Editing Germline mutation

{{short description|Inherited genetic variation}}
[[File:De novo mutations.png|thumb|292x292px|Transmittance of a [[De novo mutation|''de novo'']] mutation in germ cells to offspring.]]
A '''germline mutation''', or '''germinal mutation''', is any detectable variation within [[germ cell]]s (cells that, when fully developed, become [[sperm]] and [[Egg cell|ova]]).<ref>{{cite web|url=https://www.cancer.gov/publications/dictionaries/cancer-terms?cdrid=46384|title=NCI Dictionary of Cancer Terms|website=National Cancer Institute|access-date=2017-11-30|date=2011-02-02}}</ref> Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated [[sperm]] or [[oocyte]] come together to form a [[zygote]].<ref name="Griffiths 2000">{{cite journal|last1=Griffiths|first1=Anthony JF|last2=Miller|first2=Jeffrey H.|last3=Suzuki|first3=David T.|last4=Lewontin|first4=Richard C.|last5=Gelbart|first5=William M. | name-list-style = vanc |date=2000|title=Somatic versus germinal mutation|journal=An Introduction to Genetic Analysis|edition=7th|url=https://www.ncbi.nlm.nih.gov/books/NBK21894/}}</ref> After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every [[Somatic (biology)|somatic]] and germline cell in the offspring; this is also known as a constitutional mutation.<ref name="Griffiths 2000"/> Germline mutation is distinct from [[somatic mutation]].

Germline mutations can be caused by a variety of endogenous (internal) and exogenous (external) factors, and can occur throughout zygote development.<ref name=":0">{{cite journal | vauthors = Foulkes WD, Real FX | title = Many mosaic mutations | journal = Current Oncology | volume = 20 | issue = 2 | pages = 85–7 | date = April 2013 | pmid = 23559869 | pmc = 3615857 | doi = 10.3747/co.20.1449 }}</ref> A mutation that arises only in germ cells can result in offspring with a genetic condition that is not present in either parent; this is because the mutation is not present in the rest of the parents' body, only the germline.<ref name=":0" />

== When mutagenesis occurs ==
Germline mutations can occur before fertilization and during various stages of zygote development.<ref name=":0" /> When the mutation arises will determine the effect it has on offspring. If the mutation arises in either the sperm or the oocyte before development, then the mutation will be present in every cell in the individual's body.<ref name=":9">{{cite journal | vauthors = Samuels ME, Friedman JM | title = Genetic mosaics and the germ line lineage | journal = Genes | volume = 6 | issue = 2 | pages = 216–37 | date = April 2015 | pmid = 25898403 | doi = 10.3390/genes6020216 | pmc=4488662| doi-access = free }}</ref> A mutation that arises soon after fertilization, but before germline and somatic cells are determined, then the mutation will be present in a large proportion of the individual's cell with no bias towards germline or somatic cells, this is also called a gonosomal mutation.<ref name=":9" /> A mutation that arises later in zygote development will be present in a small subset of either somatic or germline cells, but not both.<ref name=":0" /><ref name=":9" />

== Causes ==

=== Endogenous factors ===
A germline mutation often arises due to [[Endogeny (biology)|endogenous]] factors, like errors in cellular replication and oxidative damage.<ref name=":1" /> This damage is rarely repaired imperfectly, but due to the high rate of germ cell division, can occur frequently.<ref name=":1">{{cite journal | vauthors = Crow JF | title = The origins, patterns and implications of human spontaneous mutation | journal = Nature Reviews Genetics | volume = 1 | issue = 1 | pages = 40–7 | date = October 2000 | pmid = 11262873 | doi = 10.1038/35049558 | s2cid = 22279735 }}</ref>

