The aim of this thesis is to apply recently developed technologies for genomic variation analyses, and to ensure quality of the generated information for use in preclinical cancer research. Faster access to a patients’ full genomic sequence for a lower cost makes it possible for end users such as clinicians and physicians to gain a more complete understanding of the disease status of a patient and adjust treatment accordingly. Correct biological interpretation is important in this context, and can only be provided through fast and simple access to relevant high quality data. Therefore, we here propose and validate new bioinformatic strategies for biomarker selection for prediction of response to cancer therapy. We initially explored the use of bioinformatic tools to select interesting targets for toxicity in carboplatin and paclitaxel on a smaller scale. From our findings we then further extended the analysis to the entire exome to look for biomarkers as targets for adverse effects from carboplatin and gemcitabine. To investigate any bias introduced by the methods used for targeting the exome, we analyzed the mutation profiles in cancer patients by comparing whole genome amplified DNA to unamplified DNA. In addition, we applied RNA-seq to the same patients to further validate the variations obtained by sequencing of DNA. The understanding of the human cancer genome is growing rapidly, thanks to methodological development of analysis tools. The next step is to implement these tools as a part of a chain from diagnosis of patients to genomic research to personalized treatment. / <p>QC 20121105</p>

In order to build any structure, a good supply of materials, accurate workers and quality control are needed. This is even the case when constructing DNA, the so-called “Code of Life.” For a species to continue to exist, this DNA code must be copied with incredibly high accuracy when each and every cell replicates. In fact, just one mistake in the 12 million bases that comprise the genome of budding yeast, Saccharomyces cerevisiae, can be fatal. DNA is composed of a double strand helix made up of just four different bases repeated millions of times. The building blocks of DNA are the deoxyribonucleotides (dNTPs); dCTP, dTTP, dATP and dGTP. Their production and balance are carefully controlled within each cell, largely by the key enzyme Ribonucleotide Reductase (RNR). Here, we studied how the enzymes that copy DNA, the replicative polymerases α, δ and ε, cope with the effects of an altered dNTP pool balance. An introduced mutation in the allosteric specificity site of RNR in a strain of S. cerevisiae, rnr1-Y285A, leads to elevated dCTP and dTTP levels and has been shown to have a 14-fold increase in mutation rate compared to wild type. To ascertain the full effects of the dNTP pool imbalance upon the replicative polymerases, we disabled one of the major quality control systems in a cell that corrects replication errors, the post-replicative Mismatch Repair system. Using both the CAN1 reporter assay and whole genome sequencing, we found that, despite inherent differences between the polymerases, their replication fidelity was affected very similarly by this dNTP pool imbalance. Hence, the high dCTP and dTTP forced Pol ε and Pol α/δ to make the same mistakes. In addition, the mismatch repair machinery was found to correct replication errors driven by this dNTP pool imbalance with highly variable efficiencies. Another mechanism to protect cells from DNA damage during replication is a checkpoint that can be activated to delay the cell cycle and activate repair mechanisms. In yeast, Mec1 and Rad53 (human ATR and Chk1/Chk2) are two key S-phase checkpoint proteins. They are essential as they are also required for normal DNA replication and dNTP pool regulation. However the reason why they are essential is not well understood. We investigated this by mutating RAD53 and analyzing dNTP pools and gene interactions. We show that Rad53 is essential in S-phase due to its role in regulating basal dNTP levels by action in the Dun1 pathway that regulates RNR and Rad53’s compensatory kinase function if dNTP levels are perturbed. In conclusion we present further evidence of the importance of dNTP pools in the maintenance of genome integrity and shed more light on the complex regulation of dNTP levels.

Ecological processes have the potential to influence evolution through their effects on selection. This thesis explores the effects of two ecological factors - competition and ecological opportunity. Intraspecific (within-species) competition is often expected to drive adaptation and diversification by increasing selection for the use of novel resources, thereby alleviating the detrimental effects of competition. However, this is not always the expected outcome; theory suggests that intraspecific competition can also drive convergent evolution. On the other hand, interspecific (between-species) competition is usually expected to impede adaptation and diversification because competitor species occupy potential available niches, preventing the focal species from diversifying to do so. In this thesis, I review previous experimental studies exploring the effects of competition on adaptive diversification, and then directly test these effects using experimental evolution of the bacterium Pseudomonas fluorescens. I confirm that intraspecific competition drives adaptive diversification, while the effects of interspecific competition are varied. Strong interspecific competition impedes adaptation and diversification, while the presence of weak, non-diversifying interspecific competitors drives diversification through increased resource competition. The presence of ecological opportunity is essential for adaptation and diversification, and so variation in attributes of those opportunities is expected to have important effects on the dynamics of adaptive evolution. In another evolution experiment with P. fluorescens, I tested the effects of variation in ecological opportunity on adaptive evolution and found that the type and arrangement of ecological opportunities drives adaptation but, in this system, not diversification. I also show that ecological opportunity drives differences in the degree of parallel evolution at the phenotypic and genotypic level. Finally, I explore some unexpected genetic changes identified in one of these evolved populations - two synonymous mutations that conferred fitness benefits, and show that the observed fitness improvements are the result of increased gene expression. I have shown that ecological processes can play an important role in shaping the evolutionary trajectories taken by populations. Understanding the interactions between ecological and evolutionary processes is vital for our understanding of evolutionary dynamics as a whole, and the studies laid out in this thesis represent valuable contributions to this field of study.

