Abstract Breast cancer is the most common malignancy in women and it is strongly influenced by hereditary risk factors. So far, most of the breast cancer-associated genes, including BRCA1/2, have been identified among those that encode proteins involved in DNA damage response (DDR) pathways. However, known genetic risk factors explain less than half of the familial risk of breast cancer. Identification of novel genes and mutations that predispose to breast cancer is important for the understanding of the mechanisms that contribute to the disease development and also for the identification of those individuals who are at high risk. The first aim of this study was to resolve the complementation groups of Finnish patients with Fanconi anemia (FA), which is a rare genetic disease caused by defects in a specific DDR pathway, and to study the role of the causative gene mutations in breast cancer predisposition. The second aim of this study was to identify novel breast cancer susceptibility genes and alleles by targeted next-generation sequencing (NGS) of multiple (~800) DDR related genes. In both approaches, the identified gene mutations were subjected to case-control association analysis utilizing DNA samples of over 1,000 breast cancer cases and 1,000 healthy controls. Investigation of the Finnish FA patients revealed six different disease-causing mutations in three different genes (FANCA, FANCG and FANCI). All of the studied mutations were recurrent in the Finnish population but did not associate with breast cancer. Targeted NGS identified three novel potential breast cancer susceptibility genes. A significant enrichment of TEX15 c.7253dupT and FANCD2 c.2715+1G>A mutations was observed among the hereditary breast cancer cases (P = 0.018 and P = 0.036, respectively). The strongest evidence was found for a Finnish founder mutation in MCPH1 (c.904_916del), which associated with breast cancer susceptibility both in familial (P = 0.003, OR 8.3) and unselected (P = 0.016, OR 3.3) patient cohorts. The tumor suppressive function of MCPH1 was indicated by the loss of the wild-type allele of MCPH1 in 40% of the studied carrier tumors. Furthermore, carriers exhibited a significant increase in genomic instability measured by spontaneous chromosomal rearrangements in peripheral blood lymphocytes. / Tiivistelmä Rintasyöpä on naisten yleisin syöpä. Sairastumisriskiin vaikuttavat voimakkaasti perinnölliset alttiustekijät, ja suurin osa tähän asti tunnistetuista rintasyöpäalttiusgeeneistä, kuten BRCA1/2, koodaa DNA-vauriovasteessa (DDR) toimivia proteiineja. Tunnistetut tekijät selittävät yhä kuitenkin vain alle puolet rintasyövän perinnöllisestä alttiudesta. Uusien alttiusgeenien tunnistaminen on tärkeää rintasyövän patomekanismien ymmärtämiseksi sekä korkean rintasyöpäriskin omaavien henkilöiden tunnistamiseksi. Tämän tutkimuksen tarkoituksena oli määrittää viallisesta DDR-signaalinsiirtoreitistä johtuvan Fanconin anemian (FA) komplementaatioryhmät suomalaisilta FA-potilailta sekä tutkia sairauden taustalla olevien geenimutaatioden yhteyttä rintasyöpäriskiin. Uusia alttiusgeenejä etsittiin myös kohdennetulla uuden sukupolven sekvensointimenetelmällä, jonka avulla tutkittiin yhtäaikaisesti n. 800 DDR-geeniä. Molemmilla lähestymistavoilla tunnistettujen geenimuutosten yhteyttä rintasyöpään selvitettiin tapaus-verrokkitutkimuksen avulla, jossa tutkittiin DNA-näytteitä yli tuhannelta rintasyöpäpotilaalta sekä yli tuhannelta terveeltä henkilöltä. Suomalaisten FA-potilaiden geenimuutoksia selvittävässä tutkimuksessa tunnistettiin yhteensä kuusi mutaatiota kolmessa eri geenissä (FANCA, FANCG ja FANCI). Kaikki tutkimuksessa tunnistetut mutaatiot olivat toistuvia suomalaisessa väestössä, mutta merkitsevää assosiaatiota näiden mutaatioiden ja rintasyöpäalttiuden välillä ei havaittu. DDR-geenien sekvensoinnin avulla tunnistettiin kolme uutta mahdollista rintasyöpäalttiusgeeniä. Tutkimuksessa havaittiin TEX15 c.7253dupT ja FANCD2 c.2715+1G>A mutaatioiden rikastuminen perinnöllisessä rintasyöpäaineistossa (P = 0.018 ja P = 0.036). Merkittävin yhteys rintasyöpäalttiuden kanssa todettiin MCPH1-geenin perustajamutaatiolle (c.904_916del). Tämä mutaatio assosioitui rintasyöpäalttiuden kanssa sekä perinnöllisessä (P = 0.003, OR 8.3) että valikoimattomassa potilasaineistossa (P = 0.016, OR 3.3). Useissa mutaatiokantajien tuumoreissa (40 %) normaali MCPH1 vastinalleeli oli hävinnyt, mikä viittaisi siihen, että MCPH1 toimii tuumorisuppressorina. Mutaatiokantajilla todettiin myös kohonnut määrä kromosomaalisia muutoksia veren periferaalisissa lymfosyyteissä, mahdollisesti kohonneeseen genomiseen epävakauteen liittyen.

