Signatures of Convergent Evolution and Natural Selection at the Alcohol Dehydrogenase Gene Region are Correlated with Agriculture in Ethnically Diverse Africans

The alcohol dehydrogenase (ADH) family of genes encodes enzymes that catalyze the metabolism of ethanol into acetaldehyde. Nucleotide variation in ADH genes can affect the catalytic properties of these enzymes and is associated with a variety of traits, including alcoholism and cancer. Some ADH variants, including the ADH1B*48His (rs1229984) mutation in the ADH1B gene, reduce the risk of alcoholism and are under positive selection in multiple human populations. The advent of Neolithic agriculture and associated increase in fermented foods and beverages is hypothesized to have been a selective force acting on such variants. However, this hypothesis has not been tested in populations outside of Asia. Here, we use genome-wide selection scans to show that the ADH gene region is enriched for variants showing strong signals of positive selection in multiple Afroasiatic-speaking, agriculturalist populations from Ethiopia, and that this signal is unique among sub-Saharan Africans. We also observe strong selection signals at putatively functional variants in nearby lipid metabolism genes, which may influence evolutionary dynamics at the ADH region. Finally, we show that haplotypes carrying these selected variants were introduced into Northeast Africa from a West-Eurasian source within the last ∼2,000 years and experienced positive selection following admixture. These selection signals are not evident in nearby, genetically similar populations that practice hunting/gathering or pastoralist subsistence lifestyles, supporting the hypothesis that the emergence of agriculture shapes patterns of selection at ADH genes. Together, these results enhance our understanding of how adaptations to diverse environments and diets have influenced the African genomic landscape.     

Alcohol and aldehyde dehydrogenase polymorphisms and risk for suicide: a preliminary observation in the Japanese male population

Epidemiological studies have shown that excessive alcohol consumption is a potent risk factor to develop suicidal behavior. Genetic factors for suicidal behavior have been observed in family, twin, and adoption studies. Because alcohol dehydrogenase (ADH1B) His47Arg and mitochondrial aldehyde dehydrogenase (ALDH2) Glu487Lys single nucleotide polymorphisms (SNPs), which affect alcohol metabolism, have been reported to exert significant impacts on alcohol consumption and on the risk for alcoholism in East Asia populations, we explored associations of the two functional SNPs with suicide using a case–control study of 283 completed suicides and 319 control subjects in the Japanese population. We found that the inactive ALDH2 allele (487Lys) was significantly less frequent in the completed suicides (19.3%) than in the controls (29.3%), especially in males, whereas this was not the case in females. The males bearing alcoholism‐susceptible homozygotes at both loci (inactive ADH1B Arg/Arg and active ALDH2 Glu/Glu genotypes) have a 10 times greater risk for suicide compared with the males bearing alcoholism‐protective homozygotes at both loci. Our data show the genetic impact of the two polymorphisms on suicidal behavior in the Japanese population, especially in males. Because we did not verify the daily alcohol consumption, the association of these SNPs with suicide might be due to alcoholism itself. Further studies using case–control subjects, which verifies the details of current and past alcohol consumption and diagnosis for alcoholism, are required to confirm these findings.     

Isolation of a cDNA encoding functional Drosophila alcohol dehydrogenase in Escherichia coli and purification of the bacterially produced enzyme

Because of the severe limitations on growing large quantities of Drosophila affinidisjuncta in the laboratory, direct purification of alcohol dehydrogenase (ADH) from this species has proven impossible. As an alternative source of this enzyme, a cDNA encoding functional ADH was isolated from a newly constructed cDNA library made from larval poly(A)-containing RNA. The cDNA was recovered by virtue of its hybridization to a previously isolated genomic ADH gene. Nucleotide sequence analysis confirmed the identity of the newly isolated cDNA. When the cDNA was inserted in the proper orientation downstream of the lac promoter on the vector pUC8, the cDNA directed the synthesis of functional ADH by the bacterial host. The bacterially produced enzyme was purified to homogeneity and used to elicit polyclonal antibodies in rabbits. The purified ADH has identical apparent subunit molecular weight to that of authentic ADH in larval fly extracts as determined by immunoblotting. Further, comparisons of the kinetic parameters of the bacterially produced enzyme and ADH activity in larval fly extracts indicate similar substrate preferences, pH dependencies, and Km values for 2-propanol and NAD. These results show that expression of a cDNA in Escherichia coli is a valid strategy for isolation of an ADH that would otherwise be difficult or impossible to purify.     

Alcohol and aldehyde dehydrogenase genotypes and alcoholism in Chinese men.

