Genetic studies to characterize the origin of the mutation in placental aromatase deficiency

Placental aromatase deficiency has recently been shown to be due to expression of RNA transcripts encoding abnormal aromatase molecules with 29 extra amino acids. To establish whether this aromatase deficiency is a hereditary or sporadic disease, we examined the genetic defect of the aromatase gene in the family of a patient. Direct sequencing of fragments of the aromatase gene prepared by PCR revealed that the splicing donor sequence (GT) of intron 6 in controls was mutated to GC in the patient, whereas the parents showed signals of both GT and GC. Subcloning of PCR products of the parents gave two different types of clones with GT and GC sequences in this site. Furthermore, for diagnosis of this deficiency, competitive-oligo-nucleotide-priming PCR of genomic DNA was performed in the presence of both normal and mutational oligonucleotide primers labeled with two kinds of fluorescent dyes, and the products were separated by agarose gel electrophoresis and were detected fluorometrically in the gel. Genomic DNA of the patient gave a PCR product primed only by the mutational primer, whereas that of controls gave a product primed only by the normal primer. The PCR products of the parents were primed by both primers. The results obtained by this fluorometric method were also confirmed by differential hybridizations with specific oligonucleotide probes. Thus these findings indicate that this deficiency is an autosomal hereditary disease and that the patient is a homozygote, while the parents are heterozygotes, for this mutation.