This work is a revision of the article "Die Kalkulation kalkulierbarer Mutationen” by the same author. In some chapters errors have been corrected in the mathematical representation. Chapters 6 and 7 have been re-edited. In this work, corrected excerpts from "Tumour Physics" and from "Evolution and mutation Physics" are used. To the agencies concerned should be noted.

In this study, self-catalytic maturation of heterologously expressed pro-galactose oxidase was analysed in E.coli by altering some amino acids which were supposed to play a crucial role in pro-peptide removal. Galactose oxidase (GOase / EC 1.1.3.9) from Fusarium graminearum / having a molecular mass of 68kDa, is a monomeric, copper containing enzyme with an unusual thioether bond. The enzyme is produced as a precursor with an additional 8 amino acid pre- and a 17- amino acid pro-sequence at the N terminus. Previous work has shown that the pre-peptide is removed possibly by a protease during secretion, whereas the 17 amino acid pro-peptide is removed autocatalytically by the aerobic addition of Cu2+ to the precursor, preceding the formation of the thioether bond at the active site. The pro-gao gene was on ProGON1 and ProGOMN1 constructs which were previously established on pET101/D/lacZ vector in England by directed evolution. ProGON1 contains silent mutations at the N-terminus different from native galactose oxidase whereas ProGOMN1 has six further mutations within the mature enzyme, providing high expression. The cleavage site mutations R-1P/A1P, R-1X/A1X, S2A, and the H522A mutation just against the cleavage site in the three dimensional configuration, were carried out by site-directed mutagenesis. Those and some extra mutations were confirmed by DNA sequence analysis. Next, mutant galactose oxidases were expressed in E. coli BL21 Star (DE3), and were purified by Strep-Tactin&reg / Sepharose&reg / column, operating on the basis of affinity chromatography. Subsequently, SDS-PAGE was performed to analyze self-processing by detecting molecular mass difference of protein bands resulting from pro-sequence removal or existence. When the bands obtained in SDS-PAGE were compared, it was seen that the products of original recombinant plasmids, i.e. ProGON1, ProGOMN1 / and the mutational variants showed no difference in band size, all slightly above 70kDa / indicating pro-sequence presence on all constructs. Non-mutants and some of the mutants showed galactose oxidase activity, signifying proper active site construction by thioether bond formation. ProGOMN1 was submitted for N-terminal amino acid sequencing to be able to assert that a size above 70kDa is not solely due to the existence of a 1 kDa Strep-tag II at C-terminus. Sequencing data affirmed the presence of both the pre-peptide and the pro-preptide showing that processing has not occurred at the N-terminus. Accordingly, in this study, it was shown for the first time that the existence of a pre-pro-peptide at the N-terminus of galactose oxidase does not prevent thioether bond formation at the active site. Furthermore, since the pro-peptide is cleaved autocatalytically, the lack of removal of the pre-peptide in E.coli in the presence of Cu 2+ and oxygen is very likely to be the cause of lack of pro-peptide cleavage. In future studies the region corresponding to the pre-peptide will be deleted to prove this hypothesis.

Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Στην παρούσα εργασία μελετήθηκε ο ρόλος στην ευκαρυωτική πρωτεϊνοσύνθεση των μεταλλάξεων rdn15 (A149G), η οποία βρίσκεται στο κέντρο αποκωδικοποίησης του ριβοσώματος του S. cerevisiae και sup45-R2ts, η οποία είναι εξωριβοσωματική και αφορά στην αντικατάσταση της προλίνης από αλανίνη στην θέση 86 του Τομέα 1 του eRF1. Η μελέτη έγινε με τη βοήθεια των αντιβιοτικών εδεΐνης, η οποία είναι ένας αναστολέας της έναρξης της μετάφρασης, και πακταμυκίνης, η οποία είναι ένας αναστολέας της μετατόπισης. Η μετάλλαξη rdn15 στον S.cerevisiae οδηγεί σε μικρή αύξηση του χρόνου διπλασιασμού των κυττάρων, ενώ επηρέασε μόνο ελαφρώς την πρωτεϊνοσυνθετική ενεργότητα του ριβοσώματος. Τα αποτελέσματα αυτά είναι συμβατά με το γεγονός της φυσιολογικής παρουσίας της γουανίνης στη θέση 1491 στους προκαρυώτες και σε κατώτερους ευκαρυώτες. Η παρουσία της μετάλλαξης rdn15 δεν επηρέασε σημαντικά την πιστότητα, αν και η τάση ήταν η απόδοση στα ριβοσώματα ενός ελαφρά υπερακριβούς χαρακτήρα. Η μετάλλαξη sup45-R2ts στον eRF1 φάνηκε να επιβραδύνει την ανάπτυξη των κυττάρων διπλασιάζοντας σχεδόν τον χρόνο διπλασιασμού τους. Το αποτέλεσμα αυτό θα μπορούσε να οφείλεται σε καταστολή των κωδικίων λήξης αλλά και σε επαγωγή μεταφραστικών λαθών από τον μεταλλαγμένο παράγοντα τερματισμού. Η παρουσία της μετάλλαξης sup45-R2ts in vitro επηρέασε μόνο ελαφρά την πρωτεΐνοσυνθετική ενεργότητα του ριβοσώματος, ενώ αντιθέτως μείωσε σημαντικά τη μεταφραστική πιστότητα, αυξάνοντας τη Συχνότητα Λάθους του ριβοσώματος. Το αντιβιοτικό εδεΐνη αναστέλλει την ανάπτυξη τόσο των αγρίου τύπου και όσο και των μεταλλαγμένων στελεχών, ενώ όλα τα στελέχη εμφανίζουν όμοια ευαισθησία έναντι της εδεΐνης. Ο S. cerevisiae εμφανίζει παρόμοια ευαισθησία έναντι της εδεΐνης in vivo με αυτήν του E.coli . Αναδεικνύεται έτσι ότι και στο ευκαρυωτικό κύτταρο του S. cerevisiae , οι βάσεις με τις οποίες αλληλεπιδρά η εδεΐνη έχουν τον ίδιο λειτουργικό ρόλο, όπως και στο προκαρυωτικό ριβόσωμα. Η εδεΐνη μειώνει την πρωτεϊνοσυνθετική ενεργότητα του ριβοσώματος του S. cerevisiae σε in vitro σύστημα μετάφρασης ελεύθερο κυττάρων, όπου τόσο το αγρίου τύπου όσο και τα μεταλλαγμένα στελέχη επέδειξαν παρόμοια ευαισθησία στη δράση της εδεΐνης Η έλλειψη διαφοροποίησης ανάμεσα στο αγρίου τύπου και τα μεταλλαγμένα στελέχη, μπορεί να ερμηνευθεί από το γεγονός ότι οι συγκεκριμένες μεταλλάξεις δεν εμπλέκονται στη διαδικασία της έναρξης της πρωτεϊνοσύνθεσης, που είναι ο κύριος στόχος της εδεΐνης. Η επίδραση της εδεΐνης στη μεταφραστική πιστότητα μετρήθηκε με τη Συχνότητα Λάθους (Σ.Λ.). Η εδεΐνη ελαττώνει την πιστότητα της μετάφρασης στον ίδιο βαθμό περίπου στο ευκαρυωτικό όσο και στο προκαρυωτικό ριβόσωμα, η δράση της όμως διαφοροποιείται μεταξύ των στελεχών. Το υπερακριβές στέλεχος επέδειξε ελαφρώς μικρότερη ευαισθησία στην επαγωγή μεταφραστικών λαθών από την εδεΐνη σε σχέση με το αγρίου τύπου, ενώ αντιθέτως, το επιρρεπές σε λάθη στέλεχος εμφανίστηκε εξαιρετικά ευαίσθητο στην επαγωγή λαθών από την εδεΐνη σε σχέση με το αγρίου τύπου. Φαίνεται λοιπόν ότι η δράση της εδεΐνης στην πιστότητα ακολουθεί το χαρακτήρα της μετάλλαξης. Το αντιβιοτικό πακταμυκίνη αναστέλλει την ανάπτυξη in vivo των αγρίου τύπου και των μεταλλαγμένων στελεχών. Ο S.cerevisiae εμφανίζει παρόμοια ευαισθησία έναντι της πακταμυκίνης in vivo με αυτήν του E.coli , ενώ οι υπό μελέτη μεταλλάξεις δεν επηρεάζουν την ευαισθησία των κυττάρων έναντι της πακταμυκίνης. Αυτό μπορεί να οφείλεται στο γεγονός ότι τόσο η rdn15 μετάλλαξη στο κέντρο αποκωδικοποίησης όσο και η sup45-R2ts στον eRF1 δεν παρεμβαίνουν στη διαδικασία της μετατόπισης κατά τη μετάφραση, η αναστολή της οποίας αποτελεί τον κύριο τρόπο δράσης της πακταμυκίνης. Η πακταμυκίνη προκαλεί αναστολή σύνθεσης πολυφαινυλαλανίνης in vitro τόσο σε χαμηλές όσο και σε υψηλές συγκεντρώσεις σε όλα τα στελέχη. Το αποτέλεσμα αυτό έρχεται σε αντίθεση με αυτό που ισχύει στο προκαρυωτικό κύτταρο, όπου η πακταμυκίνη διεγείρει τη σύνθεση πολυφαινυλαλανίνης (Dinos et al., 2004). Η πακταμυκίνη ακόμη και σε υψηλές συγκεντρώσεις δεν προκάλεσε σημαντικές μεταβολές στη Συχνότητα Λάθους του στελέχους αγρίου τύπου, ούτε στο υπερακριβές υπόβαθρο του rdn15 και