Molecular analysis of delta-aminolevulinate dehydratase deficiency in a patient with an unusual late-onset porphyria.

Cloning, expression, and genotype studies of the defective gene for delta-aminolevulinate dehydratase (ALAD) in a patient with an unusual late onset of ALAD deficiency porphyria (ADP) were carried out. This patient was unique in that he developed the inherited disease, together with polycythemia, at the age of 63. ALAD activity in erythrocytes of the patient was less than 1% of the normal control level. ALAD complementary DNA (cDNA) isolated from the patient's Epstein-Barr virus (EBV)-transformed lymphoblastoid cells had 2 base transitions in the same allele, G(177) to C and G(397) to A, resulting in amino acid substitutions K59N and G133R, respectively. It has been verified that the patient had no other ALAD mutations in this and in the other allele. By restriction fragment length polymorphism (RFLP) analysis, all family members of the proband who had one-half ALAD activity compared with the ALAD activity of the healthy control were shown to have the same set of base transitions. Expression of ALAD cDNA in CHO cells revealed that K59N cDNA produced a protein with normal ALAD activity, while G133R and K59N/G133R cDNA produced proteins with 8% and 16% ALAD activity, respectively, compared with that expressed by the wild type cDNA. These findings indicate that while the proband was heterozygous for ALAD deficiency, the G(397) to A transition resulting in the G133R substitution is responsible for ADP, and the clinical porphyria developed presumably due to an expansion of the polycythemic clone in erythrocytes that carried the mutant alad allele.