The dilemma of whether or not to have children may be a source of stress for women who learn of their  BRCA  mutations during their childbearing years. [ 18 ]


There is likely little or no effect of a  BRCA  gene mutation on overall  fertility , [ 19 ]  although women with a  BRCA  mutation may be more likely to have  primary ovarian insufficiency . [ 20 ]
 [ 21 ]   BRCA  mutation carriers may be more likely to give birth to girls than boys, [ 22 ]  however this observation has been attributed to  ascertainment bias . [ 23 ] [ 24 ]


If both parents are carriers of a  BRCA  mutation, then  pre-implantation genetic diagnosis  is sometimes used to prevent the birth of a child with  BRCA  mutations. [ 9 ] : 82–85    Inheriting two  BRCA1  mutations (one from each parent) has never been reported and is believed to be a lethal birth defect.  Inheriting one  BRCA1  mutation and one  BRCA2  mutation has been reported occasionally; the child's risk for any given type of cancer is the higher risk of the two genes (e.g., the ovarian cancer risk from  BRCA1  and the pancreatic cancer risk from  BRCA2 ).  Inheriting two  BRCA2  mutations produces  Fanconi anemia . [ 9 ] : 82–85


Each pregnancy in genetically typical women is associated with a significant reduction in the mother's risk of developing breast cancer after age 40. [ 18 ]  The younger the woman is at the time of her first birth, the more protection against breast cancer she receives. [ 9 ] : 113–142    Breastfeeding for more than one year protects against breast cancer. [ 9 ] : 113–142    Pregnancy also protects against ovarian cancer in genetically typical women. [ 18 ]


Although some studies have produced different results, women with  BRCA  mutations are generally not expected to receive these significant protective benefits. [ 9 ] : 113–142  [ 18 ]  Current research is too limited and imprecise to permit calculation of specific risks. [ 18 ]  However, the following general trends have been identified:


Reports of patients biallelic or homozygous for a deleterious BRCA allele conferring a greatly increased risk of breast cancer are rare. This is because deleterious BRCA alleles are  lethal alleles ; this condition is embryonically lethal in the majority of the cases. [ 26 ]  For live cases, inheriting both mutations lead to a grave prognosis, characterized by  Wilms tumors ,  leukemias , and early-onset  brain malignancies . [ 27 ]


Both  BRCA  genes are  tumor suppressor genes  that produce proteins that are used by the cell in an  enzymatic pathway  that makes very precise, perfectly matched repairs to DNA molecules that have double-stranded breaks. [ 9 ] : 39–50  [ 28 ]  The pathway requires proteins produced by several other genes, including  CHK2 ,  FANCD2  and  ATM . [ 16 ]  Harmful mutations in any of these genes disable the gene or the protein that it produces.


The cancer risk caused by  BRCA1  and  BRCA2  mutations are inherited in a  dominant  fashion even though usually only one mutated  allele  is directly inherited. [ 29 ]  This is because people with the mutation are likely to acquire a second mutation, leading to dominant expression of the cancer. A mutated  BRCA  gene can be inherited from either parent.  Because they are inherited from the parents, they are classified as   hereditary  or  germline mutations  rather than  acquired  or  somatic mutations .  Cancer caused by a mutated gene inherited from an individual's parents is a  hereditary cancer  rather than a  sporadic cancer .


Because humans have a  diploid  genome, each cell has two copies of the gene (one from each biological parent).  Typically only one copy contains a disabling, inherited mutation, so the affected person is  heterozygous  for the mutation.  If the functional copy is harmed, however, then the cell is forced to use alternate DNA repair mechanisms, which are more error-prone.  The loss of the functional copy is called  loss of heterozygosity  (LOH). [ 30 ]  Any resulting errors in DNA repair may result in cell death or a cancerous transformation of the cell. [ 9 ] : 39–50


There are many variations in  BRCA  genes, and not all changes confer the same risks.
 [ 9 ] : 39–50
Some variants are harmless; others are known to be very harmful.  Some  single nucleotide polymorphisms  may confer only a small risk, or may only confer risk in the presence of other mutations or under certain circumstances.  In other cases, whether the variant is harmful is unknown.  Variants are classified as follows: [ 9 ] : 39–50  : 109


Deleterious mutations have high, but not complete, genetic  penetrance , which means that people with the mutation have a high risk of developing disease as a result, but that some people will not develop cancer despite carrying a harmful mutation.


Genetic counseling  is recommended in women whose personal or family health history suggests a greater than average likelihood of a mutation. [ 31 ]  The purpose of genetic counseling is to educate the person about the likelihood of a positive result, the risks and benefits of being tested, the limitations of the tests, the practical meaning of the results, and the risk-reducing actions that could be taken if the results are positive.  They are also trained to support people through any emotional reactions and to be a neutral person who helps the client make his or her own decision in an  informed consent  model, without pushing the client to do what the counselor might do.  Because the knowledge of a mutation can produce substantial anxiety, some people choose not to be tested or to postpone testing until a later date. [ 9 ] : 51–74


Relative  indications  for testing for a mutation in BRCA1 or BRCA2 for newly diagnosed or family members include a  family history  among 1st (FDR), 2nd (SDR), or 3rd(TDR) degree relatives usually on the same side of the family but not limited: [ 1 ] [ 32 ]


Testing young children is considered  medically unethical  because the test results would not change the way the child's health is cared for. [ 9 ] : 82–85


Two types of tests are available. [ 9 ] : 51–74    Both commonly use a  blood sample , although testing can be done on saliva. The quickest, simplest, and lowest cost test uses positive test results from a  blood relative  and checks only for the single mutation that is known to be present in the family.  If no relative has previously disclosed positive test results, then a full test that checks the entire sequence of both  BRCA1  and  BRCA2  can be performed.  In some cases, because of the  founder effect , Jewish ethnicity can be used to narrow the testing to quickly check for the three most  common mutations seen among Ashkenazi Jews . [ 9 ] : 51–74


Testing is commonly covered by  health insurance  and public healthcare programs for people at high risk for having a mutation, and not covered for people at low risk. [ 9 ] : 51–74   The purpose of limiting the testing to high-risk people is to increase the likelihood that the person will receive a meaningful, actionable result from the test, rather than identifying a variant of unknown significance (VUS). In Canada, people who demonstrate their high-risk status by meeting specified guidelines are referred initially to a specialized program for hereditary cancers, and, if they choose to be tested, the cost of the test is fully covered.  In the US in 2010, single-site testing had a retail cost of US$400 to $500, and full-length analysis cost about $3,000 per gene, and the costs were commonly covered by private health insurance for people deemed to be at high risk.


The test is ordered by a physician, usually an  oncologist , and the results are always returned to the physician, rather than directly to the patient.  How quickly results are returned depends on the test—single-site analysis requires less lab time—and on the infrastructure in place.  In the US, test results are commonly returned within one to several weeks; in Canada, patients commonly wait for eight to ten months for test results. [ 9 ] : 51–74


A positive test result for a known deleterious mutation is proof of a predisposition, although it does not guarantee that the person will develop any type of cancer.  A negative test result, if a specific mutation is known to be present in the family, shows that the person does not have a  BRCA -related predisposition for cancer, although it does not guarantee that the person will not develop a non-hereditary case of cancer.  By itself, a negative test result does not mean that the patient has no hereditary predisposition for breast or ovarian cancer.  The family may have some other genetic predisposition for cancer, involving some other gene. [ 9 ] : 89–111


A variety of screening options and interventions are available to manage  BRCA -related cancer risks.  Screenings are adjusted to individual and familial risk factors. [ 35 ]


As these screening methods do not prevent cancer, but merely attempt to catch it early, numerous methods of prevention are sometimes practiced, with varying results. [ 9 ] : 175–207


An intensive  cancer screening  regimen is usually advised for women with deleterious or suspected deleterious  BRCA  mutations in order to detect new cancers as early as possible.  A typical recommendation includes frequent  breast cancer screening  as well as tests to detect ovarian cancer. [ 9 ] : 175–207


Breast imaging studies usually include a  breast MRI  (magnetic resonance imaging) once a year, beginning between ages 20 and 30, depending on the age at which any relatives were diagnosed with breast cancer.  Mammograms  are typically used only at advanced age as there is reason to believe that  BRCA  carriers are more susceptible to breast cancer induction by X-ray damage than general population. [ 36 ]


Alternatives include breast  ultrasonography ,  CT scans ,  PET scans ,  scintimammography ,  elastography ,  thermography ,  ductal lavage , and experimental screening protocols, some of which hope to identify  biomarkers  for breast cancer (molecules that appear in the blood when breast cancer begins). [ 9 ] : 175–207


Ovarian cancer screening usually involves ultrasonography of the pelvic region, typically twice a year. [ 9 ] : 175–207    Women may also use a  blood test  for  CA-125  and clinical  pelvic exams .  The blood test has relatively poor  sensitivity and specificity  for ovarian cancer. [ 9 ] : 175–207  [ 37 ]


In both breast and ovarian screening, areas of tissue that look suspicious are investigated with either more imaging, possibly using a different type of imaging or after a delay, or with  biopsies  of the suspicious areas.


Birth control pills  are associated with substantially lower risk of ovarian cancer in women with  BRCA  mutations. [ 38 ] [ 39 ]  A 2013  meta-analysis  found that oral contraceptive use was associated with a 42% reduction of the  relative risk  of ovarian cancer, the association was similar for BRCA1 and BRCA2 mutations. Use of oral contraceptives was not significantly associated with breast cancer risk although a small increase of risk that did not reach  statistical significance  was observed. [ 38 ] [ 39 ]  A 2011 meta-analysis found that OC use was associated with a 43% relative reduction in risk of ovarian cancer in women with  BRCA  mutations, while data on the risk of breast cancer in  BRCA  mutation carriers with oral contraceptive use were heterogeneous and results were inconsistent. [ 40 ]


Selective estrogen receptor modulators , specifically  tamoxifen , have been found to reduce breast cancer risk in women with  BRCA  mutations who do not have their breast removed. [ 9 ] : 113–142     It is effective as for  primary prevention  (preventing the first case of breast cancer) in women with  BRCA2  mutations, but not  BRCA1  mutations, and for  secondary prevention  (preventing a second, independent breast cancer) in both groups of women.  Taking tamoxifen for five years has been found to halve the breast cancer risk in women who have a high risk of breast cancer for any reason, but potentially serious adverse effects like  cataracts , blood clots, and  endometrial cancer , along with quality of life issues like hot flashes, result in some women discontinuing its use and some physicians limiting its use to women with atypical growths in the breasts.  Tamoxifen is  contraindicated  for women who are most likely to be harmed by the common complications.   Raloxifene  (Evista), which has a reduced risk of side effects, is used as an alternative, but it has not been studied in  BRCA  mutation carriers specifically.  Tamoxifen use can be combined with oophorectomy for even greater reduction of breast cancer risk, particularly in women with  BRCA2  mutations. [ 9 ] : 113–142


Aromatase inhibitors  are medications that prevent estrogen production in the  adrenal glands  and  adipose tissue .  They have fewer side effects than selective estrogen receptor modulators like tamoxifen, but do not work in premenopausal women, because they do not prevent the ovaries from producing estrogen. [ 9 ] : 113–142


Several type of  preventive surgeries  are known to substantially reduce cancer risk for women with high-risk  BRCA  mutations. [ 41 ]  The surgeries may be used alone, in combination with each other, or in combination with non-surgical interventions to reduce the risk of breast and ovarian cancer. Surgeries such as mastectomy and oophorectomy do not eliminate the chance of breast cancer; cases have reportedly emerged despite these procedures. [ 42 ]


Whether and when to perform which preventive surgeries is a complex personal decision. Current medical knowledge offers some guidance about the risks and benefits. Even carriers of the same mutation or from the same family may have substantially different risks for the kind and severity of cancer they are likely to get, as well as the age at which they may get them. Different people also have different values. They may choose to focus on total cancer prevention, psychological benefits, current quality of life, or overall survival. The possible impact of future medical developments in treatment or prognosis may also be of some importance for very young women and family planning.  The decision is individualized and is usually based on many factors, such as earliest occurrence of  BRCA -related cancer in close relatives.