στο επιρρεπές στα λάθη υπόβαθρο του sup45rdnwt. Το αποτέλεσμα αυτό δεν επιβεβαιώνει την υπερακρίβεια που προκαλεί στα προκαρυωτικά αλλά και δεν την αναιρεί. Η ταυτόχρονη παρουσία των δύο αντιβιοτικών ανέστειλε τη σύνθεση πολυφαινυλαλανίνης σε αντίθεση με το πρωκαρυωτικό (Dinos et al., 2004), ακόμη και όταν η πακταμυκίνη χρησιμοποιήθηκε σε μεγάλη περίσσεια επί της εδεΐνης. Η ταυτόχρονη παρουσία των δύο αντιβιοτικών δεν έδειξε να μεταβάλλει το χαρακτήρα της αναστολής. Τα αποτελέσματα αυτά συνηγορούν υπέρ μιας αθροιστικής δράσης των δύο αντιβιοτικών και αποκλείουν τη συνεργατικότητά τους στη σύνθεση πολυφαινυλαλανίνης από το ευκαρυωτικό ριβόσωμα. Κατά την ταυτόχρονη παρουσία ακόμη και μεγάλης περίσσειας πακταμυκίνης η εδεΐνη εξακολουθεί να προκαλεί αύξηση στη Συχνότητα Λάθους που προσομοιάζει με αυτήν που παρατηρείται κατά την παρουσία εδεΐνης μόνο. Η αλληλεπίδραση των αντιβιοτικών στην πιστότητα της μετάφρασης είναι όμοια με αυτήν που παρατηρείται στο προκαρυωτικό ριβόσωμα. / In the present study we investigated the role of two mutations, rdn15 and sup45-R2ts, in eukaryotic protein synthesis. The former is substitution A1491G and it is located in the decoding center of the S. cerevisiae ribosome. The latter is extraribosomal and involves the substitution of proline by alanine in position 86 of Domain 1 of eukaryotic release factor eRF1. These strains were then employed for the study of the mode of action of two antibiotics, edeine and pactamycin, in eukaryotic protein synthesis. In bacterial cells, edeine has been characterized as an inhibitor of translation initiation, whereas pactamycin was recently found to act as a translocation inhibitor. Mutation rdn15 leads to a slight increase of doubling times but it has no significant effect on translational fidelity although there was a slight increase of the latter indicated by slightly lower error frequencies. It should be noted that guanine is naturally present in position 1491 of prokaryotic 16S rRNA. Thus, the lack of severe impairment of ribosomal function by mutation rdn15 shows that the nature of nucleotide 1491 is not essential for ribosomal function but may be useful in the fine tuning of ribosomal activity. Omnipotent suppressor mutation sup45-R2ts in eRF1 leads to a significant increase of doubling times, almost by a factor of 2 compared to the wild type strain. This might be a result of the suppression of nonsense codons or, in our case, an increase in misincorporation of amino acids during elongation. The presence of the sup45-R2ts mutation affected marginally the synthesis of polyphenylalanine in vitro. On the contrary, it remarkably reduced translational fidelity, increasing the Error Frequency. Edeine inhibits the growth of the wild type as well as the mutant strains. All strains exhibited similar sensitivity towards edeine. Moreover, this sensitivity of S. cerevisiae cells towards edeine is similar to the sensitivity of E. coli cells towards edeine. This could indicate that the RNA bases of the yeast ribosome, with which edeine interacts, have a similar functional role as in the prokaryotic one. Edeine reduces the amount polyphenylalanine synthesis in a cell-free in vitro system and it was found that all the strains exhibit the same sensitivity towards edeine. The lack of effect may be attributed to the fact that these mutations are not involved in the initiation of translation, which is the primary target of edeine. The effect of edeine in the translational fidelity was estimated by the error frequency. Edeine highly reduces translational fidelity in the eucaryotic ribosome, almost as much as in the prokaryotic one, but differentially between the wild type strain of S. cerevisiae and the mutant strains. The hyperaccurate strain exhibited less translational errors in the presence of edeine in comparison to the wild type strain. On the contrary, the error prone strain was very sensitive and displayed a very high translational error rate. It appears that the effect of edeine in fidelity is in line with the character of the mutation. Pactamycin suppressed the growth of both the wild type and the mutant strains in vivo. S. cerevisiae exhibits a