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Inflammatory breast cancer (IBC) is an aggressive BC subtype with rapid onset and poor outcomes. After biopsy confirmation, first-line treatment is based on systemic chemotherapy. A targetable somatic PIK3CA mutation is reported in 30% of IBC, allowing for treatment by PI3Kα-specific inhibitors, such as alpélisib. The aim of this study was to evaluate the detection rate of circulating PIK3CA mutation in non-metastatic IBC patients harbouring a PIK3CA mutation on initial biopsy. This monocentric retrospective study was based on available stored plasmas and biopsies at diagnosis and included all non-metastatic IBC patients who were treated by neo-adjuvant chemotherapy. PIK3CA mutations (E542K, E545K, H1047R/L) were assessed by droplet digital PCR (ddPCR) in plasma samples and tumoral tissue at diagnosis. A total of 55 patients treated between 2008 and 2018 in Henri Becquerel Center and with sufficient DNA from tumor tissue were included. 14/55 patients (25%) had a PIK3CA mutation identified on baseline biopsy (H1047R=8; H1047L=3; E545K=2; E542K=1). All mutations were single. Among them, 11 (79%) patients had enough circulating DNA for circulating mutations analyses. Corresponding circulating PIK3CA mutations were found in 6/11 patients (55%). Beside these 6 patients, no other circulating mutation was identified among the 43 patients with fully interpretable circulating and biopsy mutational analyses. Our results revealed no prognostic or predictive value of PIK3CA mutations at diagnosis of non-metastatic IBC but highlighted a prognostic value of cfDNA rate at diagnosis. Our study showed that corresponding circulating PIK3CA mutation was identified in 55% of non-metastatic IBC patients with baseline somatic PIK3CA mutation on tumor tissue while no circulating mutation was found among non-mutated PIK3CA patients. Further studies are needed to determine the prognostic and therapeutic impact of circulating PIK3CA mutation in IBC patients.; Le cancer du sein inflammatoire (CSI) est un sous-type de cancer du sein agressif et de mauvais pronostic, caractérisé par une rapide évolutivité tumorale. Après confirmation histologique, le traitement de première ligne est basé sur la chimiothérapie. Une mutation somatique de PIK3CA est reportée dans 30% des CSI, ciblée par les inhibiteurs spécifiques de la sous-unité alpha de PI3K de développement récent, tel que l’alpélisib. L’objectif de cette étude était d’évaluer le taux de détection des mutations circulantes PIK3CA au diagnostic chez les patientes suivies pour un cancer du sein inflammatoire non métastatique présentant une mutation PIK3CA sur la biopsie initiale. Cette étude rétrospective monocentrique était basée sur du matériel tumoral et circulant disponible au diagnostic et incluait toutes les patientes prises en charge par chimiothérapie néo-adjuvante pour un CSI non métastatique. Les mutations PIK3CA (E524K, E544K, H1047R/L) étaient évaluées par droplet digital PCR (ddPCR) au sein des échantillons plasmatiques et tumoraux au diagnostic. Un total de 55 patientes traitées entre 2008 et 2018 sur le Centre Henri Becquerel, avec suffisamment d’ADN extrait des biopsies tumorales ont été inclues. 14 patientes sur 55 (25%) étaient mutées PIK3CA sur la biopsie initiale (H1047R=8 ; H1047L=3 ; E545K=2 ; E542K=1). Toutes les mutations étaient uniques. Parmi elles, 11 patientes (79%) avaient assez d’ADN plasmatique pour l’analyse mutationnelle circulante. Des mutations PIK3CA circulantes correspondantes ont été retrouvées chez 6/11 patientes (55%). Hormis ces 6 patientes, aucune autre mutation circulante PIK3CA n’a été identifiée parmi les 43 patientes présentant du matériel tumoral et circulant de qualité suffisante pour permettre une analyse mutationnelle interprétable. Nos résultats n’ont pas mis en évidence de valeur pronostique ou prédictive des mutations PIK3CA au diagnostic des CSI non métastatiques, mais ont démontré la valeur pronostique du taux d’ADN libre circulant (ADNlc) au diagnostic. Notre étude a permis d’identifier des mutations PIK3CA correspondantes circulantes chez 55% des patientes mutées sur biopsie initiale alors qu’aucune mutation circulante n’a été trouvée parmi les patientes non mutées sur tissu tumoral. De futures études prospectives de plus grande ampleur sont attendues pour déterminer l’impact pronostique et thérapeutique des mutations circulantes PIK3CA chez les patientes suivies dans le cadre d’un CSI non métastatique.

