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Congenital adrenal hyperplasia: new mutations and their effects on the enzymatic activity

Abstract

The laboratory of Human Molecular Genetics at CBMEG-UNICAMP in association with the Pediatric Endocrinology group at the Department of Pediatrics - FCM and also with the Department of Endocrinology at the FMRP-USP, is developing a research on mutation identification and characterization in the CYP21A2 and CYP11B1 genes responsible for different forms of Congenital Hiperplasia of the Adrenal. The affected alelles of CYP21A2 had been characterized for gene deleção, gene conversion and 9 point mutations (microconversions), which are frequent events for this gene. Ten new mutations had been identified. A frameshift mutation in exon 1, causing a stop codon at aminoacid 78, was found in homozygose in a child of consanguineous couple; a frameshift mutation in exon 4, in heterozygose, generating a stop codon in residue 294 and a nonsense that one creates stop codon in exon 10. As these mutations are severely deleterious they will not be tested for enzyme activity. Nevertheless, the other seven nucleotide variations will be investigated to determine the degree of reduction of the enzymatic activity they cause. Five missense mutations and two intronic nucleotide changes at donor and acceptor splice sites, respectively, will be studied. One nucleotide change was found in a patient who had already the V281L mutation in the maternal alelle, that cause the non-classic form of the disease. However, the patient presents the salt-losing classical form, therefore the first genotype did not agree with phenotype. The other was found either in homozygose or in heterozygose in eight non-related patients. Besides the cited mutations, an intron 7 nucleotide variation will be evaluate for the possible alteration in the splicing process in a patient whose phenotype do not agree with the genotype. Searching for mutations in CYP11B1 gene in individuals carrying 11beta-OH deficiency three mutations were found which two had not been described before. A missense mutation (G267S), found in homozygose in a patient, whose parents are consanguineous, resulted from G>A change in the last nucleotide of exon 4, which is also the first nucleotide of codon 267. This mutation is interesting because the affected codon is formed by the last nucleotide of exon 4 and the two first nucleotides of exon 5. Therefore, it is suspected that this mutation is deleterious for also affecting the activity of splice donor site of intron 4. In this in case, it is necessary not only evaluate the activity of the mutant enzyme, but also to study the possible alterations in the splicing process. Therefore, the aim of this project is to clone and express normal and mutant CYP21 and CYP11B1 genes and to evaluate the transcript formation to evaluate how deleterious are the missense mutations. As well as, to study the possible alterations in the splicing process for the intronic variants through the technique of mini-genes. It is our objective also to continue the search for new mutations in CYP21 and CYP11B1 gene. As new mutations are found they will be included in the study of enzymatic activity. This project is of great importance and relevance for the laboratory of Human Molecular Genetics in the CBMEG since it will promote the qualification of the laboratory for gene expression techniques introducing new methodologies which will be of extremely useful for the evaluation of biological effect caused by gene mutations. Moreover, the project will result in the formation of specialized professionals. At this time does not exist in Brazil a research group that studies the activity of mutant enzymes for genes CYP21 and CYP11B1. (AU)

Scientific publications (5)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
COELI, FERNANDA B.; SOARDI, FERNANDA C.; BERNARDI, RENAN D.; DE ARAUJO, MARCELA; PAULINO, LUCIANA C.; LAU, IVY F.; PETROLI, REGINALDO J.; DE LEMOS-MARINI, SOFIA H. V.; BAPTISTA, MARIA T. M.; GUERRA-JUNIOR, GIL; DE-MELLO, MARICILDA P. Nobel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency. BMC MEDICAL GENETICS, v. 11, JUN 29 2010. Web of Science Citations: 8.
COELI‚ F.; SOARDI‚ F.; BERNARDI‚ R.; DE ARAÚJO‚ M.; PAULINO‚ L.; LAU‚ I.; PETROLI‚ R.; DE LEMOS-MARINI‚ S.; BAPTISTA‚ M.; GUERRA-JÚNIOR‚ G.; OTHERS. Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency. BMC MEDICAL GENETICS, v. 11, n. 1, p. 104, 2010.
SOARDI, FERNANDA C.; PENACHIONI, JUNIA Y.; JUSTO, GISELLE Z.; BACHEGA, TANIA A. S. S.; INACIO, MARLENE; MENDONCA, BERENICE B.; DE CASTRO, MARGARET; DE MELLO, MARICILDA P. Novel Mutations in CYP11B1 Gene Leading to 11 beta-Hydroxylase Deficiency in Brazilian Patients. JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, v. 94, n. 9, p. 3481-3485, SEP 2009. Web of Science Citations: 14.
SOARDI, FERNANDA CAROLINE; LEMOS-MARINI, SOFIA HELENA V.; COELI, FERNANDA BORCHERS; MATURANA, VICTOR GONCALVES; BARBOSA DA SILVA, MARCIA DUARTE; BERNARDI, RENAN DARIN; JUSTO, GISELLE ZENKER; DE-MELLO, MARICILDA PALANDI. Heterozygosis for CYP21A2 Mutation Considered as 21-Hydroxylase Deficiency in Neonatal Screening. Arquivos Brasileiros de Endocrinologia e Metabologia, v. 52, n. 8, p. 1388-1392, NOV 2008. Web of Science Citations: 3.
SOARDI, F. C.; BARBARO, M.; LAU, I. F.; LEMOS-MARINI, S. H. V.; BAPTISTA, M. T. M.; GUERRA-JUNIOR, G.; WEDELL, A.; LAJIC, S.; DE MELLO, M. P. Inhibition of CYP21A2 enzyme activity caused by novel missense mutations identified in Brazilian and Scandinavian patients. JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, v. 93, n. 6, p. 2416-2420, JUN 2008. Web of Science Citations: 36.

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