| Grant number: | 12/16815-0 |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |
| Start date: | October 01, 2012 |
| End date: | February 29, 2016 |
| Field of knowledge: | Health Sciences - Medicine - Medical Clinics |
| Principal Investigator: | Maricilda Palandi de Mello |
| Grantee: | Débora de Paula Michelatto |
| Host Institution: | Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil |
| Associated scholarship(s): | 14/09844-0 - Functional analyses of novel nucleotides variations in the CYP21A2 gene identified in patients with congenital adrenal hyperplasia, BE.EP.DD |
Abstract The sex definition at birth or even before, has been essential to the society we live. When it is not possible, it become a cause of distress for the family and, later, to the child. The embrionary sex development can be divided into three stages: the undifferentiated stage, differentiation of gonads and both internal and external genitalia, the latter being dependent on the action of hormones. Defects in any of these stages can result in disorders of sex development. Among many possibilities for this to occur is Congenital Adrenal Hyperplasia whose manifestation is reflected in the development of the external genitalia. The HAC is one of the most common autosomal recessive disease that can be caused by a deficiency of one of five enzymes that catalyze each step of the adrenal steroids synthesis. StaR P450scc, 3²-hydroxysteroid dehydrogenase, 21-hydroxylase, 11²-hydroxylase and 17±-hydroxylase enzymes participate in the synthesis of adrenal steroids and, 21-hydroxylase (CYP21A2 gene) deficiency occurs in over 95% of cases.When the DNA sequencing was not accessible, first step to define affected alleles was to search for the presence or absence of deletion, gene conversion and 9 CYP21A1P pseudogene-derived mutations (microconversions). Southern blot techniques and ASO-PCR were used for that. If genotypes did not get clarifyied, the gene was sequenced in search for novel mutations or mutations not derived from pseudogene. Currently, sequencing directly the gene haplotypes can be defined, which is important since there are single nucleotide variations (SNV) that can synergistically increase mutation effects or confer important differences in the phenotype of affected individuals. In cases of homozygosity excess or whose allelic segregation does not clarify the genotype, copy number variation (CNV) can be confirmed by MLPA (Multiplex Ligation-dependent Probe Amplification). For SNVs not yet annotated that may be identified in exons and introns, it is necessary to investigate the biological effect for a complete description and to tentatively establish an adequated phenotype and genotype correlation. Continuing the study of our research group, we propose to evaluate the functional effect of exonic and intronic changes that are new or infrequent in the CYP21A2 gene identified that have been identified in families with affected individuals. The aim of the project is to clone and express the CYP21A2 gene with SNVs that lead to amino acid change, for intronic changes will used the technique of mini-genes and for 5' and 3' variations, EMSA and gene reproter expression will be used. The study will include the following changes: 1) those involving timely exchange of amino acids p.L12M, p.S202G, p.D377Y, p.L461P p.T450M and, 2) the mutation in c.1170_1178delAGGCGCCCA frame or p. (Gln389_Ala381del ), 3) combinations of mutations p.30L + p.P453S, p.V281L + + p.L461P p.D377Y p.R483Q and, 4) the new changes in non-coding regions c.-465g> C, c. -118T> C, c.289 +37 T> A (IVS2 +37 T> A), c.936 +5 G> A (IVS7 +5 G> A) and 3'UTR +56 C> G;, and 5) the rare c .290-74A> G (IVS2-74A> G), c.936 +11 G> C (IVS7 +11 G> C) and c.1222 +25 G> A (IVS9 +25 G> A). All were previously identified in patients with 21-hydroxylase deficiency both in the classical way as in non-classical. The relevance of this project is not only the molecular diagnosis of the deficiencies included, but also to provide more accurate genetic counseling and diagnostics and neo-prenatal to families involved. (AU) | |
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