The recent paradigm shift related to the development of personalized medicine in oncology requires an evolution of practices involving cancer molecular genetic laboratories. The first part of this work is based on the technological challenges of EGFR gene mutation detection to enable optimized management of patients eligible for targeted therapies in non small cell lung cancer at diagnosis and recurrence. New generation sequencing was the best alternative to identify EGFR sensitivity and acquired resistance mutations. The second part of this work concerns the biological interpretation of genetic alterations in the tumor, in particular variants in the tumor suppressor genes BRCA1 and BRCA2conditioning the sensitivity to PARP inhibitor targeted therapies. Two approaches for functional characterization of variants of uncertain significance (VUS) were used, both of which can be achieved in a time frame compatible with clinical imperatives. The first one consists in characterizing splicing defects from patient tumor RNA by RT-PCR and rapid cloning of RT-PCR products. The second is a rapid in vitro evaluation of VUS by genome editing using CRISPR-Cas9 technology and a functional score., Le récent changement de paradigme lié au développement de la médecine personnalisée en oncologie nécessite une évolution des pratiques impliquant les laboratoires de génétique moléculaire des cancers. Le premier volet de ce travail repose sur les enjeux technologiques de la détection des mutations du gène EGFR pour permettre une prise en charge optimisée des patients éligibles aux thérapies ciblées dans les cancers bronchiques non à petites cellules au diagnostic et lors des récidives. Le séquençage haut débit s’est ici présenté comme la meilleure alternative pour identifier les mutations de sensibilité et de résistance acquise de l’EGFR. Le deuxième volet de cette thèse concerne l’interprétation biologique des altérations génétiques de la tumeur, en particulier les variants des gènes suppresseurs de tumeur BRCA1 et BRCA2 conditionnant la sensibilité aux thérapies ciblées par inhibiteurs de PARP. Deux approches de caractérisation fonctionnelle de variants de signification incertaine (VSI) ont été utilisées, toutes deux réalisables dans un temps compatible avec les impératifs cliniques. La première consiste à caractériser des défauts d’épissage à partir d’ARN tumoral du patient par RT-PCR et clonage rapide des produits de RT-PCR. La deuxième est une évaluation rapide in vitro des VSI par édition du génome en utilisant la technologie CRISPR-Cas9 et un score fonctionnel.

BRAF V600E mutation in papillary thyroid carcinoma (PTC): prevalence and detection in fine needle aspiration (FNA) specimens.The activating mutation of the BRAF gene, T1799A, is the most common and specific genetic alteration in PTC. In the present study, our aims were to confirm these data and investigate the feasibility of BRAF mutation detection in FNA specimens.In a retrospective study, we examined paraffin-embedded surgical samples of 57 PTC and 51 non-PTC thyroid tumors for the presence of BRAF mutation by dideoxy sequencing. We analyzed thyroid aspirates (drop and washed-out solution) and smears from 31 patients who underwent thyroidectomy, before intraoperative frozen sections, and 25 archival thyroid FNA smears.The BRAF mutation was present in 58 % of PTC. Among non-PTC thyroid tumors, only one medullary thyroid carcinoma contained the BRAF mutation. BRAF mutation was correctly detected from the FNA-derived materials. Considering the search of BRAF mutation in preoperative FNA smears, the diagnosis of PTC would have been affirmed in 31 % (4/13) of indeterminate and suspicious FNA.BRAF mutation detection in FNA specimens is feasible and could be used as an adjunct tool for preoperative diagnosis of PTC classified as indeterminate and suspicious with conventional cytology (categories 3, 4 and 5 according to NCI/Bethesda 2008 terminology).

