Novità nella diagnosi genetica di iperplasia surrenale congenita da deficit di 21-idrossilasi

SommarioLa ricerca di varianti patogenetiche del gene CYP21A2, responsabili del 90–95% dei casi di iperplasia surrenale congenita, è resa altamente complessa dall'omologia di sequenza con lo pseudogene, dall'elevata frequenza di ricombinazione del locus e dal numero variabile di moduli ripetuti. Tuttavia, grazie alle recenti conoscenze e alle nuove metodiche si sono raggiunti ottimi livelli diagnostici importanti per una corretta diagnosi e una consulenza genetica affidabile, nonché per individuare patologie associate

Impact of molecular genetics on congenital adrenal hyperplasia management.

Congenital adrenal hyperplasia (CAH) is a family of autosomal recessive disorders caused by mutations in genes encoding the enzymes involved in one of the 5 steps of adrenal steroid synthesis or the electron donor P450 oxidoreductase (POR) enzyme. Steroid 21-hydroxylase deficiency (21-OHD), the principal focus of this review, accounts for about 90-95% of all CAH cases, and its biochemical and clinical severity depends on the underlying CYP21A2 gene disruption. Molecular genetic advancements have been achieved in recent years, and the aim of this review is to attempt to highlight its contribution to the comprehension and management of the disease. When possible, we will try to achieve this goal also by providing some results from our personal experience regarding: some aspects of CYP21A2 gene analysis, with basic genotype/phenotype relationships; its crucial role in both genetic counselling and in prenatal diagnosis and treatment in families at risk for 21-OHD; its help in the comprehension of the severity of the disease in patients diagnosed by neonatal screening and possibly treated before an evident salt-loss crisis or before performing adequate blood sampling; its usefulness in the definition of post ACTH 17-hydroxyprogesterone values, discriminating between non-classic, heterozygote and normal subjects; and finally the contribution of genes other than CYP21A2 whose function or dysfunction could influence 21-hydroxylase activity and modify the presentation or management of the disease

Improving the diagnosis of 11β-hydroxylase deficiency using home-made MLPA probes: identification of a novel chimeric CYP11B2/CYP11B1 gene in a Sicilian patient

Purpose: 11β-Hydroxylase deficiency (11OHD) represents the second most common cause of congenital adrenal hyperplasia. It is caused by mutations in the CYP11B1 gene localized about 40 kb from the CYP11B2 gene with which it shares a homology of 95 %. The asymmetric recombination of these two genes is involved both in 11OHD and in glucocorticoid-remediable aldosteronism (GRA). Our objective was to set up an easy and rapid method to detect these hybrid genes and other kinds of deletions, to improve the molecular diagnosis of 11OHD. Methods: A set of 8 specific probes for both the CYP11B1 and the CYP11B2 genes to be used for multiplex ligation-dependent probe amplification (MLPA) analysis was designed to detect rearrangements of these genes. Results: The method developed was tested on 15 healthy controls and was proved to be specific and reliable; it led us to identify a novel chimeric CYP11B2/CYP11B1 gene in one patient that carried the known A306V mutation on the other allele. Specific amplification and sequencing of the hybrid gene confirmed the breakpoint localization in the second intron. Conclusions: The MLPA kit developed enables the detection of deletions, duplications or chimeric genes and represents an optimal supplement to DNA sequence analysis in patients with 11OHD. In addition, it can also be used to show the presence of the opposite chimaera associated with GRA

Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11 beta-hydroxylase deficiency (11 beta-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11 beta-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11 beta-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11 beta-OHD showed a nearly complete loss of 11 beta-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11 beta-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11 beta-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP 11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling. published online 11 September 201

Identification of rare alleles in an Italian population of 284 patients with 21- hydroxylase deficiency by complete sequencing of the CYP21 gene

The screening of the usually tested CYP21 gene alterations (the large gene deletion/conversion and the P30L, IVS2-13A/C>G, Ex 3 D8nt, Ex6 cluster, I172N, V281L, 1766-1767insT, Q318X, R356Q, P453S) by means of Southern blotting and Allele-Specific PCR in an Italian population of 284 patients with 21-hydroxylase deficiency led us to characterize 461 of the total 506 different alleles (91.1%), while 45 (8,9 %) remained uncharacterized. Some incongruent genotype/phenotype correlation was also observed. In order to reduce the number of uncharacterized alleles and possibly to identify additional alterations in some patients’ alleles, we reanalysed the entire population (128 classic and 156 nonclassic forms selected for stimulated 17-HP level>10 ng/ml) by complete sequencing of the CYP21 gene, promoter included. Identified mutations were verified in the available parents to confirm allele segregation. Result a: of the 461 characterized alleles, 10 present an additional/different known mutation from that previously identified (2.1% false positive/negative results); 7 showed a conversion extending from the promoter to the P30L mutation (3 cases) or the IVS2 (4 cases). Result b: of the 34 uncharacterized alleles, 16 showed rare mutations (1 M283V, 1 R316X, 4 R341P, 4 R356Q, 1 R426H, 4 P482S, 1 R483P); 7 showed as far as we know unique mutations, 2 (W19X, L480Xfs) with an obvious implication in the phenotype, and 5 affecting residues L142, I171, R341, V358, L446 currently being studied; 11 remained uncharacterized. A total of 43 (23 of pseudogene origin) SNPs, 15 in the promoter, 3 in the 3’ UTR and 25 in introns were identified: further studies will verify their frequency in controls and the possible implication in the phenotypes. The high frequence of complex, rare or unique alleles in the Italian population underlines the importance to routinely analyse the CY21 gene by complete sequencing to avoid false/incomplete results, identify new mutations or sequence variations and to improve the genotype/phenotype correlation, genetic counselling and treatment