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| Author(s): |
Cibele Masotti
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. , gráficos, ilustrações, tabelas. |
| Institution: | Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) |
| Defense date: | 2007-06-26 |
| Examining board members: |
Maria Rita dos Santos e Passos Bueno;
Maria Cátira Bortolini;
Celia Priszkulnik Koiffmann;
Chin Jia Lin;
Chao Yun Irene Yan
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| Advisor: | Maria Rita dos Santos e Passos Bueno |
| Field of knowledge: | Biological Sciences - Genetics |
| Indexed in: | Banco de Dados Bibliográficos da USP-DEDALUS; Biblioteca Digital de Teses e Dissertações - USP |
| Location: | Universidade de São Paulo. Biblioteca do Instituto de Biociências; IB/D-1269 |
| Abstract | |
In the present study, we investigate two craniofacial mendelian disorders, resulting from abnormalities in the development of the first and second pharyngeal arches: the Treacher Collins Syndrome (TCS ) and the auriculo condylar syndrome (ACS). The identification of genes and molecular mechanisms associated to these conditions, in addition to contributing to the understanding of the development of structures derived from these pharyngeal arches, is fundamental for the development of molecular diagnostics, an important tool for differential diagnosis and genetic counseling. We contributed to a better clinical characterization of ACS with a description of a new family with 11 affected individuals. After excluding four candidate genes/regions for syndromes of the 1st and 2nd pharyngeal arches, we carried out linkage studies using polymorphic markers throughout the genome. We mapped the first locus associated to ACS, 1p21.1-q23.3 (lod score=3.0), and our data suggest genetic heterogeneity exists for this pathology. With respect to the study of TCS , we carried out an extensive review of the nomenclature for the pathogenic mutations described in the literature, in addition to investigating atypical mutation mechanisms in TCS , corroborating the hypothesis that mutations in the exons that undergo alternative splicing can result in the TCS phenotype. We continued the characterization of the mutation spectrum for TCOF1 and we investigated the genotype-phenotype correlation in a sample of 58 patients with TCS . The analysis of polymorphisms in the coding region allowed us to make inferences about the selective regime experienced by TCOF1 , and our results suggested that TCOF1 is under purifying selection, which acts upon weakly deleterious mutations. We also inferred the phase for a set of polymorphisms in the coding region, testing whether there was association between any haplotype and the severity of the phenotype or the susceptibility to the disease. Given the observation of no correlation between haplotype/genotype and phenotype, we tested the hypothesis that variation in the levels of expression of the normal allele could be responsible for the clinical variability observed in TCS patients. To do this, we started a functional study of the TCOF1 regulatory regions, including regions distant from the minimal promoter, predicted to be enhancers. We identified polymorphisms in the promoter region, one of which reduced the levels of transcription and affected the binding of the YY1 transcription factor to the TCOF1 promoter. Using an in vitro assay we characterized YY1 as a repressor. We also tested the hypothesis of haploinsufficiency as a mechanism associated to TCS . We quantified the levels of TCOF1 transcripts in normal and affected individuals and found a significant difference. Our study corroborates the haploinsufficiency hypothesis and for the first time shows that patients have transcript degradation. We also investigated the possibility that transcripts levels are correlated to phenotypic variability. We compared the expression data for each patient with the frequency of nine clinical signs of TCS , but no correlation was found. We investigated the pattern of methylation on the CpG island of TCOF1 in patients and controls, in order to test whether different levels of methylation among individuals were associated to the great variation in gene expression. We demonstrated that methylation of the CpG island is not the regulatory mechanisms underlying the broad variation in TCOF1 expression. (AU) | |