Les paraplégies spastiques héréditaires (PSH) font partie d’un groupe plus large de pathologies neurodégénératives associant une spasticité. J’ai exploré la variabilité clinique et moléculaire de ces pathologies à l’aide d’une cohorte de familles soudanaises. Nous avons recruté 41 familles soudanaises [337 individus/106 atteints de PSH]. J’ai extrait l’ADN génomique et constitué une banque. Le criblage de gènes candidats a été réalisé dans 4 familles en fonction du phénotype des patients. La technologie de séquençage de nouvelle génération (SNG) appliquée à 74 gènes de PSH a ensuite été appliquée aux 37 cas restants. Enfin, le séquençage de l’exome a permis de rechercher les gènes en cause dans les cas négatifs. Dans certains cas, des études fonctionnelles ont été utilisées afin de valider l’effet biologique des mutations. J’ai pu identifier la cause génétique dans 17 familles. Dans 12 familles, la mutation concernait un gène de PSH connu. Dans 3 familles, un nouveau gène a été identifié. 5 gènes candidats restent à départager dans 2 familles. Il est à noter que parfois, de multiple mutations ou maladies génétiques ségrégaient dans nos familles, dans la même branche ou dans des branches séparées. La complexité de ces familles fortement consanguines a rendu l’analyse des données du SNG difficile. Une autre particularité a été l’hétérogénéité clinique associée à des mutations du même gène entre patients de la même famille ou en comparaison avec la littérature. Ce travail est la première étude à grande échelle de patients soudanais avec PSH et rapporte de nouveaux gènes en cause, prérequis pour mieux comprendre dans le futur les mécanismes sous-jacents. / Hereditary spastic paraplegias (HSP), a heterogeneous group of spastic neurodegenerative disorders which impose diagnostic challenges. I explored the clinical varieties and genetic pathways of spastic neurodegeneration in a familial Sudanese cohort. We recruited 41 Sudanese families [337 individuals/106 HSP patients]. I have established a genomic DNA bank and when necessary, skin biopsies and fibroblasts were also obtained. A phenotype-based candidate gene approach was followed in 4 families. A targeted next generation sequencing (NGS) for 74 HSP-related genes was the main screening strategy in all-remaining 37 families. Whole exome sequencing (WES) was done in search for novel mutations in new genes in families with negative screening results. Occasionally, functional studies were conducted when feasible and relevant. I identified the genetic cause in 17/41 families. In 12 families, the mutated genes were known HSP genes. In 3 families, novel genes were identified mutated. 5 candidate genes segregated with disease in 2 other families with more experiments needed to conclude. Analysis of the NGS screening panel and of WES data imposed certain challenges as multiple genetic disorders were sometimes found running in parallel in the same/different branches of highly inbred families. We could expand the phenotypic heterogeneity of these disorders due to clinical differences observed between Sudanese patients and patients of other origins even when caused by mutations by the same gene/variant. This is the first genetic screening in a large set of HSP families in Sudan. It describes new causative genes, paving the way for further deciphering of the underlying mechanisms.

Environ 5 à 10 % des cancers du sein et de l’ovaire sont liés à des prédispositions génétiques héréditaires. Les principaux gènes responsables de telles prédispositions sont BRCA1 et BRCA2. Depuis plusieurs années, l’analyse de ces gènes est proposée dans un cadre clinique. Aux Hôpitaux Universitaires de Genève (HUG) en Suisse, une consultation d’oncogénétique a été mise sur pied dès 1994 pour les personnes concernées par leurs antécédents personnels et familiaux de cancer. Jusqu’en 2009, le seul laboratoire suisse assurant l’analyse des gènes BRCA1/BRCA2 était établi aux HUG. Ce travail de thèse intègre, d’une part, des études en lien avec la démarche clinique de conseil génétique pour les formes familiales et héréditaires de cancer du sein et de l’ovaire et, d’autre part, une évaluation détaillée des données moléculaires résultant des analyses (n= 1'163) des gènes BRCA1/BRCA2 réalisées aux HUG entre 1996 et 2009. Des perspectives quant au développement de l’oncologie prédictive aux HUG et en Suisse, et à l’activité de conseillère en génétique particulièrement dans ce domaine, sont finalement présentées. / Genetic predispositions are responsible for 5 to 10 % of all breast and ovarian cancers. The main breast/ovarian cancer predisposing genes are BRCA1 and BRCA2. For some years, the screening of pathogenic mutations in BRCA1/BRCA2 genes is provided in a clinical setting. At the Hôpitaux Universitaires de Genève (HUG, Geneva, Switzerland), a consultation in predictive oncology has been set up since 1994 for individuals concerned by the evaluation of their familial cancer risk and the probability to carry a genetic predisposition to cancer. Until 2009, the single national laboratory for BRCA1/BRCA2 testing was established in the HUG. The objectives of this work were to evaluate different aspects of the consultation process for breast/ovarian cancer predisposition syndromes provided in our Unit and to review all BRCA1/BRCA2 complete screenings (n=1’163) performed between 1996 and 2009. Results of the present study will certainly influence future activity in predictive oncology, particularly regarding the role of the genetic counselor.

