Gene regulation and the genetics of alcoholism.
The long range objective of this research project is to identify genes thatinfluence alcohol-seeking behavior. The goal of this proposal is toidentify the chromosomal location of genes that are associated with alcoholpreference. Numerous family and twin studies in the past years have shownthat genetic factors are involved in the genesis of alcoholism. Identification of the genes that influence alcohol-drinking behavior wouldhelp our understanding of the mechanisms of this disease and the approachesthat need to be taken for treatment and prevention. To better understandthe genetics of alcoholism and alcohol preference, animal studies will beused to isolate candidate regions and loci in homogeneous populationsdeveloped through selective breeding. Using quantitative trait mappingmethods, quantitative trait loci QTL with major as well as minor effectson alcohol drinking variability can be identified. First, the selectivelybred alcohol-preferring P and alcohol-nonpreferring NP rat lines will beemployed in thesearch for QTL that influence alcohol preference and othertraits that are correlated with alcohol preference. Inbreed P and NP ratswill be intercrossed to produce 400 F2 animals. The parents and F2 animalswill be tested for alcohol preference. The parental lines will then begenotyped using highly polymorphic microsatellite DNA markers. Thosemarkers that are different between the lines will be used to genotype theF2 progeny. Maximum likelihood methods using interval mapping will be usedin a genome-wide search to identify candidate QTL for alcohol preference. QTL identified in the P and NP lines will be confirmed using the noninbredfirst replicate high alcohol drinking HAD1 and low alcohol drinkingLAD1 liens. HAD1 lines. HAD1 and LAD1 animals will be matedto producethree generational families. The families will be genotyped using highlypolymorphic markers in the previously identified candidate regions. Leastsquare regression analysis will be used to determine if the candidate QTLis segregating in these families. These families will also be used for agenome-wide search to identify additional QTL influencing alcoholpreference that may not be segregating in the P and NP liens as well asother alcohol-related traits studied by other investigators. Candidate QTLdiscovered in the HAD1 and LAD1 lines will be confirmed in three- generational families from crosses of the second replicate HAD2 and lAD2lines. Using two genetically diverse rat lines PNP and HADLAD willallow us to identify chromosomal regions that are very likely influencingalcohol preference. The QTL identified in animals can then be tested inhuman studies to determine their effect on the alcoholism phenotype.
The overall goal of the Molecular Biology Core of this Alcohol ResearchCenter is to study the association between specific genes and variousaspects of alcoholism, alcohol abuse and alcoholic organ complications. The Core has developed polymerase chain reaction PCR-based genotypingassays for the alcohol metabolizing enzymes, ADH2, ADH3, and ALDH2 andhas determined the allele frequencies of these loci in variouscollaborative studies. We will continue to offer these genotypingservices to investigators in order to determine if there are furtherrelationships between these enzymes and responses to alcohol. A newfocus of the core is to establish new genotyping methods for otheralcohol metabolizing enzymes, endorphins, and the genes related todopamine and serotonin synthesis, degradation and neurotransmission. Therestriction fragment length polymorphism RFLP assay or PCR analysis ofpolymorphic repetitive DNA sequences will be employed on genes for whichpolymorphisms are known. If a new mutation in a gene of interest isfound or reported, we will develop a PCR-based genotyping assay insteadof the RFLP analysis because it requires less DNA and the assays areeasier to interpret. For genes in which polymorphisms are not yet known, we will search for polymorphic CA repeats or alternatively, amplify exonsandor introns and screen for single strand conformationpolymorphismsin order to develop genotyping assays.
Gene regulation and genetics of alcoholism
The long range objective of this research project is to identify genes thatinfluence alcohol-seeking behavior. The goal of this proposal is toidentify the chromosomal location of genes that are associated with alcoholpreference. Numerous family and twin studies in the past years have shownthat genetic factors are involved in the genesis of alcoholism. Identification of the genes that influence alcohol-drinking behavior wouldhelp our understanding of the mechanisms of this disease and the approachesthat need to be taken for treatment and prevention. To better understandthe genetics of alcoholism and alcohol preference, animal studies will beused to isolate candidate regions and loci in homogeneous populationsdeveloped through selective breeding. Using quantitative trait mappingmethods, quantitative trait loci QTL with major as well as minor effectson alcohol drinking variability can be identified. First, the selectivelybred alcohol-preferring P and alcohol-nonpreferring NP rat lines will beemployed in thesearch for QTL that influence alcohol preference and othertraits that are correlated with alcohol preference. Inbreed P and NP ratswill be intercrossed to produce 400 F2 animals. The parents and F2 animalswill be tested for alcohol preference. The parental lines will then begenotyped using highly polymorphic microsatellite DNA markers. Thosemarkers that are different between the lines will be used to genotype theF2 progeny. Maximum likelihood methods using interval mapping will be usedin a genome-wide search to identify candidate QTL for alcohol preference. QTL identified in the P and NP lines will be confirmed using the noninbredfirst replicate high alcohol drinking HAD1 and low alcohol drinkingLAD1 liens. HAD1 lines. HAD1 and LAD1 animals will be matedto producethree generational families. The families will be genotyped using highlypolymorphic markers in the previously identified candidate regions. Leastsquare regression analysis will be used to determine if the candidate QTLis segregating in these families. These families will also be used for agenome-wide search to identify additional QTL influencing alcoholpreference that may not be segregating in the P and NP liens as well asother alcohol-related traits studied by other investigators. Candidate QTLdiscovered in the HAD1 and LAD1 lines will be confirmed in three- generational families from crosses of the second replicate HAD2 and lAD2lines. Using two genetically diverse rat lines PNP and HADLAD willallow us to identify chromosomal regions that are very likely influencingalcohol preference. The QTL identified in animals can then be tested inhuman studies to determine their effect on the alcoholism phenotype.
The overall goal of the Molecular Biology Core of this Alcohol ResearchCenter is to study the association between specific genes and variousaspects of alcoholism, alcohol abuse and alcoholic organ complications. The Core has developed polymerase chain reaction PCR-based genotypingassays for the alcohol metabolizing enzymes, ADH2, ADH3, and ALDH2 andhas determined the allele frequencies of these loci in variouscollaborative studies. We will continue to offer these genotypingservices to investigators in order to determine if there are furtherrelationships between these enzymes and responses to alcohol. A newfocus of the core is to establish new genotyping methods for otheralcohol metabolizing enzymes, endorphins, and the genes related todopamine and serotonin synthesis, degradation and neurotransmission. Therestriction fragment length polymorphism RFLP assay or PCR analysis ofpolymorphic repetitive DNA sequences will be employed on genes for whichpolymorphisms are known. If a new mutation in a gene of interest isfound or reported, we will develop a PCR-based genotyping assay insteadof the RFLP analysis because it requires less DNA and the assays areeasier to interpret. For genes in which polymorphisms are not yet known, we will search for polymorphic CA repeats or alternatively, amplify exonsandor introns and screen for single strand conformationpolymorphismsin order to develop genotyping assays.
Alcohol Education, Animal Genetics/Breeding, Biological Markers, Biology, Molecular, Biomedical Research Resources, Other, Biomedical Research Training, Biotechnology, Brain, Cloning of Cells, Drug Metabolism, Gene Cloning, Gene Expression, Gene Regulation
