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.

The number of trinucleotide repeats appears to predict the progression, severity, and age of onset of Huntington's disease and similar trinucleotide repeat disorders.News Release, Weizmann Institute of Science, "Scientists at the Weizmann Institute, using computer simulations, have provided an explanation as to why certain genetic diseases caused by repeats in the code are "genetic time-bombs" whose onset and progression can be accurately predicted," November 21, 2007, at http://80.70.129.162/site/en/weizman.asp?pi=371&doc_id=5042 . Retrieved on 2007-12-30. Other human diseases in which triplet repeat expansion occurs are fragile X syndrome, several spinocerebellar ataxias, myotonic dystrophy and Friedreich's ataxia.

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.

Small-pool polymerase chain reaction (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 PCR.

Overall structure 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.

Location In terms of location, these triplet repeats can be found in both coding and non-coding regions. CAG and GCN repeats, which lead to polyglutamine and polyalanine tracts respectively, are normally found in the coding regions. At the 5' untranslated region, CGG and CAG repeats are found and responsible for fragile X syndrome and spinocerebellar ataxia 12. At the 3' untranslated region, CTG repeats are found, while GAA repeats are located in the intron region. Other disease-causing repeats, but not triplet repeats, have been located in the promoter region. Once the number of repeats exceeds normal levels, Triplet Repeat Expansions (TRE) become more likely and the number of triplet repeats can typically increase to around 100 in coding regions and up to thousands in non-coding regions. This difference is due to overexpression of glutamine and alanine, which is selected against due to cell toxicity.

Intermediates

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. CAG, CTG, and CGG repeats form a hairpin. After the hairpin forms, the primer realigns with the 3' end of the newly synthesized strand and continues the synthesis, leading to triplet repeat expansion. The structure of the hairpin is based on a stem and a loop that contains both Watson-Crick base pairs and mismatched pairs. In CTG and CAG repeats, the number of nucleotides present in the loop depends on if the number of triplet repeats is odd or even. An even number of repeats forms a tetraloop structure, while an odd number leads to the formation of a triloop.

Instability

Threshold 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. There is still not enough research found to understand the molecular nature that causes thresholds but researchers are continuing to study that the possibility could lie with the formation of the secondary structure when these repeats occur. It was found that diseases associated with trinucleotide repeat expansions contained secondary structures with hairpins, triplexes, and slipped-strand duplexes. These observations have led to the hypothesis that the threshold is determined by the number of repeats that must occur to stabilize the formation of these unwanted secondary structures, due to the fact that when these structures form there is an increased number of mutations that will form in the sequence resulting in more trinucleotide expansion.

Parental influence 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.,La Spada, A Trinucleotide Repeat Instability: Genetic Features and Molecular Mechanisms. Brain Pathology 1997; 7:943-963Fu Y.H. Kuhl, D.P. Piuuti, A. Pieretti, M. Sutcliffe, J.S. Richards, S. Verkerk, A.J. Holden, J.J. Fenwick, R.G. Jr. Warren, S.T. Oostra, B.A. Nelson, D.L. Caskey, C.T. Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 1991; 67:1047-58 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. For example, research regarding the correlation between Huntington's Disease CAG trinucleotide repeat and parental transmission has found that there is a strong correlation between the two with differences in maternal and paternal transmission. Maternal transmission has been observed to only consist of an increase in repeat units of 1 while the paternal transmission is typically anywhere from 3 to 9 extra repeats. Paternal transmission is almost always responsible for large repeat transmission resulting in the early onset of Huntington's Disease while maternal transmission results in affected individuals experiencing symptom onset mirroring that of their mother.,Ranen, N.G. Sine, O.C. Abbott, M.H. Sherr, M. Codori. A.M. Franz, M.L. Chao, N.I. Chung, A.S. Pleasant, N. Callahan, C. et al. Anticipation and instability of IT-15 (CAG)n repeats in parent-offspring pairs with Huntington disease. Am J Hum Genet 1995; 57:593-602 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.

Mechanisms

Unequal homologous exchange