Endogenous mutations are more prominent in sperm than in ova.<ref name=":2">{{cite journal | vauthors = Wong WS, Solomon BD, Bodian DL, Kothiyal P, Eley G, Huddleston KC, Baker R, Thach DC, Iyer RK, Vockley JG, Niederhuber JE | title = New observations on maternal age effect on germline de novo mutations | journal = Nature Communications | volume = 7 | pages = 10486 | date = January 2016 | pmid = 26781218 | doi = 10.1038/ncomms10486 | pmc=4735694| bibcode = 2016NatCo...710486W }}</ref> This is because [[spermatocyte]]s go through a larger number of cell divisions throughout a male's life, resulting in more replication cycles that could result in a DNA mutation.<ref name=":1" /> Errors in maternal ovum also occur, but at a lower rate than in paternal sperm.<ref name=":1" /> The types of mutations that occur also tend to vary between the sexes.<ref name=":12" /> A mother's eggs, after production, remain in stasis until each is utilized in ovulation. This long stasis period has been shown to result in a higher number of chromosomal and large sequence deletions, duplications, insertions, and transversions.<ref name=":12">{{cite journal | vauthors = Hassold T, Hunt P | title = Maternal age and chromosomally abnormal pregnancies: what we know and what we wish we knew | journal = Current Opinion in Pediatrics | volume = 21 | issue = 6 | pages = 703–8 | date = December 2009 | pmid = 19881348 | pmc = 2894811 | doi = 10.1097/MOP.0b013e328332c6ab }}</ref> The father's sperm, on the other hand, undergoes continuous replication throughout his lifetime, resulting in many small point mutations that result from errors in replication. These mutations commonly include single base pair substitutions, deletions, and insertions.<ref name=":2" />

Oxidative damage is another endogenous factor that can cause germline mutations. This type of damage is caused by [[reactive oxygen species]] that build up in the cell as a by-product of [[cellular respiration]].<ref name=":13" /> These reactive oxygen species are missing an electron, and because they are highly [[Electronegativity|electronegative]] (have a strong electron pull) they will rip an electron away from another molecule.<ref name=":13">{{cite journal | vauthors = Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ | title = Production of reactive oxygen species by mitochondria: central role of complex III | journal = The Journal of Biological Chemistry | volume = 278 | issue = 38 | pages = 36027–31 | date = September 2003 | pmid = 12840017 | doi = 10.1074/jbc.M304854200 | doi-access = free }}</ref> This can initiate DNA damage because it causes the nucleic acid guanine to shift to 8-oxoguanine (8-oxoG). This 8-oxoG molecule is then mistaken for a thymine by [[DNA polymerase]] during replication, causing a G>T [[transversion]] on one DNA strand, and a C>A transversion on the other.<ref>{{cite journal | vauthors = Ohno M, Sakumi K, Fukumura R, Furuichi M, Iwasaki Y, Hokama M, Ikemura T, Tsuzuki T, Gondo Y, Nakabeppu Y | title = 8-oxoguanine causes spontaneous de novo germline mutations in mice | journal = Scientific Reports | volume = 4 | pages = 4689 | date = April 2014 | pmid = 24732879 | pmc = 3986730 | doi = 10.1038/srep04689 | bibcode = 2014NatSR...4E4689O }}</ref>

===Male germline===

In mice and humans the spontaneous [[mutation rate]] in the male germ line is significantly lower than in [[somatic cell]]s.<ref name = Aitken2023>Aitken RJ, Lewis SEM. DNA damage in testicular germ cells and spermatozoa. When and how is it induced? How should we measure it? What does it mean? Andrology. 2023 Jan 5. doi: 10.1111/andr.13375. Epub ahead of print. PMID 36604857</ref>  Furthermore, although the spontaneous mutation rate in the male germ line increases with age, the rate of increase is lower than in somatic tissues.  Within the testicular [[spermatogonium|spermatogonial]] stem cell population the integrity of [[DNA]] appears to be maintained by highly effective [[DNA damage (naturally occurring)|DNA damage]] surveillance and protective [[DNA repair]] processes.<ref name = Aitken2023/>  The progressive increase in the mutation rate with age in the male germ line may be a result of a decline in the accuracy of the repair of DNA damages, or of an increase in [[DNA replication]] errors. Once [[spermatogenesis]] is complete, the differentiated spermatozoa that are formed no longer have the capability for DNA repair, and are thus vulnerable to attack by prevalent oxidative free radicals that cause oxidative DNA damage. Such damaged [[spermatozoon|spermatozoa]] may undergo programmed cell death ([[apoptosis]]).<ref name = Aitken2023/>