similar sensitivity as E. coli in the effect of pactamycin in vivo, whereas the mutations under study did not affect the sensitivity of the cells. This could be attributed to the fact that mutation rdn15 in the decoding center and sup45-R2ts in eRF1 do not affect translocation during translation, which is the primary target of pactamycin. Pactamycin, both at low and high concentrations, suppresses the synthesis of polyphenylalanine in vitro in all the strains under study. This result is in contrast with the effect of pactamycin in the prokaryotic cell, where pactamycin increases polyphenylalanine synthesis (Dinos et al., 2004). Pactamycin, even at high concentrations, did not have severe effects in the error frequency of the wild type strain or in that of the hyperaccurate rdn15 strain or in that of the error-prone sup45rdnwt strain. This result does not agree with the hyperaccuracy that pactamycin induces in prokaryotes but does not come in contrast to it either. In the presence of both edeine and pactamycin the synthesis of polyphenylalanine was reduced, in contrast with what is observed in the prokaryotic ribosome (Dinos et al., 2004), even when pactamycin was used in higher concentrations than edeine. Thus, the simultaneous presence of both antibiotics does not seem to alter the character of the inhibition. These results are in favor of an additive effect of these antibiotics. Moreover, in the presence of high pactamycin concentrations, edeine induces a decrease in translational fidelity which is similar to that in the presence of edeine alone. The effects of these two antibiotics on the fidelity of translation are similar to those observed in the prokaryotic ribosome.

Resumo: Os antirretrovirais (ARVs) interferem nas enzimas virais resultando na inibição da replicação do HIV. O uso combinado destas drogas tem demonstrado grande eficácia no controle da progressão da infecção pelo HIV e aumento da sobrevida do paciente. Entretanto estes benefícios podem ser comprometidos pelo desenvolvimento de resistência às drogas, consequência da emergência de mutações nas enzimas virais, representando um grande obstáculo para o sucesso do tratamento de pacientes infectados pelo HIV-1. Os testes de resistência, principalmente de genotipagem do HIV-1, permitem a orientação de novos esquemas, possibilitando o retorno da supressão viral. Este estudo teve como objetivo avaliar o perfil de mutações e a resistência genotípica aos Inibidores da Transcriptase Reversa análogos de Nucleosídeos (ITRN), Inibidores da Transcriptase Reversa Não-análogos de Nucleosídeos (ITRNN) e Inibidores da Protease (IP) em pacientes com falha terapêutica submetidos ao exame de genotipagem, realizado no Laboratório de Rotinas Diagnósticas em Biologia Molecular do Hemocentro da FMB - UNESP ponto executor da RENAGENO (Rede Nacional de Genotipagem do HIV-1). Foram analisadas sequências genômicas da região pol do HIV-1 provenientes de todos os pacientes com exame de genotipagem realizados durante os anos de 2008 e 2009. Dois grupos distintos foram formados: grupo "Adulto" (idade ≥ 18 anos), com 386 indivíduos, e grupo "Pediátrico" (<18 anos), com 45 pacientes, totalizando 431 pacientes. A genotipagem foi realizada pelo kit comercial Trugene HIV-1 Genotyping Kit (Siemens Healthcare Diagnostics). As sequências obtidas foram submetidas ao Algoritmo de Interpretação de Resistência Genotípica da Universidade de Stanford (HIVdb) e a subtipagem foi realizado pelo REGA HIV-1 Subtyping Tool e pelo programa RIP 3.0. O subtipo B foi o mais frequente nos dois grupos estudados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Antiretrovirals (ARV) interfere with viral enzymes resulting in the inhibition of HIV replication. The use of antiretroviral combinations has demonstrated highly effectiveness in controlling of the progression of HIV infection and increased of the patients' survival. However these benefits can be compromised by the development of drug resistance, as consequence of the mutations emergence in viral enzymes, representing a major obstacle to successful treatment of HIV-1 patients infected. Resistance tests, mainly HIV-1 genotyping, allow