The liver enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which are responsible for the oxidative metabolism of ethanol, are polymorphic in humans. An allele encoding an inactive form of the mitochondrial ALDH2 is known to reduce the likelihood of alcoholism in Japanese. We hypothesized that the polymorphisms of both ALDH and ADH modify the predisposition to development of alcoholism. Therefore, we determined the genotypes of the ADH2, ADH3, and ALDH2 loci of alcoholic and nonalcoholic Chinese men living in Taiwan, using leukocyte DNA amplified by the PCR and allele-specific oligonucleotides. The alcoholics had significantly lower frequencies of the ADH2*2, ADH3*1, and ALDH2*2 alleles than did the nonalcoholics, suggesting that genetic variation in both ADH and ALDH, by modulating the rate of metabolism of ethanol and acetaldehyde, influences drinking behavior and the risk of developing alcoholism.     

Allelic variation at alcohol metabolism genes (<Emphasis Type="Italic">ADH1B</Emphasis>,<Emphasis Type="Italic"> ADH1C</Emphasis>,<Emphasis Type="Italic"> ALDH2</Emphasis>) and alcohol dependence in an American Indian population

Enzymes encoded by two gene families, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), mediate alcohol metabolism in humans. Allelic variants have been identified that alter metabolic rates and influence risk for alcoholism. Specifically, ADH1B*47His (previously ADH2-2) and ALDH2-2 have been shown to confer protection against alcoholism, presumably through accumulation of acetaldehyde in the blood and a resultant 'flushing response' to alcohol consumption. In the current study, variants at ADH1B (previously ADH2), ADH1C (previously ADH3), and ALDH2 were assayed in DNA extracts from participants belonging to a Southwest American Indian tribe (n=490) with a high prevalence of alcoholism. Each subject underwent a clinical interview for diagnosis of alcohol dependence, as well as evaluation of intermediate phenotypes such as binge drinking and flushing response to alcohol consumption. Detailed haplotypes were constructed and tested against alcohol dependence and related intermediate phenotypes using both association and linkage analysis. ADH and ALDH variants were also assayed in three Asian and one African population (no clinical data) in order to provide an evolutionary context for the haplotype data. Both linkage and association analysis identified several ADH1C alleles and a neighboring microsatellite marker that affected risk of alcohol dependence and were also related to binge drinking. These data strengthen the support for ADH as a candidate locus for alcohol dependence and suggest further productive study.     

Interaction between the functional polymorphisms of the alcohol-metabolism genes in protection against alcoholism.

The genes that encode the major enzymes of alcohol metabolism, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), exhibit functional polymorphism. The variant alleles ADH2*2 and ADH3*1, which encode high-activity ADH isoforms, and the ALDH2*2 allele, which encodes the low-activity form of ALDH2, protect against alcoholism in East Asians. To investigate possible interactions among these protective genes, we genotyped 340 alcoholic and 545 control Han Chinese living in Taiwan at the ADH2, ADH3, and ALDH2 loci. After the influence of ALDH2*2 was controlled for, multiple logistic regression analysis indicated that allelic variation at ADH3 exerts no significant effect on the risk of alcoholism. This can be accounted for by linkage disequlibrium between ADH3*1 and ADH2*2 ALDH2*2 homozygosity, regardless of the ADH2 genotypes, was fully protective against alcoholism; no individual showing such homozygosity was found among the alcoholics. Logistic regression analyses of the remaining six combinatorial genotypes of the polymorphic ADH2 and ALDH2 loci indicated that individuals carrying one or two copies of ADH2*2 and a single copy of ALDH2*2 had the lowest risk (ORs 0.04-0.05) for alcoholism, as compared with the ADH2*1/*1 and ALDH2*1/*1 genotype. The disease risk associated with the ADH2*2/*2-ALDH2*1/*1 genotype appeared to be about half of that associated with the ADH2*1/*2-ALDH2*1/*1 genotype. The results suggest that protection afforded by the ADH2*2 allele may be independent of that afforded by ALDH2*2.     

Further clarification of the contribution of the ADH1C gene to vulnerability of alcoholism and selected liver diseases

The alcohol dehydrogenase 1C (ADH1C) subunit is an important member of the alcohol dehydrogenase family, a set of genes that plays a major role in the catabolism of ethanol. Numerous association studies have provided compelling evidence that ADH1C gene variation (formerly ADH3) is associated with altered genetic susceptibility to alcoholism and alcohol-related liver disease, cirrhosis, or pancreatitis. However, the results have been inconsistent, partially, because each study involved a limited number of subjects, and some were underpowered. Using cumulative data over the past two decades, this meta-analysis (6,796 cases and 6,938 controls) considered samples of Asian, European, African, and Native American origins to examine whether the aggregate genotype provide statistically significant evidence of association. The results showed strong evidence of association between ADH1C Ile350Val (rs698, formerly ADH1C *1/*2) and alcohol dependence (AD) and abuse in the combined studies. The overall allelic (Val vs. Ile or *2 vs. *1) P value was 1 × 10(-8) and odds ratio (OR) was 1.51 (1.31, 1.73). The Asian populations produced stronger evidence of association with an allelic P value of 4 × 10(-33) [OR 2.14 (1.89, 2.43)] with no evidence of heterogeneity, and the dominant and recessive models revealed even stronger effect sizes. The strong evidence remained when stricter criteria and sub-group analyses were applied, while Asians always showed stronger associations than other populations. Our findings support that ADH1C Ile may lower the risk of AD and alcohol abuse as well as alcohol-related cirrhosis in pooled populations, with the strongest and most consistent effects in Asians.     

