ABSTRACT
INTRODUCCION: La hiperplasia suprarrenal congénita se caracteriza por la disminución en la síntesis de cortisol por la glándula adrenal. El 95% de los casos se debe a la deficiencia en la enzima 21-hidroxilasa. La enzima es codificada por el gen CYP21A2, ubicado dentro del módulo RCCX; su expresión está modulada por dos regiones río arriba del gen.OBJETIVO: Obtener la caracterización molecular de la región RCCX y estudiar posibles variaciones de secuencia en las dos regiones regulatorias distales de la transcripción del gen, en pacientes de Argentina.METODOS: La caracterización molecular de la región RCCX se llevó a cabo mediante la técnica de Southern Blot, usando las restricciones con las enzimas Taq I y Bgl II y sondas para los genes CYP21, TNX y C4. El estudio de las regiones regulatorias de la transcripción se realizó por secuenciación automática de los fragmentos de interés.RESULTADOS: Se identificaron ocho haplotipos diferentes de la región RCCX y se encontró una variante de secuencia en cada región regulatoria estudiada.CONCLUSIONES: Las técnicas utilizadas fueron adecuadas para lograr los objetivos propuestos. La caracterización molecular de la región RCCX permitió evidenciar una elevada variabilidad en la composición génica y modular de la región. Por su parte ,los resultados de estudios de bioinformática indicarían que la variante alélica hallada en una de las regiones regulatorias podría modular la expresión del gen CYP21A2. Ambos estudios constituyes los reportes más detallados de la caracterización de ambas regiones en pacientes argentinos.
INTRODUCTION: The congenital adrenal hyperplasia is characterized by decreased synthesis of cortisol by the adrenal gland. 95% of the cases are fue to deficiency in the exime 21-hydroxylase. The enzyme is encoded by the CYP21A2 gene, located within the RCCX module; its expression is modulated by two regions upstream of the gene.OBJECTIVE: To obtain the molecular characterization of the RCCX region, where the CYP21A2 gene is located. Also, to study possible sequence variations in the two distal regulatory regions of gene transportation in patients of Argentina.METHODS: The molecular characterization of the RCCX region was done by Southern blot technique, using the restrictions with enzymes Taq I and Bgl II and probes for genes CYP21, TNX and C4. The study of transcriptional regulatory regions was performed by automatic sequencing of fragments of interest.RESULTS: Eight different haplotypes were identifies in the RCCX region, finding a sequence variant in each regulatory region studied.CONCLUSIONS: The techniques used were adequate to achieve the proposed objectives. The molecular characterization of the RCCX region showed a high variability in gene and modular composition. Moreover, results from bioinformatics studies indicate that the allelic variant found in one of the regulatory regions could modulate the expression of CYP21A2 gene. Both studies constitute the most detailed reports characterizing the two regions in Argentinean patients.
Subject(s)
Genetics , Adrenal Hyperplasia, Congenital , Gene Regulatory Networks/genetics , Argentina , Public HealthABSTRACT
INTRODUCCION: La hiperplasia suprarrenal congénita se caracteriza por la disminución en la síntesis de cortisol por la glándula adrenal. El 95% de los casos se debe a la deficiencia en la enzima 21-hidroxilasa. La enzima es codificada por el gen CYP21A2, ubicado dentro del módulo RCCX; su expresión está modulada por dos regiones río arriba del gen.OBJETIVO: Obtener la caracterización molecular de la región RCCX y estudiar posibles variaciones de secuencia en las dos regiones regulatorias distales de la transcripción del gen, en pacientes de Argentina.METODOS: La caracterización molecular de la región RCCX se llevó a cabo mediante la técnica de Southern Blot, usando las restricciones con las enzimas Taq I y Bgl II y sondas para los genes CYP21, TNX y C4. El estudio de las regiones regulatorias de la transcripción se realizó por secuenciación automática de los fragmentos de interés.RESULTADOS: Se identificaron ocho haplotipos diferentes de la región RCCX y se encontró una variante de secuencia en cada región regulatoria estudiada.CONCLUSIONES: Las técnicas utilizadas fueron adecuadas para lograr los objetivos propuestos. La caracterización molecular de la región RCCX permitió evidenciar una elevada variabilidad en la composición génica y modular de la región. Por su parte ,los resultados de estudios de bioinformática indicarían que la variante alélica hallada en una de las regiones regulatorias podría modular la expresión del gen CYP21A2. Ambos estudios constituyes los reportes más detallados de la caracterización de ambas regiones en pacientes argentinos.
INTRODUCTION: The congenital adrenal hyperplasia is characterized by decreased synthesis of cortisol by the adrenal gland. 95% of the cases are fue to deficiency in the exime 21-hydroxylase. The enzyme is encoded by the CYP21A2 gene, located within the RCCX module; its expression is modulated by two regions upstream of the gene.OBJECTIVE: To obtain the molecular characterization of the RCCX region, where the CYP21A2 gene is located. Also, to study possible sequence variations in the two distal regulatory regions of gene transportation in patients of Argentina.METHODS: The molecular characterization of the RCCX region was done by Southern blot technique, using the restrictions with enzymes Taq I and Bgl II and probes for genes CYP21, TNX and C4. The study of transcriptional regulatory regions was performed by automatic sequencing of fragments of interest.RESULTS: Eight different haplotypes were identifies in the RCCX region, finding a sequence variant in each regulatory region studied.CONCLUSIONS: The techniques used were adequate to achieve the proposed objectives. The molecular characterization of the RCCX region showed a high variability in gene and modular composition. Moreover, results from bioinformatics studies indicate that the allelic variant found in one of the regulatory regions could modulate the expression of CYP21A2 gene. Both studies constitute the most detailed reports characterizing the two regions in Argentinean patients.
Subject(s)
Adrenal Hyperplasia, Congenital , Steroid 21-Hydroxylase , Genetics , Gene Regulatory Networks/genetics , Argentina , Public HealthABSTRACT
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.
