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History
The first documentation of anticipation in genetic disorders was in the 1800s. However, from the eyes of geneticists, this relationship was disregarded and attributed to ascertainment bias; because of this, it took almost 200 years for a link between onset of disease and trinucleotide repeats (TNR) to be acknowledged.

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. After the breakthroughs, the four mechanisms for TNRs were determined, and more types of repeats were identified as well. Repeat composition and location are used to determine the mechanism of a given expansion. Onwards from 1995, it was also possible to observe the formation of hairpins in triplet repeats, which consisted of repeating CG pairs and a mismatch.

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. Research has shown that there is a clear inverse relationship between the length of the repeats in parents and the age of disease onset in children; therefore, the lengths of TNRs are used to predict age of disease onset as well as outcome in clinical diagnosis. In addition to this finding, another aspect of the diseases, the high variability of onset, was revealed. Although the onset of HD could be predicted by examining TNR length inheritance, the onset could vary up to fourfold depending on the patient, leading to the possibility of existence of age-modifying factors for disease onset; there were notable efforts in this search. 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. When PCR was first attempted in the detection of TNRs, multiple band artifacts were prevalent in the results, and this made recognition of TNRs troublesome; at the time, debate centered around whether disease was brought on by smaller amounts of short expansions or a small amount of long expansions. Since then, accurate methods have been established over the years. Together, the following clinically necessary protocols have 99% accuracy in measuring TNRs.


 * SP-PCR allows for recognition of repeat changes, and originated from the growing necessity for a method that would provide more accurate measurement of TNRs. It has been useful for examining how TNRs vary between human and mice in blood, sperm, and somatic cells.
 * Southern blots are used to measure CGG repeats because CG-rich regions limit polymerase movement in polymerase chain reaction (PCR).

Point of Occurrence
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.

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.

Regarding HD, the exact timing has not been determined; however there are a number proposed points during germ cell development at which expansion is thought to occur.


 * In four HD patient 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 occuring 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 (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.