Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease

Abstract

Objective: To analyse the mutational spectrum, the associated haplotypes and the genotype -phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. Design: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. Methods: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. Results: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. Conclusions: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.

Mutational spectrum of steroid 21-hydroxylase and the genotype-phenotype association in Middle European patients with congenital adrenal hyperplasia

“…This phenotypic variability of Ile172Asn mutation has also been reported in other European populations showing an otherwise good correlation between the genotype and phenotype (3,10,19,20). Similarly, many investigators have reported phenotypes more severe than expected in carriers of the Pro30Leu mutation (9,10,15,18,21). The finding of the Pro30Leu mutation linked to a gene conversion in the promoter region or with an additional mutation, Ala15Thr, could explain the increased severity of phenotypes in Slovenian patients (18).…”

“…Similarly, many investigators have reported phenotypes more severe than expected in carriers of the Pro30Leu mutation (9,10,15,18,21). The finding of the Pro30Leu mutation linked to a gene conversion in the promoter region or with an additional mutation, Ala15Thr, could explain the increased severity of phenotypes in Slovenian patients (18). The in vitro expression analysis did not demonstrate the impairment of enzyme activity by Ala15Thr mutation alone (22) but it is not excluded that this mutation has a synergistic effect on 21-hydroxylase activity when combined with the Pro30Leu mutation and/or gene conversion in the promoter region (23).…”

“…Alleles with multiple mutations were grouped according to the most severe mutation of the haplotype (Table 2). Although the impairment of enzyme activity for CYP21 gene conversion involving the promoter region alone or combined with Pro30Leu substitution has not been established in vitro these mutations were categorized in group B based on the previous reports of clinical phenotypes more severe than predicted in patients carrying these mutations (15,18). As summarized in Table 2 a good genotype -phenotype correlation was observed in patients with severe mutations categorised in mutation groups 0 and A.…”

“…High resolution genotyping by PCR-SSP, PCR-SSO, Southern blotting and sequencing was performed in Ljubljana to detect CYP21 gene deletions, conversions, point mutations or other sequence changes when mutation could not be identified on one or both alleles by low resolution genotyping or when homozygosity could not be distinguished from hemizygosity. The genotyping approach is described in detail elsewhere (17,18). The reference sequence reported by White et al (4) was used for numbering of nucleotides and amino acids.…”

Clinical Role of CYP2C19 Polymorphisms in Patients with Congenital Adrenal Hyperplasia Due to 21-hydroxylase Deficiency

“…The results of molecular analyses of 24 out of 28 patients were previously reported. 6,15 Legend: The step that can be augmented by extraadrenal hydoxylation via hepatic cytochrome P450 CYP2C19 is bolded. CYP11A1 -cholesterol side-chain cleavage enzyme; CYP17 -17-alpha-hydroxylase / 17,20-lyase; HSD3B2 -3β-hydroxysteroid dehydrogenase; CYP21A2 -steroid 21-hydroxylase; CYP11B1-steroid 11-β-hydroxylase; CYP11B2 -aldosterone synthase; 17β-HSD -17-beta-hydroxysteroid dehydrogenase.…”