Screening for Mutations of 21-Hydroxylase Gene in Hungarian Patients with Congenital Adrenal Hyperplasia1

Abstract

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders, causing impaired secretion of cortisol and aldosterone from the adrenal cortex, with subsequent overproduction of adrenal androgens. The most common enzyme defect causing CAH is steroid 21-hydroxylase deficiency. To determine the mutational spectrum in the Hungarian CAH population, the CYP21 active gene was analyzed using PCR. A total of 297 Hungarian patients with 21-hydroxylase deficiency are registered in the 2nd Department of Pediatrics, Budapest, Hungary, and their clinical status was evaluated. Blood samples for CYP21 genotype determination could be obtained from 167 patients (representing 306 unrelated chromosomes and 56.2% of the total group of patients). Eight of the most common mutations were screened [In2 (intron 2 splice mutation), I172N, Del (Del: apparents large gene conversion), Q318X, R356W, 1761Tins, ClusterE6, V281L] using allele-specific amplification. The most frequent mutation in the Hungarian CAH population was found to be In2. Our results have shown a good genotype/phenotype correlation in case of most mutations; the In2 mutation is associated mostly with the severe form of the disease, whereas I172N was expressed in a wide spectrum of phenotypes. 1999)

Genotyping the -521C/T functional polymorphism in the promoter region of dopamine D4 receptor (DRD4) gene

“…is complementary to a specific allele of a polymorphic site [21][22][23]. When genotyping with ASA, the number of necessary parallel PCR reactions equals the number of possible alleles.…”

Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix

“…Peripheral blood was collected from patients with CAH at the 2 nd Department of Pediatrics of Semmelweis University, Budapest, Hungary; genomic DNA was isolated from leukocytes by phenol-chloroform extraction as described earlier [16]. The samples were stored at ±80 o C until use.…”

“…The frequency of the classic forms is approximately 1 out of 15 000 live birth [15]. The mutation at nucleotide position 659 in intron 2 is the most common in the Hungarian population with 21-OHD (> 30%) [16] and one of the most common in other populations as well and it is usually associated with the more severe salt-wasting and simple virilizing phenotypes. In normal individuals the nucleotide at position 659 is either A or C, which is replaced by G in case of a mutation.…”

“…This technique was quite tedious since all three possible genotypes (A, C and G) had to be checked at this position after the initial PCR, requiring three different allele-specific amplifications for each patient [16]. Our group has recently implemented the method of primer extension to make the analysis of the intron 2 mutation faster and easier.…”