Molecular Genetic Analysis Of Chinese Families With 21-Hydroxylase Deficiency

Objectives:Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder with impaired cortisol secretion from the adrenal glands. The major cause of CAH is 21-hydroxylase deficiency (21-OHD), which accounts for 90%～95% of all cases in most populations. Clinical phenotype depends on the CYP21 genotype and other factors. CYP21 is. located in a locus with a complicated structure. It is found on the short arm of chromosome 6 (band 6p21.3) together with a highly homologous inactive pseudogene, CYP21P. Basically, 95% of the mutated alleles in patients with 21-OHD are due to the conversion of DNA sequences from CYP21P pseudogene to the active CYP21 gene; the remaining alleles have new mutations due to random events. Most of the rare mutations are unique for single families or are considered as population specific. This study aimed to reveal the mutations and their origins of the patients who were diagnosed as 21-OHD , and to characterize the molecular genetic basis of 21-OHD.Methods:The CYP21 gene mutations were analyzed in four patients with 21-hydroxylase deficiency and their relatives. The genomic DNA of the patients and their relatives was isolated from whole blood.Two pairs of primers were used to amplify the CYP21 gene. The amplified PCR products were purified by agarose gel and then directly sequenced; Two microsatellite loci flanking the CYP21 gene (D6S273 and D6S291) were studied in all patients and relatives who carry these mutations.Results:1.The fragments (P1-P2 and P3-P4 PCR products) of all patients were consistent with the fragments of normal individuals. So there was no the gene deletion/conversion (large deletions).2.We found six kinds of mutations. In the first family, the patient was a compound heterozygote carrying four different mutations (Cluster E6, Q318X, A391T, P459H) on CYP21 gene. Three mutations (Cluster E6, Q318X, A391T) were on her maternal allele, and a novel mutation was found: P459H. It was located at codon 459 in exon 10 and it changed a proline(CCC) to a histidine (CAC). A391T was a rare mutation. In the second family, we found two kinds of mutations: Cluster E6 and R483W. R483W was also a rare mutation. In the third family, the sequencing of the CYP21 gene of two patients revealed a homozygous T>A transition in codon 172 leading to the substitution of isoleucine by asparagine (I172N).3.Microsatellite studies showed the same haplotype in the patients and their relatives who carry these mutations.Conclusion:1.We found six kinds of mutations in the three families with 21-OHD. With direct sequencing we identified a novel mutation (P459H) and two rare mutations (A391T,R483W) of the CYP21 gene.2.Neither of these three point mutations has been observed in CYP21P pseudogene, which suggests that they were not originated from microconversion events, but probably represented random events.3.The patients with the same clinical phenotype may belong to different genotypes;The same clinical phenotype may be caused by different mutations.