T→A transversion 11 bp from a splice acceptor site in the human gene for steroidogenic acute regulatory protein causes congenital lipoid adrenal hyperplasia

Congenial lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH. Affected individuals can make no adrenal or gonadal steroids. All affected individuals are phenotypic females irrespective of gonadal sex, and frequently die in infancy if mineralocorticoid and glucocorticoid replacements are not instituted. Recent data implicate the steroidogenic acute regulatory (StAR) protein in this disorder. We now describe a 46,XY patient of Vietnamese ancestry with lipoid CAH who had a somewhat milder form of the disease. Diagnosis was at 10 weeks of age, and low levels of plasma progesterone, corticosterone, 180H-corticosterone and androstenedione were detectable. Testicular RNA for StAR was reverse transcribed, amplified, cloned and sequenced, revealing a 185 bp deletion corresponding to all of exon 5. The corresponding mRNA did not encode active protein in transfected cells. Cloned genomic DNA from the patient revealed only a T-->A transversion in intron 4,11 bp from the splice acceptor site of exon 5. This transversion destroys an NcoI site; digestion of PCR-amplified genomic DNA from the patient and both parents confirmed that the patient was homozygous and the parents were heterozygous. Expression vectors for StAR minigenes were constructed containing all StAR exons plus introns 4, 5 and 6 either with or without the T-->A mutation in intron 4. RNase protection assays showed that expression of the vector with normal intron 4 yielded correctly spliced StAR mRNA in transfected COS-1 cells, while most, but not all StAR mRNA from the vector with the T-->A transversion in intron 4 was abnormally spliced. RNase protection of the patient's testicular RNA confirmed that most, but not all StAR mRNA was similarly spliced abnormally. Splicing errors appear to be a rare cause of genetic diseases, but subtle intronic mutations may be missed when genomic DNA is the only material available for study. The low level of normal StAR mRNA produced may account for the later clinical presentation and low levels of steroid hormones detected in this patient.