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Article Evaluation: Germline Mutation[edit]

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Germline Mutation[edit]

Transmittance of a de novo mutation in germ cells to offspring.

A germline mutation, or germinal mutation, is any detectable variation within germ cells (cells that, when fully developed, become sperm and ovum)[1]. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte (egg) come together to form a zygote[2]. The germ cells, once this fertilization event occurs, divide rapidly to produce all of the cells in the body, causing this mutation to be present in every somatic and germline cell in the offspring, known as a constitutional mutation[3]

Germline mutations can also arise later in zygote development, leading to genetic mosaicism within an organism[4]. If the mutation occurs in cell prior to germline and somatic cell differentiation, then this mutation will be present in a subset of both the germline and somatic cells in the offspring (called a gonosomal mutation)[4]. On the other hand, if this mutation occurs after this division, then the mutation will be present in either the somatic or germline cells, but not both. A mutation located only in a parents' germ cells can result in offspring with a genetic condition, not present in either parent; this is because the mutation is not present in the rest of the parents' body, only the germline[4].

Causes[edit]

Endogenous Factors[edit]

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

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

Oxidative damage is another endogenous factor that can cause germline mutations. This type of damage is caused by reactive oxygen species that build up in the cell as a by-product of cellular respiration. These reactive oxygen species are missing an electron, and because they are highly electronegative (have a strong electron pull) they will rip an electron away from another molecule[8]. This can cause 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 tranversion on one DNA strand, and a C>A transversion on the other[9]

Exogenous Factors[edit]

A germline mutation can also occur due to exogenous factors. Similar to somatic mutations, germline mutations can be caused by exposure to harmful substances, which damage the DNA of germ cells. This damage can then either be repaired perfectly, and no mutations will be present, or repaired imperfectly, resulting in a variety of mutations[10]. Exogenous mutagens include harmful chemicals and ionizing radiation[11]; the major difference between germline mutations and somatic mutations is that germ cells are not exposed to UV radiation, and thus not often directly mutated in this manner[12]

Clinical Implications[edit]

Different germline mutations can affect an individual differently depending on the rest of their genome. A dominant mutation only requires 1 mutated gene to produce the disease phenotype, while a recessive mutation requires both alleles to be mutated to produce the disease phenotype[13]. 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 1 parent carries the mutant allele. This is only one example of how a child can display a recessive disease while a mutant gene is only carried by one parent.

Trisomy 21[edit]

Trisomy 21 (also known as Down Syndrome) results from a child having 3 copies of chromosome 21. This chromosome duplication occurs during germ cell formation, when both copies of chromosome 21 end up in the same daughter cell in either the mother or father, then this mutant germ cell participates in fertilization of the zygote [14]. Another, more common way this can occur is during the first cell division event after the formation of the zygote[14].

Cystic Fibrosis[edit]

Cystic Fibrosis is an autosomal recessive disorder that causes a variety of symptoms and complications, the most common of which is a thick mucus lining in lung epithelial tissue due to improper salt exchange[15], but can also affect the pancreas, intestines, liver, and kidneys[16]. 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. The most common mutation seen in this disease is ΔF508, which means a deletion of the amino acid at the 508 position[17]. If both parents have a mutated CFTR (cystic fibrosis transmembrane conductance regulator) protein, then their children have a 25% of inheriting the disease[15]. If a child inherits only 1 mutated copy of CFTR, then they will not develop the disease, but will become a carrier of the disease[15].

Sickle Cell Amenia[edit]

Sickle cell anemia is one of the most common forms of Sickle Cell Disease, as well as one of the most common disorders caused by a single gene[18]. It leads to rigid blood cells that have a reduced capacity to transport oxygen and inflammation of organs[18]. This disease is caused by inheriting 1 hemoglobin S (HbS) gene from both parents, while inheriting only a single HbS gene does not result in the same clinical disease[18].

Current Therapies[edit]

The CRISPR editing system is able to target specific DNA sequences and, using a donor DNA template, can repair mutations within this gene.

Many Mendelian disorders stem from dominant point mutations within genes, for example Cystic Fibrosis, B-Thalassemia, Sickle-Cell Anemia, and Tay Sachs Disease[19]. By inducing a double stranded break in sequences surrounding the disease-causing point mutation, a dividing cell can use the un-mutated strand as a template to repair the newly broken DNA strand, getting rid of the disease-causing mutation. Many different genome editing techniques have been extensively studied for use in genome editing, and especially germline mutation editing in germ cells and developing zygotes.

CRISPR/Cas9 Editing[edit]

This editing system also 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. 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[20]. 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)[20]. 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[21]. This method can be used to repair a point mutation by using the sister chromosome as a template, or by providing a double stranded DNA template with the CRISPR/Cas9 machinery to be used as the repair template.

This method has been used in both human and animal models (Drosophila, Mus musculus, and Arabidopsis), and current research is being focused on making this system more specific to minimize off-target cleavage sites[22].

TALEN Editing[edit]

TALEN (or 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[23]. It functions by using a specific repeated sequence of an amino acid that is 33-34 amino acids in length. 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[24]. One the DNA break is initiated, the ends can either be joined with NHEJ that induces mutations, or by HR that can fix mutations[20].

ZFN Editing[edit]

Similar to TALENs, Zinc Finger Nucleases (ZFNs) are used to create a double stranded break in the DNA at a specific locus in the genome[23]. The ZFN editing complex consists of a zinc finger protein (ZFP) and a restriction enzyme cleavage domain[25]. 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[25].

Compared to CRISPR/Cas9, the therapeutic applications of this technology are limited, due to the extensive engineering required to make each ZFN highly specific to the desired sequence[25].

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