Trinucleotide repeat expansion

DNA mutation involving an increase in number of trinucleotide repeats
A trinucleotide repeat expansion, also known as a triplet repeat expansion, is the DNA mutation responsible for causing any type of disorder categorized as a trinucleotide repeat disorder. These are labelled in dynamical genetics as dynamic mutations. Triplet expansion is caused by slippage during DNA replication, also known as "copy choice" DNA replication. Due to the repetitive nature of the DNA sequence in these regions, 'loop out' structures may form during DNA replication while maintaining complementary base pairing between the parent strand and daughter strand being synthesized. If the loop out structure is formed from the sequence on the daughter strand this will result in an increase in the number of repeats. However, if the loop out structure is formed on the parent strand, a decrease in the number of repeats occurs. It appears that expansion of these repeats is more common than reduction. Generally, the larger the expansion the more likely they are to cause disease or increase the severity of disease. Other proposed mechanisms for expansion and reduction involve the interaction of RNA and DNA molecules. In addition to occurring during DNA replication, trinucleotide repeat expansion can also occur during DNA repair. When a DNA trinucleotide repeat sequence is damaged, it may be repaired by processes such as homologous recombination, non homologous end joining, mismatch repair or base excision repair. Each of these processes involves a DNA synthesis step in which strand slippage might occur leading to trinucleotide repeat expansion. Other human diseases in which triplet repeat expansion occurs are fragile X syndrome, several spinocerebellar ataxias, myotonic dystrophy and Friedreich's ataxia. The following findings of served as support for TNR's link to onset of disease; the detection of various repeats within these diseases demonstrated this relationship. In 1991, for fragile X syndrome, the fragile X mental retardation 1 (FMR 1) gene was found to contain a CGG expansion in its 5' untranslated region (UTR). In addition, a CAG expansion was located in X linked spinal and bulbar muscular atrophy (SBMA) sequences. SMBA is the first "CAG / polygutamine" disease, which is a subcategory of repeat disorders. In 1992, for myotonic dystrophy type 1 (DM1), CTG expansion was found in the myotonic dystrophy protein kinase (DMPK) 3' UTR. In 1993, for Huntington's disease (HD), a longer than usual CAG repeat with was found in the exon 1 coding sequence. Because of these discoveries, ideas involving anticipation in disease began to develop, and curiosity formed about how the causes could be related to TNRs. During the decade after evidence that linked TNR to onset of disease was found, focus was placed on studying repeat length and dynamics on diseases, as well as investigating the mechanism behind parent child disease inheritance. In addition to this finding, another aspect of the diseases, the high variability of onset, was revealed. Currently, CAG repeat length is considered the biggest onset age modifier for TNR diseases. Detection of TNRs was made difficult by limited technology and methods early on, and years passed before the development of sufficient ways to measure the repeats. Since then, accurate methods have been established over the years. Together, the following clinically necessary protocols have 99% accuracy in measuring TNRs. Southern blots are used to measure CGG repeats because CG rich regions limit polymerase movement in PCR.

These repetitive sequences lead to instability amongst the DNA strands after reaching a certain threshold number of repeats, which can result in DNA slippage during replication. The most common and well known triplet repeats are CAG, GCG, CTG, CGG, and GAA. During DNA replication, the strand being synthesized can misalign with its template strand due to the dynamic nature and flexibility of these triplet repeats. This slippage allows for the strand to find a stable intermediate amongst itself through base pairing, forming a secondary structure other than a duplex. At the 5' untranslated region, CGG and CAG repeats are found and responsible for fragile X syndrome and spinocerebellar ataxia 12.

Depending on the sequence of the repeat, at least three intermediates with different secondary structures are known to form. A CGG repeat will form a G quadruplex due to Hoogsteen base pairing, while a GAA repeat forms a triplex due to negative supercoiling. An even number of repeats forms a tetraloop structure, while an odd number leads to the formation of a triloop.

In trinucleotide repeat expansion there is a certain threshold or maximum amount of repeats that can occur before a sequence becomes unstable. Once this threshold is reached the repeats will start to rapidly expand causing longer and longer expansions in future generations. Once it hits this minimum allele size which is normally around 30 40 repeats, diseases and instability can be contracted, but if the number of repeats found within a sequence are below the threshold it will remain relatively stable. that will form in the sequence resulting in more trinucleotide expansion.

Research suggests that there is a direct, important correlation between the sex of the parent that transmits the mutation and the degree and phenotype of disorder in the child., The degree of repeat expansion and whether or not an expansion will occur has been directly linked to the sex of the transmitting parent in both non coding and coding trinucleotide repeat disorders. While this transmission of a trinucleotide repeat expansion is regarded to be a result of "meiotic instability", the degree to which meiosis plays a role in this process and the mechanism is not clear and numerous other processes are predicted to simultaneously play a role in this process. This disability arises from a mutation at the end of the X chromosome in the FMR1 gene (Fragile X Mental Retardation Gene) which produces a protein essential for brain development called FMRP. Carriers that have repeats falling within the 53 to 200 repeat range are said to have "premutation alleles", as the alleles within this range approach 200, the likelihood of expansion to a full mutation increases, and the mRNA levels are elevated five fold. Fragile X syndrome carriers (those that fall within the premutation range) typically have unmethylated alleles, normal phenotype, and normal levels of FMR1 mRNA and FMRP protein. HD involves the basal ganglia and the cerebral cortex and manifests as symptoms such as cognitive, motor, and/or psychiatric impairment. The majority of all juvenile HD cases stem from the transmission of a high CAG trinucleotide repeat number that is a result of paternal gametogenesis. While an individual without HD has a number of CAG repeats that fall within a range between 9 and 37, an individual with HD has CAG is typically found to have repeats in a range between 37 and 102. Individuals with Myotonic Dystrophy experience severe, debilitating physical symptoms such as muscle weakness, heartbeat issues, and difficulty breathing that can be improved through treatment to maximize patients' mobility and everyday activity to alleviate some stress of their caretakers. The muscles of individuals with Myotonic Dystrophy feature an increase of type 1 fibers as well as an increased deterioration of these type 1 fibers. This (CTG)n trinucleotide repeat is located within leukocytes; the length of the repeat and the age of the individual have been found to be directly related to disease progression and type 1 muscle fiber predominance.