Orientadores: Antonio Sergio Ramalho, Luis Alberto Magna / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T02:28:10Z (GMT). No. of bitstreams: 1 Rim_PriscilaHaeHyun_M.pdf: 5489420 bytes, checksum: b193fb510af9e3efd86456897b14ae59 (MD5) Previous issue date: 2006 / Resumo: O presente trabalho tem por objetivos verificar a percepção de oftalmologistas brasileiros em relação à contribuição da Genética em sua atuação médica rotineira e a conduta frente a portadores de doenças hereditárias; investigar o nível de conhecimento e a conduta desses pacientes a respeito de sua condição e verificar a contribuição do aconselhamento genético na vida destes pacientes. Duzentos oftalmologistas que atuam na região de Campinas, SP, Brasil (universidades e/ou clínicas particulares) foram convidados a participar, por via postal, de uma entrevista sobre os seus conhecimentos, práticas e opiniões no campo da genética. A casuística de pacientes foi constituída por 53 portadores de deficiência visual importante (acuidade visual com a melhor correção menor que 0,3), de provável etiologia genética, entrevistados em um hospital universitário no período de 18 meses. O conteúdo da entrevista consistiu de perguntas sobre seus conhecimentos, opiniões, condutas e a situação ocular relativos à sua condição, utilizando-se de questionários de múltipla escolha, que permitiram a análise estatística, com comparação de proporções e testes de associação. Examinou-se também uma amostra-controle, constituída por 142 irmãos normais de pacientes, para comparação de alguns indicadores de integração social, principalmente o estabelecimento de parcerias conjugais estáveis e reprodução. Dentre os 73 profissionais que responderam o questionário (36%), foram constatadas altas taxas (49% a 88%) de respostas positivas sobre os seus conhecimentos básicos de genética, porém baixas taxas (10% a 33%) de utilização desses conhecimentos na rotina clínica. Apenas 45% dos indivíduos com oftalmopatias hereditárias receberam aconselhamento genético, geralmente fornecido pelo próprio oftalmologista. O encaminhamento a serviço de genética foi observado em 25% dos casos. O aconselhamento genético foi uma das variáveis que, juntamente com o sexo feminino e o maior nível educacional, mostraram-se associadas a mudanças no planejamento familiar. Embora os pacientes tenham revelado maior dificuldade no estabelecimento de vínculos reprodutivos estáveis, eles tiveram filhos na mesma proporção que os seus irmãos sem a deficiência visual, o que reforça a importância do aconselhamento genético. O potencial terapêutico e preventivo do aconselhamento genético não está sendo explorado adequadamente na prevenção da cegueira. A genética merece ser discutida de forma menos teórica e mais pragmática, não somente entre os oftalmologistas, mas entre os médicos e a população em geral, sobre a aplicação do potencial da genética em benefício dos pacientes, por meio de um aconselhamento genético abrangente, direcionado para a comunidade / Abstract: This study verified the perception and behavior of Brazilian ophthalmologists in relation to hereditary eye diseases and the contribuition of Genetics in their routine practice; and verified the perception and behavior of patients with severe visual impairment and the contribuition of genetic counseling in their lives. Invitations were posted to 200 ophthalmologists, who practice in the area of Campinas, SP, Brazil, to participate in an interview concerning their knowlegde, behavior and opinions in the field of Genetics. Patients sample was composed of 53 individuals with severe visual impairment (visual acuity of less than 0.3 in the better eye with the best correction) caused by genetic factors. The interviews were held in a university hospital over a period of 18 months. The multiple-choice questionnaire applied permitted statistical analysis, for proportion comparisons and associative tests. A control sample composed of 142 normal patients siblings was also examined to compare some social integration indicators - mainly the establishment of stable couples and reproduction. Of the 73 professionals who answered the questionnaire (36%), the percentage of affirmative responses regarding basic genetic knowledge was high (49% to 88%) but the application of this knowledge to regular clinical routine was poor (10% to 33%). Only 45% of the individuals with hereditary ophthalmopathies received genetic counseling, which was usually offered by the ophthalmologist. Referrals to the genetic service consisted of 25% of the cases. The variable genetic couseling, together with others such as female sex and those having a higher educational level, significantly influenced alterations in family planning. Although they had poor access to the establishment of stable conjugal unions, they had children in the same proportion as their normal siblings. This fact underscores the importance of genetic counseling. The therapeutic and preventive potential of genetic counseling in the prevention of blindness is not adequately used. Discussions on genetics held with Brazilian ophthalmologists and the community about the use of this potential, for the benefit of patients, through genetic counseling, should be more pragmatic and less theoretical / Mestrado / Oftalmologia / Mestre em Ciências Médicas

