Low initial response to alcohol has been shown to be among the best predictors of development of alcoholism. A similar phenotypic measure, difference in initial sensitivity to ethanol, has been used for the genetic selection of two mouse strains, the Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS) mice, and for the subsequent identification of four quantitative trait loci (QTLs) for alcohol sensitivity. We now report the application of high throughput comparative gene sequencing in the search for genes underlying these four QTLs. To carry out this search, over 1.7 million bases of comparative DNA sequence were generated from 68 candidate genes within the QTL intervals, corresponding to a survey of over 36,000 amino acids. Eight central nervous system genes, located within these QTLs, were identified that contain a total of 36 changes in protein coding sequence. Some of these coding variants are likely to contribute to the phenotypic variation between ILS/ISS animals, including sensitivity to alcohol, providing specific new genetic targets potentially important to the neuronal actions of alcohol.
Background:The inbred long-sleep (ILS) and inbred short-sleep (ISS) strains of mice are widely studied as a model of initial sensitivity to alcohol. Recently, a large comparative DNA sequencing study of candidate genes located within the four Lore quantitative trait loci (QTLs) associated with the ethanol-induced loss of righting reflex in ILS and ISS mice has identified eight genes that contain coding region differences corresponding to amino acid changes. Here, recently developed interval-specific congenic recombinant mice (ISCRs) have been used to map these genes in relationship to newly narrowed QTL regions.Methods: Regions of candidate genes containing DNA differences corresponding to previously identified amino acid changes between ISS and ILS mice were amplified from either genomic DNA or complementary DNA from ISCR mice using polymerase chain reaction. The products were purified and directly sequenced to determine the genotypes for each polymorphism. On the basis of these genotypic data, each candidate gene was determined to be located either within or outside of recently narrowed Lore QTL intervals.Results: Of these eight candidates with protein-coding differences, five are now excluded from their respective Lore intervals. The other three (Znf142, Ptprn, and Znf133) have been localized to the narrowed QTL intervals.Conclusions: These three central nervous system genes (Znf142, Ptprn, and Znf133) represent promising candidates for involvement in the differential sensitivity to alcohol exhibited between ILS and ISS mice. This study also demonstrates how the combination of high-throughput comparative gene sequencing and concomitant genetic fine mapping of QTL regions with ISCRs can be an effective tool for accelerating the process of moving from QTL to gene.
Coding region DNA sequence variants have been recently identified in several QTL candidate genes in a mouse model of differential sensitivity to alcohol [inbred long-sleep (ILS) and inbred short-sleep (ISS)]. This work has been extended into a human population characterized for their initial level of response to alcohol (LR). The coding region of one of the most promising of these candidate genes, zinc finger 133 (Znf133), has been sequenced completely in 50 individuals who participated in alcohol challenges at approximately age 20 and have been followed subsequently for the last 15 years. PCR products were obtained for the protein coding region of ZNF133 using human genomic DNA and directly sequenced using automated sequencers. Novel single nucleotide polymorphisms (SNPs) were detected by analyzing the sequence data using a suite of bioinformatics programs including Consed, Phred, Phrap and Polyphred. Five human SNPs were detected, two that correspond to amino acid changes in the protein, two that are silent DNA changes and one located in an intron. In this small sample, no significant association between any of the SNPs and alcohol diagnosis was detected. A follow-up of these SNPs in a larger sample should allow a more definitive conclusion to be reached. Significantly, the data presented here demonstrate the feasibility of directly testing genes in human alcoholic populations that had been identified first by comparative DNA sequencing of candidate genes located within mouse alcohol-related QTLs, even without detailed knowledge of the gene's function.
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