=== Exogenous factors ===
A germline mutation can also occur due to [[Exogeny|exogenous]] factors. Similar to somatic mutations, germline mutations can be caused by exposure to harmful substances, which damage the DNA of germ cells. This damage can then either be repaired perfectly, and no mutations will be present, or repaired imperfectly, resulting in a variety of mutations.<ref>{{cite web|url=https://evolution.berkeley.edu/evolibrary/article/evo_20|title=The causes of mutations|website=evolution.berkeley.edu|access-date=2017-11-30}}</ref> Exogenous [[mutagen]]s include harmful chemicals and [[ionizing radiation]]; the major difference between germline mutations and somatic mutations is that germ cells are not exposed to [[Ultraviolet|UV radiation]], and thus not often directly mutated in this manner.<ref>{{cite journal | vauthors = Rahbari R, Wuster A, Lindsay SJ, Hardwick RJ, Alexandrov LB, Turki SA, Dominiczak A, Morris A, Porteous D, Smith B, Stratton MR, Hurles ME | title = Timing, rates and spectra of human germline mutation | journal = Nature Genetics | volume = 48 | issue = 2 | pages = 126–133 | date = February 2016 | pmid = 26656846 | pmc = 4731925 | doi = 10.1038/ng.3469 }}</ref><ref>{{cite journal | vauthors = Cai L, Wang P | title = Induction of a cytogenetic adaptive response in germ cells of irradiated mice with very low-dose rate of chronic gamma-irradiation and its biological influence on radiation-induced DNA or chromosomal damage and cell killing in their male offspring | journal = Mutagenesis | volume = 10 | issue = 2 | pages = 95–100 | date = March 1995 | pmid = 7603336 | doi = 10.1093/mutage/10.2.95 }}</ref>

== Clinical implications ==
Different germline mutations can affect an individual differently depending on the rest of their genome. A [[Dominance (genetics)|dominant mutation]] only requires a single mutated gene to produce the disease [[phenotype]], while a recessive mutation requires both [[allele]]s to be mutated to produce the disease phenotype.<ref name=":11">{{cite web |url=http://genetics.thetech.org/about-genetics/mutations-and-disease |title=Mutations and Disease {{!}} Understanding Genetics |website=[[The Tech Interactive]] |archive-date=2021-09-14 |archive-url=https://web.archive.org/web/20210914101208/https://genetics.thetech.org/about-genetics/mutations-and-disease |url-status=dead}}</ref> For example, if the embryo inherits an already mutated allele from the father, and the same allele from the mother underwent an endogenous mutation, then the child will display the disease related to that mutated gene, even though only one parent carries the mutant allele.<ref name=":11" /> This is only one example of how a child can display a recessive disease while a mutant gene is only carried by one parent.<ref name=":11" /> Detection of chromosomal abnormalities can be found in utero for certain diseases by means of blood samples or ultrasound, as well as invasive procedures such as an [[amniocentesis]]. Later detection can be found by genome screening.

=== Cancer ===
Mutations in [[Tumor suppressor gene|tumour suppressor genes]] or [[proto-oncogenes]] can predispose an individual to developing tumors.<ref name=":10">{{Cite news|url=https://www.cancer.net/navigating-cancer-care/cancer-basics/genetics/genetics-cancer|title=The Genetics of Cancer|date=2012-03-26|work=Cancer.Net|access-date=2017-12-01}}</ref> It is estimated that inherited genetic mutations are involved in 5-10% of cancers.<ref name="genetics">{{cite web |title=The Genetics of Cancer |url=https://www.cancer.gov/about-cancer/causes-prevention/genetics |website=National Cancer Institute |publisher=NIH |access-date=23 September 2018|date=2015-04-22 }}</ref> These mutations make a person susceptible to tumor development if the other copy of the [[oncogene]] is randomly mutated. These mutations can occur in germ cells, allowing them to be [[Heritability|heritable]].<ref name=":10" /> Individuals who inherit germline mutations in [[TP53]] are predisposed to certain cancer variants because the protein produced by this gene suppresses tumors. Patients with this mutation are also at a risk for [[Li–Fraumeni syndrome]].<ref name="genetics" /> Other examples include mutations in the [[BRCA1]] and [[BRCA2]] genes which predispose to breast and ovarian cancer, or mutations in [[MLH1]] which predispose to [[hereditary nonpolyposis colorectal cancer|hereditary non-polyposis colorectal cancer]].