As Anemias Hemolíticas compreendem um grupo de doenças em que há redução acentuada na sobrevivência dos glóbulos vermelhos circulantes e a medula óssea não é capaz de compensação, mesmo aumentando sua produção, o que causa anemia desde os primeiros anos da vida da pessoa. Dentre as doenças deste grupo, a Anemia Falciforme (SCA) e a Esferocitose Hereditária (HS) destacam-se por se tratarem de enfermidades com defeitos genéticos intrínsecos das células vermelhas (RBCs) que geram complicações multissistêmicas agudas e crônicas em seus portadores. Por vias patofisiológicas distintas, reticulócitos e respectivas hemácias defeituosas de tais doenças, falciformes e esferócitos, são continuamente aprisionados e fagocitados no baço, importante órgão de destruição de células velhas e/ou defeituosas via hemólise extravascular, o que leva progressivamente à disfunção e eventual perda da função esplênica. O objetivo desse trabalho é avaliar o papel do baço em relação à habilidade e ao fenótipo adesivos de reticulócitos (ret) e eritrócitos (erit) em pacientes com SCA e HS, com e sem função esplênica preservada. Amostras de sangue de 37 pacientes (22 SCA and 15 HS) com função esplênica e 19 pacientes (13 SCA e 6 HS) sem ela foram avaliadas. Ainda, sangue de 22 crianças com SCA foi coletado em estudo longitudinal dos 6 e 29 meses de vida. Todas as amostras de sangue foram analisadas quanto à função esplênica (Contagem de células PIT e de corpúsculos de Howell-Jolly - HJB), quanto ao perfil imunofenotípico celular (em % e em média de intensidade de fluorescência - MFI) e quanto à habilidade de adesão das células vermelhas à laminina e à linhagem celular endotelial HMEC-1. A análise da transição da perda de função esplênica demonstrou que a mesma se intensificou a partir dos 3 anos de idade (PIT: r=0,8; p<0,0001; HJB: r=0,7; p<0.0001). Quanto à imunofenotipagem celular, a contagem PIT se correlacionou positivamente, principalmente com os marcadores CD147 (%ret: r=0,6; p<0,0001; MFIret: r=0,6; p<0,0001; %erit: r=0,7; p<0,0001; MFIerit: r=0,6; p<0,0001), LuBCAM (%ret: r=0,5; p=0,004; MFIret: r=0,6; p<0,0001; %erit: r=0,6; p<0,0003; MFIerit: r=0,4; p<0,004) and CD58 (%ret: r=0,4; p=0,006; MFIret: r=0,5; p<0,0013; %erit: r=0,4; p<0,009; MFIerit: r=0,6; p<0,0001). Na comparação entre ausência ou presença do baço, a perda de sua função exerceu influência no aumento da expressão de adesão de RBCs em SCA, principalmente CD147 (%ret: p=0,002; MFIret: p=0,003; %erit: p<0,0001; MFIerit: p=0,005), LuBCAM (%ret: p=0,0001; MFIret: p<0,0001; %erit: p<0,0001; MFIerit: p<0,0001) e CD58 (%ret: p=0,007; MFIret: p=0,006; %erit: p=0,003; MFIerit: p=0,0004), embora a adesão celular tenha diminuído em pacientes HS esplenectomizados. Na comparação entre as doenças, pacientes HS com o baço apresentaram maior freqüência de adesão celular em relação aos SCA, notavelmente em relação ao LuBCAM (%ret: p=0,0008; MFIret: p=0,03; %erit: p<0,0001; MFIerit: p=0,0002), CD58 (%ret: p=0,0009; %erit: p=0,003) e CD44 (%ret: p=0,009; %erit: p<0,003). No entanto, as amostras SCA sem função esplênica tiveram maior expressão de adesão celular para CD147 (%ret: p=0,006; MFIret: p=0,02; %erit: p=0,02), LuBCAM (%ret: p=0,004; MFIret: p<0,0001), CD36 (%ret: p=0,0002; MFIret: p=0,01), CD242 (%ret: p=0,0008; %erit: p=0,05) e CD49d (%ret: p=0,04). Em relação ao Ensaio de Adesão in vitro, na ausência de baço, os RBCs SCA apresentaram maior adesividade à laminina do que os RBCs SCA com função esplênica preservadaem todas as taxas de fluxo de tensão de cisalhamento empregadas (0,5 dyne/cm2: p=0,01; 1 dyne/cm2: p=0,02; 2 dynes/cm2: p=0,03; 3 dynes/cm2: p=0,03; 5 dynes/cm2: p=0,04 e 7 dynes/cm2: p=0,03). Especialmente, reticulócitos de pacientes sem baço apresentaram maior adesividade à HMEC-1 em baixas tensões de cisalhamento (1 dyne/cm2) em ambas as doenças (SCA: p=0,03; HS: p=0,03). Por fim, reticulócitos apresentaram maior habilidade adesiva à células endoteliais em indivíduos SCA do que em pacientes HS, com (0,5 dyne/cm2: p=0,04; 1 dyne/cm2: p=0,03) ou sem baço (0,5 dyne/cm2: p=0,02; 2 dynes/cm2: p=0,01; 3 dynes/cm2: p=0,03; 5 dynes/cm2: p=0,02 e 7 dynes/cm2: p=0,03). Nossos resultados indicam que embora pertençam ao grupo de Anemias Hemolíticas, as patofisiologias e evoluções clínicas distintas de SCA e de HS levam a padrões imunofenotípicos diferentes de expressão da adesão celular. Na SCA, a ausência de função esplênica teria direta relação com o aumento do fenótipo pró-adesivo e com a adesividade