the newly broken DNA strand, getting rid of the disease-causing mutation. [ 27 ]  Many different genome editing techniques have been used for genome editing, and especially germline mutation editing in germ cells and developing zygotes; however, while these therapies have been extensively studied, their use in human germline editing is limited. [ 28 ]


This editing system induces a double stranded break in the DNA, using a guide RNA and effector protein Cas9 to break the DNA backbones at specific target sequences. [ 27 ]  This system has shown a higher specificity than TALENs or ZFNs due to the Cas9 protein containing homologous (complementary) sequences to the sections of DNA surrounding the site to be cleaved. [ 27 ]  This broken strand can be repaired in 2 main ways: homologous directed repair (HDR) if a DNA strand is present to be used as a template (either homologous or donor), and if one is not, then the sequence will undergo  non-homologous end joining  (NHEJ). [ 27 ]  NHEJ often results in insertions or deletions within the gene of interest, due to the processing of the blunt strand ends, and is a way to study gene knockouts in a lab setting. [ 29 ]  This method can be used to repair a point mutation by using the  sister chromosome  as a template, or by providing a double stranded DNA template with the  CRISPR /Cas9 machinery to be used as the repair template. [ 27 ]


This method has been used in both human and animal models ( Drosophila ,  Mus musculus ,  and  Arabidopsis ), and current research is being focused on making this system more specific to minimize off-target cleavage sites. [ 30 ]


The  TALEN  (transcription activator-like effector nucleases) genome editing system is used to induce a double-stranded DNA break at a specific locus in the genome, which can then be used to mutate or repair the DNA sequence. [ 31 ]  It functions by using a specific repeated sequence of an amino acid that is 33-34 amino acids in length. [ 31 ]  The specificity of the DNA binding site is determined by the specific amino acids at positions 12 and 13 (also called the Repeat Variable Diresidue (RVD)) of this tandem repeat, with some RVDs showing a higher specificity for specific amino acids over others. [ 32 ]  Once the DNA break is initiated, the ends can either be joined with NHEJ that induces mutations, or by HDR that can fix mutations. [ 27 ]


Similar to TALENs,  zinc finger nucleases  (ZFNs) are used to create a double stranded break in the DNA at a specific locus in the genome. [ 31 ]  The ZFN editing complex consists of a  zinc finger protein  (ZFP) and a restriction enzyme cleavage domain. [ 33 ]  The ZNP domain can be altered to change the DNA sequence that the  restriction enzyme  cuts, and this cleavage event initiates cellular repair processes, similar to that of CRISPR/Cas9 DNA editing. [ 33 ]


Compared to CRISPR/Cas9, the therapeutic applications of this technology are limited, due to the extensive engineering required to make each ZFN specific to the desired sequence. [ 33 ]


Glycerol kinase deficiency  (GKD) is an X-linked recessive enzyme defect that is  heterozygous  in nature. Three clinically distinct forms of this deficiency have been proposed, namely infantile, juvenile, and adult.  National Institutes of Health  and its Office of Rare Diseases Research branch classifies GKD as a rare disease, known to affect fewer than 200,000 individuals in the United States. The responsible gene lies in a region containing genes in which deletions can cause  Duchenne muscular dystrophy  and  adrenal hypoplasia congenita . Combinations of these three genetic defects including GKD are addressed medically as Complex GKD. [ 1 ]


Glycerol Kinase Deficiency causes the condition known as hyperglycerolemia, [ 2 ]  an accumulation of  glycerol  in the  blood  and  urine .  This excess of glycerol in bodily fluids can lead to many more potentially dangerous  symptoms .   Common symptoms include vomiting and lethargy. [ 3 ]   These tend to be the only symptoms, if any, present in adult GKD which has been found to present with fewer symptoms than infant or juvenile GKD. [ 4 ]   When GKD is accompanied by  Duchenne muscular dystrophy  and  Adrenal Hypoplasia Congenita , also caused by  mutations  on the Xp21  chromosome , [ 5 ]  the symptoms can become much more severe.  Symptoms visible at or shortly after birth include:


Some other symptoms that become more noticeable with time would be:


Many of the physically visible symptoms, such as cryptorchidism, strabismus, learning disabilities, [ 5 ]  and myopathy, tend to have an added psychological effect on the subject due to the fact that they can set him or her apart from those without GKD.  Cryptorchidism, the failure of one or both of the testes to descend to the scrotum, has been known to lead to  sexual identity  confusion amongst young boys because it is such a major physiological anomaly. [ 7 ]   Strabismus is the misalignment of one's eyes.  Typically, one is focused but the other is “lazy” and is directed inward or out ward (up and down is less common but does occur).


Glycerol kinase deficiency  has two main causes.


In order to understand how this condition affects a person you must first have a basic understanding of the process called  glycolysis . This fundamental  metabolic pathway  is found in all known organisms. The process provides energy for our  cells  to carry out their daily functions.  The overall  reaction  involves a cell taking in the sugar  glucose  and converting it into the energy rich molecule  pyruvate . Inside the overall reaction there lie many steps that need to be followed in order for the original glucose molecule to be transformed into pyruvate.  The glucose first gathers a  phosphate group  from an  ATP  molecule in order to become  glucose-6-phosphate .  It is then changed into  fructose 6-phosphate , with the assistance of  phosphoglucose isomerase , which is then changed into fructose 1,6-biphosphate when the fructose molecule receives a phosphate group from another ATP.  The next step in the chain is crucial for cells in order to make more energy than they expend through the process of glycolysis; this step is when the  fructose 1,6-bisphosphate  molecule breaks down into two molecules of  dihydroxyacetone phosphate  (DHAP), so from this point on whenever  ATP  is being generated from  ADP  there are really two ATP molecules generated because there are two molecules undergoing the same reaction. [ 13 ]  One molecule that takes advantage of this second part of the metabolic process is the fatty molecule  glycerol . This is unfortunately prevented if someone is experiencing Glycerol Kinase Deficiency.


When a human's body needs to use stored  fat  for energy it will release glycerol and other  fatty acids  into the  bloodstream .  However, these glycerol molecules must contribute to the process of  glycolysis  before they can provide energy to the body, as they do not hold the necessary energy within themselves. So before  glycerol  can enter the pathway of glycolysis it must be converted into an intermediate molecule, which in this case is dihydroxyacetone phosphate (DHAP). This is where glycerol kinase comes into the picture. The enzyme is used in the first step in turning glycerol into dihydroxyacetone phosphate (DHAP). It catalyzes the transfer of a phosphate group from an ATP to a glycerol molecule forming glycerol (3) phosphate.  Then glycerol 3-phosphate, with the assistance of glycerol 3-phosphate dehydrogenase, can be dehydrogenated into DHAP. This molecule can then enter the  metabolic pathway  of glycolysis and provide more energy for the cell. [ 14 ]  Looking at the entire glycolysis pathway this conversion would yield an extra ATP for each glycerol molecule that eventually made its way into a DHAP molecule, which demonstrates the benefit of releasing glycerol into the bloodstream.  However, when suffering from a glycerol kinase deficiency many of the glycerol molecules released into the bloodstream end up not being converted to dihydroxyacetone phosphate (DHAP), because the host does not have enough of the enzyme to catalyze all of the reactions waiting to occur.  These extra molecules of glycerol are left floating around in the cell and can cause serious damage if left untreated.


GKD can be divided into three distinct forms: infantile, juvenile, and adult. Out of all of these the infantile form is the most clinically relevant because it leads to developmental delay and adrenal insufficiency. [ 11 ]


Treatments for Glycerol Kinase Deficiency are targeted to treat the symptoms because there are no permanent treatments for this disease. The main way to treat these symptoms is by using  corticosteroids , glucose  infusion , or  mineralocorticoids . Corticosteroids are steroid hormones that are naturally produced in the adrenal glands. These  hormones  regulate stress responses,  carbohydrate   metabolism , blood  electrolyte  levels, as well as other uses. The mineralocorticoids, such as  aldosterone  control many electrolyte levels and allow the  kidneys  to retain sodium. Glucose infusion is coupled with  insulin  infusion to monitor blood glucose levels and keep them stable. [ 15 ]


Due to the multitude of varying symptoms of this disease, there is no specific treatment that will cure this disease altogether. The symptoms can be treated with many different  treatments  and combinations of  medicines  to try to find the correct combination to offset the specific symptoms. Everyone with Glycerol Kinase Deficiency has varying degrees of symptoms and thereby requires different medicines to be used in combination to treat the symptoms; however, this disease is not curable and the symptoms can only be managed, not treated fully. [ 16 ]


Hemoglobin Hopkins-2  (Hb Hop-2) is a mutation of the protein  hemoglobin , which is responsible for the transportation of oxygen through the blood from the lungs to the musculature of the body in vertebrates.  The specific mutation in Hemoglobin Hopkins-2 results in two abnormal α chains (human hemoglobin consist of 2 α and 2 β polypeptides usually termed chains). [ 1 ]   The mutation is the result of  histidine  112 being replaced with  aspartic acid  in the protein's polypeptide sequence. [ 1 ]  Additionally, within one of the mutated alpha chains, there are substitutes at 114 and 118, two points on the amino acid chain. [ 2 ]  This mutation can cause  sickle cell anemia . [ 3 ]


Following the initial discovery of hemoglobin, two researchers working at  Johns Hopkins Hospital  in the mid-twentieth century, Ernest W. Smith and J.V. Torbert, discovered the Hopkins-2 mutation of hemoglobin. [ 4 ]   Work by  Harvey A. Itano  and Elizabeth A. Robinson in 1960 confirmed Smith's and Torbert's finding and emphasized the importance of the alpha loci in the mutation. [ 5 ]   Later in the twentieth century, Samuel Charache, another Hopkins affiliated scientist and doctor, studied the physiological impacts of the variant on health. [ 6 ]   His findings suggest that the variant plays no effect clinically. [ 7 ]