=== Huntington's disease ===
[[Huntington's disease]] is an [[autosomal dominant]] mutation in the HTT gene. The disorder causes degradation in the brain, resulting in uncontrollable movements and behavior.<ref name=":14">{{cite web |title=Huntington disease |url=https://ghr.nlm.nih.gov/condition/huntington-disease |website=Genetics Home Reference |publisher=NIH |access-date=23 September 2018}}</ref> The mutation involves an expansion of repeats in the Huntington protein, causing it to increase in size. Patients who have more than 40 repeats will most likely be affected. The onset of the disease is determined by the amount of repeats present in the mutation; the greater the number of repeats, the earlier symptoms of the disease will appear.<ref name=":14" /><ref>{{Cite book|title=Huntington's Disease|last=Lawrence|first=David M.|publisher=Infobase Publishing|year=2009|isbn=9780791095867|location=New York|pages=92}}</ref> Because of the dominant nature of the mutation, only one mutated allele is needed for the disease to be in effect. This means that if one parent is affected, the child will have a 50% chance of inheriting the disease.<ref name=":15">{{cite web |title=Huntington's disease |url=https://www.mayoclinic.org/diseases-conditions/huntingtons-disease/symptoms-causes/syc-20356117 |website=Mayo Clinic |access-date=23 September 2018}}</ref> This disease does not have carriers because if a patient has one mutation, they will (most likely) be affected. The disease typically has a late onset, so many parents have children before they know they have the mutation. The HTT mutation can be detected through [[Genetic testing|genome screening]].

=== Trisomy 21 ===
Trisomy 21 (also known as [[Down syndrome]]) results from a child having three copies of chromosome 21.<ref name=":3" /> This chromosome duplication occurs during germ cell formation, when both copies of chromosome 21 end up in the same [[Cell division|daughter cell]] in either the mother or father, and this mutant germ cell participates in fertilization of the zygote.<ref name=":3">{{cite journal | vauthors = Chandley AC | title = On the parental origin of de novo mutation in man | journal = Journal of Medical Genetics | volume = 28 | issue = 4 | pages = 217–23 | date = April 1991 | pmid = 1677423 | doi = 10.1136/jmg.28.4.217 | pmc=1016821}}</ref> Another, more common way this can occur is during the first cell division event after the formation of the zygote.<ref name=":3" /> The risk of Trisomy 21 increases with maternal age with the risk being 1/2000 (0.05%) at age 20 increasing to 1/100 (1%) at age 40.<ref>{{cite journal |last1=Hook |first1=EB |title=Rates of chromosome abnormalities at different maternal ages |journal=Obstetrics and Gynecology | date = September 1981 |volume=27 |issue=1 |pages=282–5 |doi=10.1016/0091-2182(82)90145-8 |pmid=6455611 }}</ref> This disease can be detected by non-invasive as well as invasive procedures prenatally. Non-invasive procedures include scanning for [[fetal DNA]] through maternal plasma via a blood sample.<ref>{{cite journal |last1=Ghanta |first1=Sujana |title=Non-Invasive Prenatal Detection of Trisomy 21 Using Tandem Single Nucleotide Polymorphisms |journal=PLOS ONE | date = October 2010 |volume=5 |issue=10 |pages=e13184 |doi=10.1371/journal.pone.0013184 |pmid=20949031 |pmc=2951898 |bibcode=2010PLoSO...513184G |doi-access=free }}</ref>

