Two of four siblings expressed the
salt-losing form of
congenital adrenal hyperplasia due to
21-hydroxylase deficiency (CAH) and had identical human lymphocyte
antigen (HLA) and
complement C4 (fourth component of
complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv
ACTH stimulation test and HLA typing. In addition, the iv
ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH.
Restriction endonuclease digests of genomic
DNA obtained from members of this family and from normal unrelated subjects were hybridized with
cDNA probes encoding human
21-hydroxylase and C4. With the
21-hydroxylase probe, Southern blots prepared from control
DNA samples revealed two major restriction fragments in each of four
restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from
DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control
DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control
DNA samples. These data indicate the presence of two different
21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive
21-hydroxylase gene. When genomic
DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4
cDNA probe, the restriction fragment hybridization patterns for all four
endonuclease digests were similar in the two groups. Hence, our results suggest that the
21-hydroxylase deficiency of our patients is due to conversion of the active
21-hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B
protein, without any detectable alterations in the restriction fragment pattern of the C4 genes.