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).