An increasing number women who test positive for faulty BRCA1 or BRCA2 genes choose to have risk-reducing surgery. At the same time the average waiting time for undergoing the procedure is two-years which is much longer than recommended. [ 44 ] [ 45 ]


The protective effect of prophylactic surgery is greater when done at young age; however, oophorectomy also has adverse effects that are greatest when done long before natural menopause.  For this reason, oophorectomy is mostly recommended after age 35 or 40, assuming childbearing is complete.  The risk of ovarian cancer is low before this age, and the negative effects of oophorectomy are less serious as the woman nears natural menopause. [ 15 ] [ 46 ]


For comparison, women in the general population have an 84% chance of living to age 70.


Research has looked into the effects of risk-reducing surgery on the psychological and social wellbeing of women with a BRCA mutation. [ 47 ]  Due to limited evidence, a 2019 meta analysis was unable to draw conclusions on whether interventions can help with the psychological effects of surgery in female BRCA carriers. More research is needed to conclude how best to support women who choose surgery. [ 47 ]


In a woman who has not developed breast cancer, removing the breasts may reduce her risk of ever being diagnosed with breast cancer by 90%, to a level that is approximately half the average woman's risk. [ 9 ] : 209–244


Bilateral mastectomy is the removal of both breasts by a  breast surgeon . [ 9 ] : 209–244   The  modified radical mastectomy  is only used in women diagnosed with invasive breast cancer.  Techniques for prophylactic mastectomies include: [ 9 ] : 209–244


Which technique is used is determined by the existence of any cancer and overall health, as well as by the woman's desire, if any, for  breast reconstruction surgery  for aesthetic purposes. [ 9 ] : 209–244    Women who choose a flat-chested appearance or use external breast prostheses typically choose simple mastectomy, with its greater risk reduction. [ 9 ] : 209–244


Breast reconstruction is usually done by a  plastic surgeon , and may be started as part of the same multi-hour surgery that removes the breasts.  Multiple techniques for reconstruction have been used, with different locations and amounts of scarring.  Some techniques use tissue from another part of the body, such as fat tissue from the lower abdomen or occasionally muscles from other parts of the torso.  Others use  breast implants , possibly preceded by tissue expanders, to provide volume.  Some reconstruction techniques require multiple surgeries.  Afterwards, some women have  tattoos  added to simulate  breast areolas  or have the skin reshaped to form a nipple. [ 9 ] : 209–244


Oophorectomy  (surgical removal of the ovaries) and  salpingectomy  (surgical removal of the fallopian tubes) are strongly recommended to women with  BRCA  mutations. [ 9 ] : 275–302   Salpingo-oophorectomy is the single most effective method of preventing ovarian and fallopian tube cancer in women with a  BRCA  mutation.  However, a small risk of  primary peritoneal cancer  remains, at least among women with  BRCA1  mutations, since the peritoneal lining is the same type of cells as parts of the ovary.  This risk is estimated to produce about five cases of peritoneal cancer per 100 women with harmful  BRCA1  mutations in the 20 years after the surgery. [ 9 ] : 275–302


BRCA2  related ovarian cancer tends to present in perimenopausal or menopausal women, so salpingo-oophorectomy is recommended between ages 45 and 50. [ 9 ] : 275–302


The surgery is often done in conjunction with a  hysterectomy  (surgical removal of the uterus) and sometimes a  cervicectomy  (surgical removal of the  cervix ), especially in women who want to take  tamoxifen , which is known to cause uterine cancer, or who have  uterine fibroids . [ 9 ] : 275–302    Multiple styles of surgery are available, including  laparoscopic  (keyhole) surgery.  Because about 5% of women with a  BRCA  mutation have undetected ovarian cancer at the time of their planned surgery, the surgery should be treated as if it were a removal of a known cancer. [ 9 ] : 275–302


Salpingo-oophorectomy makes the woman  sterile  (unable to bear children).   Infertility  services can be used to preserve her  eggs , if wanted.  However, as the benefits to the surgery are greatest close to menopause, most women simply postpone the surgery until they have already borne as many children as they choose to. [ 9 ] : 275–302


The surgery also artificially induces menopause, which causes  hot flashes , sleep disturbances, mood swings, vaginal dryness, sexual difficulties, difficulty with word recall, and other  medical signs  and  symptoms . The side effects range from mild to severe; most can be treated at least partially.  Many women with a  BRCA  take  hormone replacement therapy  to reduce these effects:  estrogen-progesterone combinations for women who have a uterus, and unopposed estrogen for women whose uterus was removed.  Estrogen can cause breast cancer, but as the amount of estrogen taken is less than the amount produced by the now-removed ovaries, the net risk is usually judged to be acceptable. [ 9 ] : 303–317


Some sources assume that oophorectomy before age 50 doubles the risk of cardiovascular disease and increases risk of hip fractures caused by osteoporosis in the relevant population. [ 15 ]


Given the high risks and the low benefit of lifestyle choices in  BRCA  mutation carriers, no lifestyle choices provide sufficient protection. [ 9 ] : 113–142


Having her first child at a younger age, having more children than average, and breastfeeding for more than one year decreases the risk of breast cancer for an average-risk woman. [ 9 ] : 113–142    Studies about this effect among  BRCA  mutation carriers have produced conflicting results, but generally speaking, having children is believed to provide little or no protection against breast cancer for women with  BRCA1  mutations, and to paradoxically increase the risk of breast cancer for women with  BRCA2  mutations. [ 9 ] : 113–142  [ 18 ]


Being physically active and maintaining a healthy body weight prevents breast and other cancers in the general population, as well as preventing heart disease and other medical conditions.  Among women with a  BRCA  mutation, being physically active and having had a healthy body weight as an adolescent has no effect on ovarian cancer and delays, but does not entirely prevent, breast cancer after menopause. [ 9 ] : 113–142  [ 48 ]  In some studies, only significant, strenuous exercise produced any benefit. [ 9 ] : 113–142    Obesity  and weight gain as an adult are associated with breast cancer diagnoses. [ 9 ] : 113–142


Studies on specific foods, diets, or  dietary supplements  have generally produced conflicting information or, in the case of  dietary fat ,  soy  consumption, and drinking  green tea , have only been conducted in average-risk women. [ 9 ] : 113–142    The only dietary intervention that is generally accepted as preventing breast cancer in  BRCA  mutation carriers is minimizing consumption of  alcoholic beverages .  Consuming more than one alcoholic drink per day is strongly associated with a higher risk of developing breast cancer, and carriers are usually encouraged to consume no more than one alcoholic drink per day, and no more than four total in a week. [ 9 ] : 113–142


In a study conducted with Ashkenazi Jewish women, it was observed that mutation carriers born before 1940 have a much lower risk of being diagnosed with breast cancer by age 50 than those born after 1940; this was also observed in the non-carrier population. [ 48 ]  The reasons for the difference is unknown.  Unlike the general population, age at  menarche  and age at menopause has no effect on breast cancer risk for  BRCA  mutation carriers. [ 9 ] : 113–142


Studies have shown that  BRCA1  mutations are not random, but under adaptive selection, indicating that although  BRCA1  mutations are linked to breast cancer, the mutations likely have a beneficial effect as well. [ 49 ]


A patent application for the isolated  BRCA1  gene and cancer-cancer promoting mutations discussed above, as well as methods to diagnose the likelihood of getting breast cancer, was filed by the University of Utah, National Institute of Environmental Health Sciences (NIEHS) and  Myriad Genetics  in 1994; [ 5 ]  over the next year, Myriad, in collaboration with investigators from Endo Recherche, Inc., HSC Research & Development Limited Partnership, and University of Pennsylvania, isolated and sequenced the  BRCA2  gene and identified key mutations, and the first  BRCA2  patent was filed in the US by Myriad and other institutions in 1995. [ 6 ]  Myriad is the exclusive licensee of these patents and has enforced them in the US against clinical diagnostic labs. [ 8 ]  This business model led to Myriad growing being a startup in 1994 to being a publicly traded company with 1200 employees and about $500M in annual revenue in 2012; [ 7 ]  it also led to controversy over high prices and the inability to get second opinions from other diagnostic labs, which in turn led to the landmark  Association for Molecular Pathology v. Myriad Genetics  lawsuit. [ 8 ] [ 50 ]  The patents began to expire in 2014.


According to an article published in the journal, Genetic Medicine, in 2010, "The patent story outside the United States is more complicated.... For example, patents have been obtained but the patents are being ignored by provincial health systems in Canada. In Australia and the UK, Myriad's licensee permitted use by health systems, but announced a change of plans in August 2008. ... Only a single mutation has been patented in Myriad's lone European-wide patent, although some patents remain under review of an opposition proceeding. In effect, the United States is the only jurisdiction where Myriad's strong patent position has conferred sole-provider status." [ 51 ] [ 52 ]  Peter Meldrum, CEO of Myriad Genetics, has acknowledged that Myriad has "other competitive advantages that may make such [patent] enforcement unnecessary" in Europe. [ 53 ]


Legal decisions surrounding the  BRCA1  and  BRCA2  patents will affect the field of  genetic testing  in general. [ 54 ]  In June 2013, in   Association for Molecular Pathology v. Myriad Genetics  (No. 12-398), the  US Supreme Court  unanimously ruled that, "A naturally occurring DNA segment is a product of nature and not  patent eligible  merely because it has been isolated," invalidating Myriad's patents on the BRCA1 and BRCA2 genes. However, the Court also held that manipulation of a gene to create something not found in nature could still be eligible for patent protection. [ 55 ]


The  C957T   gene polymorphism  is a  synonymous mutation  located within the 957th  base pair  of the  DRD2  gene. This base pair is located in  exon  7. Most synonymous mutations are  silent . However, the C957T mutation is an exception to this rule. While the 957C  allele  codes for the same  polypeptide  as the 957T allele, the  conformation  of 957T  messenger RNA  differs from the conformation of 957C messenger RNA. 957T messenger RNA is less stable and more prone to  degradation .  Dopamine D2 receptor  expression is increased among individuals who carry the 957T allele compared to individuals who carry the 957C allele (in contrast to the original article publication, which has since been corrected). [ 1 ]   [ 2 ]


Given that the C/C genotype has been associated with dissocial personality disorder one may infer that this genotype may also be associated with all four  DSM-IV  defined  cluster B personality disorders . This hypothesis, however, has not been tested.


