The Role of Single Nucleotide Polymorphism in the Matrix Metalloproteinase-1 Pro moter and Analysis By Biliesha Belvitt

Introduction Single nucleotide polymorphism is a variation within DNA that occurs whe n a single nucleotide differs from that of a chromosome pair or within a species like humans (Schork, 2000). The occurrence of single nucleotide polymorphisms takes place at a rate of every thousand base pairs in the human genome. However , of these millions of single nucleotide polymorphisms only about 60,000 of them affect gene expression (Karp, 2009). It is within the 60,000 that we find that single nucleotide polymorphism plays a significant role in the development of m alignant cancer among the population. More specifically, the role it has in the occurrence of lung cancer in relation to its presence in the matrix metalloprot einase-1 promoter. The enzyme matrix metalloproteinase (MMP) is an enzyme associated with t he degradation of the extracellular matrix within the cell (Rutter, 1998). As a result, the cell is damaged and more susceptible to causing cancer by being mor e prone to metastasis. It is this degradation, along with single nucleotide pol ymorphism on along the matrix metalloproteinase-1 promoter, which enhances the d evelopment of lung cancer caused by current exposure to the carcinogens associat ed with smoking. This particular promoter is being linked to cancer-causing act ivity due to its abundance within the human body and its known positive correlat ion with over-expression and the presence of cancer cells (Zhu,2001). The specific site of interest that contains the single nucleotide polymorphism a long the matrix metalloproteinase-1 promoter is located at base pair -1607. At this site, the nucleotide guanine (G) is inserted into the original sequence 5 -AAGA T-3 resulting in a new sequence 5 -AAGGAT-3 causing for the sequence to now code fo ectron transmitter systm (ETS) binding site with the sequence 5 -GGA-3 (Rutter, 1998). This 2G site results in increased transcription of MMP and thus increasing extr acellular matrix degradation in comparison to the original 1G site in the same c onsensus sequence. Different types of analysis are available to examine the occurrences of single n ucleotide polymorphisms in the human genome. These results can then be used to determine an individual s risk of developing certain diseases. Subsequently, an ind ividual can further understand their genomic reaction to certain environmental c onditions, like how smoking carcinogens will affect an individual with a 2G site along their matrix metalloproteinase-1 promoter consensus sequence. Methods

In order to evaluate the effect of the single nucleotide polymorphism wi thin MMP-1, experiment participants were put into two groups. One group acted a s the control by not having being diagnosed or possessing any type of cancer, wh ile the second group consisted of newly diagnosed cases of lung cancer. Each gr oup was not chosen based off of any type of demographic, only off their cancer s tatus and willingness for participation in the experiment. Among the participan ts, a genotyping was taking in order to identify who all possessed the 1G site a nd the 2G site that codes for the ETS binding site. This was determined by desi gning specific primers that allow for the 1G allele to be restricted, while the 2G alleles were not restricted due to the 5 -GGA-3 sequence causing the restriction en yme to not recognize the site(Zhu, 2001). Once genotyping of the participants MMP-1 were finalized, a correlation bet ween smoking habits and the specific alleles could then be determined. A positi ve correlation between continuous exposure to smoking carcinogens and increased

MMP transcriptional activity was observed (Zhu, 2001). Current smokers had the highest transcription activity compared to non-smokers, smokers who have smoke r oughly 100 times in their entire lives, and those that had not smoked in year pr ior to diagnosis and/or the analysis. Genotyping for analysis of single nucleotide polymorphisms is most commo nly done through the use of single-stranded DNA. This is done by denaturing the double-stranded DNA because dsDNA is more readily obtainable through polymerase chain reaction. The denaturation occurs by administering high melting temperat ures and then attaching probes. However, this technique does not provide a high percent yield when wanting to analyze the single nucleotide polymorphisms along the ssDNA. That is why a more efficient technique has been formed to be able t o perform genotyping with the use of double-stranded DNA without denaturing. Th is process involves the use of exonuclease III, nuclease S1, and peptide nucleic acid (PNA). (Ren, 2004) In this form of genotyping, the double-stranded DNA is first mixed with exonuclease III. The exonuclease III creates small cuts in the double-stranded DNA to expose single-stranded portions within the helix. Then nuclease S1 is ad ded to the mixture in order to eliminate the cut fragments of double-stranded DN A that resulted from the work of exonuclease III. This whole reaction in done i n the presence of peptide nucleic acid which is complementary to the DNA with th e exception that its backbone consists of peptides instead of the usual phosphat e groups. The newly exposed single-stranded DNA within the helix are then anal yzed through the use of a matrix-assisted laser desorption/ionization time-of-fl ight mass spectrometry (MALDI-TOF MS). This form of genotyping has proven to be more efficient than originally observing the single-stranded DNA as a result of denaturation. Keeping the double-stranded DNA that is not of interest intact, while specifically analyzing the specifc portions seems to be a more desirable a pproach. (Ren, 2004) Discussion The single nucleotide polymorphism along the matrix metalloproteinase-1 promoter has increased the presence of cancer among individuals, specifically in lung cancer. This positive correlation may be attributed to the created electr on transmitter binding site having a greater affinity for the ETS transcription factor along the 2G allele which causes the rate increase in transcription for m atrix metalloproteinase. In comparison, the presence of a 1G allele seems to di splay a weak affinity for the ETS transcription factor which results in a slower transcription rate of matrix metalloproteinase (Rutter, 1998). The analysis of this allele through genotyping using exonuclease III and nuclease S1 in the presence of peptide nucleic acid may allow for future cases of lung cancer among smokers to be detected at an earlier time. This will be th e result having a more efficient way of observing single nucleotide polymorphism s among individuals and determining if their genes code for a more active degrad ation of the extracellular matrix from high matrix metalloproteinase production. Conclusion Single nucleotide polymorphisms that occur in the human genome serve as many the basis of many different types of identifiers. There are many SNPs that occur within the genome, yet few affect us as a small amount actually occur in coding regions of the gene (Karp, 2009). These mutations that occur within the coding region of the gene, once discovered, allow us to hold a library of diseas e traits in relation to nucleotide sequences (Landegren, 1998). The discovery o f the role of single nucleotide polymorphisms within the matrix metalloproteinas e-1 promoter will now be able to form the basis of discovering the driving force s behind the progression of cancer. Future research in this particular area can now go into further detail about how the metastasis as a result of high MMP pro duction can be slowed down and/or prevented in an attempt to reverse or eliminat e the affects of cancer. More efficient means of analysis are also needed in th e laboratory in order to provide a better prediction of an individual s susceptibili ty to cell invasion due to extracellular matrix degradation.