de RBCs SCA, o que traz sérias consequências clínicas aos pacientes, enquanto na HS sem baço, de maneira geral, os eventos de adesão celular são minimizados, embora ainda apresentem reticulócitos e eritrócitos adesivos circulantes após a esplenectomia. / Hemolytic Anemias comprise a group of diseases in which there is marked reduction in the survival of circulating erythrocytes and the bone marrow is not capable of compensation, even by increasing its production, which causes anemia from the first years of the person\'s life on. Among the diseases of this group, Sickle Cell Anemia (SCA) and Hereditary Spherocytosis (HS) stand out for being diseases with intrinsic genetic defects of red blood cells (RBCs) that generate acute and chronic multisystemic complications in their patients. By distinct pathophysiological pathways, reticulocytes and these disease\'s respective defective erythrocytes, sickle and spheroid ones, are continuously trapped and phagocytosed in the spleen, important organ of destruction of old and/or defective cells via extravascular hemolysis, which progressively leads to dysfunction and eventual loss of splenic function. The objective of this study was to evaluate the role of the spleen in relation to the reticulocyte (ret) and erythrocyte (eryt) adhesive ability and adhesion phenotype in patients with SCA and HS, with and without preserved splenic function. Blood samples from 37 patients (22 SCA and 15 HS) with splenic function and 19 patients (13 SCA and 6 HS) without it were evaluated. Still, blood from 22 children with SCA was collected in a longitudinal study from 6 to 29 months of age. All blood samples were analyzed for splenic function [pitted cells (PIT) and Howell-Jolly bodies (HJB) counting], for the cellular immunophenotypic profile (in % and in mean fluorescence intensity - MFI) and for the adhesive ability of RBCs to laminin and to endothelial cell line HMEC-1. Analysis of the splenic function loss transition showed that it intensified from 3 years of age on (PIT: r=0.8, p<0.0001; HJB: r=0.7, p<0.0001). Regarding the cellular immunophenotyping, PIT count correlated positively, mainly with CD147 markers (%ret: r=0.6, p<0.0001; MFIret: r=0.6, p<0.0001; %eryt: r=0.7, p<0.0001; MFIeryt: r=0.6, p<0.0001), LuBCAM (%ret: r=0.5, p=0.004; MFIret: r=0.6, p<0.0001; %eryt: r=0.6, p<0.0003; MFIeryt: r=0.4, p<0.004) and CD58 (%ret: r=0.4, p=0.006; MFIret: r=0.5, p<0.0013; %eryt: r=0.4, p<0.009; MFIeryt: r=0.6, p<0.0001). In the comparison between spleen absence or presence, the loss of its function exerted influence on the increase of RBCs adhesion expression in SCA, mainly on CD147 (%ret: p=0.002; MFIret: p=0.003; %eryt: p<0.0001; MFIeryt: p=0.005), LuBCAM (%ret: p=0.0001; MFIret: p<0.0001; %eryt: p<0.0001; MFIeryt: p<0.0001) e CD58 (%ret: p=0.007; MFIret: p=0.006; %eryt: p=0.003; MFIeryt: p=0.0004), although cell adhesion has been decreased in splenectomized HS patients. In the comparison between diseases, HS patients with spleen showed higher cell adhesion frequency compared to SCA, notably in relation to LuBCAM (%ret: p=0.0008; MFIret: p=0.03; %eryt: p<0.0001; MFIeryt: p=0.0002), CD58 (%ret: p=0.0009; %eryt: p=0.003) and CD44 (%ret: p=0.009; %eryt: p<0.003). However, SCA samples without splenic function had higher cell adhesion expression for CD147 (%ret: p=0.006; MFIret: p=0.02; %eryt: p=0.02), LuBCAM (%ret: p=0.004; MFIret: p<0.0001), CD36 (%ret: p=0.0002; MFIret: p=0.01), CD242 (%ret: p=0.0008; %eryt: p=0.05) and CD49d (%ret: p=0.04). Concerning the in vitro Adhesion Assay, in the spleen absence, SCA RBCs showed greater adhesiveness to laminin than SCA RBCs with preserved splenic function did at all shear stress flow rates applied (0.5 dyne/cm2: p=0.01, 1 dyne/cm2: p=0.02, 2 dynes/cm2: p=0.03, 3 dynes/cm2: p=0.03, 5 dynes/cm2: p=0.04 and 7 dynes/cm2:p=0.03). Especially, reticulocytes from patients without spleen showed higher adhesiveness to HMEC-1 at low shear stresses (1 dyne/cm2) in both diseases (SCA: p=0.03; HS: p=0.03). Finally, reticulocytes showed greater adhesion ability to endothelial cells in SCA subjects than in HS patients, with (0.5 dyne/cm2: p=0.04 and 1 dyne/cm2: p=0.03) or without spleen (0.5 dyne/cm2: p=0.02, 2 dynes/cm2: p=0.01, 3 dynes/cm2: p=0.03, 5 dynes/cm2: p=0.02 and 7 dynes/cm2: p=0.03). Our results indicate that although both diseases belong to the Hemolytic Anemias group, SCA and HS distinct pathophysiologies and clinical evolution lead to different immunophenotypic patterns of cell adhesion expression. In SCA, the absence of splenic function may have a direct relation with the increase of SCA RBCs proadhesive phenotype and adhesiveness, which brings serious clinical consequences to the patients, whereas in HS without spleen, in general, cellular adhesion events are minimized, although they still present adhesive circulating reticulocytes and erythrocytes after splenectomy.

