Identification of a novel cytosolic aldehyde dehydrogenase allele, ALDH1A1*4.

This paper reports the identification of a novel cytosolic aldehyde dehydrogenase 1 ( ALDH1A1 ) allele. One hundred and sixty-two Indo-Trinidadian and 85 Afro-Trinidadian individuals were genotyped. A novel ALDH1A1 allele, ALDH1A1*4 , was identified in an Indo-Trinidadian alcoholic with an A inserted at position -554 relative to the translational start site, +1. It was concluded that a wider cross-section of individuals needs to be evaluated in order to determine the representative frequency of the allele, and to see if it is associated with risk of alcoholism.

Introduction

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 Km (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 (Kcat/Km)[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 Km of 0.4 μM and 1.5 μM for 3,4-dihydroxyphenylacetaldehyde and phenylacetaldehyde, respectively, and near equivalent Kcat values for these substrates.[14] ALDH1A1 has also displayed a Km of 0.06 μM in retinaldehyde metabolism, with the Kcat 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.

Materials and methods

Subjects

Patients were recruited from admissions to the substance abuse centres at Caura, San Fernando General and Scarborough Regional hospitals. There were no differences in admission or treatment based on ethnicity. Control subjects of both ethnic groups were matched by age, sex and ethnicity to the alcohol-dependent participants, and were recruited through fliers distributed in the communities and also by word of mouth. Whole-blood samples for genotyping were taken from a total of 247 individuals (162 Indo-Trinidadian and 85 Afro-Trinidadian individuals), which included both alcohol dependent (n = 139) and non-alcohol-dependent (n = 108) subjects. Diagnosis of alcohol dependence was assessed using the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA).[25,26] The study was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association, and approval for the study was obtained from the ethics committees of the participating hospitals (San Fernando General, Caura and Scarborough Regional), the Faculty of Medical Sciences at the University of the West Indies and the Institutional Review Board (IRB) at The Scripps Research Institute. Informed, written consent was obtained from all participants before inclusion into the study.

Genotyping

Genomic DNA was isolated from dried blood spots.[27] The primers, ALDHlA-forward (5'-GCACTGAAAATACACAAGACTGAT-3') and ALDHlA-reverse (5'-AGAATTTGAGGATTG AAAAGAGTC-3'), were designed on the basis of human ALDH1A1 exon 1 and promoter sequences (accession number M31982), and used in polymer-ase chain reaction (PCR) reactions to obtain [α-33P] deoxycytidine triphosphate-radiolabelled fragments. Products were electrophoresed on 6 per cent acrylamide denaturing gels and scored on the basis of the mobility of each resulting PCR fragment.

Results and discussion

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]

Figure 1

Autoradiogram of four human . PCR was used to generate [a-33P] deoxycytidine triphosphate-radiolabelled fragments, which were separated by size on a 6 per cent acrylamide gel by electrophoresis. ALDHIAI*I is 209 bp, ALDHIAI*2 192 bp, ALDHIAI*3 212 bp and ALDHIAI*4 210 bp in length. The ALDHIAI*4 allele is a new finding reported here. The inserted nucleotide was confirmed by sequence analysis. (a) A heterozygous genotype ALDHIAI*11*2. (b) A heterozygous genotype ALDHIAI*2/*4. (c) A homozygous genotype ALDHIAI*I*I . (d) A heterozygous genotype ALDHIAI*I*3.

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.

Figure 2

Schematic representation of the three human . The region designates the fragment that was amplified for the genotyping assay from -361 to -578. The two grey boxes represent the forward and reverse primers. The relative positions of the three ALDHIAI polymorphisms are indicated with black boxes: the 17 bp deletion (-4I6/-432), the 3 bp insertion (-524) and the 1 bp insertion (-554). The sequence flanking the inserted A (in bold) is: ACTGATAACGATA.

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 Km. 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.