Highly parallel genetic approaches in Mendelian and complex diseases

The recent advance in high-throughput sequencing and genotyping protocols allows rapid investigation of Mendelian and complex diseases on a scale not previously been possible. In my thesis research I took advantage of these modern techniques to study retinitis pigmentosa (RP), a rare inherited disease characterized by progressive loss of photoreceptors and leading to blindness; and hypertension, a common condition affecting 30% of the adult population. Firstly, I compared the performance of different next generation sequencing (NGS) platforms in the sequencing of the RP-linked gene PRPF31. The gene contained a mutation in an intronic repetitive element, which presented difficulties for both classic sequencing methods and NGS. We showed that all NGS platforms are powerful tools to identify rare and common DNA variants, also in case of more complex sequences. Moreover, we evaluated the features of different NGS platforms that are important in re-sequencing projects. The main focus of my thesis was then to investigate the involvement of pre-mRNA splicing factors in autosomal dominant RP (adRP). I screened 5 candidate genes in a large cohort of patients by using long-range PCR as enrichment step, followed by NGS. We tested two different approaches: in one, all target PCRs from all patients were pooled and sequenced as a single DNA library; in the other, PCRs from each patient were separated within the pool by DNA barcodes. The first solution was more cost-effective, while the second one allowed obtaining faster and more accurate results, but overall they both proved to be effective strategies for gene screenings in many samples. We could in fact identify novel missense mutations in the SNRNP200 gene, encoding an essential RNA helicase for splicing catalysis. Interestingly, one of these mutations showed incomplete penetrance in one family with adRP. Thus, we started to study the possible molecular causes underlying phenotypic differences between asymptomatic and affected members of this family. For the study of hypertension, I joined a European consortium to perform genome-wide association studies (GWAS). Thanks to the use of very informative genotyping arrays and of phenotipically well-characterized cohorts, we could identify a novel susceptibility locus for hypertension in the promoter region of the endothelial nitric oxide synthase gene (NOS3). Moreover, we have proven the direct causality of the associated SNP using three different methods: 1) targeted resequencing, 2) luciferase assay, and 3) population study. - Le récent progrès dans le Séquençage à haut Débit et les protocoles de génotypage a permis une plus vaste et rapide étude des maladies mendéliennes et multifactorielles à une échelle encore jamais atteinte. Durant ma thèse de recherche, j'ai utilisé ces nouvelles techniques de séquençage afin d'étudier la retinite pigmentale (RP), une maladie héréditaire rare caractérisée par une perte progressive des photorécepteurs de l'oeil qui entraine la cécité; et l'hypertension, une maladie commune touchant 30% de la population adulte. Tout d'abord, j'ai effectué une comparaison des performances de différentes plateformes de séquençage NGS (Next Generation Sequencing) lors du séquençage de PRPF31, un gène lié à RP. Ce gène contenait une mutation dans un élément répétable intronique, qui présentait des difficultés de séquençage avec la méthode classique et les NGS. Nous avons montré que les plateformes de NGS analysées sont des outils très puissants pour identifier des variations de l'ADN rares ou communes et aussi dans le cas de séquences complexes. De plus, nous avons exploré les caractéristiques des différentes plateformes NGS qui sont importantes dans les projets de re-séquençage. L'objectif principal de ma thèse a été ensuite d'examiner l'effet des facteurs d'épissage de pre-ARNm dans une forme autosomale dominante de RP (adRP). Un screening de 5 gènes candidats issus d'une large cohorte de patients a été effectué en utilisant la long-range PCR comme étape d'enrichissement, suivie par séquençage avec NGS. Nous avons testé deux approches différentes : dans la première, toutes les cibles PCRs de tous les patients ont été regroupées et séquencées comme une bibliothèque d'ADN unique; dans la seconde, les PCRs de chaque patient ont été séparées par code barres d'ADN. La première solution a été la plus économique, tandis que la seconde a permis d'obtenir des résultats plus rapides et précis. Dans l'ensemble, ces deux stratégies se sont démontrées efficaces pour le screening de gènes issus de divers échantillons. Nous avons pu identifier des nouvelles mutations faux-sens dans le gène SNRNP200, une hélicase ayant une fonction essentielle dans l'épissage. Il est intéressant de noter qu'une des ces mutations montre une pénétrance incomplète dans une famille atteinte d'adRP. Ainsi, nous avons commencé une étude sur les causes moléculaires entrainant des différences phénotypiques entre membres affectés et asymptomatiques de cette famille. Lors de l'étude de l'hypertension, j'ai rejoint un consortium européen pour réaliser une étude d'association Pangénomique ou genome-wide association study Grâce à l'utilisation de tableaux de génotypage très informatifs et de cohortes extrêmement bien caractérisées au niveau phénotypique, un nouveau locus lié à l'hypertension a été identifié dans la région promotrice du gène endothélial nitric oxide sinthase (NOS3). Par ailleurs, nous avons prouvé la cause directe du SNP associé au moyen de trois méthodes différentes: i) en reséquençant la cible avec NGS, ii) avec des essais à la luciférase et iii) une étude de population.