Many steps go into developing a clinical viral gene therapy. The course starts with appropriate disease selection and moves through the many hurdles of in-vitro testing, animal model validation and proof-of-concept studies, all the way through pre-clinical large animal studies. In this thesis, I propose to outline the process of developing a translation pathway for a gene therapy using recombinant adeno-associated virus (rAAV). I will expand on this outline using data that I have generated during the course of my Ph.D. that ranges from animal model validation all the way through pre-clinical vector stability studies. Two disease models will be discussed throughout this thesis, Cockayne Syndrome (CS) and Alpha-1 Antitrypsin Deficiency (AATD). Cockayne Syndrome is a rare autosomal recessive genetic disorder involving mutations in either the CSA or CSB gene, leading to defects in DNA repair. Clinically this presents as progressive degeneration of the central nervous system, retina, cardiovascular system, and cochlea, which leads to mental retardation, post-natal growth defects, ocular abnormalities, and shortened life expectancy. Alpha-1 antitrypsin is a serine protease inhibitor largely produced in the liver that mainly functions to inhibit neutrophil elastase within the lung. AATD leads to an increased risk of emphysema, with shortened life expectancy, and also results in accumulations of mutant AAT polymers in the liver, sometimes leading to liver failure. Using these two disease models I will outline the upstream and downstream pre-clinical work as well as the transition to clinical trials of a rAAV based gene therapy.

Une des plus grandes avancées en cancérologie et en génétique au cours des vingt dernières années fût l'identification des formes héréditaires de cancer et des gènes deprédisposition impliqués. Chez une majorité de patients soupçonnés de présenter une formehéréditaire de cancer, les analyses centrées sur les gènes connus pour être impliqués dansles prédispositions mendéliennes au cancer restent bien souvent négatives. Aujourd'hui,grâce à l'émergence du séquençage à haut-débit (NGS), il est possible de séquencerl'ensemble des exons (exome) d'un individu ou plusieurs centaines de gènes dans un lapsde temps court et à des coûts raisonnables. Dans ce contexte, nous avons appliqué plusieurs stratégies basées sur ces nouveaux outils, avec l'objectif d'identifier de nouvellesbases moléculaires des cancers héréditaires à survenue précoce. Tout d’abord, nous avons employé une stratégie d'analyse exomique intrafamiliale dans une famille atypique présentant des chondrosarcomes de localisation thoracique pour lesquels aucune base moléculaire n'avait pu être mise en évidence. Grâce à cette stratégie, nous avons pu identifier une altération tronquante du gène EXT2 (NM_000401.3; c.237G>A; p.Trp79*). Les altérations perte de fonction documentées pour ce gène sont impliquées dans la maladie des ostéochondromes multiples (OM), des tumeurs bénignes. Or, dans cette famille, aucun signe clinique d'OM n'était présent. Ces travaux nous ont donc permis d'étendre le spectre phénotypique des mutations EXT2 et de modifier la prise en charge clinique de cette famille. Nous avons ensuite employé une stratégie d'analyse exomique soustractive de trio enfant malade / parents sains dans le but d’identifier des mutations de novo potentiellement responsables de la prédisposition génétique au cancer observée chez un jeune patient ayant développé un médulloblastome du cervelet à l’âge de 8 ans, suivi d’un méningiome à 22 ans. L’analyse exomique du trio a révélé l’existence chez ce patient d'une mutation de novo faux-sens affectant un acide aminé très conservé de la protéine HID-1. Cette dernière est particulièrement exprimée dans les cellules neuronales et sécrétrices, et semble fonctionner autour de l’appareil de Golgi pour réguler le tri des vésiculesnouvellement formées. Ainsi, notre hypothèse est qu’un défaut de la protéine HID-1, lié à une mutation du gène HID-1, perturberait la voie de sécrétion et participerait à la genèse du médulloblastome. Ces travaux, toujours en cours, démontrent à la fois la force de la stratégie exomique de trio pour identifier rapidement des mutations de novo et illustre toute la difficultéd'interprétation des variants détectés dans des gènes non impliqués dans le cancer. Par ailleurs, nous avons appliqué une stratégie exomique soustractive et interfamiliale à une cohorte de dix patients ayant développé un corticosurrénalome à un âge très précoce