In the mid-1900s, many factors prompted hemoglobin research in  Baltimore, Maryland  and ultimately led to the discovery of Hemoglobin Hopkins-2.  First, the development of new technology, including  x-ray crystallography  and protein chemistry, that could be utilized in molecular biology studies catalyzed research. [ 8 ]   Furthermore, the large presence of  thalassemia  (Hb H), a disorder in which the alpha gene is dysfunctional, in Southeast Asia and southern China further concerned researchers as, if left untreated, the mutation could result in bone deformities,  swelling of the spleen , slowed growth rate, or  cardiac dysfunction . [ 9 ] [ 10 ]   Additionally,  Max Perutz 's, a Cambridge researcher, discovery of hemoglobin's basic  tertiary structure  in 1962 catalyzed research in hematology. [ 11 ]   Vernon Ingram's  research surrounding  sickle cell anemia  in 1956 revealed that variants, or mutations, in hemoglobin's RNA resulted in the  sickle cell disease . [ 8 ] [ 3 ]


Ernest W. Smith and J.V. Norbert examined Ingram's discovery and, in 1958, they discovered the hemoglobin Hopkins-2 mutation.  Smith and Torbert, research fellows working in Lockard Conley's  Hematology  Research Department at Johns Hopkins University, proved that recombination of  non-allelic genes  resulted in two variants of the gene that produced hemoglobin.  Variants of the "normal" hemoglobin gene result in mutation in the hemoglobins produced. [ 4 ]    In other words, the researchers discovered two versions of  hemoglobin :  the [Hb-Hop2] and  S variants . [ 8 ]


Smith's and Torbert's findings were confirmed and built upon by  Harvey A. Itano  and Elizabeth A. Robinson in 1960.   In their paper,  Genetic Control of the a- and B-Chains of Hemoglobin ,  Itano  and Robinson explained that both the alpha and beta  loci  are involved in the regulation of hemoglobin.  The recombination of the alpha loci on the gene coding for hemoglobin results in a mutation of the hemoglobin protein.  Researchers refer to this mutated protein as a Hopkins-2 variation of hemoglobin. [ 5 ]


Further research surrounding the Hopkins-2 genetic mutation was conducted by Johns Hopkins doctors, who remained engaged in research in the hematologic field despite a general change in focus away from hematology in the medical community.  Prominent Hopkins researcher Samuel Charache was one of many scientists at Hopkins who investigated  blood  and its components during the late twentieth century. [ 8 ]  Specifically, Charache is well known for his work with George Dover, another Hopkins researcher; together, they discovered a treatment option for  sickle cell anemia .  However, Charache was also engaged in hemoglobin Hopkins-2 research.  In his  Nature  article, titled  Clinical Studies and Physiological Properties of Hopkins Hemoglobin-2,  Charache announced his discoveries surrounding the hemoglobin variant.  Charache focused on physical implications of the variant, rather than on genetics themselves. Ultimately, Charache asserts in his paper that the variant is not prominent  phenotypically  and plays an unnoticeable or no effect on human health. [ citation needed ]


Hematologic studies were prominent at Johns Hopkins Hospital prior to the discovery of the hemoglobin Hopkins-2 variant. After observing the cells under a  microscope , Sir William Osler connected the concept of dysfunctional platelets to the development of ulcerative endocarditis and  thrombosis .  Osler's initial observations of platelets catalyzed the study of blood and hematology at Hopkins and in other research environments. [ 12 ]  Platelets were discovered independently by Max Schultze in 1865 [ 13 ]  and G. Bizzozero in 1882. [ 14 ]


Beginning in 1920, doctors at Johns Hopkins Hospital conducted research on  sickle cell anemia , or sickle cell disease.  Although their conclusions surrounding the disease are outdated, Doctors Taliaferro and Huck discovered a  latent  form of sickle cell anemia.  Their study on sickle cell anemia was the first of many to occur at Hopkins.  In 1940, Irving Sherman, a medical student at Johns Hopkins, correctly identified the  deoxygenation  of hemoglobin in sickle cell patients after he noted  refraction  patterns characteristic of deoxygenation when light was passed through the protein. [ 15 ]   The deoxygenation of hemoglobin in sickle cell patients has severe implications on those who carry the mutation.  The hemoglobin proteins, present in those with sickle cell disease, cannot carry oxygen to the organs and other tissues of the human body.  This results in  pain crises  and the disease results in an abbreviated  life expectancy  of 40–60 years. [ 16 ]


Ernest W. Smith and Torbert were integral in the discovery of Hopkins Hemoglobin-2, in addition to many other hematologic mutations and conditions. [ 4 ]   The two scientists worked together at Hopkins to identify the N-Baltimore mutation of Hemoglobin in 1958. [ 17 ]   Also referred to as the Hopkins-I, Jenkins, N-Memphis, or Kenwood mutation, the N-Baltimore mutation is a point mutation in which a  glycine  codon is replaced with an  adenosine  codon.  The N-Baltimore mutation is associated with the C and S mutations of hemoglobin. [ 18 ]


Smith conducted extensive research in conjunction with Locklard Conley, one of Smith's bosses at the time of the Hopkins Hemoglobin-2 variant discovery.  Lockard Conley, commonly referred to as "Lock," was a  Johns Hopkins  undergraduate and  Columbia  trained doctor.  In 1947, Conley became the first director of the Hematology Department at Hopkins and remained in the position for 33 years.  While there, he studied blood-related diseases, such as  blood coagulation  and  sickle cell anemia , and invented machinery to analyze molecular species. [ 19 ]   Specifically, he and Smith created a device that allowed for the separation of hemoglobin variants from standard hemoglobin molecules.  Conley's impact on hematology, therefore, was not only scientific discovery; but, also, technological discovery that allowed the hematologic field to expand. [ 20 ]   Conley remained a doctor and professor at  Johns Hopkins Hospital  until his death in 2010. [ 19 ]


Although a significant amount of hematologic research was completed during the 1950s and 1960s, scientists questioned whether more research could be completed without the development of more advanced technology. Ultimately, this doubt resulted in fewer scientists pursuing research in hematology in the mid twentieth century. [ 8 ]   Despite the scarcity of researchers, new discoveries surrounding genetics and hemoglobin were made.  However, research continued in major medical laboratories, like Hopkins. [ 21 ]


In the mid and late twentieth century, both doctors George J. Dover and Samuel Charache studied sickle cell anemia's pathology at Johns Hopkins Hospital. [ 7 ]   Together, they implemented treatment of the disease through the use of a cancer drug,  hydroxyurea ; the drug was successful in alleviating some of the painful spurts associated with  sickle cell anemia , in addition to  pulmonary  symptoms associated with the disease.  Dover, a pediatric hematologist and expert on  fetal hemoglobin , initiated the use of the protein as a way to treat sickle cell anemia in adults. [ citation needed ]


The Hopkins-2 variant of hemoglobin has an oxygen affinity within the body, meaning that there is an increase of oxygen spreading through the body due to the fact that Ho-2 carries a higher amount of oxygen. [ 22 ]  There is no red cell effect with Ho-2 compared to  Hemoglobin S , which changes the shape of the cell to become sickled. The Hopkins-2 variant of hemoglobin is not involved in forming  sickle cells . There is a lack of phenotypic expression of Ho-2 in terms of sickle cell, so a person with sickle cell and hemoglobin Hopkins-2 would be asymptomatic. [ citation needed ]


The Hopkins 2 variant of  hemoglobin  is the result of a mutation.  Specifically, the mutated protein is composed of two alpha chains.  The mutation for the Hopkins-2 variant of hemoglobin is located on the surface of the molecule.  Within the Ho-2 variant,  aspartic acid  replaces  histidine  at position 112 on the alpha chain. [ 2 ]  Dr.  Max Perutz  examined the aspartic acid in alpha 112 claiming that the carboxyl group of the amino acid forms a hydrogen bond with phenolix hydroxyl, which in turn stabilizes the structure of the molecule and increases oxygen affinity.  Ho-2 hemoglobin has high levels of oxygen, which replaces the lack of oxygen within red blood cells.  Ho-2 is similar to  Hemoglobin A , which is normal hemoglobin and also contains two α-chains.  This provides an explanation as to how the Hopkins-2 variant of hemoglobin is related to the symptoms of  sickle cell . [ citation needed ]


Hopkins-2 can also interact specifically with Hemoglobin S.   Hemoglobin S  is the most common abnormal hemoglobin variant.  Hemoglobin S is the variant that causes  sickle cell , which is a disorder in which red blood cells break down and become abnormally shaped.  Hemoglobin S has two beta chains, whereas hemoglobin Hopkins-2 has two alpha chains.  Hopkins-2 makes up 22% of hemolysates in single heterozygotes; therefore, there is the normal version of the gene in these patients.  Hemolysates are the products of the destruction of red blood cells.  Ho-2 also comprises 11% of hemolysates in ‘double’ heterozygotes, which are when the gene contains both Hopkins-2 and Hemoglobin S. [ 23 ]


There are currently multiple possible explanations as to how the Hemoglobin variant Hopkins-2 works. One explanation that scientists have come up with is that Ho-2 is created due to a point mutation causing the substitution of histidine by aspartic acid. [ 23 ]  The other explanation is that there is an unequal crossing over between two α genes which explains the replacement of histidine by aspartic acid. [ 23 ]  This then led a deletion occurring within the chromosome housing Ho-2. This deletion removes the region where the N-terminus is located in the major α-chain and where the c-terminus is located on the minor α-chain. [ 23 ]  Deletion of the N-terminus inactivates enzymes and halting their ability to cut chains at certain areas, which inevitably damages the chain. [ citation needed ]


Case studies were completed on some patients who carried the Hemoglobin Hopkins-2 genetic mutation by Samuel Charache and others. Researchers concluded after completing the studies that many of carriers of the mutation were asymptomatic to sickle cell and were overall quite healthy.  Any medical issues that occurred had no correlation to sickle cell.  These studies occurred in the 1970s. [ 8 ]


There were five carriers of Hemoglobin Hopkins 2 in the Fuller-Carr family and ten double heterozygotes of Ho-2 and Hemoglobin S. [ 24 ]  All the carriers were in good health and had normal hematology test results. Out of those carrying  hemoglobin S  and Ho-2, none were  anemic ; but, a few of those studied displayed elevated reticulocyte counts. [ 24 ]  This is measured through a blood test that analyzes the speed of production of red blood cells by bone marrow and its release into the blood. There was no suggestion of symptomatic sickle cell anemia in the family. [ citation needed ]


There was a study on a three-year-old that was a carrier of the hemoglobin variant of Hopkins-2. The child had mild anemia and reticulocytosis, which is commonly seen in anemia. [ 25 ]  There were, however, no sickled cells found in the blood and they had no symptoms relating to sickle cell. There was also a reduced mean corpuscular volume (MCV), which is the average volume of  red blood cell  count. [ 25 ]


Heteroplasmy  is the presence of more than one type of  organellar   genome  ( mitochondrial DNA  or  plastid  DNA) within a cell or individual. It is an important factor in considering the severity of  mitochondrial diseases . Because most  eukaryotic cells  contain many hundreds of mitochondria with hundreds of copies of mitochondrial DNA, it is common for  mutations  to affect only some mitochondria, leaving most unaffected.