Given the importance of the C957T and  TaqIA  polymorphisms for the diagnosis and treatment of psychological disorder, a rapid screening protocol for these genes has been developed. [ 14 ]  Since the DRD2 1101A allele nulls the effects of the 957T allele, [ 15 ]  screens for the 957C allele which ignore the 1101A allele can sometimes lead to false negatives.


Calico sheep  is a type of  sheep  that has more than one colour within its coat. [ 1 ]   The colouring is due to a piebald  mutation , but the calico sheep is not a  recognized   breed .


These sheep tend to be very small due to intensive inbreeding and are often mislabeled as a true breed. Rather, they are mere crossbred sheep that have interesting colour patterns.


The calico mutation is due to an  autosomal recessive  gene, first reported by Roberts. [ 1 ]


Calico sheep are not related to the black and white sheep that are native to ancient  Sudan [ 2 ]  and to  South Africa . [ 3 ]


The modern mutation was developed by breeding  Shetland sheep  with  Barbados Blackbelly Sheep . [ citation needed ]


Cat coat genetics  determine the coloration, pattern, length, and texture of feline fur. The variations among cat coats are physical properties and should not be confused with cat breeds. A cat may display the coat of a certain breed without actually being that breed. For example, a  Neva Masquerade  ( Siberian  colorpoint) could wear  point coloration , the stereotypical coat of a  Siamese .


The browning gene  B/b/b l  codes for  TYRP1  ( Q4VNX8 ), an enzyme involved in the metabolic pathway for eumelanin pigment production. Its dominant form,  B , will produce black eumelanin.  It has two recessive variants,  b  (chocolate) and  b l  (cinnamon), with  b l  being recessive to both  B  and  b . [ 1 ]  Chocolate is a rich dark brown color, and is referred to as chestnut in some breeds. Cinnamon is a light brown which may be reddish.


The  sex-linked  red  "Orange"  locus,  O/o , determines whether a cat will produce eumelanin. In cats with orange fur,  phaeomelanin  (red pigment) completely replaces  eumelanin  (black or brown pigment). [ 2 ]  This gene is located on the  X chromosome . The orange allele is O, and non-orange is o. Males can typically only be orange or non-orange due to only having one X chromosome. Since females have two X chromosomes, they have two alleles of this gene. OO results in orange fur, oo results in fur without any orange (black, brown, etc.), and Oo results in a  tortoiseshell cat , in which some parts of the fur are orange and other areas non-orange. [ 3 ]  One in three thousand tortoiseshell cats are male, making the combination possible but rare- however, due to the nature of their genetics, male tortoiseshells often exhibit chromosomal abnormalities. [ 4 ]  In one study, less than a third of male calicos had a simple XXY  Klinefelter's  karyotype, slightly more than a third were complicated XXY mosaics, and about a third had no XXY component at all. [ 4 ]


The pelt color commonly referred to as "orange" is scientifically known as red. Other common names include yellow, ginger, and marmalade. Red show cats have a deep orange color, but it can also present as a yellow or light ginger color. Unidentified "rufousing  polygenes " are theorized to be the reason for this variance.
 Orange  is  epistatic  to  nonagouti , so all red cats are tabbies. "Solid" red show cats are usually low contrast ticked tabbies. [ 5 ]


The precise identity of the gene at the  Orange  locus is unknown. It has been narrowed down to a 3.5 Mb stretch on the X chromosome in 2009. [ 5 ]


The  Dense pigment  gene,  D/d , codes for  melanophilin  ( MLPH ;  A0SJ36 ), a protein involved in the transportation and deposition of pigment into a growing hair. [ 5 ]  When a cat has two of the recessive  d  alleles (Maltese dilution), black fur becomes "blue" (appearing gray), chocolate fur becomes "lilac" (appearing light, almost grayish brown-lavender), cinnamon fur becomes "fawn", and red fur becomes "cream". [ 6 ]  Similar to red cats, all cream cats are tabbies. The  d  allele is a single-base deletion that truncates the protein. If the cat has d/d genes, the coat is diluted. If the genes are D/D or D/d, the coat will be unaffected. [ 5 ]


Tabby cats have a range of variegated and blotched coats, consisting of a dark pattern on a lighter background. This variety is derived from the interplay of multiple genes and resulting phenotypes. Most tabbies feature thin dark markings on the face, including the 'M' on the forehead and an eyeliner effect, pigmented lips and paws, and a pink nose outlined in darker pigment. [ citation needed ]  However, the following different coat patterns are all possible: [ citation needed ]


The agouti factor determines the "background" of the tabby coat, which consists of hairs that are banded with dark eumelanin and lighter phaeomelanin along the length of the hair shaft. The  Agouti  gene , with its dominant  A  allele and recessive  a  allele, controls the coding for  agouti signaling protein  (ASIP;  Q865F0 ). The wild-type dominant  A  causes the banding and thus an overall lightening effect on the hair, while the recessive  non-agouti  or "hypermelanistic" allele  a  does not initiate this shift in the pigmentation pathway. As a result, homozygous  aa  have pigment production throughout the entire growth cycle of the hair and therefore along its full length. [ 10 ]  These homozygotes are solidly dark throughout, which obscures the appearance of the characteristic dark tabby markings—sometimes a suggestion of the underlying pattern, called "ghost striping", can be seen, especially in bright slanted light on kittens and on the legs, tail and sometimes elsewhere on adults.


A major exception to the solid masking of the tabby pattern exists, as the  O  allele of the  O/o  locus is  epistatic  over the  aa  genotype. That is, in red or cream colored cats, tabby marking is displayed regardless of the genotype at the agouti locus. However, some red and most cream tabbies do have a fainter pattern when lacking an agouti allele, indicating that the  aa  genotype does still have a faint effect even if it does not induce complete masking. The mechanism of this process is unknown.


The  Tabby  locus on chromosome A1 accounts for most tabby patterns seen in domestic cats, including those patterns seen in most breeds. The dominant allele  Ta M  produces mackerel tabbies, and the recessive  Ta b  produce classic (sometimes referred to as blotched) tabbies. [ 11 ]  The gene responsible for this differential patterning has been identified as  transmembrane aminopeptidase Q  ( Taqpep ,  M3XFH7 ). [ citation needed ]  A  threonine  to  asparagine   substitution  at  residue  139 ( T139N ) in this protein is responsible for producing the tabby phenotype in domestic cats. In  cheetahs , a  base pair insertion  into  exon 20  of the protein replaces the 16 C-terminal residues with 109 new ones (N977Kfs110), generating the  king cheetah  coat variant. [ 12 ]


The wild-type (in  African wildcats ) is the mackerel tabby (stripes look like thin fishbones and may break up into bars or spots). The most common variant is the  classic  tabby pattern (broad bands, whorls, and spirals of dark color on pale background usually with bulls-eye or oyster pattern on flank). [ 12 ]  Spotted tabbies have their stripes broken up into spots, which may be arranged vertically or horizontally. A 2010 study suggests that spotted coats are caused by the modification of mackerel stripes, and may cause varying phenotypes such as "broken mackerel" tabbies via multiple loci. If the genes are Sp/Sp or Sp/sp the tabby coat will be spotted or broken. If it is an sp/sp gene, the tabby pattern will remain either mackerel or blotched. This gene has no effect on cats with a ticked coat. [ 11 ]


The  Ticked  ( Ti ) locus on chromosome B1 controls the generation of "ticked coats", agouti coats with virtually no stripes or bars. Ticked tabbies are rare in the random-bred population, but fixed in certain breeds such as the  Abyssinian  and  Singapura . [ 13 ]   Ti A  is the dominant allele that produces ticked coats;  Ti +  is the recessive one. The causative gene for ticked tabby markings is  Dickkopf-related protein 4  ( DKK4 ). [ 14 ]  Both a  cysteine  to  tyrosine   substitution  at  residue  63 ( C63Y ) and an  alanine  to  valine   substitution  at  residue  18 ( A18V ) result in decreased  DKK4 , which is associated with . Both variants are present in the  Abyssinian  breed, and the A18V variant is found in the  Burmese  breed. [ 13 ]  Stripes often remain to some extent on the face, tail, legs, and sometimes the chest ("bleeding through"). Traditionally, this has been thought to happen in heterozygotes ( Ti A Ti + ) but be nearly or completely nonexistent in homozygotes ( Ti A Ti A ). The ticked tabby allele is epistatic to and therefore completely (or mostly) masks all the other tabby alleles, "hiding" the patterns they would otherwise express. [ 11 ]


It was once thought that  Ti A  was an allele of the  Tabby  gene, called  T a , dominant to all other alleles at the locus. [ 15 ]


Tortoiseshells have patches of orange fur (pheomelanin based) and black or brown (eumelanin based) fur, caused by  X-inactivation . Because this requires two X chromosomes, the vast majority of tortoiseshells are female, with approximately 1 in 3,000 being male. [ 17 ]  Male tortoiseshells can occur as a result of chromosomal abnormalities such as  Klinefelter syndrome , by  mosaicism , or by a phenomenon known as  chimerism , where two early stage embryos are merged into a single kitten.


Tortoiseshells with a relatively small amount of  white spotting  are known as "tortoiseshell and white", while those with a larger amount are known in the United States as  calicos . Calicos are also known as tricolor cats,  mi-ke  (meaning "triple fur") in Japanese, and  lapjeskat  (meaning "patches cat") in Dutch. The factor that distinguishes tortoiseshell from calico is the pattern of eumelanin and pheomelanin, which is partly dependent on the amount of white, due to an effect of the white spotting gene on the general distribution of melanin. A cat which has both an orange and non-orange gene, Oo, and little to no white spotting, will present with a mottled blend of black/red and blue/cream, reminiscent of  tortoiseshell  material, and is called a tortoiseshell cat. An Oo cat with a large amount of white will have bigger, clearly defined patches of black/red and blue/cream, and is called a calico in the US.