et pour lesquels aucune base moléculaire n'a pu être mise en évidence. Malheureusement, nous n'avons pas pu identifier de nouvelles bases moléculaires du corticosurrénalome de l'enfant par ces techniques. Enfin, sous l'hypothèse que des mutations rares ou privées dans un nombre limité de gènes impliqués dans le cancer contribueraient à des formes héréditaires de cancer, nous avons entrepris un projet visant à séquencer à haut débit 201 gènes fortement impliqués dans le cancer chez des patients ayant développé des tumeurs à un âge pédiatrique. Les premiers résultats de ce projet toujours en cours ont permis de confirmer la robustesse de cette technique et suggèrent une extension phénotypique du spectre des mutations DICER1 ainsi qu'une contribution oligogénique des gènes de réparation de l'ADN dans les tumeurs pédiatriques. L'ensemble de ces résultats seront bientôt compilés au sein d'une base de données et bénéficieront d'une analyse statistique fine avec l'objectif d'identifier des enrichissements en variants rares dans des gènes ou voies biologiques. / One of the greatest advances in oncology and genetics over the past 20 years has been the identification of hereditary forms of cancer and of the cancer genes. Nevertheless, in a majority of patients suspected to present an inherited form of cancer, analyses of the genes known to be involved in the Mendelian predispositions to cancer often remain negative. Today, thanks to the emergence of high-throughput sequencing (NGS), it is now possible to sequence all exons of an individual (exome) or several hundred genes in a short period of time and for a reasonable cost. In this context, we have applied several strategiesbased on these new tools in order to identify new molecular basis of early-onset cancers. First, we applied an intra-familial exome analysis strategy to an atypical family with chondrosarcomas of the chest, for which no molecular basis could be identified. Using this strategy, we were able to identify a truncating alteration of the EXT2 gene NM_000401.3; c.237G> A; p.Trp79 *). The documented loss of function alterations of this gene are implicated in a disease called multiple osteochondromas (OM), associated with benign lesions. Interestingly, these patients showed no clinical signs of OM indicating a potential phenotypic extension of EXT2 mutations. Plus, this work allowed us to change the clinical management of this family. We then used a strategy of subtractive exomic analysis of trio sick child/healthy parents in order to identify de novo mutations in a young patient who developed a medulloblastoma of the cerebellum at 8 years-old followed by a meningioma at 22 years-old. The analysis of the trio revealed the existence of a de novo mutation affecting a highly conserved amino acid of the HID-1 protein. HID-1 is specifically expressed in neuronal and secretory cells, and seems to function around the Golgi apparatus to regulate the sorting of newly formed vesicles. Our hypothesis is that a defect of the HID-1 protein linked to a mutation of the HID-1 gene, could alter the secretory pathway therefore contributing to the development of the tumor. This work, which is still ongoing, demonstrates both the strength of the trio strategy for the rapid identification of de novo mutations and illustrates all the difficulty of interpreting variants detected in genes not yet involved in cancer. Then, thanks to the recruitment of the Laboratory of Molecular Genetics of the CHU of Rouen, we have collected a cohort of 10 patients who developed an adrenocortical carcinoma (ACC) at a very early age and for which no molecular basis could be identified. Despite subtractive and inter-familial exomic analyses, we were unable to highlight new molecular bases for these cases of pediatric ACC. Finally, under the assumption that rare or private mutations in a limited number of genes involved in cancer could contribute to inherited forms of cancer, we undertook a project to sequence 201 genes involved in cancer in patients who developed tumors at a pediatric age. The first results of this project confirmed the robustness of this technique and suggested a phenotypic extension of the DICER1 mutation spectrum as well as an oligogenic contribution of DNA repair genes in pediatric tumors. Soon, these results will be compiled in a database and will benefit from a statistical analysis with the objective to identify enrichment of rare variants in specific genes or biological pathways in these patients compared to control individuals.

