Ching-Yu Lin

Deletion of the C4-CYP21 repeat module leading to the formation of a chimeric CYP21P/CYP21 gene in a 9.3-kb fragment as a cause of steroid 21-hydroxylase deficiency

Gross gene deletions have been reported in 20% of alleles in patients with congenital adrenal hyperplasia (CAH) involving a 21-hydroxylase deficiency (1). This type of deletion occurs in the RCCX module, including the CYP21P , tenascin A ( TNXA ), RP2 , C4B , CYP21 , and tenascin B ( TNXB ) genes, as evidenced by a 30-kb deletion identified by pulse-field electrophoresis (2). Inactivation of the CYP21 gene may also occur through intergenic recombination with transfer of deleterious mutations from the neighboring CYP21P pseudogene. The frequency of gene deletions or conversions in CAH is controversial (3)(4)(5) and is dependent on the population studied. Evidence for gene deletions and/or conversions is traditionally obtained by Southern blot analysis. Multiple probes and separate restriction endonuclease digestions are used. Taq I generates 3.7-kb (functional) and 3.2-kb (pseudogene) fragments, and Bgl II produces 11-kb (functional) and 12-kb (pseudogene) fragments. These analyses have been used since 1984 (1)(3)(5)(6)(7)(8)(9). However, the method is indirect and time-consuming, and densitometry of fragments can be prone to error. To identify the interchange region and improve detection of gene deletions and conversions in the RCCX module (10)(11)(12)(13), we have developed a novel Southern blot analysis that uses two restriction endonucleases, Ase I and Nde I, and requires only one probe. In addition, we use a PCR product amplified with locus-specific primers covering the TNXB gene to the 5′ end of CYP21P or CYP21 to directly analyze the 3.2/3.7-kb Taq I fragment and the status of the CYP21 gene. For the novel Southern blot analysis, 10 μg of genomic DNA was digested with the restriction endonucleases Ase I and Nde I, resolved on a 0.65% agarose gel, blotted on a Hybond-N+ membrane …

Structural analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency

AbstractCongenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed “apparent gene conversion,” from the neighboring CYP21P pseudogene. A chimeric CYP21P/CYP21 gene with its 5′ end corresponding to CYP21P and 3′ end corresponding to CYP21 has been identified. This type of gene is nonfunctional because it produces a truncated protein. We found two distinct chimeric genes in CAH patients. Both genes had a sequence with −300 nucleotides of the 5′ head as the CYP21P gene. The coding region consisted of a fusion molecule with the CYP21P gene in two different regions. One of the junctions was located in the chi-like sequence of GCTGGGC in the third intron and the other was in the minisatellite consensus TGGCAGGAGG of exon 5 of the CYP21P gene. In addition, analysis of restriction fragment length polymorphism for these two 3.3-kb chimeric molecules showed that these sequences arose as a consequence of unequal crossover between the CYP21P and CYP21 genes. It is plausible that both consensus sequences are responsible for the gene conversion of these two chimeric genes.