Alcohol and aldehyde dehydrogenase genotypes and drinking behavior of Chinese living in Shanghai

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the principal enzymes responsible for oxidative metabolism of ethanol, exist in multiple, genetically determined molecular forms. Widely different kinetic properties in some of these isozymes account for the individual differences in alcohol sensitivity. In this study we used the polymerase chain reaction/restriction fragment length polymorphism method to determine the genotypes of the ADH2 and ALDH2 loci of alcoholic and nonalcoholic Chinese living in Shanghai. We also investigated the subjects' drinking patterns by means of semistructured interviews. The alcoholics had significantly lower frequencies of the ADH2² and ALDH2² alleles than did the nonalcoholics, suggesting the inhibitory effects of these alleles for the development of alcoholism. In the nonalcoholic subjects, ADH2² had little, if any, effect, despite the significant effect of the ALDH2² allele in decreasing the alcohol consumption of the individual. Taken together, these results fit the proposed hypothesis for the development of alcoholism, i.e., drinking behavior is greatly influenced by the individual's gentoypes of alcohol-metabolizing enzymes, and the risk of becoming alcoholic is proportionate with the ethanol consumption of the individual.     

Drosophila alcohol dehydrogenase: a novel reporter gene for use in mammalian embryos.

A reporter gene construct containing the Drosophila alcohol dehydrogenase (ADH) gene under regulation of the Rous sarcoma virus long terminal repeat (RSV LTR) was microinjected into mouse zygote pronuclei. ADH activity after injection of the RSV-ADH construct was visualized in cultured embryos by means of a simple histochemical enzyme assay. The RSV LTR was an efficient promoter that led to abundant ADH activity at all stages of preimplantation development. There was a high incidence of mosaicism among stained embryos.     

Replication Study of ESCC Susceptibility Genetic Polymorphisms Locating in the ADH1B-ADH1C-ADH7 Cluster Identified by GWAS

China was one of the countries with highest esophageal squamous cell carcinoma (ESCC) incidence and mortality worldwide. Alcohol drinking has been identified as a major environmental risk-factor related to ESCC. The alcohol dehydrogenase (ADH) family are major enzymes involved in the alcohol-metabolizing pathways, including alcohol dehydrogenase 1B (ADH1B) and ADH1C. Interestingly, ADH1B and ADH1C genes locate tandemly with ADH7 in a genomic segment as a gene cluster, and are all polymorphic. Several ESCC susceptibility single nucleotide polymorphisms (SNPs) of the ADH1B-ADH1C-ADH7 cluster have been identified previously through a genome-wide association study (GWAS). In the study, we examined the association between five ADH1B-ADH1C-ADH7 cluster SNPs (rs1042026, rs17033, rs1614972, rs1789903 and rs17028973) and risk of developing ESCC. Genotypes were determined in two independent case-control sets from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Our data demonstrated that these ADH1B-ADH1C-ADH7 cluster SNPs confer susceptibility to ESCC in these two case-control sets, which were consistent to results of the previous GWAS.     

Association of the sEH gene promoter polymorphisms and haplotypes with preeclampsia

BackgroundThe epoxyeicosatrienoic acids (EETs) have antihypertensive, anti-inflammatory, and organ protective properties and their circulation levels are related to hypertension, diabetes mellitus, cardiovascular diseases, and preeclampsia. Soluble epoxide hydrolase (sEH) catalyses the degradation of EETs to less biologically active dihydroxyeicosatrienoic acids. Here, we sequenced the promoter region of EPHX2 to investigate the association between promoter sequence alterations that we thought to affect the expression levels of the enzyme and preeclampsia (PE).MethodsNucleotide sequencing of the promoter region of the EPHX2, spanning from position -671 to +30, was performed on 100 pregnant women with PE and, 20 or more weeks pregnant normotensive, healthy women (n=100).ResultsPregnant women who carry rs4149235, rs4149232, rs73227309, and rs62504268 polymorphisms have 4.4, 2.4, 2.3, and 2.8 times significantly increased risk of PE, respectively. CCGG (OR: 3.11; 95% CI: 1.12-8.62) and CCCA (OR: 0.45; 95% CI: 0.36-0.55) haplotypes were associated with an increased and decreased risk of PE, respectively.ConclusionsFour SNPs (rs4149232, rs4149235, rs73227309, and rs62504268) in the promoter region of the EPHX2, and CCGG and CCCA haplotypes of these 4 SNPs were significantly associated with PE. These SNPs in the promoter region may affect sEH expression and thus enzyme activity and may play a role in PE pathogenesis by causing individual differences in EET levels. However, future studies are needed to confirm our findings and examine the effect of these SNPs on the sEH expression and/or enzyme activity.     