Although detrimental scenarios are well-studied, heteroplasmy can also be beneficial. For example,  centenarians  show a higher than average degree of heteroplasmy. [ 1 ]


At birth, all copies of mitochondrial DNA are thought to be identical in most humans. [ 2 ]  Microheteroplasmy is mutations of up to about 2−5% of mitochondrial genomes, and is present in most adults. This refers to hundreds of independent mutations in one organism, with each mutation found in about 1–2% of all mitochondrial genomes. [ 3 ]  Very low-level heteroplasmic variance is present in essentially all individuals, even those who are healthy, and is likely to be due to both inherited and somatic single base substitutions. [ 2 ]


In order for heteroplasmy to occur,  organelles  must contain a genome and, in turn, a  genotype . In animals,  mitochondria  are the only organelles that contain their own genomes, so these organisms will only have mitochondrial heteroplasmy. In contrast, photosynthetic plants contain mitochondria and  chloroplasts , each of which contains  plastid genomes . Therefore, plant heteroplasmy occurs in two dimensions. [ 4 ]


Microheteroplasmy is the presence of mutations levels of up to about 2−5% of mitochondrial genomes. In  human mitochondrial DNA , microheteroplasmy constitutes hundreds of independent mutations in one organism, with each mutation usually found in 1–2% of all mitochondrial genomes. [ 5 ]


The distinction of microheteroplasmy and more gross heteroplasmy is dictated by technical considerations - classical  DNA sequencing  of mitochondrial DNA by the use of  PCR  is capable only of detecting mutations at levels of 10% or more, as a result of which mutations at lower levels were never systematically observed until the work of Lin et al. [ 6 ]


As it became apparent after the use of Lin's  cloning  and sequencing strategy, capable of detecting mutations at levels of 1% or less, such low-level heteroplasmy, or microheteroplasmy, is exceedingly common, and is in fact the most common form of mutational damage to human DNA found to date. In aged adults, each copy of mitochondrial DNA has on average 3.3 mutations changing  protein  structure. This exceeds previous estimates by more than three orders of magnitude.


The discovery of microheteroplasmy lends support to the mitochondrial theory of aging, and has already been linked to the causation of  Parkinson's disease . [ 7 ]


In 1909, while studying chloroplast genomes,  Erwin Baur  made the first observations about organelle inheritance patterns. Organelle genome inheritance differs from  nuclear genome , and this is illustrated by four violations of  Mendel's laws . [ 8 ]


Vegetative segregation , the random partitioning of cytoplasm, is a distinguishable characteristic of organelle heredity. During cell division, the organelles are divided equally, providing each daughter cell with a random selection of plasmid genotypes. [ 8 ]


Uniparental inheritance refers to the fact that, in most organisms, many offspring inherit organelle genes from only one parent. However, this is not a general law. Many organisms that have the ability to differentiate maternal and paternal sexes will produce offspring with a mixture of maternal, paternal, and biparental mitochondrial DNA. [ 8 ]


Entities undergoing uniparental inheritance and with little to no recombination may be expected to be subject to  Muller's ratchet , the inexorable accumulation of deleterious mutations until functionality is lost. Animal populations of mitochondria avoid this buildup through a developmental process known as the mtDNA bottleneck. The bottleneck exploits  stochastic processes in the cell  to increase in the cell-to-cell variability in mutant load as an organism develops: a single egg cell with some proportion of mutant mtDNA thus produces an embryo where different cells have different mutant loads. Cell-level selection may then act to remove those cells with more mutant mtDNA, leading to a stabilisation or reduction in mutant load between generations. The mechanism underlying the bottleneck is debated, [ 10 ] [ 11 ] [ 12 ]  with a recent mathematical and experimental metastudy providing evidence for a combination of random partitioning of mtDNAs at cell divisions and random turnover of mtDNA molecules within the cell. [ 13 ]


The mitochondrial bottleneck concept refers to the  classic evolutionary term , which is used to explain an event that reduces and specifies a population. It was developed to describe why mitochondrial DNA in an embryo might be drastically different from that of its mother. When a large population of DNA is subsampled, each sample population will receive a slightly different proportion of mitochondrial genotypes. Consequently, when paired with a high degree of replication, a rare or mutated allele can begin to proportionally dominate. In theory, this makes possible a single-generation shift of overall mitochondrial genotype. [ 9 ]


Although it is not well characterized, selection can occur for organelle genomes in heteroplasmic cells.  Intracellular  ("within cells") selection occurs within individual cells. It refers to the selective segregation of certain genotypes in mitochondrial DNA that allows the favoured genotype to thrive. Intercellular ("between cells") selection occurs on a larger scale, and refers to the preferential growth of cells that have greater numbers of a certain mitochondrial genotype. [ 8 ]  Selective differences can occur between naturally occurring, non-pathological mtDNA types when mixed in cells, and may depend on tissue type, age, and genetic distance. [ 14 ]  Selective differences between naturally occurring mtDNA types may pose challenges for gene therapies. [ 15 ]


In mitochondrial DNA, there is evidence for potent germline  purifying selection , as well as  purifying selection  during embryogenesis. Additionally, there is a dose-dependent decrease in reproduction ability for females that have mutations in mitochondrial DNA. This demonstrates another selection mechanism to prevent the evolutionary preservation of harmful mutations. [ 9 ]


It is very rare for organelle genes from different lineages to recombine. These genomes are usually inherited uniparentally, which does not provide a  recombination  opportunity. If they are inherited biparentally, it is unlikely that the organelles from the parents will fuse, meaning they will not share genomes.


However, it is possible for organelle genes from the same lineage to recombine. Intramolecular and intermolecular recombination can cause inversions and repeats in chloroplast DNA, and can produce subgenomic circles in mitochondrial DNA. [ 8 ]


Mutations in mitochondrial DNA are usually single nucleotide substitutions, single base insertions, or deletions.


Because each cell contains thousands of mitochondria, nearly all organisms house low levels of mitochondrial variants, conferring some degree of heteroplasmy. Although a single mutational event might be rare in its generation, repeated mitotic segregation and clonal expansion can enable it to dominate the mitochondrial DNA pool over time. When this occurs, it is known as reaching threshold, and it usually results in physiological consequences. [ 9 ]


Symptoms of severe heteroplasmic mitochondrial disorders do not usually appear until adulthood. Many cell divisions and a great deal of time are required for a cell to accumulate enough mutant mitochondria to cause symptoms.  An example of this phenomenon is  Leber optic atrophy .  Generally, individuals with this condition do not experience vision difficulties until they have reached adulthood.  Another example is  MERRF syndrome  (or Myoclonic Epilepsy with Ragged Red Fibers). In  MELAS , heteroplasmy explains the variation in severity of the disease among siblings.


Preimplantation genetic screening  (PGS) can be used to quantitate the risk of a child of being affected by a mitochondrial disease. In most cases, a muscle mutation level of approximately 18% or less confers a 95% risk reduction. [ 16 ]


One notable example of an otherwise healthy individual whose heteroplasmy was discovered incidentally is  Nicholas II of Russia , whose heteroplasmy (and that of his  brother ) served to convince  Russian  authorities of the authenticity of his remains. [ 18 ]


In  evolutionary developmental biology ,  homeosis  is the transformation of one  organ  into another, arising from  mutation  in or misexpression of certain developmentally critical  genes , specifically  homeotic genes . In animals, these developmental genes specifically control the development of organs on their anteroposterior axis. [ 1 ]  In plants, however, the developmental genes affected by homeosis may control anything from the development of a  stamen  or petals to the development of chlorophyll. [ 2 ]  Homeosis may be caused by mutations in  Hox genes , found in animals, or others such as the  MADS-box  family in plants. Homeosis is a characteristic that has helped  insects  become as successful and diverse as they are. [ 3 ]


Homeotic mutations work by changing segment identity during development. For example, the  Ultrabithorax   genotype  gives a  phenotype  wherein metathoracic and first  abdominal  segments become mesothoracic segments. [ 4 ]  Another well-known example is  Antennapedia : a  gain-of-function   allele  causes legs to develop in the place of  antennae . [ 5 ]


In  botany ,  Rolf Sattler  has revised the concept of homeosis (replacement) by his emphasis on partial homeosis in addition to complete homeosis; [ 6 ]  this revision is now widely accepted.


Homeotic mutants in  angiosperms  are thought to be rare in the wild: in the annual plant  Clarkia  ( Onagraceae ), homeotic mutants are known where the petals are replaced by a second whorl of sepal-like organs, originating in a mutation of a single gene. [ 7 ]  The absence of lethal or deleterious consequences in floral mutants resulting in distinct morphological expressions has been a factor in the evolution of  Clarkia , and perhaps also in many other plant groups. [ 8 ]


Following the work on homeotic mutants by  Ed Lewis , [ 9 ]  the phenomenology of homeosis in  animals  was further elaborated by discovery of a conserved DNA binding sequence present in many  homeotic proteins. [ 10 ]
Thus, the 60 amino acid DNA binding protein domain was named the  homeodomain , while the 180  bp  nucleotide sequence encoding it was named the  homeobox . The homeobox gene clusters studied by Ed Lewis were named the  Hox genes , although many more homeobox genes are encoded by animal  genomes  than those in the Hox gene clusters.