The recent paradigm shift related to the development of personalized medicine in oncology requires an evolution of practices involving cancer molecular genetic laboratories. The first part of this work is based on the technological challenges of EGFR gene mutation detection to enable optimized management of patients eligible for targeted therapies in non small cell lung cancer at diagnosis and recurrence. New generation sequencing was the best alternative to identify EGFR sensitivity and acquired resistance mutations. The second part of this work concerns the biological interpretation of genetic alterations in the tumor, in particular variants in the tumor suppressor genes BRCA1 and BRCA2conditioning the sensitivity to PARP inhibitor targeted therapies. Two approaches for functional characterization of variants of uncertain significance (VUS) were used, both of which can be achieved in a time frame compatible with clinical imperatives. The first one consists in characterizing splicing defects from patient tumor RNA by RT-PCR and rapid cloning of RT-PCR products. The second is a rapid in vitro evaluation of VUS by genome editing using CRISPR-Cas9 technology and a functional score., Le récent changement de paradigme lié au développement de la médecine personnalisée en oncologie nécessite une évolution des pratiques impliquant les laboratoires de génétique moléculaire des cancers. Le premier volet de ce travail repose sur les enjeux technologiques de la détection des mutations du gène EGFR pour permettre une prise en charge optimisée des patients éligibles aux thérapies ciblées dans les cancers bronchiques non à petites cellules au diagnostic et lors des récidives. Le séquençage haut débit s’est ici présenté comme la meilleure alternative pour identifier les mutations de sensibilité et de résistance acquise de l’EGFR. Le deuxième volet de cette thèse concerne l’interprétation biologique des altérations génétiques de la tumeur, en particulier les variants des gènes suppresseurs de tumeur BRCA1 et BRCA2 conditionnant la sensibilité aux thérapies ciblées par inhibiteurs de PARP. Deux approches de caractérisation fonctionnelle de variants de signification incertaine (VSI) ont été utilisées, toutes deux réalisables dans un temps compatible avec les impératifs cliniques. La première consiste à caractériser des défauts d’épissage à partir d’ARN tumoral du patient par RT-PCR et clonage rapide des produits de RT-PCR. La deuxième est une évaluation rapide in vitro des VSI par édition du génome en utilisant la technologie CRISPR-Cas9 et un score fonctionnel.

If hormone therapy is a key treatment for hormone receptor positive advanced breast cancers, secondary resistance occurs as a rule. Recently, acquired alterations of the ESR1 gene have been identified as a mechanism of resistance on aromatase inhibitor (AI) treatment. The selective pressure by AI exposure during the metastatic setting triggers the emergence of ESR1 activating mutations. In that context, the "liquid biopsy" concept has been used to detect this molecular resistance before progression. Thus, the ESR1 circulating mutation detection will soon be used in daily practice to help monitoring patients on AI treatment and provide an early change for specific therapies that still have to be determined in prospective clinical trials. This review will present the acquired ESR1 mutations, as well as the methods used for their detection in blood and the potential clinical impact of this approach for hormone receptor positive breast cancer management.

Before molecular analysis is performed, morphological control with an estimation of the tumour cell percentage (%TC) could have a major impact on mutation detection. Accreditation according to NF EN ISO 15189 commands an authorization through evaluation of skills. The objective of this work was to validate the empowerment of pathologists to estimate %TC in tissue sample prior to molecular analysis. The accreditation technical guidance methods in Medical biology and histopathology were taken as references. %TC was the ratio of tumour cell nuclei on all nuclei within the area selected for the DNA extraction. External evaluations quality scores were used for accuracy. In order to assess the intermediate precision, 35 %TC estimation were performed 15 days apart in 4 samples (biopsies, transparietal biopsies or surgical specimen, either fixed or frozen) by 7 pathologists. Three other cases with interference (inflammation, mucus, necrosis) were evaluated. A result was satisfactory if %TC were within ±20 % of expected percentage obtained by the average of 35 estimates. The performances were satisfactory since no estimate was made more than 20 % of the expected percentage. Low interpathologists reproducibility has been reported in the literature and can have a consequence on molecular analysis in samples with low %TC, where the value reach the analytical sensitivity thresholds of molecular techniques. The current report is an example of a step of the accreditation process, which is a challenge for pathologists' activity in the future.

