Identification of a novel cytosolic aldehyde dehydrogenase allele, ALDHIAI*4

The human cytosolic enzyme aldehyde dehydrogenase 1 (ALDH1A1) functions mainly in acet-aldehyde and neurotransmitter metabolism. It is also reported to play a major role in the production of retinoic acid, which is important for gene expression and tissue differentiation, and also in cyclophosphamide detoxification. [1–3] It is found in various tissues, including the central nervous system (CNS),[4] with highest levels in the liver.[2] Research has implicated the enzyme in the development of alcohol dependence and other alcohol-use disorders, alcohol-induced flushing and sensitivity to alcohol.[3, 5, 6]

Normal ALDH1A1, is tetrameric and predominantly of cytosolic origin. This enzyme has a relatively high K_m (50-100 μM),[7, 8] which is far greater than recorded physiological concentrations (0.4-2.5 μM).[9–12] In addition, this enzyme has low catalytic efficiency (K_cat/K_m)[13] for acet-aldehyde metabolism and hence exhibits its importance in ethanol elimination. Research into ALDH1A1 kinetics with respect to various neuro transmitter aldehydes reveals a Km of 2.4 μM for 5-hydroxyindole acetaldehyde and a K_m of 0.4 μM and 1.5 μM for 3,4-dihydroxyphenylacetaldehyde and phenylacetaldehyde, respectively, and near equivalent K_cat values for these substrates.[14] ALDH1A1 has also displayed a K_m of 0.06 μM in retinaldehyde metabolism, with the K_cat value equivalent to the value in acetaldehyde metabolism [8]. These functions are so important that some researchers believe that this enzyme is essential for life, and this belief is supported by the evidence that no individual has yet been identified with a total absence of ALDH1A1 catalytic activity.[15]

The gene coding for this enzyme, ALDH1A1, encodes 501 amino acid residues and is located on human chromosome 9q21.13. Its transcript (NM_000698) contains 13 exons and the gene is approximately 52 kilobases (kb) in length (NW_924484).[16, 17] In the promoter region, an ATA box and a CCAAT box are located 32 and 74 base pairs (bp), respectively, upstream from the transcription initiation site. The transcription initiation site is located 53 bp upstream from the A of the initiation codon (ATG).[18–20] Two polymorphisms have been previously identified in the promoter region of ALDH1A1. The ALDH1A1*2 allele contains a 17 bp deletion from position --416 to -432 compared with the ALDH1A1*1 allele, and the ALDH1A1*3 allele has a three bp insertion at -- 524.[3]ALDH1A1*2 was observed at frequencies of 0.035, 0.023, 0.023 and 0.012 in Asian, Caucasian, Jewish and African-American individuals, respectively, while ALDH1A1*3 was only observed in African-American individuals, at a frequency of 0.029.[3] These polymorphisms also have been observed in Mission Indians of Southwest California, where an allele frequency of 0.03 was detected for ALDH1A1*2. Two subjects possessed the ALDH1A1*2 allele and one subject displayed both the ALDH1A1*2 and ALDH1A1*3 alleles.[21]

Trinidad and Tobago is a twin island country, located at the southern end of the Caribbean chain of islands, 10 km (seven miles) north-east of the coast of Venezuela. The population of the country is multi-ethnic but the two largest ethnic groups are those of East Indian (40.0 per cent, Indo-Trinidadians) and African (37.5 per cent, Afro-Trinidadians) descent.[22] The ancestors of the Afro-Trinidadians were originally from West Africa and the Indo-Trinidadians came mainly from northern and southern India. The estimated rate of alcohol problems in this country is approximately 47 per cent for Indo-Trinidadians and 33 per cent for Afro-Trinidadians.[23] The frequency of genotypes of alcohol metabolism in this population was unknown until recently. A study was undertaken that evaluated associations of ALDH1A1 promoter polymorphisms with alcohol-related phenotypes in this population [24]. In that study, the allele frequencies for ALDH1A1*1, ALDH1A1*2 and ALDH1A1*3 in Afro-Trinidadians were found to be 0.941, 0.035 and 0.024, respectively, and 0.926, 0.074 and 0.000 in Indo-Trinidadians. The present paper reports the sequence of a novel allele identified in that study.

PCR analysis, using the ALDH1A1 forward and reverse PCR primers, generated products with corresponding sizes as follows: ALDH1A1*1 = 209 bp; ALDH1A1*2 = 192 bp; ALDH1A1*3 = 212 bp and ALDH1A1*4 = 210 bp (Figure 1). These genetic variations have been previously detected in other populations. The ALDH1A1*2 allele has been identified in diverse ethnic populations, including, Asians, Caucasians and African-Americans, while ALDH1A1*3 has only been discovered thus far in African-Americans, Mission Indians and Afro-Trinidadians.[3, 21, 24]

In our sample, for one individual, an Indo-Trinidadian alcohol-dependent subject, a slightly different allele size was detected on the autoradio-gram; it appeared to be slightly larger than the ALDH1A1*1 allele and smaller than the ALDH1A1*3 allele (Figure 1). By sequencing the PCR product, this unique allele was confirmed to have an A insertion at position -554 relative to the transcriptional start site (M31982). This new allele was named ALDH1A1*4, in accordance with the nomenclature rules, and, therefore, the genotype of the subject would be ALDH1A1*2/*4. The relative positions of the three polymorphisms are shown in Figure 2.

This new ALDH1A1*4 allele was not discovered in any of our Afro-Trinidadian subjects and its frequency in other populations is not known. Therefore, future research in relation to this allele would have to incorporate a wider cross-section of the population of Trinidad and Tobago in order to determine the representative frequency ofthis allele in the respective ethnic groups. In addition, further analyses would be required to determine the expression difference,if any, of the ALDH1A1*4 isozyme.

Establishing the function and kinetics of the ALDH1A1*4 isozyme will also be valuable for the future. Cloning the mRNA and expressing, as well as isolating, the protein for kinetic analysis would lead ultimately to determining the K_m. These data will definitely add to the body of knowledge of aldehyde dehydrogenase enzymes and their associated genetic influences. Differences in expression may produce altered acetaldehyde, neurotransmitter and retinoic acid metabolism, and have an impact on the development of alcohol dependence and alcohol-related disorders, as well as on other physiological functions. Conducting similar studies in the populations of origin of our inhabitants (ie India and West Africa) could provide genotypic and phenotypic associations relating to the presence of these polymorphisms, and perhaps serve as predictors of alcohol disorders and other pathologies.