=== Cystic fibrosis ===
Cystic fibrosis is an [[Dominance (genetics)|autosomal recessive]] disorder that causes a variety of symptoms and complications, the most common of which is a thick mucous lining in lung [[Epithelium|epithelial]] tissue due to improper salt exchange, but can also affect the [[pancreas]], [[Gastrointestinal tract|intestines]], [[liver]], and [[kidney]]s.<ref name=":4">{{cite web|url=http://www.cysticfibrosis.ca/about-cf|title=Cystic Fibrosis Canada|website=www.cysticfibrosis.ca|access-date=2017-11-30}}</ref><ref>{{cite journal | vauthors = O'Sullivan BP, Freedman SD | title = Cystic fibrosis | journal = Lancet | volume = 373 | issue = 9678 | pages = 1891–904 | date = May 2009 | pmid = 19403164 | doi = 10.1016/S0140-6736(09)60327-5 | s2cid = 46011502 }}</ref> Many bodily processes can be affected due to the hereditary nature of this disease; if the disease is present in the DNA of both the sperm and the egg, then it will be present in essentially every cell and organ in the body; these mutations can occur initially in the germline cells, or be present in all parental cells.<ref name=":4" /> The most common mutation seen in this disease is ΔF508, which means a deletion of the amino acid at the 508 position.<ref>{{cite web|url=https://ghr.nlm.nih.gov/gene/CFTR#conditions|title=CFTR gene|last=Reference|first=Genetics Home|website=Genetics Home Reference|access-date=2017-11-30}}</ref> If both parents have a mutated [[Cystic fibrosis transmembrane conductance regulator|CFTR]] (cystic fibrosis transmembrane conductance regulator) protein, then their children have a 25% of inheriting the disease.<ref name=":4" /> If a child has one mutated copy of CFTR, they will not develop the disease, but will become a carrier of the disease.<ref name=":4" /> The mutation can be detected before birth through amniocentesis, or after birth via prenatal genetic screening.<ref name=":16">{{cite web |title=Prenatal Diagnosis |url=https://www.hopkinscf.org/what-is-cf/diagnosis/presentations/prenatal-diagnosis/ |website=Johns Hopkins Cystic Fibrosis Center |access-date=23 September 2018}}</ref>

== Current therapies ==
Many Mendelian disorders stem from [[Dominance (genetics)|dominant]] point mutations within genes, including [[cystic fibrosis]], [[Thalassemia|beta-thalassemia]], [[Sickle-cell disease|sickle-cell anemia]], and [[Tay–Sachs disease]].<ref name=":11"/> By inducing a double stranded break in sequences surrounding the disease-causing point mutation, a dividing cell can use the non-mutated strand as a template to repair the newly broken DNA strand, getting rid of the disease-causing mutation.<ref name=":6" /> Many different genome editing techniques have been used for genome editing, and especially germline mutation editing in germ cells and developing zygotes; however, while these therapies have been extensively studied, their use in human germline editing is limited.<ref>{{cite web|url=https://www.geneticsandsociety.org/internal-content/about-human-germline-gene-editing|title=About Human Germline Gene Editing {{!}} Center for Genetics and Society|website=www.geneticsandsociety.org|access-date=2017-12-01}}</ref>

=== CRISPR/Cas9 editing ===
[[File:DNA Repair after CRISPR-Cas9 cut.svg|thumb|254x254px|The CRISPR editing system is able to target specific DNA sequences and, using a donor DNA template, can repair mutations within this gene.]]
This editing system induces a double stranded break in the DNA, using a guide RNA and effector protein Cas9 to break the DNA backbones at specific target sequences.<ref name=":6" /> This system has shown a higher specificity than TALENs or ZFNs due to the Cas9 protein containing homologous (complementary) sequences to the sections of DNA surrounding the site to be cleaved.<ref name=":6">{{cite journal | vauthors = Sander JD, Joung JK | title = CRISPR-Cas systems for editing, regulating and targeting genomes | journal = Nature Biotechnology | volume = 32 | issue = 4 | pages = 347–55 | date = April 2014 | pmid = 24584096 | doi = 10.1038/nbt.2842 | pmc=4022601}}</ref>&nbsp;This broken strand can be repaired in 2 main ways: homologous directed repair (HDR) if a DNA strand is present to be used as a template (either homologous or donor), and if one is not, then the sequence will undergo [[non-homologous end joining]] (NHEJ).<ref name=":6" /> NHEJ often results in insertions or deletions within the gene of interest, due to the processing of the blunt strand ends, and is a way to study gene knockouts in a lab setting.<ref name=":5">{{cite journal | vauthors = Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F | title = Genome-scale CRISPR-Cas9 knockout screening in human cells | journal = Science | volume = 343 | issue = 6166 | pages = 84–87 | date = January 2014 | pmid = 24336571 | doi = 10.1126/science.1247005 | pmc=4089965| bibcode = 2014Sci...343...84S }}</ref> This method can be used to repair a point mutation by using the [[Chromatid|sister chromosome]] as a template, or by providing a double stranded DNA template with the [[CRISPR]]/Cas9 machinery to be used as the repair template.<ref name=":6" />