Mémoire de DES (Diplôme d'Etudes Spécialisées) tenant lieu de thèse d'exercice de pharmacie. Thèse proposée pour un prix de thèse.; Directly-acting antivirals brought considerable progress to hepatitis C treatment, but their activity varies depending on viral genotypes and subtypes. Therefore, accurate genotyping is still of major importance, as well as the detection of resistance-associated mutations. To answer these goals, an approach to amplify hepatitis C virus near-complete genome with a single long-range PCR and sequence it with a next-generation technique was developed. The overall amplification success rate was of 73% for usual genotypes (i.e. HCV1a, 1b, 3a and 4a, n = 16/22) and 45% for recombinant forms RF_2k/1b (n = 5/11). After pyrosequencing and de novo assembly, a near-full-length genomic consensus sequence was obtained for 19/21 samples. Genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Circulation of this particular strain, difficult to identify, is probably underestimated. Ten cases have been detected in the Rhône-Alpes region between January and August 2014 in patients from the Caucasus area. All viral types considered, resistance-associated mutations were detected in 7/13 patients at baseline, in NS3 (n = 3) or NS5A region (n = 4). One of them was treated with daclatasvir and experienced a relapse. Viral sequences from pre- and post-treatment samples of four patients who had relapsed after a sofosbuvir-based therapy were compared: the selected variants seem too far from NS5B active site to be held responsible. Although tested on a limited set of samples and with technical improvements still needing to be made, this assay has proved to be successful both for genotyping and mutation detection on several HCV types.; Les antiviraux à action directe ont apporté des progrès considérables au traitement de l’hépatite C, mais leur activité varie selon les génotypes et sous-types viraux. Un typage fiable et précis est donc indispensable, de même que la détection des variants résistants. Une approche d’amplification du génome quasi-complet du virus de l’hépatite C par une PCR unique suivie d’un séquençage de nouvelle génération a été développée pour répondre à ces objectifs. Le taux de réussite de la RT-PCR était de 73 % pour les génotypes classiques (1a, 1b, 3a, et 4a, n = 16/22) et 45 % pour les formes recombinantes RF_2k/1b (n = 5/11). Après pyroséquençage puis assemblage de novo, les génomes quasi-complets ont pu être obtenus pour 19/21 échantillons. L’analyse phylogénétique a confirmé les génotypes et sous-types de tous les échantillons, incluant les recombinants. Cette souche, difficile à mettre en évidence, a été identifiée rétrospectivement chez dix patients en région Rhône-Alpes entre janvier et août 2014. Tous types viraux confondus, des mutations de résistance ont été détectées chez 7/13 patients naïfs de traitement, dans les régions NS3 (n = 3) ou NS5A (n = 4). L’un d’eux a ensuite rechuté après un traitement par daclatasvir. La comparaison chez quatre patients des séquences virales pré- et post-traitement par sofosbuvir a montré l’apparition de certains variants, mais trop éloignés du site actif de NS5B pour être responsables de la rechute. Bien que testée sur un nombre limité d’échantillons, et nécessitant encore des améliorations techniques, cette méthode a montré son efficacité pour le typage comme pour la détection des variants résistants sur différents types viraux du VHC.