the orientation of new schemes, enabling the return of viral suppression. This study aimed to evaluate the profile of mutations and genotypic resistance to the Nucleoside Reverse Transcriptase Inhibitors (NRTI), Nonnucleoside Reverse Transcriptase Inhibitors (NNRTI) and Protease Inhibitors (PI) in the patients in therapeutic failure submitted to the genotyping test, performed in Diagnostic Routine Laboratory in Molecular Biology at the Blood Center of FMB - UNESP, part of RENAGENO (National Network of HIV-1 Genotyping). HIV-1 pol region genomic sequences from all patients with genotype test performed during the years 2008 and 2009 were analyzed. The patients were separated in two distinct groups: "Adult group" (age ≥ 18 years), with 386 individuals, and "Pediatric group" (<18 years), with 45 patients, in a total of 431 patients. Genotyping was performed by Trugene HIV-1 Genotyping Kit (Siemens Healthcare Diagnostics). The sequences obtained were submitted to the Genotypic Resistance Interpretation Algorithm of Stanford University (HIVdb) and the subtyping was performed by REGA HIV-1 Subtyping Tool and by RIP 3.0 program. Subtype B was the most frequent in both groups followed by hybrid forms B and F (BF or FB) and subtype F1. Resistance mutations were found in 97.15% of patients in the adult group... (Complete abstract click electronic access below) / Orientador: Rejane Maria Tommasini Grotto / Coorientador: Maria Inês de Moura Campos Pardini / Banca: Dennis Armando Bertolini / Banca: Paulo Inácio da Costa / Mestre

A falência ovariana prematura (FOP) é uma patologia rara, definida como a falência da função ovariana antes dos 40 anos de idade, causando amenorréia, hipogonadismo e níveis elevados de gonadotrofinas. Na maioria dos casos, apresenta-se na forma esporádica, pois apenas 5% apresentam história familial. Com relativa freqüência, a causa etiológica não é obtida, sendo então denominada de idiopática. Entre as causas conhecidas estão as alterações dos genes ligados ao cromossomo X e cromossomos autossômicos, doenças autoimunes, alterações tóxicas e iatrogênicas. Vários estudos têm sugerido que a FOP possa ser uma desordem genética, sendo o gene do receptor do FSH (FSHR) considerado um dos principais genes candidato. A primeira mutação inativadora do gene do receptor do FSH (FSHR) foi descrita por Aittomaki et al., 1995, em mulheres de famílias finlandesas que apresentavam amenorréia primária e uma mutação em ponto C566T no exon 7. Posteriormente, outras mutações inativadoras foram descritas: Ile169Thr e Arg573Cys (BEAU et al.,1998), Asp224Val e Leu601Val (TOURAINE et al.,1999), Ala419Thr (DOHERTY et al., 2002), Pro348Arg (ALLEN et al., 2003) e Pro519Thr (MEDURI et al., 2003). Por outro lado, duas variantes polimórficas, Ala307Thr e Ser307Asn, também, foram identificadas em pacientes com FOP (DA FONTE KOHEK et al., 1998, SUNDBLAD et al., 2004), embora uma relação com o fenótipo não tenha sido sistematicamente investigada.Assim, estudou-se uma coorte de 39 mulheres com FOP (casos esporádicos e familiais) que estão em acompanhamento na Unidade de Endocrinologia Ginecológica, Serviço de Endocrinologia do Hospital de Clínicas de Porto Alegre, com o objetivo de determinar a presença de mutações e/ou polimorfismos no gene do FSHR e verificar se estão associadas com o fenótipo clínico. Para este fim foram realizados 2 trabalhos, o primeiro analisando 36 casos com FOP esporádica e incluindo 2 pacientes probantes de 2 familias com FOP. O segundo estudo descrevendo o genótipo e fenótipo de 5 pacientes oriundas de 2 famílias com FOP. Variáveis clínicas e hormonais foram determinadas de todas pacientes. O DNA foi isolado de leucócitos periféricos. Os exons 6, 7, 9 e 10 do gene do FSHR foram analisados pela reação de polimerização em cadeia (PCR), seguidos por análise de restrição enzimática, eletroforese em gel com gradiente de desnaturação (DGGE) e seqüenciamento direto. Também foram medidos o volume uterino, espessura endometrial e volume ovariano porultrassonografia pélvica transvaginal. Embora não se tenha encontrado nenhuma mutação no gene do FSHR, identificou-se uma alta prevalência dos polimorfismos