With intermediate amounts of white, a cat may exhibit a calico pattern, a tortie pattern, or something in between, depending on other epigenetic factors. Blue tortoiseshell, or  diluted calico , cats have a lighter coloration (blue/cream) and are sometimes called  calimanco  or  clouded tiger . [ 18 ]


A true tricolor must consist of three colors: white, a red-based color like ginger or cream, and black-based color like black or blue. Tricolor should not be mistaken for the natural gradations in a tabby pattern. The shades which are present in the pale bands of a tabby are not considered to constitute a separate color. [ 19 ]


Tortoiseshell cats with small white patches are called  tortico  cats, a portmanteau of  calico  and  tortoiseshell . [ 21 ] [ failed verification ]


The  KIT  gene determines whether or not there will be any white in the coat, except when a solid white coat is caused by albinism. White spotting and epistatic white (also known as dominant white) were long thought to be two separate genes (called  S  and  W  respectively), [ 22 ]  but in fact they are both on the  KIT  gene. The two have been combined into a single  white spotting  locus ( W ). White spotting can take many forms, from a small spot of white to the mostly-white pattern of the Turkish Van, while epistatic white produces a fully white cat (solid or self white). The KIT gene  W  locus has the following alleles: [ 22 ] [ 23 ] [ 24 ]


The  colorpoint  pattern is most commonly associated with  Siamese  cats, but due to crossbreeding may also appear in any (non-pedigree) domesticated cat. A colorpoint cat has dark colors on the face, ears, feet, and tail, with a lighter version of the same color on the rest of the body, and possibly some white. The exact name of the colorpoint pattern depends on the actual color. A few examples are seal points (dark brown to black), chocolate points (warm, lighter brown), blue points (gray), lilac or frost points (silvery gray-pink), red or flame points (orange), and tortie (tortoiseshell mottling) points. This pattern is the result of a  temperature sensitive mutation  in one of the  enzymes  in the  metabolic pathway  from  tyrosine  to pigment, such as  melanin ; thus, little or no pigment is produced except in the extremities or points where the skin is slightly cooler. For this reason, colorpoint cats tend to darken with age as bodily temperature drops; also, the fur over a significant injury may sometimes darken or lighten as a result of temperature change. More specifically, the  albino  locus contains the gene  TYR  ( P55033 ). [ 5 ]  Two distinct alleles causing blue-eyed and pink-eyed albinism respectively have been previously theorized.


Although the Siamese colorpoint pattern is the most famous coloration produced by  TYR , there are color mutations at the locus.


The tyrosine pathway also produces  neurotransmitters , thus mutations in the early parts of that pathway may affect not only pigment, but also neurological development. This results in a higher frequency of  cross-eyes  among colorpoint cats, as well as the  high frequency of cross-eyes in white tigers . [ 33 ]


The  silver  series is caused by the  Melanin inhibitor  gene  I/i . The dominant form causes melanin production to be suppressed, but it affects phaeomelanin (red pigment) much more than eumelanin (black or brown pigment). On tabbies, this turns the background a sparkling silver color while leaving the stripe color intact, making a cold-toned  silver tabby . On solid cats, it turns the base of the hair pale, making them  silver smoke . [ 34 ]  The term  cameo  is commonly used for red silver and cream silver (inhibitor gene (I-O-)) colored coats in cats.


Silver agouti cats can have a range of phenotypes, from silver tabby, to silver shaded (under half the hair is pigmented, approx. 1/3 of hair length), to tipped silver also called chinchilla or shell (only the very tip of the hair is pigmented, approx. 1/8 of hair length). This seems to be affected by hypothetical  wide band  factors, which make the silver band at the base of the hair wider. Breeders often notate wide band as a single gene  Wb/wb , but it is most likely a  polygenic  trait.


If a cat has the wide band trait but no silver melanin inhibitor, the band will be golden instead of silver. These cats are known as  golden  tabbies, or in  Siberian cats   sunshine  tabbies. The golden color is caused by the  CORIN  gene. Shaded golden and tipped golden are also possible, in the same hair length distribution as the silver-gene. However, there is no golden smoke, because the combination of wide band and nonagouti simply produces a solid cat. [ 35 ] [ unreliable source ] [ 36 ]


The genetics involved in producing the ideal tabby,  tipped  [ fr ] , shaded, or smoke cat is complex. Not only are there many interacting genes, but genes sometimes do not express themselves fully, or conflict with one another. For example, the silver melanin inhibitor gene in some instances does not block pigment, resulting in a grayer undercoat, or in  tarnishing  (yellowish or rusty fur). The grayer undercoat is considered less desirable to fanciers.


Likewise, poorly-expressed non-agouti or over-expression of melanin inhibitor will cause a pale, washed out black smoke. Various polygenes (sets of related genes), epigenetic factors, or modifier genes, as yet unidentified, are believed to result in different phenotypes of coloration, some deemed more desirable than others by fanciers.


The genetic influences on tipped or shaded cats are:


Fever coat  is an effect known in domestic cats, where a pregnant female cat has a  fever  or is stressed, causing her unborn kittens' fur to develop a silver-type color (silver-grey, cream, or reddish) rather than what the kitten's  genetics  would normally cause. After birth, over some weeks the silver fur is replaced naturally by fur colors according to the kitten's genetics. [ 37 ] [ 38 ] [ 39 ]


Cat fur can be short, long, curly, or hairless. Most cats are short-haired, like their ancestor. [ 40 ]  The fur can naturally come in three types of hairs;  guard ,  awn , and  down hair . The length, density and proportions of these three hairs varies greatly between breeds, and in some cats only one or two types are found. [ 40 ] [ 41 ]


Most oriental breeds only express one single layer of silky coat. [ 40 ]  However, cats can also have double-layered coats out of two hair types in which the down hairs form the soft, insulating undercoat, and the guard hairs form the protective outer coat. [ 40 ]


A typical cat coat exists of all three natural hair types, but due to the equal lengths of two of these hairs, the coat is still considered double-layered. [ 40 ]  Typically, the down hairs comprise the undercoat while the guard and awn hairs make up the basic top coat. [ 40 ] [ 41 ]  Double-coated cats with thick undercoats require daily grooming as these coats are more prone to matting. [ 40 ]  Double coats are found in for example the  Persian ,  British Shorthair ,  Maine Coon  and  Norwegian Forest cat .


Additionally, there even exist cats which express all three natural types of cat hair in different lengths and structures, which form three different layers. These cats are called triple-coated.  Siberians  and  Neva Masquerades  are known for their unique triple coats, [ 40 ]  which provides double insulation to withstand their natural cold climate.


There have been many genes identified that result in unusual cat fur. These genes were discovered in random-bred cats and selected for. Some of the genes are in danger of going extinct because the cats are not sold beyond the region where the mutation originated or there is simply not enough demand for cats expressing the mutation.


In many breeds, coat gene mutations are unwelcome. An example is the rex allele which appeared in Maine Coons in the early 1990s. Rexes appeared in America, Germany and the UK, where one breeder caused consternation by calling them "Maine Waves". Two UK breeders did test mating which indicated that this was probably a new rex mutation and that it was recessive. The density of the hair was similar to normally coated Maine Coons, but consisted only of down type hairs with a normal down type helical curl, which varied as in normal down hairs. Whiskers were more curved, but not curly. Maine Coons do not have awn hairs, and after moulting, the rexes had a very thin coat. [ citation needed ]


Cat fur length is governed by the  Length  gene in which the dominant form,  L , codes for short hair, and the recessive  l  codes for long hair. In the longhaired cat, the transition from anagen (hair growth) to catagen (cessation of hair growth) is delayed due to this mutation. [ 42 ]  A rare recessive shorthair gene has been observed in some lines of Persian cat (silvers) where two longhaired parents have produced shorthaired offspring.


The  Length  gene has been identified as the  fibroblast growth factor 5  (FGF5;  M3X9S6 ) gene. The dominant allele codes for the short coat is seen in most cats. Long coats are coded for by at least four different recessively inherited mutations, the alleles of which have been identified. [ 43 ]  The most ubiquitous is found in most or all long haired breeds while the remaining three are found only in Ragdolls, Norwegian Forest Cats, and Maine Coons.


There are various genes producing curly-coated or "rex" cats. New types of rex arise spontaneously in random-bred cats now and then. Some of the rex genes that breeders have selected for are:


There are also genes for hairlessness:


Some rex cats are prone to temporary hairlessness, known as baldness, during moulting.


Here are a few other genes resulting in unusual fur:


Name


A  clastogen  is a  mutagenic  agent that disturbs normal  DNA  related processes or directly causes  DNA  strand breakages, thus causing the  deletion ,  insertion , or rearrangement of entire  chromosome  sections. [ 1 ]  These processes are a form of  mutagenesis  which if left unrepaired, or improperly repaired, can lead to  cancer . [ 1 ]  Known clastogens include  acridine yellow ,  benzene ,  ethylene oxide ,  arsenic ,  phosphine ,  mimosine ,  actinomycin D ,  camptothecin ,  methotrexate ,  methyl acrylate ,  resorcinol  and  5-fluorodeoxyuridine . [ 2 ]  Additionally,  1,2-dimethylhydrazine  is a known colon  carcinogen  and shows signs of possessing clastogenic activity. [ 3 ]  There are many clastogens not listed here and research is ongoing to discover new clastogens. Some known clastogens only exhibit clastogenic activity in certain  cell  types, such as caffeine which exhibits clastogenic activity in plant cells. [ 4 ]  Researchers are interested in clastogens for researching  cancer , as well as for other human health concerns such as the inheritability of clastogen effected paternal  germ cells  that lead to  fetus  developmental defects. [ 5 ]


There is not one all encompassing method by which clastogens damage chromosomal  DNA , instead different clastogens have unique ways they interact with DNA, or DNA associated  proteins , and disrupt normal function. Broadly these different types of clastogenic activity can be organized into three classes: ‘classic’ breaks theory; ‘mis-repair of breaks’ theory and ‘repair-created breaks’ theory. [ 4 ]  It may not always be known how a clastogen causes chromosomal damage.


Radiation was the earliest known clastogen that caused direct DNA damage, following the classic breaks theory. [ 6 ]  DNA is frequently damaged and there are many  DNA repair  pathways that combat this, but repair does not always work perfectly resulting in mistakes (called a misrepair). [ 7 ]  A widely studied class of clastogens are  alkylating  agents which do not break DNA at all, but instead form  DNA adducts , and these have often eluded the common theories for DNA breaks leading to misrepair. [ 4 ]  The final theory encompasses clastogens that do not interact with DNA but instead impair  DNA synthesis   proteins  or  DNA repair   proteins  causing damage to occur through loss of normal function of the protein. [ 4 ]


Clastogen damage in certain areas of the  chromosome  can lead to instability, such as loss or damage to  telomeres . [ 8 ]  Studies have shown that rat cells that were exposed to chemical clastogens express telomeric irregularities in function and can remain for several cell generations after treatment has been attempted. [ 8 ]


There are many different methods for testing for clastogenic activity. Two of the most common methods are listed below, but this is not a comprehensive guide.


There have been studies done that work with the usage of the deletion (DEL)  assay  to screen for clastogens.


The micronucleus test is another type of  assay  that uses gut cells to observe clastogens, and there are a few different types. The  micronucleus  test on gut cells is useful because in the case of the bone marrow  micronucleus  test there is not much activity seen after there has been oral exposure therefore more activity is seen in the gut cells.  In vitro   micronucleus  assay (IVMN) can screen for clastogen activity, this method is useful because it can pick up clastogen activity and be used to foresee  chromosome aberration  activity. The IVMN assay can pick up on fragments that were membrane bound to DNA that were split from nuclei throughout the process of cell division.


