ALAD Alleles Research Articles

ALAD Porphyria Is a Conformational Disease

ALAD porphyria is a rare porphyric disorder, with five documented compound heterozygous patients, and it is caused by a profound lack of porphobilinogen synthase (PBGS) activity. PBGS, also called "delta-aminolevulinate dehydratase," is encoded by the ALAD gene and catalyzes the second step in the biosynthesis of heme. ALAD porphyria is a recessive disorder; there are two common variant ALAD alleles, which encode K59 and N59, and eight known porphyria-associated ALAD mutations, which encode F12L, E89K, C132R, G133R, V153M, R240W, A274T, and V275M. Human PBGS exists as an equilibrium of functionally distinct quaternary structure assemblies, known as "morpheeins," in which one functional homo-oligomer can dissociate, change conformation, and reassociate into a different oligomer. In the case of human PBGS, the two assemblies are a high-activity octamer and a low-activity hexamer. The current study quantifies the morpheein forms of human PBGS for the common and porphyria-associated variants. Heterologous expression in Escherichia coli, followed by separation of the octameric and hexameric assemblies on an ion-exchange column, showed that the percentage of hexamer for F12L (100%), R240W (80%), G133R (48%), C132R (36%), E89K (31%), and A274T (14%) was appreciably larger than for the wild-type proteins K59 and N59 (0% and 3%, respectively). All eight porphyria-associated variants, including V153M and V275M, showed an increased propensity to form the hexamer, according to a kinetic analysis. Thus, all porphyria-associated human PBGS variants are found to shift the morpheein equilibrium for PBGS toward the less active hexamer. We propose that the disequilibrium of morpheein assemblies broadens the definition of conformational diseases beyond the prion disorders and that ALAD porphyria is the first example of a morpheein-based conformational disease.

Interaction between blood lead concentration and δ‐amino‐levulinic acid dehydratase gene polymorphisms increases the odds of essential tremor

Blood lead (BPb) concentrations are elevated in essential tremor (ET) cases. The delta-amino-levulinic acid dehydratase (ALAD) gene codes for ALAD, the principal enzyme involved in lead kinetics. Carriers of the ALAD-2 allele may be more susceptible to lead toxicity. The objective of this study was to test, using a case-control design, whether an interaction between BPb concentration and ALAD allele status increases the odds of ET. Mean log BPb concentration was significantly higher in 63 cases than 101 controls (0.47+/-0.26 vs. 0.35+/-0.25 microg/dl). Eighteen (28.6%) cases vs. 17 (16.8%) controls had an ALAD-2 allele (OR=1.98; 95% CI=0.93--4.21). In an adjusted logistic regression analysis, the interaction term (ALAD allele status x log BPb concentration) was associated with increased odds for ET. In stratified analyses, log BPb concentration was not associated with odds of ET in individuals with two ALAD-1 alleles (OR=2.69; 95% CI=0.61--11.82), but in individuals with an ALAD-2 allele, BPb concentration was significantly associated with odds of ET (OR=80.29; 95% CI=3.08--2,096.36). There was an interaction between BPb concentration and ALAD allele status; the odds of ET were greatly elevated in individuals with both an ALAD-2 allele and an elevated BPb concentration. The presence of increased circulating BPb concentrations along with a greater potential for lead toxicity (ALAD-2 allele) could result in greater cerebellar damage, thereby increasing the risk of developing tremor.

Novel molecular defects of the δ-aminolevulinate dehydratase gene in a patient with inherited acute hepatic porphyria

Cloning and expression of the defective gene for delta-aminolevulinate dehydratase (ALAD) from the second of 2 German patients with ALAD deficiency porphyria (ADP), who had been originally reported by Doss et al. in 1979, were performed. Cloning of cDNAs for the defective ALAD were performed using Epstein-Barr virus (EBV)-transformed lymphoblastoid cells of the proband, and nucleotide sequences of cloned cDNA were determined. Two separate mutations of ALAD cDNA were identified in each ALAD allele. One was G457A, termed "H1," resulting in V153M substitution, while the other was a deletion of 2 sequential bases at T(818) and C(819), termed "H2," resulting in a frame shift with a premature stop codon at the amino acid position of 294. Using allele-specific oligonucleotide hybridization, the mother of the proband was shown to have an H1 defect, while using genomic DNA analysis, the father was shown to have an H2 defect. Expression of H1 cDNA in Chinese hamster ovary cells produced an ALAD protein with only a partial activity (10.65% +/- 1.80% of the normal), while H2 cDNA encoded no significant protein. These data thus demonstrate that the proband was associated with 2 novel molecular defects of the ALAD gene, 1 in each allele, and account for the extremely low ALAD activity in his erythrocytes ( approximately 1% of normal).

Erythrocyte delta-aminolevulinic acid dehydratase genotype and other mechanisms affecting workers' susceptibility to lead.

In this study, the role of delta-aminolevulinic acid dehydratase (ALAD) variants in lead susceptibility was examined. The study subjects comprised 223 male workers, and the relationship between their blood lead level and erythrocyte ALAD activity or plasma/urine delta-aminolevulinic acid level was studied. Leukocyte specimens from 11 workers, whose erythrocyte ALAD activities were as low as one-fifth that of the other normal workers, were subjected to analyses of their ALAD and ALAD alleles. Further, the entire exon fragment of the ALAD gene was analyzed by polymerase chain reaction, and the reaction product was used as a target for direct DNA sequencing. Genomic DNA analysis revealed that all 11 workers had the ALAD allele, whereas the entire ALAD gene analysis failed to indicate other variants, except for the Rsa I site. The depletion in erythrocyte ALAD activity was not found to be caused by the ALAD allele.