Ala307Thr e Ser680Asn, que se encontram em desequilíbrio de ligação. Não foram observadas associações entre a presença destes polimorfismos com os níveis séricos de FSH, LH, estradiol, bem como com volume ovariano e presença de folículos. No entanto, as pacientes com FOP esporádicas, com o polimorfismo Ala307Thr, apresentaram a última menstruação mais precocemente (A: idade=33.3 ± 7.1 anos vs. T: 28.6 ± 11.4 anos, p=0.04). A genotipagem dos casos de FOP familial evidenciou a presença dos 2 polimorfismos do gene do FSHR nas 5 pacientes e o fenótipo foi semelhante ao apresentado pelas mulheres com FOP esporádica. Em conclusão, a presença do polimorfismo Ala307Thr pode estar associada com um início mais precoce das manifestações clínicas em pacientes com FOP. Entretanto, estudos longitudinais são necessários para confirmar os resultados do presente estudo. / Premature ovarian failure (POF) is a rare pathology, defined as the failure of ovarian function before age 40, causing amenorrhea, hypogonadism and high gonadotropin levels. In most cases, premature ovarian failure is sporadic and only 5% of the affected individuals have a family history. Relatively often, the etiological cause is not determined and POF is thus labeled idiopathic. Among the known causes are alterations associated with the X chromosome and autosomal chromosomes, autoimmune diseases, and toxic and iatrogenic alterations. Several studies have suggested that POF may be a genetic disorder, the FSH receptor (FSHR) gene being considered one of the main candidate genes.The first inactivating mutation in the FSHR gene was described by Aittomaki et al., in 1995, in women of Finnish families with primary amenorrhea and a C566T point mutation in exon 7. Other inactivating mutations have since then been described: Ile169Thr and Arg573Cys (BEAU et al., 1998), Asp224Val and Leu601 Val (TOURAINE et al., 1999), Ala419Thr (DOHERTY et al., 2002), Pro348Arg (ALLEN et al., 2003), and Pro519Thr (MEDURI et al., 2003). On the other hand, two polymorphic variants, Ala307Thr and Ser307Asn, were also identified in POF patients (DA FONTE KOHEK et al., 1998, SUNDBLAD et al., 2004), although a relation with the phenotype has not been systematically investigated. Thus, a cohort was studied comprising 39 women with POF (sporadic and familial cases) being followed at the Gynecological Endocrinology Unit, Service of Endocrinology, Hospital de Clínicas de Porto Alegre, in order to determine the presence of mutations and/or polymorphisms in the FSHR gene and to ascertain if these are associated with the clinical phenotype. For this purpose, 2 studies were conducted, the first assessing 36 cases with sporadic POF, including 2 patients from 2 families with POF and the second describing the genotype and phenotype of 5 patients from 2 families with POF. Clinical and hormonal variables were determined for all patients. The DNA was isolated from peripheral leukocytes. Exons 6, 7, 9 and 10 of the FSHR gene were analyzed by polymerase chain reaction (PCR), followed by restriction enzyme analysis, denaturating gradient gel electrophoresis (DGGE), and direct sequencing. Also, uterine size, endometrial thickness and ovarian size were measured by transvaginal pelvic ultrasonography. Although no mutation of the FSHR gene was found, a high prevalence for Ala307Thr and Ser680Asn polymorphisms was found, that are in linkagedisequilibrium. No association was observed between the presence of these polymorphisms and the serum levels of FSH, LH and estradiol, as well as ovarian size and presence of follicles. However, patients with sporadic POF, presenting Ala307Thr polymorphism, had their latest menses earlier (A: age=33.3 ± 7.1 years vs. T: 28.6 ± 11.4 years, p = 0.04). The genotyping of cases with familial-related POF showed the presence of 2 polymorphisms in the FSHR gene in 5 patients, and the phenotype was similar to that presented by women with sporadic POF. In conclusion, the presence of Ala307Thr polymorphism may be associated with an earlier onset of clinical manifestations in POF patients. However, longitudinal studies are needed to confirm the results of the present study.