These assays are time-consuming so novel methods for monitoring clastogens and aneuploidogens are highly desirable. One example is the use of the monochromosomal hybrid cell for the detection of mis-segregating chromosomes.


There is a possibility of clastogens affecting telomeres. There can be uncertainty with telomeres that occur short term during the first round of cell division in which there can be chromosomal damage by clastogens. Clastogens (which break chromosomes) contribute to telomeric instability because it leads to chromosome end loss or true telomere loss. Clastogens can bring on issues with telomeres and cause them to fail to function as intended, most often seen anomalies are seen to occur in human lymphocytes, cancer cell lines, and non-human established cell lines where there is telomere loss and copies of anomalies in the exposed cells, thus, the problems that arise in telomeres can be duplicated and seen in exposed cells.


In addition, studies have shown that rat cells that were exposed to chemical clastogens express telomeric irregularities in function and can remain for several cell generations after treatment has been attempted. [ 8 ]


In terms of resistance, for a specific clastogen known as "Zeocin", an amino acid residue known as XLF-L115D mutant is flawed in terms of being resistant thus the clastogen activity shows no amount of decreasing. [ 9 ]


In plants and mice cells studies have found that  purine   receptor   agonists   adenosine ,  ATP ,  ADP ,  cyclohexyladenosine , phenylisopropyladenosine and dimethylaminopurine riboside can lower the amount of clastogen damage seen in  chromosomes  and reduce the amount of  micronuclei  affected brought on by  ethyl methanesulfonate  and  cyclophosphamide . Some  ligands  more than others can stop or reduce the clastogen activity of ethyl methanesulfonate such as  adenosine ,  ADP  or DAP. [ 10 ]


In a study where rats were treated with Brevetoxin B (PbTx2), there was a noticeable 2-3 fold growth in the amount of DNA seen in comet tails which tell us that Brevetoxin B shows in vivo clastogenic activity. This clastogen activity was seen after Brevetoxin B was injected by way of intratracheal administering in the rat. [ 11 ]


D145E  is a  point mutation  on  troponin C  that leads to  hypertrophic cardiomyopathy  disease. This mutation is caused by the change of  nucleotide  C to A at nucleotide 435, [ 1 ]  switching the amino acid  aspartic acid  to  glutamic acid , which is located at the  C-terminal  tail. [ 2 ]  Patients with this mutation have different structure on the thin filament and alter the binding of  Ca 2+  at the troponin C site IV. [ 1 ] [ 3 ]  Further, D145E causes increase in development of force and activation of  ATPase  in the presence of Ca 2+ . [ 4 ]


The  duplicated gene nucleotide variant database  ( dbDNV ) is a database of  duplicated-gene   nucleotide variants  in the human genome  [ 1 ]


In  genetics , a  deletion  (also called  gene deletion ,  deficiency , or  deletion mutation ) (sign:  Δ ) is a  mutation  (a genetic aberration) in which a part of a  chromosome  or a sequence of  DNA  is left out during DNA replication. Any number of  nucleotides  can be deleted, from a single  base  to an entire piece of chromosome. [ 1 ]  Some  chromosomes  have fragile spots where breaks occur, which result in the  deletion  of a part of the chromosome. The breaks can be induced by heat, viruses, radiation, or chemical reactions. When a  chromosome  breaks, if a part of it is deleted or lost, the missing piece of chromosome is referred to as a deletion or a deficiency. [ 2 ]


For  synapsis  to occur between a chromosome with a large intercalary deficiency and a normal complete homolog, the unpaired region of the normal homolog must loop out of the linear structure into a  deletion  or  compensation loop .


The smallest single base deletion mutations occur by a single  base flipping  in the template DNA, followed by template DNA strand slippage, within the DNA polymerase active site. [ 3 ] [ 4 ] [ 5 ]


Deletions can be caused by errors in  chromosomal crossover  during  meiosis , which causes several serious  genetic diseases . Deletions that do not occur in multiples of three bases can cause a  frameshift  by changing the 3-nucleotide protein reading frame of the genetic sequence. Deletions are representative of  eukaryotic  organisms, including humans and not in  prokaryotic  organisms, such as bacteria.


Causes include the following:


Types of deletion include the following:


Micro-deletion is usually found in children with physical abnormalities. A large amount of deletion would result in immediate abortion (miscarriage).


The  International System for Human Cytogenomic Nomenclature  (ISCN) is an international standard for  human chromosome   nomenclature , which includes band names, symbols and abbreviated terms used in the description of human chromosome and chromosome abnormalities. Abbreviations include a minus sign (−) for chromosome deletions, and  del  for deletions of parts of a chromosome. [ 7 ]


Small deletions are less likely to be fatal; large deletions are usually fatal – there are always variations based on which genes are lost. Some medium-sized deletions lead to recognizable human disorders, e.g.  Williams syndrome .


Deletion of a number of pairs that is not evenly divisible by three will lead to a  frameshift mutation , causing all of the  codons  occurring after the deletion to be read incorrectly during  translation , producing a severely altered and potentially nonfunctional  protein . In contrast, a deletion that is evenly divisible by three is called an  in-frame  deletion. [ 8 ]


Deletions are responsible for an array of genetic disorders, including some cases of male  infertility , two thirds of cases of  Duchenne muscular dystrophy , [ 1 ]  and two thirds of cases of  cystic fibrosis  (those caused by  ΔF508 ). [ 9 ]  Deletion of part of the short arm of chromosome 5 results in  Cri du chat  syndrome. [ 1 ]  Deletions in the  SMN -encoding gene cause  spinal muscular atrophy , the most common genetic cause of infant death.


Microdeletions are associated with many different conditions, including Angelman Syndrome, Prader-Willi Syndrome, and DiGeorge Syndrome. [ 10 ]   Some syndromes, including Angelman syndrome and Prader-Willi syndrome, are associated with both microdeletions and genomic imprinting, meaning that same microdeletion can cause two different syndromes depending on which parent the deletion came from. [ 11 ]


Recent work suggests that some deletions of highly conserved sequences (CONDELs) may be responsible for the evolutionary differences present among closely related species. Such deletions in humans, referred to as  hCONDELs , may be responsible for the anatomical and behavioral differences between humans,  chimpanzees  and other varieties of mammals like  ape  or  monkeys. [ 12 ]


Recent comprehensive patient-level classification and quantification of driver events in  TCGA  cohorts revealed that there are on average 12 driver events per tumor, of which 2.1 are deletions of  tumor suppressors . [ 13 ]


The introduction of molecular techniques in conjunction with classical cytogenetic methods has in recent years greatly improved the diagnostic potential for chromosomal abnormalities. In particular, microarray-comparative genomic hybridization (CGH) based on the use of BAC clones promises a sensitive strategy for the detection of DNA copy-number changes on a genome-wide scale. The resolution of detection could be as high as >30,000 "bands" and the size of chromosomal deletion detected could as small as 5–20 kb in length. [ 14 ]  Other computation methods were selected to discover DNA sequencing deletion errors such as  end-sequence profiling . [ 15 ] [ 16 ]


In the yeast  Saccharomyces cerevisiae , the nuclear genes  Rad51 p,  Rad52 p and  Rad59p  encode proteins that are necessary for recombinational repair and are employed in the repair of  double strand breaks  in  mitochondrial DNA . [ 17 ]    Loss of these proteins decreases the rate of spontaneous DNA deletion events in mitochondria. [ 17 ]   This finding implies that the repair of DNA double-strand breaks by homologous recombination is a step in the formation of mitochondrial DNA deletions.


Depurination  is a chemical reaction of  purine   deoxyribonucleosides ,  deoxyadenosine  and  deoxyguanosine , and  ribonucleosides ,  adenosine  or  guanosine , in which the β-N- glycosidic bond  is  hydrolytically  cleaved releasing a nucleic base,  adenine  or  guanine , respectively. The second product of depurination of deoxyribonucleosides and ribonucleosides is sugar, 2'- deoxyribose  and  ribose , respectively. More complex compounds containing nucleoside residues,  nucleotides  and  nucleic acids , also suffer from depurination. Deoxyribonucleosides and their derivatives are substantially more prone to depurination than their corresponding ribonucleoside counterparts. Loss of  pyrimidine  bases ( cytosine  and  thymine ) occurs by a similar mechanism, but at a substantially lower rate. [ 1 ] [ 2 ]


When depurination occurs with  DNA , it leads to the formation of  apurinic site  and results in an alteration of the structure. Studies estimate that as many as 5,000 purines are lost this way each day in a typical human cell. [ 3 ]  In cells, one of the main causes of depurination is the presence of endogenous metabolites undergoing chemical reactions. Apurinic sites in double-stranded DNA are efficiently repaired by portions of the  base excision repair  (BER) pathway. Depurinated bases in single-stranded DNA undergoing  replication  can lead to  mutations , because in the absence of information from the complementary strand, BER can add an incorrect base at the apurinic site, resulting in either a  transition  or  transversion  mutation. [ 4 ]


Depurination is known to play a major role in cancer initiation. [ 5 ]


Hydrolytic depurination is one of the principal forms of damage to  ancient DNA  in fossil or subfossil material, since the base remains unrepaired. This results in both loss of information (the base sequence), and difficulties in recovery and  in vitro  replication of the damaged molecule by the  polymerase chain reaction .


Depurination is not uncommon because purine is a good  leaving group  via the 9N-nitrogen (see the structure of a  purine ). Furthermore, the  anomeric carbon  is especially reactive towards  nucleophilic substitution  (effectively making the carbon-oxygen bond shorter, stronger and more polar, while making the carbon-purine bond longer and weaker). This makes the bond especially susceptible to hydrolysis.


In chemical  synthesis  of  oligonucleotides , depurination is one of the major factors limiting the length of synthetic oligonucleotides. [ 6 ]


Disease gene identification  is a process by which scientists identify the mutant  genotypes  responsible for an inherited  genetic disorder .  Mutations  in these genes can include single nucleotide substitutions, single nucleotide additions/deletions, deletion of the entire gene, and other genetic abnormalities.


Knowledge of which genes (when non-functional) cause which disorders will simplify diagnosis of patients and provide insights into the functional characteristics of the mutation.  The advent of modern-day  high-throughput sequencing  technologies combined with insights provided from the growing field of  genomics  is resulting in more rapid disease gene identification, thus allowing scientists to identify more complex mutations.


Disease gene identification techniques often follow the same overall procedure.  DNA is first collected from several patients who are believed to have the same genetic disease.  Then, their DNA samples are analyzed and screened to determine probable regions where the mutation could potentially reside.  These techniques are mentioned below.  These probable regions are then lined-up with one another and the overlapping region should contain the mutant gene.  If enough of the genome sequence is known, that region is searched for  candidate genes .  Coding regions of these genes are then sequenced until a mutation is discovered or another patient is discovered, in which case the analysis can be repeated, potentially narrowing down the region of interest.


