Molecular characterization of the human delta-aminolevulinate dehydratase 2 (ALAD2) allele: implications for molecular screening of individuals for genetic susceptibility to lead poisoning

Abstract

The second enzyme in the heme biosynthetic pathway, delta-aminolevulinate dehydratase (ALAD), is a homooctameric protein encoded by a gene localized to human chromosome 9q34. Expression of the two common alleles, ALAD1 (p = .9) and ALAD2 (q = .1), results in a polymorphic enzyme system with three distinct charge isozymes, designated 1-1, 1-2, and 2-2. Individuals heterozygous (2pq = .18) or homozygous (q2 = .01) for the ALAD2 allele have significantly higher blood lead levels than do ALAD1 homozygotes, when exposed to low or high levels of lead in the environment. To investigate the molecular nature of this common polymorphism, total RNA from an ALAD2 homozygote was oligo-dT primed and reverse transcribed, and then the ALAD2 cDNA was amplified, subcloned, and sequenced. Compared with the ALAD1 sequence, the only difference in the ALAD2 cDNA was a G-to-C transversion of nucleotide 177 in the coding region, which created an MspI restriction site. This base substitution predicted the replacement of a positively charged lysine by a neutral asparagine (K59N), an amino acid change consistent with the more electronegative charge of the ALAD-2 subunit. The ALAD1 and ALAD2 alleles were easily detected by amplification of a 916-bp region of genomic DNA and MspI digestion which results in 582- and 511-bp products, respectively. Molecular analysis of 85 ALAD1/ALAD2 heterozygotes and of eight ALAD2 homozygotes revealed no discrepancy between the predicted genotype and the erythrocyte isozyme phenotype, indicating that all the ALAD2 alleles analyzed had the G-to-C transversion.(ABSTRACT TRUNCATED AT 250 WORDS)