Microsatellite instability

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Micrograph showing tumor-infiltrating lymphocytes in a case of colorectal cancer with evidence of MSI-H on immunostaining. H&E stain.

Microsatellite instability (MSI) is the condition of genetic hypermutability that results from impaired DNA Mismatch Repair (MMR). In other words, MSI is the phenotypic evidence that MMR is not functioning normally. DNA MMR corrects errors that spontaneously occur during DNA replication like single base mismatches or short insertions and deletions. The proteins involved in MMR form a complex that binds to the mismatch, identifies the correct strand of DNA, then subsequently excises the error and repairs the mismatch.[1] Cells with abnormally functioning MMR tend to accumulate errors rather than correcting those errors. As a result, gene sequences are not preserved faithfully through DNA replication, and novel Microsatellites fragments are created. Microsatellite instability is detected by PCR based assays that reveal these novel microsatellites.

Microsatellites are repeated sequences of DNA. These sequences can be made of repeating units of 1-6 base pairs in length. Although the length of these microsatellites is highly variable from person to person (part of DNA "fingerprint"), each individual has microsatellites of a set length. The most common microsatellite in humans is a dinucleotide repeat of CA, which occurs tens of thousands of times across the genome. Microsatellites are also known as simple sequence repeats (SSRs).

Example of Microsatellite Instability in a DNA Electropherogram Trace

Microsatellite Instability Structure[edit]

The Microsatellite Instability (MSI) structure consists of repeated nucleotides, most often seen as GT/CA repeats.[2]

Although, researchers have yet to confirm the (MSI) structure has been precisely defined. Some researchers believe that Microsatellites are short tandem DNA repeat sequences of 1-6 base pairs -distributed throughout the genome. All researchers agree that they are repeat sequences, but the length of the sequences remain in question.[3]

Although researchers do not agree on a specific threshold for the number of tandem repeats that constitute a Microsatellite, there is a consensus of relative size. Bigger sequences are called Mini-Satellites and even bigger sequences - Satellite DNA sites. Some scientists determine this by a minimum number of base pairs and others use a minimum number of repeated units. The majority of repeats occur in untranslated regions (UTR), specifically introns. However, MSI that occur in coding regions, often inhibit the expansion of most downstream events. Microsatellites makeup approximately 3% of the human genome or more than 1 million fragments of DNA. Microsatellite density increases with genome size and is seen twice more at the ends of chromosome arms than in the chromosome bodies.[4]

Microsatellite Instability Formation and Function[edit]

MSI was discovered in the 1970s and 1980s. The first human disease attributed to MSI was xeroderma pigmentosa. This disease resulted from two alleles activating mutations on nucleotide excision repair. In a broad sense, MSI results from the inability of the mismatch repair (MMR) proteins to fix a DNA replication error. DNA replication occurs in the “S” phase of the cell cycle and the faulty event creating an MSI region occurs during the second replication event. The original strand is unharmed, but the daughter strand experiences a frame-shift mutation due to DNA polymerase slippage. Specifically, DNA polymerase slips (slippage) creating a temporary insertion-deletion loop (IDL) which is usually recognized by MMR proteins. However, when the MMR proteins are ineffective, as in the case of MSI, this loop results in frame-shift mutations either through insertions or deletions yielding non-functioning proteins.[5]

MSI is unique to DNA polymorphisms in that they vary in length instead of sequence. The rate and direction of the mutations yielding MSI’s are the major components in determining genetic differences. “Long MSI’s have been associated with virulence pathways acting as transcriptional “switches."” To date, scientists agree that the mutation rates differ in loci position. The greater the length of the MSI the greater the mutation rate is deriving the MSI.[4]

Although most mutations of MSI are result of frameshift mutations, occasionally the mutation events leading to MSI are derived from the hypermethylation of the hMLH1 (MMR protein) promoter. Hypermethylation occurs when a methyl group is added to a DNA nucleotide resulting in gene silencing thus yielding MSI.[6]

Researchers have shown oxidative damage yield frameshift mutations thus yielding MSI. It is shown that the more oxidative stress is placed on the system, the more likely mutations will occur. Additionally, catalase reduces mutations whereas Copper and Nickel increase mutations by increasing reduction of peroxides. Although researchers have demonstrated that oxidative damage causes increased mutations leading to MSI, they have yet to agree on a precise mechanism. However, some researchers believe that the oxidative stress on specific loci results in DNA polymerase pausing at those sites creating an environment for DNA slippage to occur.[7]

Researchers first believed that MSI was random, however, there is evidence suggesting that MSI targets include a growing list of genes. Examples include transforming growth factor Beta receptor gene and BAX gene. Each target leads to different phenotypes and pathologies.[8]

Clinical Significance of Microsatellite Instability[edit]

Microsatellite instability (MSI) may result in colon cancer, gastric cancer, endometrium cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer and skin cancers. MSI is most prevalent in the cause of colon cancers. Each year there are over 500,000 colon cancer cases worldwide. Based on findings from over 7,000 patients stratified for MSI-High (MSI-H), MSI-Low (MSI-L) or Microsatellite Stable (MSS) colon cancers, those with MSI-H had a more positive prognosis by 15% compared to MSI-L or MSS tumors.[9]