Usher syndrome is a genetic disorder combining sensorineural hearing loss (HL) and retinitis pigmentosa (RP). Some patients will also exhibit vestibular areflexia (VA). Clinical and genetic heterogeneity is recognized as the 3 clinical subgroups, defined mainly on the degree of HL and VA, can be caused by mutations in one of the 10 known genes. It is important to use all accessible genetic tools to identify and characterize molecular origin in order to improve the knowledge of the physiopathological mechanisms causing Usher Syndrome.In this context, we have developed an exhaustive approach. In a first step, we have implemented the analysis and established the mutational spectrum of the 2 minor USH2 genes (GPR98 and DFNB31). In addition, we have developed several tools, in particular to study variants susceptible to alter splicing or lying in the promoter regions of the USH2 genes.Thanks to this work, the USH2 mutation detection rate has now been raised to 90%, similar to that of USH1.We have then designed a targeted exome of the Usher genes to be sequenced using the GS Junior system (Roche 454). The aim of the study was to test the feasibility of this new technics for a possible transfer to diagnostic facilities. Quality criteria and variant priorization were set up on a control cohort (previously studied in one of the USH gene). The study has then been extended on a patient cohort. Our results indicate that NGS Usher-exome can be used in molecular diagnostics but improvement of the reliability of the sequencing technology, bioinformatics tools and dedicated databases is essential.; Le syndrome de Usher est une maladie génétique associant surdité congénitale et rétinopathie pigmentaire (RP), auxquelles peuvent s'ajouter des troubles vestibulaires. Les différences phénotypiques, distinguées en 3 types cliniques, s'accompagnent d'une hétérogénéité génétique impliquant au moins 10 gènes. Identifier et caractériser les causes moléculaires grâce aux outils d'analyses génétiques disponibles permet d'améliorer la compréhension des mécanismes physiopathologiques à l'origine des symptômes du syndrome de Usher. Dans ce cadre, nous nous sommes inscrits dans une recherche d'exhaustivité des études moléculaires. Dans un premier temps, nous avons ainsi mis en place l'analyse et défini le spectre mutationnel des gènes minoritairement impliqués dans le type II (GPR98 et DFNB31). Nous avons également développé différents outils, notamment pour l'analyse de variants altérant le mécanisme d'épissage ou touchant les régions promotrices des gènes USH2.Ces travaux permettent d'obtenir un taux de détection des altérations conduisant au syndrome de Usher type 2 de 90 %. Ce taux est maintenant similaire à celui observé pour le type 1, qui constituait jusqu'ici la référence.Nous avons, dans un second temps, développé le séquençage nouvelle génération (NGS) appliqué à l'exome Usher. L'objectif de cette analyse était de tester la faisabilité et l'efficacité de cette approche, en vue de son éventuelle utilisation en diagnostic moléculaire. La définition des critères de qualité et la mise en place de la priorisation des variants ont été réalisées sur un groupe contrôle. L'étude a ensuite été étendue sur une cohorte de patients. Les résultats obtenus montrent qu'une utilisation en diagnostic est possible mais restera dépendante de l'amélioration de la technique du séquençage, de son analyse et des outils bioinformatiques pour interpréter le volume de données ainsi généré.

International audience; Criteria have been proposed for genetic testing of breast and ovarian cancer susceptibility genes BRCA1 and BRCA2. Using simulations, this study evaluates the efficiency (sensitivity, positive predictive value [PPV] and specificity) of the various criteria used in France. The efficiency of the criteria published in 1998, which are largely used, is not optimal. We show that some extensions of these criteria provide an increase in sensitivity with a low decrease in specificity and PPV. The study shows that scoring systems (Manchester, Eisinger) have similar efficiency that may be improved. In this aim, we propose a new scoring system that takes into account unaffected individuals and kinship coefficients between family members. This system increases sensitivity without affecting PPV and specificity. Finally, we propose a two-step procedure with a large screening by the physician for recommending genetic counselling, followed by a more stringent selection by the geneticist for prescribing genetic testing. This procedure would result in an increase of genetic counselling activity but would allow the identification of almost 80% of mutation carriers among affected individuals, with a mutation detection rate of 15% and a specificity of 88%.

To screen hepatitis B virus (HBV) genotypes and associated basal core promoter (BCP; T1762A/A1764) and precore (PC; A1896) mutations among the 100 HBV surface antigen (HBsAg) positive voluntary blood donors in France.HBV genotypes were determined by using direct sequence analysis. Three methods were used to detect G1896A mutation: non-commercial real-time PCR (PCRTR°, line probe assay (InnoLiPA HBV PreCore, INNOGENETICS(®)) and direct sequencing of precore gene. HBV viral load was quantified with two commercial real-time PCR (COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV Test/Roche and Real Time HBV/M2000/Abbott).The mean age of donors was 30 (18-64). Patients were from Africa (42%), Europa (50%), and Asia (8%). HBV/D was the most predominant (37%) genotype followed by HBV/A (31%) and HBV/E (22%). PC and BCP mutants were found in 57% with Inno-LIPA HBV test and 59% with both PCRTR and sequencing methods. A significant difference in the viral load of blood donors with wild and PC mutants was observed with the Taqman Cobas real time PCR (3,19 Log(10) UI/ml versus 4,93 Log(10) UI/ml, p 0.05). Precore phenotype determination was in agreement with the three PC mutation detection methods in 56% of cases.Non-Caucasian genotype E was present in the French blood donors. PC mutation was more common than BCP mutations in this study. As HBV infected blood donors were more often asymptomatic carriers, we could speculate that the G1896A mutation may favour the asymptomatic state, supporting previous observations.

