Alcohol addiction and associated maladies constitute significant health issues for America. Substantial progress has been made to the symptomatic treatment of both the physiological and psychological effects of alcohol abuse, but there is a lot left to learn about the formation of an addiction on the molecular level. It is known that there is a genetic component to alcoholism6 and there is an obvious environmental component7 (e.g. exposure to ethanol). The question that we must attempt to solve is how the two combine to create an alcoholic.
There is a noticeable difference in the behavioral response to acute ethanol treatment between a subject that is predisposed to overly self-administer ethanol and one that is not. This is true even if the subject is na•ve to ethanol (has not had it previously administered)3. These behavorial differences are caused either by differences in basal gene expression between the two types of subject or by differences in the changes the ethanol evokes in gene expression between the two subject groups4. By characterizing the underlying gene expression and gene expression changes, we will be better able to understand and ultimately intervene in the development of alcoholism.
Oligonucleotide arrays seem an ideal method to solve this problem, as they are highly efficient for characterizing expression levels in large numbers of genes and for comparing levels of expression between samples9, 5. Unfortunately, because oligionucleotide arrays measure gene expression by the quantity of mRNA annealed to a probe of DNA, a measured difference could be caused by an actual difference in expression levels, or it could be caused by the mRNA from one subject being more complementary to the DNA probe than that from the other subject. Although hypotheses have been made as to the effect that polymorphisms (typically a substitution mutation of a singe nucleotide) have on annealing of mRNA to oligionucleotide arrays1,8, the extent of the effect, and whether that extent is dependent on where in the mRNA the polymorphism is relative to the probe strand are questions that have yet to be thoroughly tested.
The unanswered question, therefore, is what the differences on a molecular level are between subjects predisposed to significant ethanol self-administration and those that are not. In order to address this question, the reliability of short oligionucleotide arrays to compare samples from slightly varying genomes must be determined.
Two primary methods will be used to address these questions. The gene expression after initial exposure to ethanol has been measured using Affymetrix GeneChip mU74Av2 arrays for two inbred strains of mice -- DBA/2J (non-alcoholic model) and C57BL/6 (alcoholic model) mice. Genes that the array indicated were expressed differently between the strains at basal levels are either differently expressed between C57BL/6 and DBA/2J mice or they have polymorphisms that are affecting the array results. Genes that appear to be expressed at different basal levels across three brain regions (the prefrontal cortex, nucleus accumbens and ventral tegmental area) are considered to be candidates for data-affecting polymophisms for the purposes of this work. mRNA for these genes taking from each strain after saline has been administered will be subjected to real time Polymerase Chain Reaction (PCR) to verify the relative expression levels between the strains.
PCR results are based entirely on the initial amount of DNA, and are not sensitive to polymorphisms that are not in the primer area. Therefore, the PCR results will be used as a standard to measure the effect that the polymorphisms had on the results of the oligonucleotide experiments.
A small program will also be written that, in conjunction with BLAST, will identify genes in which there are polymorphisms between the DBA/2J mice and C57BL/6 mice.
Results
There are a number of possible results. We expect to see that in genes with polymorphisms between DBA/2J and C57BL/6 mice PCR will show less of a difference in gene expression than was identified by short oligonucleotide arrays, indicating that the polymorphisms did affect the annealing of mRNA to the probes. We expect that the discrepancy between the results of the PCR and the oligonucleotide array experiments will depend on the number of polymorphisms and to what part of the probe the polymorphism corresponds. We expect that the more polymorphisms between the genes for each strain and the closer that polymorphisms are to the middle of the probe8, the greater the difference between the results of the two measurements. This would mean that the reliability of oligonucleotide data in comparing gene expression is most significantly hampered in these cases.
We may see no significant difference between measured gene expression from PCR and oligonucleotide arrays for some genes, which will give an indication of the frequency of polymorphisms having an effect on microarray data.
In addition, the genes that PCR confirms as significantly different in expression between the two strains are clear candidates for further research on their involvment with the development of alcoholism.
This project should take until August 2003 (approximately two months).
1. Chee M, Yang R, Hubbell E, Berno A, Huang X, Stern D, Winkler
J, Lockhart D, Morris M, Fodor S.
Accessing genetic information with high-density DNA arrays. Science 274 610-614 (1996)
2. Schuckit MA. Low
level of response to alcohol as a predictor of future alcoholism. American Journal of Psychiatry 151, 184-189 (1994)
3. Thibault C, Lai C, Wilke N, Duong B, Olive MF, Rahman S, Dong
H, Hodge C, Lockhart D, Miles M.
Expression profiling of neural cells reveals specific patterns of
ethanol-responsive gene expression.
Molecular Phramacology 52 1593-1600 (2000)
4. Thibault C, Wang L, Zhang L, Miles M. DNA arrays and functional genomics in neurobiology. International Review of Neurobiology 48, 219-253 (2001)
5. Tyndale RF. Genetics of alcohol and tobacco use in humans. Annals of Medicine 35, 94-121 (2003).
6. Viken RJ, Rose RJ, Morzorati SL,
Christian JC, Li TK. Subjective intoxication in
response to alcohol challenge: heritability and covariation with personality,
breath alcohol level, and drinking history. Alcoholism, clinical and experimental research 27, 795-803 (2003).
7. Zhang L, Miles M, Aldape K. (unpublished)
8. Zhang L, Wang L, Ravindranathan A, Miles M. A new algorithm for analysis of
oligonucleotide arrays: application to expression profiling in mouse brain
regions. Journal of Molecular
Biology 00 1-11 (2002)