Genetic characterization of a large cohort of Argentine 21-hydroxylase Deficiency.

CONTEXT: 21-hydroxylase deficiency is the most common cause of Congenital Adrenal Hyperplasia. It presents as severe or classical forms-salt wasting and simple virilizing-and a mild or nonclassical (NC). Several studies have reported the frequency of pathogenic variants in different populations, although few of them included a large number of NC patients. OBJECTIVE: To analyse the CYP21A2 gene defects in a large cohort of Argentine patients. DESIGN: Molecular characterization of 628 patients (168 classical, 460 nonclassical, representing 1203 nonrelated alleles), 398 relatives, 126 partners. METHODS: Genetic variants were assessed by allele-specific PCR, PCR-RFLP or direct sequencing. Deletions, duplications and large gene conversions (LGC) were studied by Southern blot/MLPA or long-range PCR. Biological implications of novel variants were analysed by structure-based in silico studies. RESULTS: The most frequent pathogenic variants were p.V282L (58%) in NC alleles and c.293-13C>G (31.8%) and p.I173N (21.1%) in classical. Deletions and LGC were found at low frequency (6.2%), 57 alleles had rare pathogenic variants, and 3 had novel variants: p.(S166F); p.(P189R), p.(R436L). Genotype-phenotype correlation was observed in 98.6% of the cases, 11 asymptomatic first-degree relatives had pathogenic variants in both alleles, and 21/126 partners were carriers. CONCLUSIONS: We conducted a comprehensive genetic characterization of the largest cohort of 21-hydroxylase patients from the region. In particular, we add to the molecular characterization of a large number of NC patients and to the estimation of the disease carrier's frequency in our population.

Functional studies of p.R132C, p.R149C, p.M283V, p.E431K, and a novel c.652-2A>G mutations of the CYP21A2 gene.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90-95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A>G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A>G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.

[Pediatric emergency: adrenal insufficiency and adrenal crisis]. / Emergencia pediátrica: insuficiencia suprarrenal aguda.

Adrenal insufficiency is defined by impaired secretion of adrenocortical hormones. It is classified upon the etiology in primary and secondary. Rapid recognition and therapy of adrenocortical crisis are critical to survival. Patients often have nonspecific symptoms: anorexia, vomiting, weakness, fatigue and lethargy. They are followed by hypotension, shock, hypoglicemia, hyponatremia and hyperkalemia. All patients with adrenal insufficiency require urgent fluid reposition, correction of hypoglycemia and glucocorticoid replacement, in order to avoid serious consequences of adrenal crisis. After initial crisis treatment, maintenance dose of corticoids should be indicated. Mineralocorticoids replacement, if necessary, should also be initiated.

Emergencia pediátrica: insuficiencia suprarrenal aguda / Pediatric emergency: adrenal insufficiency and adrenal crisis

La insuficiencia suprarrenal aguda es un cuadro originado por deficiencia mineralocorticoidea o glucocorticoidea, cuyo no diagnóstico y adecuado tratamiento lleva a una emergenciagrave con riesgo para la vida del paciente. Se clasifica en insuficiencia suprarrenal primaria, que presenta en general compromiso glucocorticoideo y mineralocorticoideo, y secundaria,sin deficiencia mineralocorticoidea. Los pacientes pueden no presentar síntomas que alerten precozmente, comoanorexia, náuseas, astenia, vómitos y dolor abdominal. De no corregirse, aparecen hipotensión, hipoglucemia, hiponatremia con hipercaliemia, deshidratación y shock. Es indispensable,aun en caso de duda, corregir la hipovolemia, el desequilibrioelectrolítico y la hipoglucemia, y administrar glucocorticoidesa dosis de estrés. Superada la fase aguda, administrar la dosis de corticoides de mantenimiento y, en caso necesario, añadir mineralocorticoides.

Classical and nonclassical 21-hydroxylase deficiency: a molecular study of Argentine patients.

OBJECTIVE: To characterize the molecular basis of the 21-hydroxylase deficiency in a group of Argentine patients presenting the classical and nonclassical forms of the disease. DESIGN: To analyse the frequency of point mutations in the CYP21 gene by DNA amplification and mutation detection. PATIENTS: Forty-one patients from 36 nonrelated families: 25 nonclassical (NC), 11 salt-wasting (SW) and five simple virilizing (SV). A total of 27 parents and 13 nonaffected siblings were also analysed. MEASUREMENTS: Basal steroid hormones and 17-hydroxyprogesterone levels following adrenal stimulation with adrenocorticotrophic hormone were measured, together with an analysis of 10 point mutations in the CYP21 gene. RESULTS: A total of 83% and 74.4% classical and nonclassical chromosomes, respectively, were characterized. The intron 2 mutation was the most prevalent among classical alleles. In addition, a high frequency for R356W was observed in both groups (13.3 and 6.9%, respectively), while V281L was the most frequent mutation among the nonclassical patients with a frequency of 39.5%. No alleles containing P30L were observed, and one de novo mutation (R356W) was found. A total of 68.3% patients were fully genotyped, and all but one showed no genotype/phenotype discrepancy. Though the cut-off value for post-ACTH 17-hydroxyprogesterone stimulation was 30.25 nmol/l (10.00 microg/l), the lowest value observed in the fully genotyped nonclassical group was 42.35 nmol/l (14.00 microg/l). CONCLUSIONS: The high number of unidentified alleles in the nonclassical group suggests that less frequent mutations, or the presence of new ones, might be the cause of the disease in the Argentine population. Alternatively, the cut-off value in the ACTH-stimulated 17-hydroxyprogesterone test might overestimate the diagnosis of the nonclassical form by including some patients with heterozygous status.