The differences between most disease gene identification procedures are in the second step (where DNA samples are analyzed and screened to determine regions in which the mutation could reside).


Without the aid of the whole-genome sequences, pre-genomics investigations looked at select regions of the genome, often with only minimal knowledge of the gene sequences they were looking at.  Genetic techniques capable of providing this sort of information include  Restriction Fragment Length Polymorphism  (RFLP) analysis and  microsatellite  analysis.


Loss of heterozygosity (LOH)  is a technique that can only be used to compare two samples from the same individual.  LOH analysis is often used when identifying cancer-causing  oncogenes  in that one sample consists of (mutant) tumor DNA and the other (control) sample consists of genomic DNA from non-cancerous cells from the same individual.  RFLPs and microsatellite markers provide patterns of DNA polymorphisms, which can be interpreted as residing in a  heterozygous  region or a  homozygous  region of the genome.  Provided that all individuals are affected with the same disease resulting from a manifestation of a deletion of a single copy of the same gene, all individuals will contain one region where their control sample is heterozygous but the mutant sample is homozygous - this region will contain the disease gene. [ 1 ] [ 2 ]


With the advent of modern laboratory techniques such as  High-throughput sequencing  and software capable of genome-wide analysis, sequence acquisition has become increasingly less expensive and time-consuming, thus providing significant benefits to science in the form of more efficient disease gene identification techniques.


Identity by descent  (IBD) mapping generally uses  single nucleotide polymorphism  (SNP) arrays to survey known polymorphic sites throughout the genome of affected individuals and their parents and/or siblings, both affected and unaffected.  While these SNPs probably do not cause the disease, they provide valuable insight into the makeup of the genomes in question. A region of the genome is considered identical by descent if contiguous SNPs share the same genotype.  When comparing an affected individual to his/her affected sibling, all identical regions are recorded (ex. Shaded in red in above figure).  Given that an affected sibling and an unaffected sibling do not have the same disease phenotype, their DNA must by definition be different (barring the presence of a genetic or environmental  modifier ).  Thus, the IBD mapping results can be further supplemented by removing any regions that are identical in both affected individuals and unaffected siblings. [ 3 ]   This is then repeated for multiple families, thus generating a small, overlapping fragment, which theoretically contains the disease gene.


Homozygosity/Autozygosity mapping is a powerful technique, but is only valid when searching for a mutation segregating within a small, closed population.  Such a small population, possibly created by the  founder effect , will have a limited gene pool, and thus any inherited disease will probably be a result of two copies of the same mutation segregating on the same  haplotype .  Since affected individuals will probably be homozygous in the regions, looking at SNPs in a region is an adequate marker of regions of homozygosity and heterozygosity.  Modern day  SNP arrays  are used to survey the genome and identify large regions of homozygosity.  Homozygous blocks in the genomes of affected individuals can then be laid on top of each other, and the overlapping region should contain the disease gene. [ 4 ]


This analysis is often extended by analyzing  autozygosity , an extension of homozygosity, in the genomes of affected individuals. [ 5 ]   This can be accomplished by plotting a cumulative  LOD score  alongside the overlaid blocks of homozygosity.  By taking into consideration the population allele frequencies for all SNPs via autozygosity mapping, the results of homozygosity can be confirmed. [ 5 ]   Furthermore, if two suspicious regions appear as a result of homozygosity mapping, autozygosity mapping may be able to distinguish between the two (ex. If one block of homozygosity is a result of a very non-diverse region of the genome, the LOD score will be very low).


Tools for Homozygosity Mapping


Genome-wide  knockdown  studies are an example of the  reverse genetics  made possible by the acquisition of whole genome sequences, and the advent of genomics and gene-silencing technologies, mainly  siRNA  and  deletion mapping .  Genome-wide knockdown studies involve systematic knockdown or deletion of genes or segments of the genome. [ 7 ]   This is generally done in  prokaryotes  or in a  tissue culture  environment due to the massive number of knockdowns that must be performed. [ 8 ]   After the systematic knockout is completed (and possibly confirmed by mRNA expression analysis), the phenotypic results of the knockdown/knockout can be observed.  Observation parameters can be selected to target a highly specific phenotype. [ 8 ]   The resulting dataset is then queried for samples which exhibit phenotypes matching the disease in question – the gene(s) knocked down/out in said samples can then be considered candidate disease genes for the individual in question.


Whole  exome sequencing  is a brute-force approach that involves using modern day sequencing technology and  DNA sequence assembly  tools to piece together all coding portions of the genome.  The sequence is then compared to a  reference genome  and any differences are noted.  After filtering out all known benign polymorphisms, synonymous changes, and  intronic  changes (that do not affect splice sites), only potentially pathogenic variants will be left.  This technique can be combined with other techniques to further exclude potentially pathogenic variants should more than one be identified. [ 9 ]


Natural DNA damage  is an alteration in the chemical structure of  DNA , such as a break in a strand of DNA, a  nucleobase  missing from the backbone of DNA, or a chemically changed base such as  8-OHdG . DNA damage can occur naturally or via environmental factors, but is distinctly different from  mutation , although both are types of error in  DNA . DNA damage is an abnormal chemical structure in DNA, while a mutation is a change in the sequence of base pairs. DNA damages cause changes in the structure of the genetic material and prevents the replication mechanism from functioning and performing properly. [ 1 ]  The DNA damage response (DDR) is a complex signal transduction pathway which recognizes when DNA is damaged and initiates the cellular response to the damage. [ 2 ]


DNA damage and mutation have different biological consequences.  While most DNA damages can undergo  DNA repair , such repair is not 100% efficient.  Un-repaired DNA damages accumulate in non-replicating cells, such as cells in the brains or muscles of adult mammals, and can cause aging. [ 3 ] [ 4 ] [ 5 ]  (Also see  DNA damage theory of aging .)  In replicating cells, such as cells lining the colon, errors occur upon replication of past damages in the  template  strand of DNA or during repair of DNA damages.  These errors can give rise to  mutations  or  epigenetic  alterations. [ 6 ]   Both of these types of alteration can be replicated and passed on to subsequent cell generations.  These alterations can change gene function or regulation of gene expression and possibly contribute to progression to cancer.


Throughout the cell cycle there are various checkpoints to ensure the cell is in good condition to progress to mitosis. The three main checkpoints are at G1/s, G2/m, and at the spindle assembly checkpoint regulating progression through anaphase.  G1  and  G2  checkpoints involve scanning for damaged DNA. [ 7 ]   During S phase the cell is more vulnerable to DNA damage than any other part of the cell cycle. G2 checkpoint checks for damaged DNA and DNA replication completeness.


Damage to DNA that occurs naturally can result from  metabolic  or  hydrolytic  processes.  Metabolism releases compounds that damage DNA including  reactive oxygen species ,  reactive nitrogen species , reactive  carbonyl  species,  lipid peroxidation  products, and  alkylating agents , among others, while hydrolysis cleaves chemical bonds in DNA. [ 8 ]  Naturally occurring oxidative DNA damages arise at least 10,000 times per cell per day in humans and as much as 100,000 per cell per day in rats [ 9 ]  as documented below.


Oxidative DNA damage can produce more than 20 types of altered bases [ 10 ] [ 11 ]  as well as single strand breaks. [ 12 ]


Other types of endogeneous DNA damages, given below with their frequencies of occurrence, include  depurinations ,  depyrimidinations ,  double-strand breaks ,  O6-methylguanines , and  cytosine deamination .


DNA can be damaged via environmental factors as well. Environmental agents such as UV light, ionizing radiation, and genotoxic chemicals. Replication forks can be stalled due to damaged DNA and double strand breaks are also a form of DNA damage. [ 13 ]


The list below shows some frequencies with which new naturally occurring DNA damages arise per day, due to endogenous cellular processes.


Another important endogenous DNA damage is  M1dG , short for (3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one).  The excretion in urine (likely reflecting rate of occurrence) of M1dG may be as much as 1,000-fold lower than that of 8-oxodG. [ 26 ]  However, a more important measure may be the steady-state level in DNA, reflecting both rate of occurrence and rate of DNA repair. The steady-state level of M1dG is higher than that of 8-oxodG. [ 27 ]   This points out that some DNA damages produced at a low rate may be difficult to repair and remain in DNA at a high steady-state level. Both M1dG [ 28 ]  and 8-oxodG [ 29 ]  are  mutagenic .


Steady-state levels of DNA damages represent the balance between formation and repair.  More than 100 types of oxidative DNA damage have been characterized, and 8-oxodG constitutes about 5% of the steady state oxidative damages in DNA. [ 18 ]  Helbock et al. [ 14 ]  estimated that there were 24,000 steady state oxidative DNA adducts per cell in young rats and 66,000 adducts per cell in old rats.  This reflects the accumulation of DNA damage with age.  DNA damage accumulation with age is further described in  DNA damage theory of aging .


Swenberg et al. [ 30 ]  measured average amounts of selected steady state endogenous DNA damages in mammalian cells. The seven most common damages they evaluated are shown in Table 1.


Evaluating steady-state damages in specific tissues of the rat, Nakamura and Swenberg [ 31 ]  indicated that the number of abasic sites varied from about 50,000 per cell in liver, kidney and lung to about 200,000 per cell in the brain.


Proteins promoting endogenous DNA damage were identified in a 2019 paper as the DNA "damage-up" proteins (DDPs). [ 32 ]  The DDP mechanisms fall into 3 clusters:


The DDP human homologs are over-represented in known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. [ 32 ]


In the presence of DNA damage, the cell can either repair the damage or induce cell death if the damage is beyond repair.


The seven main types of DNA repair and one pathway of damage tolerance, the lesions they address, and the accuracy of the repair (or tolerance) are shown in this table.  For a brief description of the steps in repair see  DNA repair mechanisms  or see each individual pathway.