X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder. ALD is characterized by progressive demyelination within the central and peripheral nervous system, adrenal insufficiency (Addison's disease) and accumulation of very-long-chain fatty acids (VLCFA) in plasma, fibroblasts and tissues. The overall incidence of ALD is 1:17,000 including hemizygotes and heterozygotes who are frequently symptomatic. There are two main ALD phenotypes: 1) a cerebral demyelinating form which affects boys between 5-12 years, but also 35% of adult males; 2) a form that mainly involves the spinal cord (adrenomyeloneuropathy, AMN) in adult males between 20-50 years and 50% of heterozygous women after the age of 40 years. AMN presents with progressive spastic paraparesis. Addison's disease may be the first symptom of ALD in boys and adult males. These patients are at risk to develop cerebral ALD or AMN for life. ALD results from mutations in the ABCD1 gene without correlation between genotype and phenotype. The diagnosis of ALD relies upon the measurement of plasma VLCFA levels that allows the identification of 100% affected males and of 80-95% heterozygous women. Because of these false-negative, it is therefore mandatory to search for a mutation in the ABCD1 gene in all women at risk to be heterozygous for ALD. The ABCD1 gene encodes a peroxisomal transmembrane protein (ALD protein) with the structure of an half ATP-binding cassette transporter. It is possible that ALD protein imports VLCFA or VLCFA-CoA into peroxisomes in which they are degraded by a peroxisomal beta-oxidation system. Elongation of VLCFAs is enhanced in fibroblasts from ALD patients and likely contributes to the load of VLCFA in tissues. The underlying mechanisms that lead to cerebral demyelination, axonal degeneration in spinal cord and adrenal insufficiency are unknown. The "toxic" role of VLCFA accumulation remains to be demonstrated. The mechanisms that lead to the inflammatory reaction in cerebral ALD might involve abnormal acylation of gangliosides and phospholipids by VLCFA that would result in immune reaction of brain macrophages and astrocytes bearing CD1 molecules that recognize lipid antigens. De novo mutation of ABCD1 occurs in less than 8% of ALD patients. The genetic counseling aims to identify: 1) women who are at risk to be heterozygous; 2) neurologically asymptomatic boys. It is only at this stage that allogeneic bone marrow transplantation has clinical benefit; 3) ALD patients who have Addison's disease that can lead to sudden death. Prenatal diagnosis (chorionic villus samples, cultured amniotic fluid cells) relies upon DNA based mutation detection techniques, expression of ALD protein and measurement of VLCFA levels. Allogeneic bone marrow transplantation is the only treatment that provides a permanent cure when the procedure is performed at an early stage of cerebral demyelination, i.e when the patients are asymptomatic despite abnormal brain MRI. Treatment of Addison's disease is mandatory but does not modify the course of neurological symptoms. Dietary therapy failed to halt the neurologic progression in cerebral ALD and AMN. It might have a partial preventive effect in boys treated before 6 years of age.

Soft tissue sarcomas are rare and may be a source of problems for diagnosis and treatment. Four types of genetic disorders can be distinguished: translocations, gene amplifications, mutations and complex genetic imbalances. Detection of these disorders may help in diagnosis and in determining prognosis. Detection of specific translocation is recommended in synovial sarcoma, alveolar rhabdomyosarcoma or PNET diagnosis because of therapeutic consequences; in case of rarer histologic type (low grade fibromyxoid sarcoma, clear cell sarcoma, infantile fibrosarcoma...), it may confirm the diagnosis. In some cases, some translocations have a prognostic value (alveolar rhabdomyosarcoma) whereas it is discussed in others (synovial sarcoma). The techniques used to detect these translocations are very sensitive so it may be used to detect microscopical metastasis (bone marrow metastasis of alveolar rhabdomyosarcoma for example). Detection of MDM2 and CDK4 genes amplifications (FISH or quantitative PCR) may be sometimes useful in well differentiated and dedifferentiated liposarcomas diagnosis. Mutation detection of KIT or PDGFRA may help in GIST diagnosis and type of mutation is predictive of response to treatment. Study of complex genomic imbalances in sarcomas is not used in routine practice but remains useful in research.

Congenital disorders of glycosylation are a group of inherited disorders, characterized by a central nervous system dysfunction and multiorgan failure associated with defective N-glycosylation. CDG-I comprises all defects in the assembly of the dolichol-linked glycan and its transfer to the protein, whereas CDG-II refers to defects in the processing of the protein-bound glycans. The diagnosis is done by the presence of hypoglycosylated glycoproteins in the serum and typing by enzymatic assay (available for CDG-Ia and Ib) and/or mutation detection. We give an overview of the latest results of molecular diagnosis from the French CDG I families. We report novel mutations and their functional study. In addition we looked for a founder effect for the most frequent mutations observed in the French population.