The homeotic-function of certain proteins was first postulated to be that of a "selector" as proposed by  Antonio Garcia-Bellido . [ 11 ]
By definition selectors were imagined to be ( transcription factor ) proteins that stably determined one of two possible cell fates for a cell and its cellular descendants in a  tissue .
While most animal homeotic functions are associated with homeobox-containing factors, not all homeotic proteins in animals are encoded by homeobox genes, and further not all homeobox genes are necessarily associated with homeotic functions or ( mutant ) phenotypes.
The concept of homeotic selectors was further elaborated or at least qualified by  Michael Akam  in a so-called "post-selector gene" model that incorporated additional findings and "walked back" the "orthodoxy" of selector-dependent stable binary switches. [ 12 ]


The concept of  tissue compartments  is deeply intertwined with the selector model of homeosis because the selector-mediated maintenance of cell fate can be restricted into different organizational units of an animal's  body plan . [ 13 ]
In this context, newer insights into homeotic mechanisms were found by  Albert Erives  and colleagues by focusing on  enhancer DNAs  that are co-targeted by homeotic selectors and different combinations of developmental signals. [ 14 ]
This work identifies a protein biochemical difference between the  transcription factors  that function as homeotic selectors versus the  transcription factors  that function as effectors of developmental signaling pathways, such as the  Notch signaling pathway  and the  BMP signaling pathway . [ 14 ]
This work proposes that homeotic selectors function to "license"  enhancer DNAs  in a restricted  tissue compartment  so that the enhancers are enabled to read-out developmental signals, which are then integrated via  polyglutamine -mediated aggregation. [ 14 ]


Like the complex  multicellularity  seen in  animals , the multicellularity of  land plants  is developmentally organized into  tissue  and  organ  units via  transcription factor  genes with homeotic effects. [ 15 ]
Although plants have homeobox-containing genes, plant homeotic factors tend to possess  MADS-box  DNA binding domains.
Animal genomes also possess a small number  MADS-box  factors.
Thus, in the independent evolution of  multicellularity  in plants and animals, different  eukaryotic  transcription factor families were  co-opted  to serve homeotic functions.
MADS-domain factors have been proposed to function as co-factors to more specialized factors and thereby help to determine organ identity. [ 15 ]
This has been proposed to correspond more closely to the interpretation of animal homeotics outlined by  Michael Akam . [ 16 ]


Homoplasmy  is a term used in  genetics  to describe a  eukaryotic cell  whose copies of  mitochondrial DNA  are all identical. [ 1 ]  In normal and healthy tissues, all cells are homoplasmic. [ 2 ]  Homoplasmic mitochondrial DNA copies may be normal or mutated; [ 1 ]  however, most mutations are  heteroplasmic [ 2 ] [ 3 ]  (only occurring in some copies of mitochondrial DNA). It has been discovered, though, that homoplasmic mitochondrial DNA mutations may be found in human tumors. [ 4 ]


The term may also refer to uniformity of plant  plastid  DNA, whether occurring naturally or otherwise. [ citation needed ]


In almost every species, mitochondrial DNA is  maternally inherited . [ 2 ]  This means that all of the offspring of a female will have identical and homoplasmic mitochondrial DNA. It is very rare for females to pass on heteroplasmic or homoplasmic mutations because of the genetic bottleneck, where only a few out of many mitochondria actually are passed on to offspring. [ 2 ]


The mussel  Mytilus edulis  is an anomaly in terms of mitochondrial DNA inheritance. Unlike almost all animals, this species has  biparental inheritance  for mitochondrial DNA, meaning that both the male and the female contribute mitochondria to the offspring. This was discovered when researchers realized that most individuals of a  Mytilus edulis  population were heteroplasmic. [ 5 ]  Researchers also believe that this could be a by-product of species hybridization. [ 5 ]


There is evidence of both homoplasmic and heteroplasmic inherited mutations that lead to disease, though heteroplasmic mutations typically are a precursor to homoplasmic disease. [ 6 ] [ 7 ]
Many diseases resulting from mutations in mitochondrial DNA are not inherited but developed as the untranslated region of mitochondrial DNA (mtDNA) is thought to be particularly susceptible to mutation. [ 8 ]  Many cancer types are the result of mutations in the mtDNA. For example, a specific type of mutation in one specific area of mtDNA was found to be in several different tumor types. [ 9 ]


Mitochondria  often undergo  fission and fusion , which means that different  organelles  in the same cell can fuse together to become one mitochondria, or can break apart and become two. [ 10 ]  This process can be used to mitigate the effects of heteroplasmic mutations. Each mitochondria has multiple  nucleoids , which consist of several copies of mtDNA, and when mitochondria fuse together, these nucleoids do not exchange DNA; therefore, if two mitochondria that have different DNA fuse together, they will have only two types of nucleoids. This means that fusion can be used to generate complementary nucleoids if a mutation causes one mitochondria to no longer be functional. Additionally, fission can cause one mitochondria with two different nucleoids to become two mitochondria each with only one type of nucleoid. [ 10 ]  Some researchers believe that this could be a useful tool to treat diseases caused by mutations in mtDNA. [ 10 ]


Leber's hereditary optic neuropathy  (LHON) is the disease in humans that is most frequently associated with homoplasmy. [ 7 ]  This condition is characterized by the atrophy of  retinal ganglion cells , which leads to central  blindness  and eventually total blindness. [ 11 ]  Although it is passed down maternally, it is seen more often in young men than in other ages or sexes, which leads researchers to believe that there are many other genetic or environmental factors that contribute to developing the disease. [ 11 ]  Specifically, researchers have thought that the genetic component outside of the mitochondria would be on the X chromosome; however, in multiple studies, there have been no findings that suggest this. [ 12 ] [ 13 ]  Environmental factors,  cigarette smoke  in particular, have been shown to affect LHON's  penetrance . [ 11 ]  In one study, cigarette smoke condensate was used to demonstrate the effects of smoking on cells with the LHON mutation. All cells were homoplasmic, but some were from individuals who were afflicted with LHON and some were from individuals who were just carriers. The researchers found that cigarette smoke condensate lowered the amount of mitochondria in the cells, but that carrier individuals were better able to compensate than those from individuals with LHON. [ 11 ]  Though there is an additive environmental effect, [ 11 ]  there is more to learn about why certain homoplasmic individuals have the disease and others do not.


Some research has shown that an inherited heteroplasmic mutation can cause cancer in older age as cells become homoplasmic. [ 6 ]  In one study, doctors found that a cancer patient's tumor consisted of only homoplasmic cells with mutant mtDNA and that healthy cells in his body were heteroplasmic for mutant mtDNA. [ 6 ]  Additionally, researchers found that the patient's siblings had the same heteroplasmic mutation. This indicates that the heteroplasmic mutation was inherited, and over time led to homoplasmic cells that caused cancer. [ 6 ]


The term  host cell reactivation  or  HCR  was first used to describe the survival of  UV -irradiated bacteriophages, that were transfected to UV-pretreated cells. [ 1 ]  This phenomenon was first thought to be the result of homologous recombination between both bacteria and phage, but later recognized as enzymatic repair. [ 2 ] [ 3 ] [ 4 ]   Modifications of the assay were later developed, using transient expression plasmid DNA vectors on  immortalized fibroblasts , [ 5 ]  and lately on human  lymphocytes . [ 6 ]


The  HCR  assay known also as plasmid reactivation assay, indirectly monitors cellular transcriptional repair system, that is activated by the transcriptional-inhibited damage inflicted by UV-Radiation into the  plasmid . Given that  UV-induced  DNA damage is used as  mutagen , the cell uses nucleotide excision repair  NER  pathway, that is activated by distortion in the DNA helix. [ 1 ]


The  Host-Cell Reactivation Assay  or  HCR  is a technique used to measure the  DNA repair  capacity of cell of a particular DNA alteration. In the  HCR  assay  the ability of an intact cell to repair exogenous DNA is measured [ 7 ]  The host cell is  transfected  with a damaged  plasmid  containing a  reporter gene , usually   luciferase , which has been deactivated due to the damage. The ability of the cell to repair the damage in the plasmid, after it has been introduced to the cell, allows the reporter gene to be reactivated.  Earlier versions of this assay were based on the chloramphenicol acetyltransferase  (CAT)  gene, [ 5 ]  but the version of the assay using luciferase as  reporter gene  is as much as 100-fold more sensitive. [ 1 ]


Human somatic variations  are  somatic mutations  ( mutations  that occur in  somatic cells ) both at early stages of development and in adult cells. These variations can lead either to pathogenic  phenotypes  or not, even if their function in healthy conditions is not completely clear yet. [ 1 ]


The term  mosaic  (from medieval Latin  musaicum , meaning "work of the Muses") [ 2 ]  has been used since antiquity to refer to an artistic patchwork of ornamental stones, glass, gems, or other precious material. At a distance, the collective image appears as it would in a painting; only on close inspection do the individual components become recognizable. [ 2 ]  In biological systems, mosaicism implies the presence of more than one genetically distinct cell line in a single  organism . Occurrence of this phenomenon not only can result in major  phenotypic  changes but also reveal the expression of otherwise lethal  genetic mutations . [ 3 ]


Genetic mutations involved in mosaicism may be due to endogenous factors, such as transposons and ploidy changes, or exogenous factors, such as UV radiation and nicotine. [ 4 ]


Somatic mosaicism  arises a result of  somatic mutations : genomic (or even  mitochondrial ) alterations of different sizes ranging from a single  nucleotide  to  chromosome  gains or loss within somatic cells. These alterations within somatic cells begin at an early stage (pre- implantation  or conception) and continue during aging, giving rise to phenotypic heterogeneity within cells, which may lead to the development of diseases such as cancer. [ 4 ]  Novel  array  based techniques for screening  genome-wide  copy number variants and loss of heterozygosity in single cells showed that chromosome  aneuploidies , uniparental disomies, segmental  deletions ,  duplications , and  amplifications  frequently occur during  embryogenesis . [ 5 ]  Yet not all somatic mutations are propagated to the adult individual, due to the phenomenon of  cell competition . [ 6 ]