The schematic diagram indicates the roles of insufficient DNA repair in aging and cancer, and the role of apoptosis in cancer prevention. An excess of naturally occurring DNA damage, due to inherited deficiencies in particular DNA repair enzymes, can cause premature aging or increased risk for cancer (see  DNA repair-deficiency disorder ). On the other hand, the ability to trigger  apoptosis  in the presence of excess un-repaired DNA damage is critical for prevention of cancer. [ 42 ]


DNA repair  proteins are often activated or induced when DNA has sustained damage. However, excessive DNA damage can initiate  apoptosis  (i.e., programmed cell death) if the level of DNA damage exceeds the repair capacity. Apoptosis can prevent cells with excess DNA damage from undergoing mutagenesis and progression to cancer. [ 43 ]


Inflammation  is often caused by infection, such as with  hepatitis B virus  (HBV),  hepatitis C virus  (HCV) or  Helicobacter pylori . Chronic inflammation is also a central characteristic of obesity. [ 44 ] [ 45 ] [ 46 ] [ 47 ]  Such inflammation causes oxidative DNA damage. This is due to the induction of  reactive oxygen species  (ROS) by various intracellular inflammatory mediators. [ 48 ] [ 49 ] [ 50 ]  HBV and HCV infections, in particular, cause 10,000-fold and 100,000-fold increases in intracellular ROS production, respectively. [ 51 ]  Inflammation-induced ROS that cause DNA damage can trigger apoptosis, [ 52 ] [ 53 ]  but may also cause cancer if repair and apoptotic processes are insufficiently protective. [ 45 ]


Bile acids , stored in the gall bladder, are released into the small intestine in response to fat in the diet.  Higher levels of fat cause greater release. [ 54 ]   Bile acids cause DNA damage, including oxidative DNA damage, double-strand DNA breaks, aneuploidy and chromosome breakage. [ 55 ]   High-normal levels of the bile acid deoxycholic acid cause apoptosis in human colon cells, [ 56 ]  but may also lead to colon cancer if repair and apoptotic defenses are insufficient. [ 57 ]


Apoptosis serves as a safeguard mechanism against tumorigenesis. [ 58 ]   It prevents the increased mutagenesis that excess DNA damage could cause, upon replication. [ 59 ]


At least 17 DNA repair proteins, distributed among five DNA repair pathways, have a "dual role" in response to DNA damage.  With moderate levels of DNA damage, these proteins initiate or contribute to DNA repair.  However, when excessive levels of DNA damage are present, they trigger apoptosis. [ 43 ]


The packaging of eukaryotic DNA into  chromatin  is a barrier to all DNA-based processes that require enzyme action.  For most DNA repair processes, the chromatin must be  remodeled .  In eukaryotes,  ATP -dependent  chromatin remodeling  complexes and  histone-modifying enzymes  are two factors that act to accomplish this remodeling process after DNA damage occurs. [ 60 ]   Further DNA repair steps, involving multiple enzymes, usually follow.  Some of the first responses to DNA damage, with their timing, are described below.  More complete descriptions of the DNA repair pathways are presented in articles describing each pathway.  At least 169 enzymes are involved in DNA repair pathways. [ 61 ]


Oxidized bases in DNA are produced in cells treated with Hoechst dye followed by micro-irradiation with 405 nm light. [ 62 ]   Such oxidized bases can be repaired by  base excision repair .


When the 405 nm light is focused along a narrow line within the nucleus of a cell, about 2.5 seconds after irradiation, the chromatin remodeling enzyme  Alc1  achieves half-maximum recruitment onto the irradiated micro-line. [ 63 ]   The line of chromatin that was irradiated then relaxes, expanding side-to-side over the next 60 seconds. [ 63 ]


Within 6 seconds of the irradiation with 405 nm light, there is half-maximum recruitment of  OGG1  to the irradiated line. [ 62 ]  OGG1 is an enzyme that removes the oxidative DNA damage  8-oxo-dG  from DNA. Removal of 8-oxo-dG, during base excision repair, occurs with a half-life of 11 minutes. [ 18 ]


Ultraviolet  (UV) light induces the formation of DNA damages including  pyrimidine dimers  (such as thymine dimers) and 6,4 photoproducts.  These types of "bulky" damages are repaired by  nucleotide excision repair . [ citation needed ]


After irradiation with UV light,  DDB2 , in a complex with  DDB1 , the  ubiquitin ligase  protein  CUL4A  and the RING finger protein ROC1, associates with sites of damage within chromatin. Half-maximum association occurs in 40 seconds. [ 64 ]   PARP1  also associates within this period. [ 65 ]   The PARP1 protein attaches to both DDB1 and DDB2 and then  PARylates  (creates a poly-ADP ribose chain) on DDB2 that attracts the DNA remodeling protein  ALC1 . [ 65 ]   ALC1 relaxes chromatin at sites of UV damage to DNA.  In addition, the ubiquitin E3 ligase complex DDB1-CUL4A carries out ubiquitination of the core  histones  H2A, H3, and H4, as well as the repair protein XPC, which has been attracted to the site of the DNA damage. [ 66 ]   XPC, upon ubiquitination, is activated and initiates the  nucleotide excision repair  pathway.  Somewhat later, at 30 minutes after UV damage, the  INO80  chromatin remodeling complex is recruited to the site of the DNA damage, and this coincides with the binding of further nucleotide excision repair proteins, including  ERCC1 . [ 67 ]


Double-strand breaks (DSBs) at specific sites can be induced by transfecting cells with a plasmid encoding  I-SceI endonuclease  (a  homing endonuclease ).  Multiple DSBs can be induced by irradiating sensitized cells (labeled with  5'-bromo-2'-deoxyuridine  and with Hoechst dye) with 780 nm light.  These DSBs can be repaired by the accurate  homologous recombinational repair  or by the less accurate  non-homologous end joining  repair pathway.  Here we describe the early steps in homologous recombinational repair (HRR). [ citation needed ]


After treating cells to introduce DSBs, the stress-activated protein kinase,  c-Jun N-terminal kinase (JNK) , phosphorylates  SIRT6  on serine 10. [ 68 ]   This  post-translational modification  facilitates the mobilization of SIRT6 to DNA damage sites with half-maximum recruitment in well under a second. [ 68 ]   SIRT6 at the site is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to a DNA break site and for efficient repair of DSBs. [ 68 ]   PARP1  protein starts to appear at DSBs in less than a second, with half maximum accumulation within 1.6 seconds after the damage occurs. [ 69 ]   This then allows half maximum recruitment of the DNA repair enzymes  MRE11  within 13 seconds and  NBS1  within 28 seconds. [ 69 ]   MRE11 and NBS1 carry out early steps of the HRR pathway.


γH2AX, the phosphorylated form of  H2AX  is also involved in early steps of DSB repair.  The  histone  variant H2AX constitutes about 10% of the H2A histones in human chromatin. [ 70 ]   γH2AX (H2AX phosphorylated on serine 139) can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute. [ 70 ]   The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break. [ 70 ]   γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of irradiation,  RNF8  protein can be detected in association with γH2AX. [ 71 ]   RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with  CHD4 , [ 72 ]  a component of the nucleosome remodeling and deacetylase complex  NuRD .


After rapid  chromatin remodeling ,  cell cycle   checkpoints  may be activated to allow DNA repair to be completed before the cell cycle progresses.  First, two  kinases ,  ATM  and  ATR , are activated within 5 or 6 minutes after DNA is damaged.  This is followed by phosphorylation of the cell cycle checkpoint protein  Chk1 , initiating its function, about 10 minutes after DNA is damaged. [ 73 ]


The DNA damage  8-oxo-dG  does not occur randomly in the genome.  In  mouse embryonic fibroblasts , a 2 to 5-fold enrichment of 8-oxo-dG was found in genetic control regions, including  promoters ,  5'-untranslated regions  and  3'-untranslated regions  compared to 8-oxo-dG levels found in  gene  bodies and in  intergenic regions . [ 74 ]   In rat pulmonary artery endothelial cells, when 22,414 protein-coding genes were examined for locations of 8-oxo-dG, the majority of 8-oxo-dGs (when present) were found in promoter regions rather than within gene bodies. [ 75 ]   Among hundreds of genes whose expression levels were affected by hypoxia, those with newly acquired promoter 8-oxo-dGs were  upregulated , and those genes whose promoters lost 8-oxo-dGs were almost all  downregulated . [ 75 ]


As reviewed by Wang et al., [ 76 ]  oxidized guanine appears to have multiple regulatory roles in gene expression.  In particular, when oxidative stress produces 8-oxo-dG in the promoter of a gene, the oxidative stress may also inactivate  OGG1 , an enzyme that targets 8-oxo-dG and normally initiates repair of 8-oxo-dG damage.  The inactive OGG1, which no longer excises 8-oxo-dG, nevertheless targets and complexes with 8-oxo-dG, and causes a sharp (~70 o ) bend in the DNA.  This allows the assembly of a transcriptional initiation complex, up-regulating transcription of the associated gene. [ 76 ] [ 77 ]


When 8-oxo-dG is formed in a guanine rich,  potential G-quadruplex-forming sequence  (PQS) in the coding strand of a promoter, active OGG1 excises the 8-oxo-dG and generates an  apurinic/apyrimidinic site  (AP site). The AP site enables melting of the duplex to unmask the PQS, adopting a  G-quadruplex  fold (G4 structure/motif) that has a regulatory role in transcription activation. [ 76 ] [ 78 ]


When 8-oxo-dG is complexed with active OGG1 it may then recruit  chromatin remodelers  to modulate gene expression.   Chromodomain helicase DNA-binding protein 4 (CHD4) , a component of the  (NuRD) complex , is recruited by OGG1 to oxidative DNA damage sites.  CHD4 then attracts DNA and histone methylating enzymes that repress transcription of associated genes. [ 76 ]


Oxidation of guanine, particularly within  CpG sites , may be especially important in learning and memory.  Methylation of cytosines occurs at 60–90% of CpG sites depending on the tissue type. [ 79 ]  In the mammalian brain, ~62% of CpGs are methylated. [ 79 ]   Methylation of CpG sites  tends to stably silence genes. [ 80 ]   More than 500 of these CpG sites are de-methylated in neuron DNA during  memory formation  and  memory consolidation  in the  hippocampus [ 81 ] [ 82 ]  and  cingulate cortex [ 82 ]  regions of the brain.  As indicated below, the first step in de-methylation of methylated cytosine at a CpG site is oxidation of the guanine to form 8-oxo-dG.


The figure in this section shows a CpG site where the cytosine is methylated to form  5-methylcytosine  (5mC) and the guanine is oxidized to form  8-oxo-2'-deoxyguanosine  (in the figure this is shown in the tautomeric form 8-OHdG).  When this structure is formed, the  base excision repair  enzyme  OGG1  targets 8-OHdG and binds to the lesion without immediate excision.  OGG1, present at a 5mCp-8-OHdG site recruits  TET1 , and TET1 oxidizes the 5mC adjacent to the 8-OHdG.  This initiates de-methylation of 5mC. [ 83 ]   TET1  is a key enzyme involved in de-methylating 5mCpG.  However, TET1 is only able to act on 5mCpG if the guanine was first oxidized to form  8-hydroxy-2'-deoxyguanosine  (8-OHdG or its  tautomer  8-oxo-dG), resulting in a 5mCp-8-OHdG dinucleotide (see figure in this section). [ 83 ]   This initiates the de-methylation pathway on the methylated cytosine, finally resulting in an unmethylated cytosine (see  DNA oxidation  for further steps in forming unmethylated cytosine).


