trinucleotide repeat sequences. research has found that this hairpin formation depends on the orientation of the trinucleotide repeats within each cag/ctg trinucleotide strand. strands that have duplex formation by ctg repeats in the leading strand are observed to result in extra repeats, while those without ctg repeats in the leading strand result in repeat deletions. these intermediates can pause activity of the replication fork based on their interaction with dna polymerases through strand slippage. contractions occur when the replication fork skips over the intermediate on the okazaki fragment. expansions occur when the fork reverses and restarts, which forms a chicken-foot structure. this structure results in the unstable intermediate forming on the nascent leading strand, leading to further tre. furthermore, this intermediate can avoid mismatch repair due to its affinity for the msh-2-msh3 complex, which stabilizes the hairpin instead of repairing it. in non-dividing cells, a process called flap-ligation can be responsible for tre. 8-oxo-guanine dna glycosylase removes a guanine and forms a nick in the sequence. the coding strand then forms a flap due to displacement, which prevents removal by an endonuclease. when the repair process finishes for either mechanism, the length of the expansion is equivalent to the number of triplet repeats involved in the formation of the hairpin intermediate. large scale expansions two mechanisms have been proposed for large scale repeats: template switching and break-induced replication. template switching, a mechanism for large scale gaa repeats that can double the number of triplet repeats, has been proposed. gaa repeats expand when their repeat length is greater than the okazaki fragment's length. these repeats are involved in the stalling of the replication fork as these repeats form a triplex when the 5' flap of ttc repeats fold back. okazaki fragment synthesis continues when the template is switched to the nascent leading strand. the okazaki fragment eventually ligates back to the 5' flap, which results in tre. a different mechanism, based on break-induced replication, has been proposed for large scale cag repeats and can also occur in non-dividing cells. at first, this mechanism follows the same process as the small scale strand slippage mechanism until replication fork reversal. an endonuclease then cleaves the chicken-foot structure, which results in a one-ended double strand break. the cag repeat of this broken daughter strand forms a hairpin and invades the cag strand on the sister chromatid, which results in expansion of this repeat in a migrating d-loop dna synthesis. this synthesis continues until it reaches the replication fork and is cleaved, which results in an expanded sister chromatid. disorders background fragile x syndrome is the second most common form of intellectual disability affecting 1 in 2,000-4,000 women and 1 in 4,000-8,000 men, women being twice as likely to inherit this disability due to their xx chromosomes. 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. individuals with fragile x syndrome experience a variety of symptoms at varying degrees that depend on gender and mutation degree such as attention deficit disorders, irritability, stimuli sensitivity, various anxiety disorders, depression, and/or aggressive behavior. some treatments for these symptoms seen in individuals with fragile x syndrome include ssri's, antipsychotic medications, stimulants, folic acid, and mood stabilizers. genetic causation sizable expansions of a cgg trinucleotide element are the singular cause of the male genetic disorder called fragile x syndrome. in males without fragile x syndrome, the cgg repeat number ranges from 53 to 200 while those affected have greater than 200 repeats of this trinucleotide sequence located at the end of the x chromosome on band xq28.3.1. 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. research has shown that individuals with premutation alleles in the range of 59-69 repeats have about a 30% risk of developing full mutation and compared to those in the high range of ≥ 90 repeats. 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. fragile x syndrome men possess alleles in the full mutation range (>200 repeats) with fmrp protein levels much lower than normal and experience hypermethylation of the promoter region of the fmr1 gene. some men with alleles in the full mutation range experience partial or no methylation which results in only slightly abnormal phenotypes due to only slight down-regulation of fmr1 gene transcription. unmethylated and partially methylated alleles in the mutation range experience increased and normal levels of fmr1 mrna when compared to normal controls. in contrast, when unmethylated alleles reach a repeat number of approximately 300, the transcription levels are relatively unaffected and operate at normal levels; the transcription levels of repeats greater than 300 is currently unknown. promoter silencing the cgg trinucleotide repeat expansion is present within the fmr1 mrna and its interactions are responsible for promoter silencing. the cgg trinucleotide expansion resides within the 5' untranslated region of the mrna, which undergoes hybridization to form a complementary cgg repeat portion. the binding of this genomic repeat to the mrna results in silencing of the promoter. beyond this point, the mechanism of promoter silencing is unknown and still being further investigated. background huntington's disease (hd) is a dominantly, paternally transmitted neurological disorder that affects 1 in 15,000-20,000 people in many western populations. hd involves the basal ganglia and the cerebral cortex and manifests as symptoms such as cognitive, motor, and/or psychiatric impairment. causation this autosomal dominant disorder results from the expansions of a trinucleotide repeat which involves cag in exon 1 of the it15 gene. 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. research has shown an inverse relationship between the number of trinucleotide repeats and age of onset, however, no relationship between trinucleotide repeat numbers and rate of hd progression and/or effected individual's body weight has been observed. severity of functional decline has been found to be similar across a wide range of individuals with varying numbers of cag repeats and differing ages of onset, therefore, it is suggested that the rate of disease progression is also linked to factors other than the cag repeat such as environmental and/or genetic factors. background myotonic dystrophy is a rare muscular disorder in which numerous bodily systems are affected. there are four forms of myotonic dystrophy: mild phenotype and late-onset, onset in adolescence/young adulthood, early childhood featuring only learning disabilities, and a congenital form. 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. in addition to these physical ailments, individuals with myotonic dystrophy have been found to experience varying internalized disorders such as anxiety and mood disorders as well as cognitive delays, attention deficit disorders, autism spectrum disorders, lower iq's, and visual-spatial difficulties. research has shown that there is a direct correlation between expansion repeat number, iq, and an individual's degree of visual-spatial impairment. causation myotonic dystrophy results from a (ctg)n trinucleotide repeat expansion that resides in a 3' untranslated region of a serine/threonine kinase coding transcript. 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. age and (ctg)n length only have small correlation coefficients to disease progression, research suggests that various other factors play a role in disease progression such as changes in signal transduction pathway, somatic expression, and cell heterogeneity in (ctg)n repeats. background friedreich's ataxia is a progressive neurological disorder. individuals experience gait and speech disturbances due to degeneration of the spinal cord and peripheral nerves. other symptoms may include cardiac complications and diabetes. typical age at symptom onset is 5–15, with symptoms progressively getting worse over time. causation 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. point of occurrence fragile x syndrome 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. myotonic dystrophy type 1 many similarities can be drawn between dm1 and fxs involving aspects of mutation. full maternal inheritance is present within dm1, repeat expansion length is linked to maternal age and the earliest instance of expansions is seen in the two-cell stage of preimplantation embryos. 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. huntington's disease in huntington's disease (hd), the exact timing has not been determined; however there are a number of proposed points during germ cell development at which expansion is thought 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. repeat expansions have been observed to occur before the completion of meiosis in humans, specifically the first division. 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. spinocerebellar ataxia type 1 spinocerebellar ataxia type 1 (sca1) cag repeats are most often passed down through paternal inheritance and similarities can be seen with hd. the tract size for offspring of mothers with these repeats does not display any degree of change. 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. therapeutics trinucleotide repeat expansion, is a dna mutation