Genetic alterations involving gains or loss of entire chromosomes predominantly occur during  anaphase  stage of cell division. But these are uncommon in somatic cells because they are usually selected against due to their deleterious consequences. [ 7 ]  Somatic variations during embryonic development can be represented by monozygous twins since they carry different copy number profiles and  epigenetic  marks that keep on increasing with age. [ 8 ]


Early research on  somatic mutations  in aging showed that deletions, inversion, and translocations of genetic material are common in aging mice and aging genomes tend to contain visible chromosomal changes, mitotic  recombination , whole gene deletions, intragenic deletions, and  point mutations . Other factors include the loss of  methylation , increasing gene expression heterogeneity correlating to genomic abnormalities, [ 4 ]  and telomere shortening. [ 9 ]  It is uncertain if transcription-based DNA repair takes part in the maintaining of somatic mutations in aging tissues. [ 4 ]


In some cells, the somatically acquired alterations can be reversed back to wild type alleles by  reversion  mosaicism. This can be due to endogenous mechanism such as  homologous recombination ,  codon  substitution, second-site suppressor mutations, DNA slippage, and  mobile elements . [ 10 ]


The advent of  Next-Generation Sequencing  technologies has increased the resolution of mutation detection and has led to the revelation that older individuals not only accumulate chromosomal alterations but also abundant mutations in cancer driver genes. [ 11 ]


Age-associated accumulation of chromosomal alterations has been documented with a variety of cytogenetic approaches, from  chromosome painting  to single nucleotide polymorphism ( SNP ) arrays. [ 12 ]


Numerous studies demonstrated that the clonal populations might lead to loss of organismal health through the functional decline of tissue and/or the promotion of disease processes, such as cancer. This is the reason why the aberrant clonal expansion (ACE) resulting from cancer-associated mutations are common in noncancerous tissue and accumulate with age. This is universal in most organisms and affects multiple tissues. [ 11 ]


In the hematopoietic compartment mutations include both large structural  chromosomal alterations  and point mutations affecting cancer-associated genes. Some translocations appear to occur very early in life. The frequency of these events is low in people younger than 50 years (<0.5%), but this frequency rapidly increases to 2% to 3% of individuals in their 70s and 80s. This phenomenon was termed  clonal hematopoiesis . A number of environmental factors, such as smoking, viral infections, and pesticide exposure, may contribute not only through mutation induction but also by modulation of clonal expansion. [ 13 ]


Otherwise, the detection of somatic variants in normal solid tissues has historically proved difficult. The main reasons are the generally slower replicative index, clonally restrictive tissue architecture, difficulty of tissue access, and low frequency of mutation occurrence. Recently, the analysis of somatic mutations in benign tissues adjacent to tumors revealed that 80% of samples harbors clonal mutations, with increased frequency associated with older age, smoking, and concurrent mutations in DNA repair genes. With the advent of NGS, it has become increasingly clear that somatic mutations accumulate with aging in normal tissue, even in individuals who are  cancer -free. [ 11 ]


This suggested that clonal expansions driven by cancer genes are a near-universal feature of aging. NGS technologies revealed that the clonal expansions of cancer-associated mutations are very common condition in somatic tissues. [ 11 ]


Through several recent studies a prevalence of somatic variations, both in pathological and healthy nervous systems, has been highlighted. [ 14 ] [ 15 ]


Somatic  aneuploidy  such as SNVs ( single-nucleotide variations ) and CNVs ( copy number variations ) have been particularly observed and linked to brain disfunctions when arising in prenatal brain development; anyway those somatic aneuploidy have been observed in rates of 1,3-40%, potentially increasing with age and for this reason they have been proposed as a mechanism to generate normal genetic diversity among  neurons . [ 16 ]


The confirmation of that hypothesis has been obtained through studies of  single-cell sequencing , which allow a direct assessment of single neuronal genomes, so that a systematic characterization of somatic aneuploidies and subchromosomal CNVs of these cells is possible. Using postmortem brains of both healthy and diseased humans it has been possible to study how CNVs change among these two groups. It emerged that somatic aneuploidies in healthy brains are quite rare, but somatic CNVs instead aren't. [ 17 ]


These studies also showed that clonal CNVs exist in both pathological and healthy brains. This means that some CNVs can arise in early development without causing diseases, even though, when compared to the CNVs arising in other cell types such as  lymphoblast , the brain's ones are more often private. This evidence could be given by the fact that, while lymphoblasts can generate clonal CNVs for a long period as they continue to proliferate, adult neurons do not replicate anymore, so the clonal CNVs they are carrying must have been generated in an early development stage. [ 17 ]


Data highlighted a tendency in neurons for the loss, rather than for the gain of copies when compared to lymphoblasts. These differences could suggest that the molecular mechanisms of CNVs arising in that two cell types are completely different. [ 17 ]


The  retrotransposon  LINE-1 (long interspersed element 1, L1) is a transposable element that has colonized the mammalian germline. L1 retrotransposition can happen also in somatic cells causing mosaicism (SLAVs – L1-associated variations) and in cancer. Retrotransposition is a copy and paste process in which the RNA template is retrotranscribed in DNA and integrated randomly in the genome. In humans there are around 500.000 copies of L1 and occupy 17% of genome. Its  mRNA  encodes for two proteins; one of them in particular has a  reverse transcriptase  and  endonuclease  activity that allows the retrotransposition in cis. Anyway most part of these copies are rendered immobile by  mutations  or 5’ truncation, leaving just about 80–100 mobile L1 per human genome and just about 10 are considered hot L1s so able to mobilize efficiently. [ 18 ]


L1 transpose using a mechanism called TPRT (target primed reverse transcription) it's able to insert a L1 endonuclease motif, target site duplications (TSD) and a  poly-A  tail with a cis preference. [ 19 ]


It has been seen in the past that there's L1 mobilization in neural  progenitors  during foetal and adult  neurogenesis  suggesting that the brain may be a L1 mosaicism hotspot. Moreover, some studies suggested that also non-dividing neurons can support L1 mobilization. This has been confirmed by  single-cell  genomic studies. [ 18 ]


Single-cell  paired-end sequencing  experiments found out that SLAVs are present both in  neurons  and  glia  of  hippocampus  and  frontal cortex . Any neural cell has a similar probability to contain a SLAV, suggesting that somatic variations are a random phaenomenon, not focused on a specific group of cells. SLAVs occurrence in the brain is estimated to be of 0.58–1 SLAVs per cell and to involve 44–63% of the brain cells. [ citation needed ]


Since experiments showed that a half of the analyzed SLAVs lack  target site duplication  (TSD), another kind of L1-associated variant might occur. In fact those sequences don't have an endonuclease activity, but still have endonuclease motifs so that they can be retrotransposed  in trans . [ citation needed ]


An application of the study of somatic mosaicism in the brain could be the tracing of specific brain cells. Indeed, if the somatic L1 insertions occurs in a  progenitor cell , the unique variant could be used to trace the progenitor cell's development, localization, and spreading through the brain. On the contrary, if the somatic L1 insertion occurs late in development, it will be present just in a single cell or in a small group of cells. Therefore, tracing somatic variations could be useful to understand at which point of development they have occurred. Further experiments are necessary to understand the role of somatic mosaicism in brain function, since small groups of cells or even single cells can affect  network  activity. [ 20 ]


Human somatic mutations (HSMs) are intensively exploited by the  immune system  for the production of antibodies. HSMs, recombination in particular, are indeed the reason why antibodies can identify an  epitope  with such high specificity and sensitivity. [ 21 ]


Antibodies are encoded by B cells. Each antibody is composed of two  heavy chains  (IgH, encoded by IGH gene) and two light chains (IgL, encoded by either IGL or IGK gene). Each chain is then composed of a constant region (C) and a variable region (V). The constant region (C) on the heavy chain is important in the BCR signaling and determines the type of immunoglobuline (IgA, IgD, IgE, IgG, or IgM). The variable region (V) is responsible for the recognition of the target epitope and is the product of recombination processes in the related loci. [ 22 ]


After exposure of an  antigen , B cells start developing. B cells genome undergoes repeated recombination processing on the Ig genes until the recognition of the epitope is perfectioned. The recombination involves the IGH locus first and then the IGL and IGK loci. All IGL, IGK, and IGH genes are the product of the V(D)J recombination process. This recombination involves the variable (V), diversity (D) and joining (J) segments. All three segments (V, D, J) are involved in the formation of the heavy chain, while only V and J recombination products encode for the light chain. [ 23 ]


The recombination between these regions allows the formation of 10 12 –10 18  potential different sequences. However, this number is an overestimation, since many factors contribute to limit the diversity of the B cell repertoire, first of all the actual number of B cell in the organism. [ 23 ]


Somatic mosaicism  has been noted in the heart. Sequencing suggested mosaic variation in the gap junction protein connexin in three patients out of 15 might contribute to  atrial fibrillation [ 24 ]  although subsequent reports in larger numbers of patients found no examples among a large panel of genes. [ 25 ]  At  Stanford , a team led by  Euan Ashley  demonstrated somatic mosaicism in the heart of a newborn presenting with life threatening arrhythmia. Family-based  genome sequencing  as well as tissue  RNA sequencing  and single cell genomics techniques were used to verify the finding. A model combining partial and ordinary differential equations with inputs from heterologous single channel electrophysiology experiments of the genetic variant recapitulated certain aspects of the clinical presentation. [ 26 ]


Indel  ( in sertion- del etion) is a molecular biology term for an  insertion  or  deletion  of  bases  in the  genome  of an organism. Indels ≥ 50 bases in length are classified as  structural variants . [ 1 ] [ 2 ]