Altered protein expression in neurons, due to changes in methylation of DNA, (likely controlled by 8-oxo-dG-dependent de-methylation of CpG sites in gene promoters within neuron DNA) has been established as central to memory formation. [ 84 ]


Double-stranded breaks (DSBs) in regions of DNA related to neuronal activity are produced by a variety of mechanisms within and around the genome. The enzyme  topoisomerase II , or TOPIIβ plays a key role in DSB formation by aiding in the  demethylation  or loosening of histones wrapped around the double helix to promote  transcription . [ 85 ]  Once the chromatin structure is opened, DSBs are more likely to accumulate, however, this is normally repaired by TOPIIβ through its intrinsic religation ability that rejoins the cleaved DNA ends. [ 85 ]


Failure of TOPIIβ to religase can have drastic consequences on protein synthesis, where it is estimated that "blocking TOPIIβ activity alters the expression of nearly one-third of all developmentally regulated genes," such as neural  immediate early genes  (IEGs) involved in  memory consolidation . [ 85 ] [ 86 ]  Rapid expression of  egr-1 ,  c-Fos , and  Arc  IEGs have been observed in response to increased neuronal activity in the hippocampus region of the brain where memory processing takes place. [ 87 ]  As a preventative measure against TOPIIβ failure, DSB repair molecules are recruited via two different pathways:  non-homologous end joining  (NHEJ) pathway factors, which perform a similar religation function to that of TOPIIβ, and the  homologous recombination (HR)  pathway, which uses the non-broken sister strand as a template to repair the damaged strand of DNA. [ 85 ] [ 88 ]


Stimulation of neuronal activity, as previously mentioned in IEG expression, is another mechanism through which DSBs are generated. Changes in level of activity have been used in studies as a biomarker to trace the overlap between DSBs and increased  histone H3K4   methylation  in promoter regions of IEGs. [ 85 ] [ 88 ]  Other studies have indicated that  transposable elements (TEs)  can cause DSBs through endogenous activity that involves using  endonuclease  enzymes to insert and cleave target DNA at random sites. [ 89 ] [ 90 ]


While accumulation of DSBs generally inhibits  long-term memory  consolidation, the process of  reconsolidation , in contrast, is DSB-dependent. Memory reconsolidation involves the modification of existing memories stored in long-term memory. [ 91 ]  Research involving  NPAS4 , a gene that regulates  neuroplasticity  in the  hippocampus  during  contextual learning  and memory formation, has revealed a link between deletions in the  coding region  and impairments in recall of fear memories in  transgenic  rats. [ 85 ]  Moreover, the enzyme H3K4me3, which catalyzes the demethylation of the H3K4 histone, was  upregulated  at the promoter region of the NPAS4 gene during the reconsolidation process, while  knockdown (gene knockdown)  of the same enzyme impeded reconsolidation. [ 85 ]  A similar effect was observed in TOPIIβ, where knockdown also impaired the  fear memory  response in rats, indicating that DSBs, along with the enzymes that regulate them, influence memory formation at multiple stages. [ citation needed ]


Buildup of DSBs more broadly leads to the degeneration of neurons, hindering the function of memory and learning processes. Due to their lack of cell division and high  metabolic activity , neurons are especially prone to DNA damage. [ 88 ]  Additionally, an imbalance of DSBs and DNA repair molecules for neuronal-activity genes has been linked to the development of various human  neurodegenerative  diseases including  Alzheimer's disease  (AD),  Parkinson's disease  (PD), and  amyotrophic lateral sclerosis  (ALS). [ 88 ]  In patients with Alzheimer's disease, DSBs accumulate in neurons at early stages and are the driving force behind memory loss, a key characteristic of the disease. [ 88 ]  Other external factors that result in increased levels of activity-dependent DSBs in people with AD are  oxidative damage  to neurons, which can result in more DSBs when multiple  lesions  occur close to one another. Environmental factors such as viruses and a high-fat diet have also been associated with disrupted function of DNA repair molecules. [ citation needed ]


One targeted therapy for treating patients with AD has involved suppression of the  BRCA1 gene  in human brains, initially tested in transgenic mice, where DSB levels were observed to have increased and memory loss had occurred, suggesting that BRCA1 could "serve as a therapeutic target for AD and AD-related dementia." [ 88 ]  Similarly, the protein  ATM  involved in DNA repair and  epigenetic modifications  to the genome is positively correlated with neuronal loss in AD brains, indicating the protein is another key component in the intrinsically linked processes of neurodegeneration, DSB production, and memory formation. [ 88 ]


Most damage can be repaired without triggering the damage response system, however more complex damage activates ATR and ATM, key protein kinases in the damage response system. [ 92 ]  DNA damage inhibits M-CDKs which are a key component of progression into  mitosis .


In all eukaryotic cells, ATR and ATM are protein kinases that detect DNA damage. They bind to DNA damaged sites and activate  Chk1 ,  Chk2 , and, in animal cells,  p53 . Together, these proteins make up the DNA damage response system. Some DNA damage does not require the recruitment of ATR and ATM, it is only difficult and extensive damage that requires ATR and ATM. ATM and ATR are required for NHEJ, HR, ICL repair, and NER, as well as replication fork stability during unperturbed DNA replication and in response to replication blocks. [ 2 ]


ATR is recruited for different forms of damage such as nucleotide damage, stalled replication forks and double strand breaks. ATM is specifically for the damage response to double strand breaks. The MRN complex (composed of Mre11, Rad50, and Nbs1) form immediately at the site of double strand break. This MRN complex recruits ATM to the site of damage. ATR and ATM phosphorylate various proteins that contribute to the damage repair system. The binding of ATR and ATM to damage sites on DNA lead to the recruitment of Chk1 and Chk2. These protein kinases send damage signals to the cell cycle control system to delay the progression of the cell cycle. [ 13 ]


Chk1 leads to the production of DNA repair enzymes. Chk2 leads to reversible cell cycle arrest. Chk2, as well as ATR/ATM, can activate p53, which leads to permanent cell cycle arrest or apoptosis. [ citation needed ]


When there is too much damage, apoptosis is triggered in order to protect the organism from potentially harmful cells.7 p53, also known as a tumor suppressor gene, is a major regulatory protein in the DNA damage response system which binds directly to the promoters of its target genes. p53 acts primarily at the G1 checkpoint (controlling the G1 to S transition), where it blocks cell cycle progression. [ 7 ]  Activation of p53 can trigger cell death or permanent cell cycle arrest. p53 can also activate certain repair pathways such was NER. [ 92 ]


In the absence of DNA damage, p53 is regulated by Mdm2 and constantly degraded. When there is DNA damage, Mdm2 is phosphorylated, most likely caused by ATM. The phosphorylation of Mdm2 leads to a reduction in the activity of Mdm2, thus preventing the degradation of p53. Normal, undamaged cell, usually has low levels of p53 while cells under stress and DNA damage, will have high levels of p53. [ 13 ]


p53 serves as a transcription factors for both  bax , a proapoptotic protein as well as  p21 , a CDK inhibitor. CDK Inhibitors result in cell cycle arrest. Arresting the cell provides the cell time to repair the damage, and if the damage is irreparable, p53 recruits bax to trigger apoptosis. [ 92 ]


p53 is a major key player in the growth of cancerous cells. Damaged DNA cells with mutated p53 are at a higher risk of becoming cancerous. Common chemotherapy treatments are genotoxic. These treatments are ineffective in cancer tumor that have mutated p53 since they do not have a functioning p53 to either arrest or kill the damaged cell. [ citation needed ]


One indication that DNA damages are a major problem for life is that DNA repair processes, to cope with DNA damages, have been found in all cellular organisms in which DNA repair has been investigated.  For example, in bacteria, a regulatory network aimed at repairing DNA damages (called the SOS response in  Escherichia coli ) has been found in many bacterial species.   E. coli  RecA, a key enzyme in the SOS response pathway, is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. [ 93 ]  Genes homologous to  RecA  and to other central genes in the SOS response pathway are found in almost all the bacterial genomes sequenced to date, covering a large number of phyla, suggesting both an ancient origin and a widespread occurrence of recombinational repair of DNA damage. [ 94 ]   Eukaryotic  recombinases that are homologues of RecA are also widespread in eukaryotic organisms.  For example, in fission yeast and humans, RecA homologues promote duplex-duplex DNA-strand exchange needed for repair of many types of DNA lesions. [ 95 ] [ 96 ]


Another indication that DNA damages are a major problem for life is that cells make large investments in DNA repair processes.  As pointed out by Hoeijmakers, [ 4 ]  repairing just one double-strand break could require more than 10,000  ATP  molecules, as used in signaling the presence of the damage, the generation of repair foci, and the formation (in humans) of the RAD51 nucleofilament (an intermediate in homologous recombinational repair).  (RAD51 is a homologue of bacterial RecA.) If the structural modification occurs during the G1 phase of DNA replication, the G1-S checkpoint arrests or postpones the furtherance of the cell cycle before the product enters the S phase. [ 1 ]


Differentiated somatic cells of adult mammals generally replicate infrequently or not at all.  Such cells, including, for example, brain neurons and muscle myocytes, have little or no cell turnover.  Non-replicating cells do not generally generate mutations due to DNA damage-induced errors of replication.  These non-replicating cells do not commonly give rise to cancer, but they do accumulate DNA damages with time that likely contribute to aging ( see  DNA damage theory of aging ).  In a non-replicating cell, a single-strand break or other type of damage in the  transcribed  strand of DNA can block RNA polymerase II-catalysed transcription. [ 97 ]  This would interfere with the synthesis of the protein coded for by the gene in which the blockage occurred. [ citation needed ]


Brasnjevic et al. [ 98 ]  summarized the evidence showing that single-strand breaks accumulate with age in the brain (though accumulation differed in different regions of the brain) and that single-strand breaks are the most frequent steady-state DNA damages in the brain.  As discussed above, these accumulated single-strand breaks would be expected to block transcription of genes.  Consistent with this, as reviewed by Hetman et al., [ 99 ]  182 genes were identified and shown to have reduced transcription in the brains of individuals older than 72 years, compared to transcription in the brains of those less than 43 years old. When 40 particular proteins were evaluated in a muscle of rats, the majority of the proteins showed significant decreases during aging from 18 months (mature rat) to 30 months (aged rat) of age. [ 100 ]


Another type of DNA damage, the double-strand break, was shown to cause cell death (loss of cells) through  apoptosis . [ 101 ]  This type of DNA damage would not accumulate with age, since once a cell was lost through apoptosis, its double-strand damage would be lost with it. Thus, damaged DNA segments undermine the DNA replication machinery because these altered sequences of DNA cannot be utilized as true templates to produce copies of one's genetic material. [ 1 ]


When DNA is damaged, the cell responds in various ways to fix the damage and minimize the effects on the cell. One such response, specifically in eukaryotic cells, is to delay cell division—the cell becomes arrested for some time in the G2 phase before progressing through the rest of the cell cycle. Various studies have been conducted to elucidate the purpose of this G2 arrest that is induced by DNA damage. Researchers have found that cells that are prematurely forced out of the delay have lower cell viability and higher rates of damaged chromosomes compared with cells that are able to undergo a full G2 arrest, suggesting that the purpose of the delay is to give the cell time to repair damaged chromosomes before continuing with the cell cycle. [ 102 ]  This ensures the proper functioning of mitosis.