点突变对高加索乳杆菌醇脱氢酶酶活的影响

从高加索乳杆菌基因组中克隆醇脱氢酶基因,构建重组表达菌后发现不同转化子具有不同的活性,测序结果表明在部分位点发生了点突变.结合生物信息学知识通过对醇脱氢酶结构与作用机理分析,认为在酶关键位点的变变对酶的活性影响较大,而非关键位点的突变对酶活的影响虽明显降低,但其突变的数目可能对酶活的影响呈现一定的累加效应.其中活性最高的重组菌表达了一个可将苯乙酮高选择对映还原成(S)-笨乙醇的醇脱氢酶,该研究结果为酶的定向进化研究提供了理论依据.     

The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) protein is necessary for amebic growth and survival and requires an intact C-terminal domain for both alcohol dahydrogenase and acetaldehyde dehydrogenase activity

The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N- and C-terminal halves of the molecule.     

Genetic variation in the expression of ADH in Drosophila melanogaster

Several chromosomes derived from natural populations have been identified that affect the expression of alcohol dehydrogenase (ADH). Second chromosomes, which also carry the structural gene Adh, show a great deal of polymorphism of genetic elements that determine how much enzyme protein accumulates. The level of enzyme was measured in third instar larvae, 6-to-8-day-old males and in larval fat bodies and alimentary canals. In general, activities in the different organs and stages are highly correlated with one another. One line was found, however, in which the ADH level in the fat body is more than twice the level one would expect on the basis of the activity in alimentary canal. We have also found evidence of third-chromosome elements that affect the level of ADH.     

Expression inEscherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation

Human alcohol dehydrogenase (ADH, tiff isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the fl-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3 ~o of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic fl-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.     

Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies.

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.     

Linkage disequilibrium at the ADH2 and ADH3 loci and risk of alcoholism

Two of the three class I alcohol dehydrogenase (ADH) genes (ADH2 and ADH3) encode known functional variants that act on alcohol with different efficiencies. Variants at both these genes have been implicated in alcoholism in some populations because allele frequencies differ between alcoholics and controls. Specifically, controls have higher frequencies of the variants with higher Vmax (ADH2*2 and ADH3*1). In samples both of alcoholics and of controls from three Taiwanese populations (Chinese, Ami, and Atayal) we found significant pairwise disequilibrium for all comparisons of the two functional polymorphisms and a third, presumably neutral, intronic polymorphism in ADH2. The class I ADH genes all lie within 80 kb on chromosome 4; thus, variants are not inherited independently, and haplotypes must be analyzed when evaluating the risk of alcoholism. In the Taiwanese Chinese we found that, only among those chromosomes containing the ADH3*1 variant (high Vmax), the proportions of chromosomes with ADH2*1 (low Vmax) and those with ADH2*2 (high Vmax) are significantly different between alcoholics and controls (P<10-5). The proportions of chromosomes with ADH3*1 and those with ADH3*2 are not significantly different between alcoholics and controls, on a constant ADH2 background (with ADH2*1, P=.83; with ADH2*2, P=.53). Thus, the observed differences in the frequency of the functional polymorphism at ADH3, between alcoholics and controls, can be accounted for by the disequilibrium with ADH2 in this population.     

Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase

The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 ? resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.     

Cloning and sequence analysis of the gene encoding an NADP-dependent alcohol dehydrogenase in Mycobacterium bovis BCG.

The nucleotide sequence of a 1619-bp fragment of Mycobacterium bovis BCG containing the gene that encodes an alcohol dehydrogenase (ADH) has been determined. The M(r) calculated from the deduced amino acid (aa) sequence, as well as the N terminus, are in good accordance with those determined for the ADH purified from M. bovis BCG extracts. The M. bovis BCG cloned adh gene was expressed in Escherichia coli by its own promoter and the synthesized product shows ADH activity in the butane-1-ol-NADP system. Based on comparison of the aa sequence, this enzyme belongs to the zinc-containing, long-chain alcohol/polyol dehydrogenase family, which has been primarily described in eukaryotes. Of the 22 strictly conserved residues in this group, 19 are also conserved in M. bovis BCG ADH (BCGADH).