Colorectal tumors with MSI are found in the right colon, associated with poor differentiated tissue, high mucinogens, tumor infiltrating lymphocytes and a presence of a Crohn’s like host response. MSI-H tumors contributing to colorectal cancer exhibit less metastasis than other derived colorectal cancer. This is demonstrated by previous research showing MSI-H tumors are more representative in Stage II rather than Stage III cancers.[3]

Scientists have explored Vascular Sorting Proteins (VPS) proteins connection to MSI. VPS is similarly linked to gastric and colon cancers as MSI. One study reports that VPS proteins were linked to MSI-H cancers but not MSI-L cancers thus restricting VPS to MSH-H specific cancers. “Frameshift mutations of VPS37B and VPS33A in cancers with MSI-H may result in truncated proteins and altered interactions with tumor suppressors.” [10]

Furthermore, researchers agree that MSI-H status indicates the diagnosis of Lynch syndrome, non-metastatic prognosis and less aggressive treatment regimens. Lynch syndrome is caused by MSI and increases the risk for colon, endometrium, ovary, stomach, small intestine, hepatobiliary tract, urinary tract, brain and skin cancers.[3]

One study conducted over 120 Lynch syndrome patients attributing Crohn’s Like Syndrome (CLR) associated with MSI to “tumor specific neopeptides generated during MSI-H carcinogenesis.” This study further corroborated that the “presence of antimetastatic immune protection in MSI-H CRC patients may explain recent findings that adjuvant 5-FU chemotherapy has no beneficial or even adverse effects in this collective.” The researchers assume that there is a protective role of lymphocytes against the MSI-H CRC that prevents tumor metastasis.[11]

MSI tumors in 15% of sporadic colorectal cancer result from the hypermethylation of the MLH 1 gene promoter. Whereas, MSI tumors in Lynch Syndrome are caused by germline mutations in MLH 1, MSH 2, MSH 6 and PMS2.[3]

MSI has been evident in the cause of sebaceous carcinomas. Sebaceous carcinomas are a subset of a larger pathology, Muir-Torre syndrome. MSI is variably expressed in Muir-Torre syndrome, most often expressed with shared pathologies in patients with colon cancer. Furthermore, MMR proteins MSH 1, MSH 2, MSH6, and PMS2, are instrumental in periocular sebaceous carcinoma which is seen on the eyelid in 40% of sebaceous carcinomas.[12]

Microsatellite Instability Diagnostics[edit]

MSI is a good marker for determining Lynch syndrome and determining a prognosis for cancer treatments. The NCI has agreed on five microsatellite markers necessary to determine MSI presence. Two mononucelotides – BAT25/26 and three dinucelotide repeats – D2S123,D5S346,D17S250. MSI-High (MSI-H) tumors result from MSI of greater than 30% of unstable MSI biomarkers. MSI-Low tumors result from less than 30% of unstable MSI biomarkers. MSI-L tumors are classified as tumors of alternative etiologies. Several studies demonstrate that MSI-H patients respond best to surgery alone, rather than chemotherapy and surgery, thus preventing the needless patient experience with chemotherapy.[3]

Direct and indirect mechanisms contribute to chemotherapy resistance. Direct mechanisms include pathways that metabolize the drug while indirect mechanisms include pathways that respond to the chemotherapy treatment. The NER DNA repair pathway plays a substantial role in reversing cell damage caused by chemotherapeutic agents such as 5-FU.[13]

Recent Discoveries[edit]

Additionally, researchers have found that another MSI exists, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). However, EMAST is unique in that it is not derived from MMR and it is commonly associated with P53 mutations.[5]

EMAST is seen in a variety of cancers including lung, head and neck, colorectal, skin, urinary tract, and the reproductive organs. External organ sites have more potential for EMAST. Some researchers believe EMAST maybe a consequence of mutagenesis. EMAST positive margins in otherwise negative cancer margins suggest disease relapse for patients.[14]

List of MSI-H Genes Causing Different Cancers[edit]

A List of MSI-H genes are listed here.[15]

References[edit]

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  2. ^ http://web.udl.es/usuaris/e4650869/docencia/segoncicle/genclin98/recursos_classe_(pdf)/revisionsPDF/MicrosatInstab.pdf
  3. ^ a b c d e Dig Liver Dis. 2013 Jun;45(6):441-9. doi: 10.1016/j.dld.2012.10.006. Epub 2012 Nov 26.
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  5. ^ a b Gastroenterology. 2010 Jun;138(6):2073-2087.e3. doi: 10.1053/j.gastro.2009.12.064.
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  7. ^ Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12468-73.
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  11. ^ Br J Cancer. 2005 May 9;92(9):1746-53.
  12. ^ Am J Ophthalmol. 2014 Mar;157(3):640-7.e1-2. doi: 10.1016/j.ajo.2013.12.002. Epub 2013 Dec 7.
  13. ^ J Surg Res. 2012 May 1;174(1):1-6. doi: 10.1016/j.jss.2011.06.016. Epub 2011 Jul 7.
  14. ^ Br J Cancer. 2014 Apr 1. doi: 10.1038/bjc.2014.167.
  15. ^ Ann Genet. 2002 Apr-Jun;45(2):71-5.