Friedreich's ataxia is an autosomal recessive disorder cause by a GAA expansion in the intron of the FXN gene. This gene codes for the protein frataxin, a mitochondrial protein involved in iron homeostasis. The mutation impairs transcription of the protein, so affected cells produce only 5 10% of the frataxin of healthy cells. This leads to iron accumulation in the mitochondria, and makes cells vulnerable to oxidative damage. Research shows that GAA repeat length is correlated with disease severity.

The precise timing of TNR occurrence varies by disease. Although the exact timing for FXS is not certain, research has suggested that the earliest CGG expansions for this disorder are seen in primary oocytes. It has been proposed that the repeat expansion happens in the maternal oocyte during meiotic cell cycle arrest in prophase I, however the mechanism remains nebulous. Maternally inherited premutation alleles may expand into full mutation alleles (greater than 200 repeats), resulting in decreased production of the FMR 1 gene product FMRP and causing fragile X mental retardation syndrome. For females, the large repeat expansions are based upon repair, while for males, the shortening of long repeat expansions is due to replication; therefore, their sperm lack these repeats, and paternal inheritance of long repeat expansions does not occur. Between weeks 13 and 17 of human fetal development, the large CGG repeats are shortened. There is a positive correlation between male inheritance and allele length. A study of mice found the exact timing of CTG repeat expansion to be during development of spermatogonia. In DM1 and FXS, it is hypothesized that expansion of TNRs occurs by means of multiple missteps by DNA polymerase in replication. An inability of DNA polymerase to properly move across the TNR may cause transactivation of translesion polymerases (TLPs), which will attempt to complete the replication process and overcome the block. It is understood that as the DNA polymerase fails in this way, the resulting single stranded loops left behind in the template strand undergo deletion, affecting TNR length. This process leaves the potential for TNR expansions to occur. In four HD samples examined, CAG repeat expansion lengths were more variable in mature sperm than that of sperm in development in the testes, leading to the conclusion that repeat expansions had a likelihood of occurring later in sperm development. In germ cells undergoing differentiation, evidence suggests it is possible for expansions to generate after the completion of meiosis as well, as larger HD mutations have been found in postmeiotic cells. Because TNR instability is not present in young female mice, and female SCA1 patient age and instability are directly related, expansions must occur in inactive oocytes. A trend has seemed to emerge of larger expansions occurring in cells inactive in division and smaller expansions occurring in actively dividing or nondividing cells. with emphasis on the research and studying of genomic manipulation. The most advanced available therapies aim to target mutated gene expression by using antisense oligonucleotides (ASO) or RNA interference (RNAi) to target the messenger RNA (mRNA). Another synthetic RNA is the short hairpin RNAs (shRNA) Some problems that may occur is if the guide single strand siRNA within the RISC complex may become unstable when cleaved and begin to unwind, resulting in binding to an unfavorable mRNA strand. The perfect complementary guides for the targeted RNAs are easily recognized and will be cleaved within the RISC complex; if there is only partial complementary pairing between the guide strand and the targeted mRNA may cause the incorrect translation or destabilization at the target sites. The goal of using these antisense oligonucleotides are the decrease in protein expression of a specific target usually by the inhibition of the RNase H endonuclease, as well as inhibition of the 5' cap formation or alteration of the splicing process. In the native state ASOs are rapidly digested, this requires the use of phosphorylation order for the ASO to go through the cell membranes. Despite the obvious benefits that antisense therapeutics can bring to the world with their ability to silence neural disease, there are many issues with the development of this therapy. One problem is the ASOs are highly susceptible to degradation by the nucleases within the body. This results in a high amount of chemical modification when altering the chemistry to allow for the nucleases to surpass the degradation of these synthetic nucleic acids. Native ASOs have a very short half life even before being filtered throughout the body especially in the kidney and with the a high negative charge makes the crossing through the vascular system or membranes very difficult when trying to reach the targeted DNA or mRNA strands. With all these barriers, the chemical modifications may lead to devastating effects when being introduced into the body making each problem develop more and more side effects. The synthetic oligonucleotides are negatively charged molecules that are chemically modified in order for the molecule to regulate the gene expression within the cell. Some issues that come about this process is the toxicity and variability that can come about with chemical modification. The goal of the ASO is to modulate the gene expression through proteins which can be done in 2 complex ways; a)the RNase H dependent oligonucleotides, which induce the degradation of mRNA, and (b) the steric blocker oligonucleotides, which physically prevent or inhibit the progression of splicing or the translational machinery. The majority of investigated ASOs utilize the first mechanism with the Rnase H  enzyme that hydrolyzes an RNA strand, when this enzyme is assisted using the oligonucleotides the reduction of RNA expression is efficiently reduced by 80 95% and can still inhibit expression on any region of the mRNA.