O câncer de mama é uma das neoplasias malignas mais comuns que afetam mulheres de todo o mundo. No Brasil, o Estado do Rio Grande do Sul tem índices de incidência e mortalidade por câncer de mama que situam-se entre os maiores do país. Aproximadamente 5-10% dos diagnósticos são causados por mutações germinativas em genes de predisposição entre os quais estão BRCA1 e BRCA2, associados à Síndrome de Câncer de mama e Ovário Hereditários (Hereditary Breast and Ovarian Cancer Syndrome ou HBOC, OMIM #114480).A identificação dos casos hereditários de câncer de mama é importante porque indivíduos afetados apresentam risco cumulativo vital muito superior ao da população para o desenvolvimento de câncer, porque familiares de um afetado podem estar igualmente em risco porque há medidas de rastreamento intensivo e intervenções preventivas que podem diminuir significativamente o risco de câncer em portadores de mutação. O diagnóstico molecular da síndrome HBOC é laborioso e caro devido à heterogeneidade molecular da doença. Famílias que apresentam características indicativas de uma síndrome de predisposição ao câncer de mama e ovário hereditários, mas que são negativas para mutações pontuais em BRCA1/2 vêm sendo testadas para grandes rearranjos visto que essas anormalidades têm sido consideradas como respondendo por, no mínimo, 10% do todos os casos HBOC com mutação identificável, incluindo grandes deleções ou duplicações. Um estudo recente de Portugal, demonstrou que um rearranjo fundador no exon 3 de BRCA2 ocorre em por 8% das famílias HBOC do Norte do país. Os objetivos deste trabalho incluíram a verificação da freqüência e caracterização de rearranjos gênicos nos genes BRCA1 e BRCA2, incluindo a mutação fundadora c.156_157insAlu no exon 3 de BRCA2 em famílias brasileiras dealto risco para a síndrome HBOC. Em um grupo de 145 indivíduos em risco nãorelacionados rastreados para a mutação fundadorac.156_157insAlu no exon 3 de BRCA2 foram encontrados 3 portadores da mutação (prevalência de 2%). Em um grupo de 145 indivíduos de risco não-relacionados rastreados para rearranjos gênicos em BRCA1 e BRCA2 pela técnica de MLPA (multiplex ligation-dependent probe amplification) foram identificados 4 portadores de mutação germinativa, sendo a mutação em dois deles um rearranjo gênico no gene BRCA1 (1,4%) envolvendo sequencias Alu. Rearranjos gênicos em BRCA1 e BRCA2 são responsáveis por uma parcela das mutações em famílias HBOC Brasileiras. O presente estudo, envolvendo uma série grande de famílias com o fenótipo da síndrome HBOC, não identificou novos rearranjos fundadores, no entanto, demonstrou a presença de rearranjos tanto em BRCA1 quanto em BRCA2, reiterando a importância da busca ativa por estas alterações, que dificilmente são identificadas por técnicas convencionais de sequenciamento gênico. A técnica de MLPA associada a um protocolo específico para detecção da mutação fundadora Portuguesa c.156_157insAlu podem ser utilizadas como estratégia inicial de rastreamento de mutações em famílias Brasileiras com a síndrome. Os resultados apresentados aqui, no entanto, indicam que mutações serão identificadas em menos de 10% dos casos utilizando esta estratégia. / Breast cancer is one of the most common malignancies affecting women worldwide. In Brazil, the State of Rio Grande do Sul has incidence rates and mortality from breast cancer are among the largest in the country. Approximately 5-10% of the cases are caused by germline mutations in predisposing genes including BRCA1 and BRCA2 are associated with the syndrome of breast and ovarian cancer Hereditary (Hereditary Breast and Ovarian Cancer Syndrome or HBOC, OMIM # 114480). The identification of inherited cases of breast cancer is important because affected individuals have cumulative risk life much higher than the population for developing cancer because of an affected family may also be at risk because there are measures of intensive screening and preventive interventions that can significantly decrease the risk of cancer in mutation carriers. The molecular diagnosis of HBOC syndrome is laborious and expensive due to the molecular heterogeneity of the disease. Families that have characteristics indicative of a cancer predisposition syndrome of hereditary breast and ovarian cancers, but are negative for mutations in BRCA1/2 have been tested for large rearrangements because these abnormalities have been identified as accounting for at least 10 % of all cases HBOC identifiable mutation, including large deletions or duplications. A recent study from Portugal, the founder showed that a rearrangement in exon 3 of BRCA2 occurs in 8% of HBOC families of the north. The objectives of this work included the verification of the frequency and characterization of gene rearrangements in BRCA1 and BRCA2 genes, including c.156_157insAlu founder mutation in exon 3 of BRCA2 mutations in Brazilian families at high risk for HBOC syndrome. In a group of 145 individuals at risk unrelated traced to c.156_157insAlu founder mutation in exon 3 of 3 found BRCA2 mutation carriers (prevalence 2%). In a group of 145 individuals at risk unrelated screened for gene rearrangements in BRCA1 and BRCA2 by the technique of MLPA (multiplex ligationdependent probe amplification) identified four carriers of germline mutation, and two of the mutation in a gene rearrangement in the gene BRCA1 (1.4%) involving Alu sequences. Gene rearrangements in BRCA1 and BRCA2 account for a portion of HBOC mutations in Brazilian families. This study, involving a large series of families with HBOC syndrome phenotype, no new rearrangements identified founders, however, showed the presence of rearrangements in both BRCA1 and BRCA2, reiterating the importance of active search for these changes, which hardly are identified by conventional techniques of gene sequencing. The technique of MLPA protocol associated with a specific mutation detection founder Portuguese c.156_157insAlu strategy can be used as initial screening for mutations in families with Brazilian syndrome. The results presented here, however, indicate mutations that will be identified in less than 10% of the cases using this strategy.