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

Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
European Journal of Endocrinology (Impact Factor: 3.69). 08/2005; 153(1):99-106. DOI: 10.1530/eje.1.01944
Source: PubMed

ABSTRACT To analyze the mutational spectrum of steroid 21-hydroxylase (CYP21) and the genotype- phenotype correlation in patients with congenital adrenal hyperplasia (CAH) registered in the Middle European Society for Pediatric Endocrinology CAH database, and to design a reliable and rational approach for CYP21 mutation detection in Middle European populations.
Molecular analysis of the CYP21 gene was performed in 432 CAH patients and 298 family members. Low-resolution genotyping was performed to detect the eight most common point mutations. High-resolution genotyping, including Southern blotting and sequencing was performed to detect CYP21 gene deletions, conversions, point mutations or other sequence changes.
CYP21 gene deletion and In2 and Ile172Asn mutation accounted for 72.7% of the affected alleles in the whole study group. A good genotype-phenotype correlation was observed, with the exception of Ile172Asn and Pro30Leu mutations. In 37% of patients low resolution genotyping could not identify the causative mutation or distinguish homozygosity from hemizygosity. Using high-resolution genotyping, the causative mutations could be identified in 341 out of 348 analyzed patients. A novel mutation Gln315Stop was found in one simple virilising CAH (SV-CAH) patient from Austria. In the remaining seven patients polymorphisms were identified as the leading sequence alteration. The presence of elevated basal and ACTH-stimulated 17-hydroxyprogesterone, premature pubarche, advanced bone age and clitoral hypertrophy directly implicated Asn493Ser polymorphism in the manifestation of nonclassical- (NC) and even SV-CAH.
By genotyping for the most common point mutations, CYP21 gene deletion/conversion and the 8 bp deletion in exon 3, it should be possible to identify the mutation in 94-99% of the diseased alleles in any investigated Middle European population. In patients with a mild form of the disease and no detectable mutation CYP21 gene polymorphisms should be considered as a plausible disease-causing mutation.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The objective was to seek evidence on the prevalence and consequences of heterozygous CYP21A2 mutations in girls, adolescent, and adult females with clinical manifestation of androgen excess. The study included 64 girls diagnosed with premature adrenarche (PA) in childhood and 141 females with clinical hyperandrogenemia manifested in adolescence or adulthood. Direct DNA sequencing and multiplex ligation-dependent probe amplification analysis were used to identify mutations in the CYP21A2 gene. (1) Thirty-four patients were diagnosed with nonclassical-congenital adrenal hyperplasia (NC-CAH) based on the 17-hydroxyprogesterone (17-OHP) levels and the presence of two mutations in CYP21A2 and therefore were excluded from the study, 66 were found to be heterozygotes and finally 105 had no identifiable mutations. The most frequent mutations among the carriers were the mild p.Val281 Leu and p.Qln318stop. Higher levels of mean stimulated 17-OHP were found in the carriers of the p.Val281 Leu. (2) A notable increased allelic frequency for the known p.Asn493 Ser polymorphism was observed in the pool of females with hyperandrogenemia in whom no mutation was identified. (3) In girls, who presented early with PA, 26.6% were diagnosed with NC-CAH and carried two mutations, 28.7% were identified as heterozygotes 43.7% had no identifiable genetic defect in the translated region of the CYP21A2 gene. On the contrary, in the group of 141 females with late onset hyperandrogenemia, the presence of 2 mutations was detected in 12%, 1 mutation in 33.4% and no mutation in 54.6%. The carrier status for 21-OHD, may be an important factor in the variable phenotype of hyperandrogenism and may be a contributing factor for the early manifestation of the disease.
    Indian journal of endocrinology and metabolism. 11/2014; 18(Suppl 1):S72-9.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: About 95% of the cases of congenital adrenal hyperplasia (CAH) are caused by mutations in CYP21A2 encoding 21-hydroxylase (21OH). Recently we reported four novel CYP21A2 variants in the Norwegian population of patients with CAH, of which p.L388R and p.E140K were associated with salt-wasting (SW), p.P45L with simple virilising (SV) and p.V211M + p.V281L with SV to non-classical (NC) phenotypes. To characterise the novel variants functionally utilising a newly designed in vitro assay of 21OH enzyme activity and structural simulations, and compare the results with clinical phenotypes. CYP21A2 mutations and variants were expressed in vitro. Enzyme activity was assayed by assessing conversion of 17-hydroxyprogesterone to 11-deoxycortisol by liquid chromatography tandem mass spectroscopy. PyMOL 1.3 was used for structural simulations, and PolyPhen2 and PROVEAN for predicting severity of the mutants. The CYP21A2 mutants, p.L388R and p.E140K, exhibited 1.1 % and 11.3%, of wild type (wt) 21OH enzyme activity, respectively, in vitro. We could not detect any functional deficiency of the p.P45L variant in vitro; although prediction tools suggest p.P45L to be pathogenic. p.V211M displayed enzyme activity equivalent to the wt in vitro, which was supported by in silico analyses. p.A159T was proposed to be a normal variant by both evaluation methods. We found good correlations between phenotype and the in vitro enzyme activities of the SW mutants, but not for the SV p.P45L variant. p.V211M might have a synergistic effect together with p.V281L explaining a phenotype between SV and NC CAH.
    Endocrine connections. 03/2014;
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To seek evidence on the prevalence of CYP21A2 genetic defects and consequences in girls with premature adrenarche (PA). The study included 59 girls diagnosed with PA. Direct DNA sequencing and MLPA analysis were performed to identify mutations in CYP21A2 gene. Twelve girls were diagnosed with non-classic congenital adrenal hyperplasia (NC-CAH) based on stimulated 17-hydroxyprogesterone (17-OHP) levels and the presence of two mutations in CYP21A2, 19 were heterozygotes. The most frequent mutations detected were the mild p.Val281Leu and p.Pro453Ser. Higher levels of mean stimulated 17-OHP were found in the carriers of the p.Val281Leu mutation. The detection rate for two CYP21A2 mutations was higher in girls with PA than in adult females with hyperandrogenemia in our studied population. A notable increased allelic frequency for the known p.Asn493Ser polymorphism was observed in the pool of the 28 girls with PA in whom no mutation was identified. In girls with PA, the frequency of the underlying CYP21A2 genetic defects is similar to that observed in other populations. The carrier status is likely a contributing factor in the genotype-phenotype correlation in NC-CAH. However, polymorphisms and other genes may be implicated in the clinical manifestation of the disease.
    Journal of endocrinological investigation 12/2014; · 1.65 Impact Factor