In  coding regions  of the genome, unless the length of an indel is a multiple of 3, it will produce a  frameshift mutation . For example, a common microindel which results in a frameshift causes  Bloom syndrome  in the Jewish or Japanese population. [ 3 ]  Indels can be contrasted with a  point mutation .  An indel inserts or deletes nucleotides from a sequence, while a point mutation is a form of substitution that  replaces  one of the  nucleotides  without changing the overall number in the DNA. Indels can also be contrasted with Tandem Base Mutations (TBM), which may result from fundamentally different mechanisms. [ 4 ]  A TBM is defined as a substitution at adjacent nucleotides (primarily substitutions at two adjacent nucleotides, but substitutions at three adjacent nucleotides have been observed). [ 5 ]


Indels, being either insertions, or deletions, can be used as genetic markers in natural populations, especially in  phylogenetic  studies. [ 6 ] [ 7 ]  It has been shown that genomic regions with multiple indels can also be used for species-identification procedures. [ 8 ] [ 9 ] [ 10 ]


An indel change of a single base pair in the coding part of an  mRNA  results in a frameshift during mRNA translation that could lead to an inappropriate (premature)  stop codon  in a different frame. Indels that are not multiples of 3 are particularly uncommon in coding regions but relatively common in non-coding regions. [ 11 ] [ 12 ]  There are approximately 192-280 frameshifting indels in each person. [ 13 ]  Indels are likely to represent between 16% and 25% of all sequence polymorphisms in humans. [ 14 ]  In most known genomes, including humans, indel frequency tends to be markedly lower than that of  single nucleotide polymorphisms (SNP) , except near highly repetitive regions, including  homopolymers  and  microsatellites . [ 15 ]


The term "indel" has been co-opted in recent years by genome  scientists  for use in the sense described above. This is a change from its original use and meaning, which arose from  systematics . In systematics, researchers could find differences between sequences, such as from two different species. But it was impossible to infer if one species lost the sequence or the other species gained it. For example, species  A  has a run of 4  G  nucleotides at a locus and species  B  has 5 G's at the same locus. If the mode of selection is unknown, one can not tell if species A lost one G (a "deletion" event") or species B gained one G (an "insertion" event). When one cannot infer the  phylogenetic  direction of the sequence change, the sequence change event is referred to as an "indel". [ citation needed ]


Using passenger-immunoglobulin mouse models, a study found that the most prevalent indel events are the activation-induced cytidine deaminase (AID)-dependent ±1-base pair (bp) indels, which can lead to deleterious outcomes, whereas longer in-frame indels were rare outcomes. [ 16 ]


In  genetics , an  insertion  (also called an  insertion mutation ) is the addition of one or more  nucleotide   base pairs  into a  DNA  sequence. This can often happen in  microsatellite  regions due to the  DNA polymerase  slipping.  Insertions can be anywhere in size from one base pair incorrectly inserted into a DNA sequence to a section of one chromosome inserted into another. The mechanism of the smallest single base insertion mutations is believed to be through base-pair separation between the template and primer strands followed by non-neighbor base stacking, which can occur locally within the DNA polymerase active site. [ 1 ]  On a  chromosome  level, an  insertion  refers to the insertion of a larger sequence into a chromosome. This can happen due to unequal  crossover  during  meiosis .


N region addition  is the addition of non-coded nucleotides during  recombination  by  terminal deoxynucleotidyl transferase .


P nucleotide insertion  is the insertion of  palindromic sequences  encoded by the ends of the recombining gene segments.


Trinucleotide repeats  are classified as insertion mutations [ 2 ] [ 3 ]  and sometimes as a separate class of mutations. [ 4 ]


Zinc finger nuclease(ZFN) ,  Transcription activator-like effector nucleases (TALEN) , and  CRISPR gene editing  are the three main methods used in the former research to achieve gene insertion. And  CRISPR/Cas tools  have already become one of the most used methods to present research.


Based on  CRISPR/Cas tools , different systems  have already been developed to achieve specific functions. For example, one strategy is double-strand nucleases cutting system, using the normal  Cas9  protein with single  guide RNA  (sgRNA) and then achieving the gene insertion through end-joining or dividing cells with the  DNA repair  system. [ 5 ]  Another example is the  prime editing  system, which uses Cas9 nickase and the prime editing guide RNA (pegRNA) carrying the target genes. [ 5 ]


One limitation of current technology is that the size for DNA precise insertion is not large enough [ 6 ]  to meet the demand for genome research. RNA-guided DNA transposition is an emerging area to solve this problem. [ 7 ]  More efficient methods are expected to be developed and applied in the genome engineering area.


Insertions can be particularly hazardous if they occur in an  exon , the  amino acid  coding region of a  gene .  A  frameshift mutation , an alteration in the normal  reading frame  of a gene, results if the number of inserted nucleotides is not divisible by three, i.e., the number of nucleotides per  codon .  Frameshift mutations will alter all the amino acids encoded by the gene following the mutation.  Usually, insertions and the subsequent frameshift mutation will cause the active  translation  of the gene to encounter a premature  stop codon , resulting in an end to translation and the production of a truncated protein.  Transcripts carrying the frameshift mutation may also be degraded through  Nonsense-mediated decay  during translation, thus not resulting in any protein product.  If translated, the truncated proteins frequently are unable to function properly or at all and can result in any number of genetic disorders depending on the gene in which the insertion occurs. [ 8 ]


In-frame insertions occur when the reading frame is not altered as a result of the insertion; the number of inserted nucleotides is divisible by three.  The reading frame remains intact after the insertion and translation will most likely run to completion if the inserted nucleotides do not code for a stop codon.  However, because of the inserted nucleotides, the finished protein will contain, depending on the size of the insertion, multiple new amino acids that may affect the function of the protein.


In Muller's classification, an  isomorph  is described as a  gene mutation  that expresses a  nonsense   point mutant , with  expression  identical to the original  allele .


Therefore, in respect to the relationships between the original and mutated genes, it is difficult to ascertain the effects of  dominanceness  and/or  recessiveness . [ 1 ] [ 2 ]
 [ 3 ]


Lavender  or  self-blue  refers to a  plumage  color pattern in the  chicken  ( Gallus gallus domesticus ) characterized by a uniform, pale bluish grey color across all  feathers . The distinctive color is caused by the action of an autosomal recessive gene, commonly designated as " lav ", which reduces the expression of  eumelanin  and  phaeomelanin  so that black areas of the plumage appear pale grey instead, and red areas appear a pale buff. [ 1 ]


The "lavender" gene ( lav ) in the chicken causes the dilution of both black (eumelanin) and red/brown (phaeomelanin) pigments, so according to color background, dilution due to "lavender" gives a sort of plumage color patterns: On an  extended black  background, this condition causes the entire surface of the body an even shade of light slaty blue, which is the typical phenotype known as '"self-blue"'.


On a red/brown color plumage background, lavender gene degrades color to beige, like in some  Pekin Bantams  as in the picture set aside. On the color background of the  Belgian Bearded d'Uccle Bantams , frequently referred to as the "Mille Fleur" in the United States, [ 2 ]  lavender causes the pattern known as "porcelain". The resulting "porcelain" pattern is beige with each feather tipped with a V-shaped of  slate blue  near the end of the feather and the feather tipped with a V-shaped white spangle. [ 3 ]


Light and  electron microscope  studies have revealed that, although lavender  melanocytes  possess relatively normal dendrite morphology, there is defective peripheral accumulation of  melanosomes  to the dendrites. [ 4 ]  This results in the patchy transfer of melanosomes into the  keratinocytes  of the growing feather. The dilution effect is essentially the result of a mixture of pigmented and unpigmented regions within the  feather barbs .


The lavender gene was first discovered in the Porcelain variety of  Belgian Bearded d'Uccle bantams  in 1972, [ 5 ]  and verified in 1980. [ 1 ]  Porcelain colored d'Uccle bantams were around as early as 1909, [ 6 ] [ 7 ]  though the Porcelain variety was not recognized by the  American Poultry Association  until 1964. [ 8 ]  Whether from the  Porcelain d'Uccle  or other, unknown sources, the lavender gene has been introduced to a number of new chicken breeds over the years, including the  Polish  and the  Silkie .


"Lavender" is an  autosomal recessive  mutation of the chicken affecting the  neural crest  derived  melanocytes . It causes the dilution of both  eumelanin  and  phaeomelanin  to a light grey or buff, respectively. It has been assigned the symbol  lav . [ 5 ] [ 9 ]


The ultimate goal of the modern genetic studies is to find out the underlying genes involved in these traits. Lavender in chickens has been found to be a mutation caused by a single base-pair change in  exon  1 of MLPH  (melanophilin) gene. [ 10 ]


In genetic linkage studies,  Lav  locus has been assigned to a linkage group known as Cp-R-U group (Creeper-Rose comb-Uropygial). Although Lavender locus is linked to the R ( rose comb ) locus by 32.5%, its position has not yet mapped. [ 11 ]


Until now, all the reported causal mutations in MLPH (melanophilin) of humans, mice, and other species have been single-base substitutions or small  deletions , the effects of which were limited to the dilution of hair [ 12 ] [ 13 ]  or feather colour. [ 10 ]  The MLPH-associated dilution of coat or plumage pigmentation should then result in the defective transport of  melanosomes . This produces a diluted, leaden or lavender blue-grey colour and has been reported in several mammals: humans ( Griscelli syndrome type 3 ), [ 12 ]  mice, [ 14 ]  cats, [ 15 ]  dogs [ 16 ]  and minks. [ 17 ]


The lavender phenotype in  Japanese quail  ( Coturnix coturnix japonica ) is a dilution of both eumelanin and phaeomelanin in feathers that produces a blue-grey colour on a wild-type feather pattern background. Studies of intergeneric  hybridization  proved that the lavender mutation in quail is homologous to the same phenotype in chickens. [ 18 ]


In this species, the lavender phenotype is associated with a non-lethal complex mutation involving three consecutive overlapping chromosomal changes (two  inversions  and one  deletion ) that have consequences on the genomic organization of four genes (MLPH and the neighbouring PRLH, RAB17 and LRRFIP1). The deletion of PRLH has no effect on the level of circulating  prolactin . Lavender birds have lighter body weight, lower body temperature and increased feed consumption and residual feed intake than wild-type plumage quail, indicating that this complex mutation is affecting the  metabolism  and the regulation of  homeothermy . [ 19 ]


In other bird species, similar feather colour dilutions have been described, including the autosomal recessive slate turkey ( Meleagris gallopavo ), [ 9 ] [ 20 ]  milky pigeon ( Columba livia ), [ 21 ]  and the lavender muscovy duck ( Cairina moschata ). [ 22 ]  It is not yet known which genes are responsible for these dilution mutations in these bird species.