Comparaison des coûts du diagnostic moléculaire des rétinites pigmentaires entre le séquençage selon Sanger et un séquençage à haut débit

La rétinite pigmentaire (RP) et l'amaurose congénitale de Leber (LCA) sont deux maladies héréditaires classées dans le groupe des rétinopathies pigmentaires. Plus de 100 gènes ou loci ont étés identifiés dans les RP (comptant pour 60% des patients) et 14 gènes pour les LCA (responsables de 70% des cas). A elles-deux et en ne prenant que les plus fréquentes, ces maladies représentent 948 exons à analyser lorsqu'on recherche une mutation chez un patient. La recherche de mutations au moyen du séquençage génomique classique (séquençage selon Sanger) de tous les gènes de ces deux maladies implique des délais d'analyse importants et des coûts très élevés. La méthode de séquençage à haut débit (avec le séquenceur Roche GS Junior) permet grâce à une puce à ADN le séquençage simultané des 948 exons. Le but de mon travail de Master est de comparer ces deux approches afin de déterminer celle qui est la plus économique et la plus efficiente en temps. Pour cela, j'ai d'abord établi la liste de tous les gènes impliqués dans les RP et LCA, puis identifié tous les exons ainsi que les promoteurs et séquences 3' non traduites. J'ai ensuite calculé le coût théorique d'une analyse de tous les gènes avec chacune des méthodes. J'ai également estimé les coûts à facturer à l'assurance concernant le séquençage à haut débit sur la base des coûts facturables à l'assurance de la méthode Sanger et des bénéfices du laboratoire. Le séquençage des 948 exons par le séquençage à haut débit (avec le GS Junior) représente la technique de séquençage la plus économique et la plus efficiente en temps et constitue donc la méthode de choix dans le screening diagnostic des gènes impliqués dans les RP et LCA. Cette méthode est plus rapide, les réactifs et la machine sont moins coûteux et la laborantine peut analyser un nombre plus important d'exons en un temps moindre, donc elle coûtera moins cher au laboratoire. Cette méthode est donc d'un grand intérêt pour les patients, les assurances et le laboratoire. Cette nouvelle technique de séquençage soulève de nouvelles interrogations telles que la décision de savoir quelle information doit être donnée aux médecins, aux assurances et aux patients. Interrogations auxquelles il devient de plus en plus pressant de répondre.

Identification of an RP1 Prevalent Founder Mutation and Related Phenotype in Spanish Patients with Early-Onset Autosomal Recessive Retinitis.

OBJECTIVE: To identify the genetic causes underlying early-onset autosomal recessive retinitis pigmentosa (arRP) in the Spanish population and describe the associated phenotype. DESIGN: Case series. PARTICIPANTS: A total of 244 unrelated families affected by early-onset arRP. METHODS: Homozygosity mapping or exome sequencing analysis was performed in 3 families segregating arRP. A mutational screening was performed in 241 additional unrelated families for the p.Ser452Stop mutation. Haplotype analysis also was conducted. Individuals who were homozygotes, double heterozygotes, or carriers of mutations in RP1 underwent an ophthalmic evaluation to establish a genotype-phenotype correlation. MAIN OUTCOME MEASURES: DNA sequence variants, homozygous regions, haplotypes, best-corrected visual acuity, visual field assessments, electroretinogram responses, and optical coherence tomography images. RESULTS: Four novel mutations in RP1 were identified. The new mutation p.Ser542Stop was present in 11 of 244 (4.5%) of the studied families. All chromosomes harboring this mutation shared the same haplotype. All patients presented a common phenotype with an early age of onset and a prompt macular degeneration, whereas the heterozygote carriers did not show any signs of retinitis pigmentosa (RP). CONCLUSIONS: p.Ser542Stop is a single founder mutation and the most prevalent described mutation in the Spanish population. It causes early-onset RP with a rapid macular degeneration and is responsible for 4.5% of all cases. Our data suggest that the implication of RP1 in arRP may be underestimated. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

A large deletion in the adRP gene PRPF31: evidence that haploinsufficiency is the cause of disease.

PURPOSE: To report a large deletion that encompasses more than 90% of PRPF31 gene and two other neighboring genes in their entirety in an adRP pedigree that appears to show only the typical clinical features of retinitis pigmentosa. METHODS: To identify PRPF31 mutation in a dominant RP family (ADRP2) previously linked to the RP11 locus, the 14 exons of PRPF31 were screened for mutations by direct sequencing. To investigate the possibility of a large deletion, microsatellite markers near PRPF31 gene were analyzed by non-denaturing PAGE. RESULTS: Initial screening of PRPF31 gene in the ADRP2 family did not reveal an obvious mutation. A large deletion was however suspected due to lack of heterozygosity for nearly all PRPF31 intragenic single nucleotide polymorphysm (SNPs). In order to estimate the size of the deletion, SNPs and microsatellite markers spanning and flanking PRPF31 were analyzed in the entire ADRP2 family. Haplotype analysis with the above markers suggested a deletion of approximately 30 kb that included the putative promoter region of a novel gene OSCAR, the entire genomic content of genes NDUFA3, TFPT and more than 90% of PRPF31 gene. Sequence analysis of the region flanking the potential deletion showed a high presence of Alu elements implicating Alu mediated recombination as the mechanism responsible for this event. CONCLUSIONS: This mutation provides evidence that haploinsufficiency rather than aberrant function of mutated proteins is the cause of disease in these adRP patients with mutations in PRPF31 gene.