Congenital Adrenal Hyperplasia (CAH) encompasses several disorders related to disruptions in the adrenal steroid production pathway. These disruptions may cause virilization of the external female sex organs, incorrect gender assignment, precocious puberty, and in the most severe form, may cause life-threatening salt wasting and adrenal crisis if not detected and treated early in the newborn period. 17α-Hydroxyprogesterone (17-OHP) is the primary target for immunofluorescence detection of CAH from dried blood spots in newborn screening (NBS). Unfortunately, current immunoassay techniques for the detection of CAH suffer from high false positive rates. The primary factors contributing to false positive determinations can include the natural increase of 17-OHP due to stress stimuli as well as cross-reactivity of the immunoassay antibody with other hormones and endogenous compounds in blood. Analysis of the adrenal steroid profile and corresponding analyte ratios using high performance liquid chromatography (HPLC)or ultra-high pressure liquid chromatography (UHPLC)combined with tandem mass spectrometry (MS/MS) has been shown to be a sensitive and selective technique for the significant reduction of the false positive reporting rate for CAH in newborn screening. In working toward optimization, validation, and implementation of an HPLC-MS/MS steroid profile for use by Virginia’s Newborn Screening laboratory as a 2nd tier analysis for CAH screening, a commercially-available core-shell HPLC column with a biphenyl stationary phase was determined to offer adequate retention and selectivity to achieve baseline resolution of isobaric target analytes under rapid reversed phase gradient conditions. Method linearity, precision, and accuracy were assessed using enriched dried blood spot materials. Double-blinded analyses of over 300 newborn dried blood spot specimens were used to determine clinical sensitivity and specificity of the assay, which is projected to substantially reduce the false positive reporting rate for CAH screening while meeting target sample turnaround times.

A l'heure où la famille moderne se conçoit au pluriel, à l'heure où les ascendants organisent en amont la transmission et la dévolution de leur patrimoine, à l'heure où la mondialisation prône l'uniformisation du droit et la fin des spécificités étatiques, il est nécessaire de s'interroger sur l'avenir de la réserve héréditaire. Les parents ne souhaitent plus être privés de leur pouvoir de disposer librement de leur patrimoine, même pour cause de mort. Que répondre à un père qui angoisse à l'idée de voir son patrimoine dilapidé par un enfant ingrat avec lequel il ne vit que de conflits ? Que dire à cet enfant qui a vu son frère avantagé par décision maternelle, de manière abusive et arbitraire ? Quels conseils donner à ces individus qui ouvrent chaque jour la porte de nos offices pour demander l'aide de leur notaire ? Que dire à cet époux qui veut protéger son conjoint de la véhémence d’enfants d’un premier lit ? Afin de leur répondre dans la pratique, appliquons-nous à étudier les moyens de contourner cette réserve héréditaire. Attachons-nous à vérifier leur efficacité. Au-delà des mécanismes classiques du Code civil tels que le testament et la donation, l'intérêt de cette thèse est de se tourner vers des mécanismes plus complexes, afin de répondre aux parents déçus par leur progéniture, mais aussi à eux, enfants lésés, pour leur donner des moyens juridiques de contrer une injuste privation. Il est utile de comprendre et de connaître ce qu’il est possible de faire pour transmettre son patrimoine avec une liberté recouvrée sans risquer les foudres de la loi. A travers l’étude de ces instruments, de leur efficacité et de leur traitement tant légal que familial, la question de la modernisation de la réserve ressurgit. La famille change, et avec elle, la conception traditionnelle de la réserve héréditaire. / At a time when the modern family is conceived in the plural, at a time when ascendants organize upstream transmission and devolution of their heritage, at a time when globalization advocates the standardization of law and the end of specificities States, it is necessary to question the future of the hereditary reserve. Parents no longer wish to be deprived of their power to freely dispose of their patrimony, even for reasons of death. What to answer to a father who is anxious about seeing his heritage squandered by an ungrateful child with whom he lives only conflicts ? What to say to this child who saw his brother favored by maternal decision, in an abusive and arbitrary way ? What advice can be given to those individuals who open the door of our offices every day to ask the help of their notary ? What to say to this husband who wants to protect his spouse from the angry of children from a first bed ? In order to answer them in practice, let us study ways of circumventing this hereditary reserve. Let's focus on checking their effectiveness. Beyond the classical mechanisms of the Civil Code such as the will and the gift, the interest of this thesis is to turn to more complex mechanisms, in order to answer the parents disappointed by their offspring, but also to them, children wronged to give them legal means to counter unfair deprivation. It is useful to understand and know what can be done to transmit one's heritage with a recovered freedom without risking the wrath of the law. Through the study of these instruments, their efficiency and their legal and family treatment, the question of the modernization of the reserve resurfaces. The family changes, and with it, the traditional conception of the hereditary reserve.

In Introduction, the author of this dissertation deals with postoperative venous thromboembolism (VTE), hereditary and acquired risk factors, prophylaxis regimens and recent recommendation of VTE prevention in surgery. In Practical part of this work the author assesses the risk of VTE in surgical patients according to risk assessment model. Genetic testing is carried out in all patients to determine the incidence of hereditary thrombophilia and coagulation markers are measured in 28-days postoperative period. Prevalence of VTE in 1-year postoperative period is observed. The results are analysed in group of patients with benign disease (hernia and gallstone disease) and group of patients with malignancy (colorectal cancer and pancreatic cancer) separately. The objective of this work is to determine the incidence of the most frequent thrombophilic mutations (factor V Leiden mutation and protrombin G20210A mutation) and assess the impact of hereditary thrombophilia on incidence of postoperative venous thromboembolism in general surgery. Validation of venous thrombosis risk assessment model recommended by Czech Society for Thrombosis and Hemostasis is further objective.