LEAFY  (abbreviated  LFY ) is a  plant   gene  that causes groups of undifferentiated  cells  called  meristems  to develop into  flowers  instead of leaves with associated  shoots . [ 1 ]


LEAFY  is involved in floral meristem identity.


LEAFY  encodes a plant-specific  transcription factor , is found in all land plants and in  charophytes [ 2 ]  and one of its  exons  have been used extensively in phylogenetic work on  spermatophytes . [ 3 ]  When the gene is  overexpressed , the plant is less sensitive to environmental signals and flowers earlier. [ 4 ]


The LEAFY protein has two conserved domains: the DNA binding domain, a  Helix-Turn-Helix  motif buried inside a unique 7-helix fold [ 5 ]  and a  Sterile Alpha Motif . [ 6 ]  It binds DNA as a dimer and its binding site has been identified both in vivo and in vitro. [ 7 ] [ 8 ]  The F-box protein Unusual Floral Organs (UFO) is able to redirect LFY to binding sites that LFY cannot access alone and, together, they regulate genes involved in petal and stamen development (such as APETALA3, PISTILLATA or RABBIT EARS). [ 9 ]   [ 10 ]


A  genetic lineage  includes all descendants of a given genetic sequence, typically following a new  mutation . It is not the same as an  allele  because it excludes cases where different mutations give rise to the same allele, and includes descendants that differ from the ancestor by one or more  mutations . The genetic sequence can be of different sizes, e.g. a single  gene  or a  haplotype  containing multiple adjacent genes along a  chromosome . Given  recombination , each gene can have a separate genetic lineages, even as the  population  shares a single  organismal lineage . In  asexual   microbes  or  somatic cells ,  cell lineages  exactly match genetic lineages, and can be  traced . [ 1 ]


Incomplete lineage sorting  describes when the  phylogenetic tree  for a gene does not match that of the  species . For example, while most human gene lineages  coalesce  first with chimpanzee lineages, and then with gorilla lineages, other configurations also occur. [ 2 ]


Lineage selection  occurs when the frequency of members of one lineage changes relative to another lineage. It is useful for studying alleles with complex effects that play out over multiple generations, e.g. alleles that affect  recombination ,  evolvability , or  altruism . [ 3 ] [ 4 ]  Lineage selection is also useful in determining the effects of mutations in highly structured environments such as tumors. [ 5 ]


Long-term  stochastic  outcomes of competition among lineages can be quantified within  mathematical models  as the ratio of  fixation probability  : counterfixation probability. [ 6 ]   Inclusive fitness  is equal to the average organismal  fitness  of individuals across the  probability distribution  of possible lineages. [ 7 ]


Tree sequence recording describes efficient methods to record surviving lineages while conducting  computer simulations  of  population genetics . [ 8 ]  Resulting 'forward time'  computer simulations  offer an alternative to 'backward time'  coalescent theory . Tree sequence recording has been incorporated into the population simulation software  SLiM . [ 9 ]


Cats , like all living organisms, occasionally have  mutations  that affect their body type. Sometimes, these mutations are striking enough that  humans  select for and perpetuate them. However, in relatively small or isolated feral cat populations the mutations can also spread without human intervention, for example on islands. Cat breeders exploit the naturally occurring mutations by  selectively breeding  them in a small gene pool, resulting in the creation of new cat breeds with unusual physical characteristics. [ 1 ]  The term  designer cat  is often used to refer to these cat breeds. [ 1 ]  This is not always in the best interests of the cat, as many of these mutations are harmful; some are even  lethal  in their  homozygous  form. To protect the animal’s welfare it is illegal in several countries or states to breed with parent cats that bear certain of these hypertype mutations.


This article gives a selection of cat body type mutant  alleles  and the associated mutations with a brief description.


A germ-cell mutation occurred in a male Persian cat called Treker in 1995, resulting in diminutive, but healthy and normally-proportioned, offspring. Treker and the females with which he was mated were normally sized, but 75% of the kittens sired by Treker inherited diminutive stature, but of normal proportions. The gene was found to be dominant and the diminutive offspring were sold as teacup or toy Persians.  Toy and Teacup Persians  are a separate breed and not all cats advertised under those names result from Treker's dominant mutation. Most teacup and toy size Persian kittens raised now are descendants from Silver and Golden color division to reduce cat size and are in no way related to Treker. [ 13 ]


Many of the major  cat registries  do not allow the registry and breeding of cat breeds which bear certain of these genetic mutations, nor recognise them, due to concerns about the animal’s welfare.


Breeding with parent animals that bear hypertype mutations is effectively banned in Europe under the  European Convention for the Protection of Pet Animals : [ 14 ]


Any person who selects a pet animal for breeding shall be responsible for having regard to the anatomical, physiological and behavioural characteristics which are likely to put at risk the health and welfare of either the offspring or the female parent.


Since 2014, it is illegal in the  Netherlands  to breed with parent cats which bear genetic mutations or other physical abnormalities that cause health or welfare issues in their offspring. [ 15 ] [ 16 ]  Examples of these genetic mutations and physical abnormalities are  dwarfism , the lack of protecting fur in hairless cats, and fold-eared cats.


Furthermore, several countries or states have prohibited breeding with certain of the genetic mutations in cats, including  Scotland , [ 17 ] [ 18 ]   Victoria  and the  Australian Capital Territory  ( Australia ), [ 19 ] [ 20 ]   Austria , [ 21 ] [ 22 ]  and  Flanders  ( Belgium ). [ 23 ]


Locus heterogeneity  occurs when mutations at multiple genomic loci are capable of producing the same  phenotype  (ie. a single trait, pattern of traits, or disorder), and each individual mutation is sufficient to cause the specific phenotype independently. [ 1 ]  Locus heterogeneity should not be confused with  allelic heterogeneity , in which a single phenotype can be produced by multiple mutations, all of which are at the same locus on a chromosome. [ 1 ]  Likewise, it should not be confused with  phenotypic heterogeneity , in which different phenotypes arise among organisms with identical  genotypes  and environmental conditions. [ 2 ]  Locus heterogeneity and allelic heterogeneity are the two components of  genetic heterogeneity . [ 3 ]


Locus heterogeneity may have major implications for a number of human diseases. For instance, it has been associated with  retinitis pigmentosa , [ 4 ]   hypertrophic cardiomyopathy , [ 5 ]   osteogenesis imperfecta , [ 6 ]   familial hypercholesterolemia , [ 7 ]  and  hearing loss . [ 8 ]  Heterogenous loci involved in formation of the same phenotype often contribute to similar biological pathways. [ 1 ]  The role and degree of locus heterogeneity is an important consideration in understanding disease phenotypes and in the development of therapeutic treatment for these diseases. [ 1 ]


The detection of causal genes for diseases impacted by locus heterogeneity is difficult with genetic analysis methods such as  linkage  analysis and  genome sequencing . [ 9 ]  These methods rely on comparison of affected family members, but when different family members have different disease-causing genes, such genes may not be accurately identified. [ 9 ]  Existing techniques have been modified and new techniques have been developed to overcome these challenges. [ 9 ] [ 10 ] [ 11 ]


Retinitis pigmentosa  is a condition that causes damage to the light-sensitive cells of the retina. [ 12 ]  There have been over 60 genes identified whose mutations independently cause retinitis pigmentosa, and these can be inherited in an  autosomal dominant ,  autosomal recessive , or  X-linked  pattern. [ 13 ]  Examples of such genes include the  rhodopsin  gene (RHO), the gene encoding for  retinitis pigmentosa GTPase regulator  (RGPR), and the gene encoding retinitis pigmentosa 2 protein (RP2). [ 14 ]


In  genetics ,  loss of heterozygosity  ( LOH ) is a type of  genetic abnormality  in  diploid  organisms in which one copy of an entire  gene  and its surrounding  chromosomal  region are lost. [ 1 ]  Since diploid cells have two copies of their genes, one from each parent, a single copy of the lost gene still remains when this happens, but any  heterozygosity  (slight differences between the versions of the gene inherited from each parent) is no longer present.


The loss of heterozygosity is a common occurrence in  cancer  development. Originally, a heterozygous state is required and indicates the absence of a functional  tumor suppressor gene  copy in the region of interest. However, many people remain healthy with such a loss, because there still is one functional gene left on the other chromosome of the  chromosome pair . The remaining copy of the tumor suppressor gene can be inactivated by a  point mutation  or via other mechanisms, resulting in a loss of heterozygosity event, and leaving no tumor suppressor gene to protect the body. Loss of heterozygosity does not imply a homozygous state (which would require the presence of two identical alleles in the cell). The exact targets for LOH are not characterised for all chromosomal losses in cancer, but certain are very well mapped. Some examples are 17p13 loss in multiple cancer types where a copy of  TP53  gene  gets inactivated, 13q14 loss in retinoblastoma with  RB1  gene  deletion or 11p13 in  Wilms' tumor  where  WT1  gene  is lost. [ 2 ]  Other commonly lost chromosomal loci are still being investigated in terms of potential tumor suppressors located in those regions.


Copy-neutral LOH is thus called because no net change in the copy number occurs in the affected individual. Possible causes for copy-neutral LOH include acquired  uniparental disomy  (UPD) and gene conversion. In UPD, a person receives two copies of a chromosome, or part of a chromosome, from one parent and no copies from the other parent due to errors in meiosis I or meiosis II. This acquired homozygosity could lead to development of cancer if the individual inherited a non-functional allele of a tumor suppressor gene.