This method has been used in both human and animal models (''[[Drosophila]]'', ''[[House mouse|Mus musculus]]'', ''and [[Arabidopsis]]''), and current research is being focused on making this system more specific to minimize off-target cleavage sites.<ref>{{cite journal | vauthors = Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, Wang Y, Brodsky RA, Zhang K, Cheng L, Ye Z | title = Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs | language = en | journal = Cell Stem Cell | volume = 15 | issue = 1 | pages = 12–3 | date = July 2014 | pmid = 24996165 | doi = 10.1016/j.stem.2014.06.011 | pmc=4338993}}</ref>

=== TALEN editing ===
The [[Transcription activator-like effector nuclease|TALEN]] (transcription activator-like effector nucleases) genome editing system is used to induce a double-stranded DNA break at a specific locus in the genome, which can then be used to mutate or repair the DNA sequence.<ref name=":7">{{cite journal | vauthors = Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG, Tan W, Penheiter SG, Ma AC, Leung AY, Fahrenkrug SC, Carlson DF, Voytas DF, Clark KJ, Essner JJ, Ekker SC | title = In vivo genome editing using a high-efficiency TALEN system | journal = Nature | volume = 491 | issue = 7422 | pages = 114–8 | date = November 2012 | pmid = 23000899 | doi = 10.1038/nature11537 | pmc=3491146| bibcode = 2012Natur.491..114B }}</ref> It functions by using a specific repeated sequence of an amino acid that is 33-34 amino acids in length.<ref name=":7" /> The specificity of the DNA binding site is determined by the specific amino acids at positions 12 and 13 (also called the Repeat Variable Diresidue (RVD)) of this tandem repeat, with some RVDs showing a higher specificity for specific amino acids over others.<ref>{{cite journal | vauthors = Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM | title = TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery | journal = Acta Naturae | volume = 6 | issue = 3 | pages = 19–40 | date = July 2014 | pmid = 25349712 | pmc = 4207558 | doi = 10.32607/20758251-2014-6-3-19-40 }}</ref> Once the DNA break is initiated, the ends can either be joined with NHEJ that induces mutations, or by HDR that can fix mutations.<ref name=":6" />

=== ZFN editing ===
Similar to TALENs, [[zinc finger nuclease]]s (ZFNs) are used to create a double stranded break in the DNA at a specific locus in the genome.<ref name=":7" /> The ZFN editing complex consists of a [[Zinc finger|zinc finger protein]] (ZFP) and a restriction enzyme cleavage domain.<ref name=":8">{{cite journal | vauthors = Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD | title = Genome editing with engineered zinc finger nucleases | journal = Nature Reviews Genetics | volume = 11 | issue = 9 | pages = 636–46 | date = September 2010 | pmid = 20717154 | doi = 10.1038/nrg2842 | s2cid = 205484701 }}</ref> The ZNP domain can be altered to change the DNA sequence that the [[restriction enzyme]] cuts, and this cleavage event initiates cellular repair processes, similar to that of CRISPR/Cas9 DNA editing.<ref name=":8" />

Compared to CRISPR/Cas9, the therapeutic applications of this technology are limited, due to the extensive engineering required to make each ZFN specific to the desired sequence.<ref name=":8" />

== See also ==
* [[Somatic mutation]]
* [[Genome editing|Genome Editing]]

== References ==
{{reflist}}

[[Category:Germ line cells]]
[[Category:Mutation]]