Ret! syndrome (RTT) IS a progressIVe neurodevelopmentaI disorder affecting almost exclusively females with an incidence of 1 in 15,000. Defined by accurate clinical criteria, RTT is characterized by a period of early normal growth and development followed by regression with loss of speech and acquired motor skills, stereotypical hand movements, and seizures. Althougb 99,5% of cases are sporadic, exclusion mapping using rare familial cases allowed the assignment of the gene responsable for RTT to Xq28. Systematic mutation analysis of candidate genes in the critical region resulted in the identification of mutations inthe MECP2 gene. During this work, we carried out a mutation analysis of MECP2 gene in 255 females with RTT and 15 mentally retarded boys. To identify MECP2 mutations, we developped several complementary approaches (FISH, Southem blot, semi-quantitative PCR, CSGE-sequencing, then DHPLC-sequencing) and provided insights into an heterogeneous spectrum of mutationsat the MECP2 locus in RTT patients. Accordingly, we showed that in addition of point mutations, rearrangements involving several hundreds base pairs can be detected. Moreover and for the first time in RTT girls, we reported cases of somatic mosaicism. Finally, a study at the chromosome level of the MECP2 gene did not reveal any deletion in RTT patients. We found 79% of mutations in RTT patients, our mutation detection rate is in agreement with theIiterature data. Our mutation screening strategy of the ]MECP2 gene is an essential molecular diagnosis tool to confirm the c1inical diagnosis. The link between MECP2 function (global repressor of methylated genes transcription) and disease expression is still unknoWll. Many studies are necessary to understand thephysiological mechanism leading to RTT and eventually to develop therapeutic strategies., La méthylation des cytosines et l'acétylationJdésacétylation des histones constituent deux modifications épigénétiques importantes du génome eucaryote. Elle sont, toutes les deux, impliquées dans la répression transcriptionnelle. La découverte de la famille protéique MeCP (Methyl-CpG-binding protein) et notamment de la protéine MeCP2, archétype de cettefamille, a permis d'établir un lien entre ces deux modifications épigénétiques. En effet, la protéine MeCP2, une fois liée aux cytosines méthylées des dinucléotides 5'CpG3' réprime indirectement la transcription par l'intermédiaire d'un complexe multiprotéique comprenant en plus du co-répresseur Sin-3A, les histones désacétylases 1 et 2 (HDACI et 2). Les HDACI et 2, en désacétylant les histones du nucléosome entraînent la condensation de la chromatine.Récemment, le gène MECP2 codant la protéine MeCP2 a été impliqué dans un retard mental syndromique lié au chromosome X : le syndrome de Rett. Le syndrome de Rett, pathologie rare (1 fille sur 10 000 à 15 000) se caractérise par une période périnatale normale (jusqu'à 618 mois) avant l'apparition des premiers symptômes correspondant à une régression des acquisitions psychomotrices.Nous avons recherché les mutations dans le gène MECP2 chez 255 patientes atteintes d'un syndrôme de Rett et chez 15 garçons retardés mentaux. Nous avons mis en évidence un spectre mutationnel hétérogène du gène MECP2 chez les patientes atteintes d'un syndrome de Rett. En effet, en plus de mutations ponctuelles, le gène MECP2 peut être remanié sur plusieurs centaines de paires de bases. D'autre part, nous rapportons, pour la première fois chez des filles, l'existence de mutations en mosaïque somatique.

More than 10 genetic diseases, including 3 frequent ones (the fragile X mental retardation syndrome, myotonic dystrophy and Huntington's disease) are caused by unstable expansions of trinucleotide repeats. The instability of these mutations and their strong tendency to further expansion, account for the special characteristics of the mode of inheritance of these diseases. Unlike most other genetic diseases, that can be caused by a large number of different mutations, the trinucleotide expansion diseases are due to a single type of mutation. Detection of the expansion by DNA analysis allows thus easy and reliable diagnosis of these diseases. For several of them, the late but generally very deleterious clinical expression, raises the very delicate problem of genetic counselling and presymptomatic diagnosis.