In tumor cells copy-neutral LOH can be biologically equivalent to the second hit in the Knudson hypothesis. [ 3 ]  Acquired UPD is quite common in both hematologic and solid tumors, and is reported to constitute 20 to 80% of the LOH seen in human tumors. [ 4 ] [ 5 ] [ 6 ] [ 7 ]  Determination of  virtual karyotypes  using SNP-based arrays can provide genome-wide copy number and LOH status, including detection of copy-neutral LOH. Copy-neutral LOH cannot be detected by arrayCGH, FISH, or conventional cytogenetics. SNP-based arrays are preferred for virtual karyotyping of tumors and can be performed on fresh or paraffin-embedded tissues.


The classical example of such a loss of protecting genes is hereditary  retinoblastoma , in which one parent's contribution of the tumor suppressor  Rb1  is flawed. Although most cells will have a functional second copy, chance loss of heterozygosity events in individual cells almost invariably lead to the development of this retinal cancer in the young child.


The genes  BRCA1  and  BRCA2  show loss of heterozygosity in samplings of tumors from patients who have  germline mutations . [ citation needed ]   BRCA1/2  are genes that produce proteins which regulate the DNA repair pathway by binding to  Rad51 . [ citation needed ]


In  breast ,  ovarian ,  pancreatic , and  prostate  cancers, a core enzyme employed in  homologous recombination  repair (HRR) of  DNA damage  is often defective due to LOH, that is genetic defects in both copies (in the diploid human genome) of the  gene  encoding an enzyme necessary for HRR. [ 8 ]  Such LOH in these different cancers was found for DNA repair genes  BRCA1 ,  BRCA2 ,  BARD1 ,  PALB2 ,  FANCC ,  RAD51C  and  RAD51D . [ 8 ]  Reduced ability to accurately repair DNA damages by homologous recombination may lead to compensating inaccurate repair, increased mutation and progression to cancer.


Loss of heterozygosity can be identified in cancers by noting the presence of  heterozygosity  at a genetic  locus  in an organism's  germline   DNA , and the absence of heterozygosity at that locus in the cancer cells. This is often done using  polymorphic  markers, such as  microsatellites  or  single-nucleotide polymorphisms , for which the two parents contributed different  alleles . Genome-wide LOH status of fresh or paraffin embedded tissue samples can be assessed by  virtual karyotyping  using SNP arrays.


It has been proposed that LOH may limit the longevity of asexual organisms. [ 9 ] [ 10 ]  The minor allele in heterozygous areas of the genome is likely to have mild fitness consequences compared to de-novo mutations because selection has had time to remove deleterious alleles. When  allelic gene conversion  removes the major allele at these sites organisms are likely to experience a mild decline in fitness. Because LOH is much more common than de-novo mutation, and because the fitness consequences are closer to neutrality, this process should drive  Muller's ratchet  more quickly than de-novo mutations. While this process has received little experimental investigation, it is known that major signature of asexuality in metazoan genomes appears to be genome wide LOH, a sort of anti- meselson effect .


Low copy repeats  ( LCRs ), also known as  segmental duplications  ( SDs ), or  duplicons , are DNA sequences present in multiple locations within a genome that share high levels of sequence identity.


The repeats, or duplications, are typically 10–300  kb  in length, and bear greater than 95%  sequence identity . Though rare in most mammals, LCRs comprise a large portion of the  human genome  owing to a significant expansion during  primate evolution . [ 1 ]  In humans,  chromosomes Y  and  22  have the greatest proportion of SDs: 50.4% and 11.9% respectively. [ 2 ]   SRGAP2  is an SD.


Misalignment of LCRs during  non-allelic homologous recombination  (NAHR) [ 3 ]  is an important mechanism underlying the  chromosomal microdeletion disorders  as well as their reciprocal duplication partners. [ 4 ]  Many LCRs are concentrated in "hotspots", such as the 17p11-12 region, 27% of which is composed of LCR sequence. NAHR and  non-homologous end joining  (NHEJ) within this region are responsible for a wide range of disorders, including  Charcot–Marie–Tooth syndrome type 1A , [ 5 ]   hereditary neuropathy with liability to pressure palsies , [ 5 ]   Smith–Magenis syndrome , [ 6 ]  and  Potocki–Lupski syndrome . [ 3 ]


The two widely accepted methods for SD detection [ 7 ]  are:


Methylenetetrahydrofolate reductase deficiency  is the most common genetic cause of elevated serum levels of  homocysteine  ( hyperhomocysteinemia ). It is caused by genetic defects in  MTHFR , which is an important enzyme in the methyl cycle. [ 1 ]


Common variants of MTHFR deficiency are asymptomatic and have only minor effects on disease risk. [ 2 ]  Severe variants (from nonsense mutations) are rare. [ 3 ]


The common MTHFR deficiencies are usually asymptomatic, although the 677T variant can cause a mildly increased risk of some diseases. [ citation needed ]


For individuals homozygous in the 677T variant, there is a mildly elevated risk of thromboembolism ( odds ratio  1.2), [ 4 ]  and stroke (odds ratio 1.26). [ 5 ]  There is also an elevated risk of neural tube defects among children of individuals with the C677T polymorphism (odds ratio 1.38). [ 6 ]


Common MTHFR deficiencies were once thought to be associated with cardiovascular risk, but meta-analyses  indicate that correlation this was an artifact of publication bias. [ clarification needed ] [ 7 ] [ 8 ]


MTHFR is the rate-limiting enzyme in the methyl cycle, which includes the conversion of homocysteine into methionine. Defects in variants of  MTHFR  can therefore lead to  hyperhomocysteinemia . [ 9 ]


There are two common variants of  MTHFR  deficiency. In the more significant of the two, the individual is homozygous for the 677T polymorphism. This variant in particular is the most common genetic cause of hyperhomocysteinemia. [ 9 ]  The resulting enzyme is  thermolabile  and in homozygotes, enzymatic activity is depressed to 35% of its usual level. [ 10 ]  The second variant is a milder one, caused by a homologous 1298C polymorphism. This leads to 68% of the control values of enzyme activity, [ 10 ]  and it normally does not lead to low serum folate. [ 9 ]


MTHFR deficiency is diagnosed by  genetic testing . [ citation needed ]


In common forms of MTHFR deficiency, elevated plasma homocysteine levels have sometimes been treated with  Vitamin B12  and low doses of  folic acid . [ 2 ]  Although this treatment significantly decreases the serum levels of homocysteine, this treatment is not thought to improve health outcomes. [ 11 ] [ 12 ] [ 13 ]


Due to the ineffectiveness of these treatments, it was no longer considered clinically useful to test for MTHFR in most cases of thrombophilia or recurrent pregnancy loss. [ 14 ] [ 15 ]  A more recent evaluation from a case series recommends testing for  MTHFR  in case of long lasting impaired fertility and repeat miscarriages. [ citation needed ]  Treatment with high doses of  folic acid  (5 mg/day) are deemed unsuitable for MTHFR isoform carriers, who could alternatively be treated with the metabolically active form,  5-methyltetrahydrofolate . [ 16 ]  5-MTHF was shown to induce significantly higher plasma folate concentrations compared to folic acid in  homozygous  MTHFR  mutation  carriers in this case series. [ citation needed ]


A different study corroborates these results and suggests a physiological dose (800 μg) of  5-methyltetrahydrofolate  can bypass MTHFR C677T and A1298C isoforms in couples with fertility problems. [ 16 ]  This treatment with  5-MTHF  also avoids un-metabolized folic acid syndrome, which can occur with folic acid intakes of 5 mg per day. [ 16 ]


Whether MTHFR deficiency has any effect at all on all-cause mortality is unclear. One Dutch study showed that the MTHFR mutation was more prevalent in younger individuals (36% relative to 30%), and found that elderly men with MTHFR had an elevated mortality rate, attributable to cancer. Among women, however, no difference in life expectancy was seen. [ 17 ]  More recently, however, a meta-analysis has shown that overall cancer rates are barely increased with an odds ratio of 1.07, which suggests that an impact on mortality from cancer is small or zero. [ 18 ]


The prevalence of 677T homozygosity varies with race. 18-21% of Hispanics and Southern Mediterranean populations have this variant, as do 6-14% of North American Whites and <2% of Blacks living outside of Africa.
 [ 9 ]


The prevalence of the 1298C mutation is lower, at 4-12% for most tested populations. [ 9 ]


A study in 2000 had identified only 24 cases of severe MTHFR deficiency (from nonsense mutations) across the whole world. [ 3 ]


A  micronucleus test  is a test used in toxicological screening for potential  genotoxic  compounds. The assay is now recognized as one of the most successful and reliable assays for genotoxic  carcinogens , i.e., carcinogens that act by causing genetic damage and is recommended by the  OECD  guideline for the testing of chemicals. [ 1 ]   There are two major versions of this test, one  in vivo  and the other  in vitro .


The  in vivo  test normally uses mouse  bone marrow  or mouse peripheral blood. When a bone marrow  erythroblast  develops into a polychromatic  erythrocyte , the main nucleus is extruded; any  micronucleus  that has been formed may remain behind in the otherwise anucleated  cytoplasm . Visualisation of  micronuclei  is facilitated in these cells because they lack a main  nucleus . An increase in the frequency of micronucleated  polychromatic   erythrocytes  in treated animals is an indication of induced  chromosome  damage. [ 2 ]


Micronuclei  were first used to quantify chromosomal damage by H.J. Evans et al., in root tips of the  Broad Bean ,  Vicia faba .  Subsequently, the  in vivo  assay was developed independently by W. Schmid and by J.A. Heddle and their colleagues.  The mouse peripheral blood assay was developed by J.T. MacGregor and has now been adapted for measurement by  flow cytometry  by A. Tometsko and colleagues.  The first use of  micronuclei  in  cultured cells  was by J.A. Heddle and colleagues in human  lymphocytes . The assay has been improved by M. Fenech and colleagues for use in  lymphocytes  and other cells in  culture cells .