Chromatic pupillometry in patients with retinitis pigmentosa.

OBJECTIVE:: To evaluate the chromatic pupillary response as a means of assessing outer and inner retinal function in patients with retinitis pigmentosa (RP). DESIGN:: Evaluation of diagnostic technology. PARTICIPANTS:: Thirty-two patients with RP and visual loss and 43 normal subjects. METHODS:: Patients were tested with a chromatic pupillometer using red and blue lights (1, 10, and 100 cd/m(2)), and their pupil responses were compared with those from 43 normal subjects (reported previously). Visual field and electroretinography (ERG) results were examined and compared with the pupil responses. MAIN OUTCOME MEASURES:: The percent pupil contraction of the transient response to a low-intensity (1 cd/m(2)) blue light and high-intensity (100 cd/m(2)) red light and the sustained response to a high-intensity blue light was calculated for 1 eye of each subject. RESULTS:: The pupil responses to red and blue light at all intensities were recordable in all patients except 1, whose pupil responded only to bright blue light. There was a significant difference of the pupil response between patients with RP and normal subjects in testing conditions that emphasized rod (1 cd/m(2) blue light) or cone (100 cd/m(2) red light) contribution (P<0.001). Patients with a non-recordable scotopic ERG showed significantly reduced pupil responses (P<0.001) to low-intensity blue light (1 cd/m(2)). Patients with a non-recordable or abnormal photopic ERG showed significantly reduced pupil responses (P<0.05) to high-intensity red light (100 cd/m(2)). Patients with a nonrecordable ERG had the most visual field loss and reduced pupil responses. Unexpectedly, patients with RP showed a slower re-dilation of the pupil after termination of bright blue light compared with red light, a pattern not observed in normal subjects. CONCLUSIONS:: Pupil responses to red and blue light stimuli weighted to favor cone or rod input are significantly reduced in patients with RP but are still recordable in patients having a non-recordable ERG. In addition, outer photoreceptor disease appears to unmask a post-illumination pupillary constriction to bright blue light, most likely mediated by intrinsic activation of melanopsin ganglion cells. Chromatic pupillometry provides a novel, noninvasive method for following retinal functional status, particularly in patients with severe RP and non-recordable ERG. FINANCIAL DISCLOSURE(S):: Proprietary or commercial disclosure may be found after the references.

How Much Chemotherapy Are Patients With Advanced Pancreatic Cancer Receiving at the End of Life?

Background: Advanced pancreatic adenocarcinoma (APC) is a chemoresistant cancer with poor prognosis. We evaluated the use of chemotherapy in the last months of life.Methods: Retrospective analysis of patients with APC treated from 1993 to 2010 at the Oncology Institute of Southern Switzerland. Clinical and laboratory parameters starting from 28 days prior to the last administration of chemotherapy were recorded, including ECOG performance status, presence of ascites, haemoglobin (Hb), white blood cell (WBC) count, platelets, total bilirubin, albumin, LDH, C-reactive protein (C-rp) and Ca 19.9.Results: The characteristics of the 231 patients were: males/females 53%/47%; metastatic/locally advanced disease 80%/20%; median age 66 years (range 32−85). Median overall survival calculated from diagnosis was 6.1 months (95% CI: 5.1−7.2); death was due to disease progression in all cases. At last chemotherapy administration, ECOG performance status was 0−1 in 38% and 2−3 in 62%. Fifty-nine percent of pts received first-line chemotherapy only (gemcitabine in 70%; gemcitabine-based doublets or 5FU in 30%), whilst 32%, 8% and 1% had second- (5FU 37%; oxaliplatinbased doublets 57%; phase I trial 6%), third- and fourth-line therapy (single agent or phase I trial), respectively. The interval between last chemotherapy administration and death was <4 weeks in 24%, _4−12 weeks in 47% and >12 weeks in 29%. Table 1 summarizes the proportion of patients treated according to the interval between last chemotherapy and death refered to chemotherapy line. Median survival from last chemotherapy delivery to death was 7.5 weeks (95% CI 6.7−8.4). In univariate analysis, presence of ascites, elevated WBC, total bilirubin, LDH, C-rp and Ca 19.9, and reduced albumin were found to predict shorter survival (p < 0.05 for each). However, none of them was an independent predictor in the multivariate analysis.Conclusions: A significant proportion of patients with APC received chemotherapy in the last months of life. In our study, none of the clinical and laboratory parameters recorded 28 days priorto the last chemotherapy delivery were found to predict survival.

Nonsense Mutations in FAM161A Cause RP28-Associated Recessive Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a degenerative disease of the retina leading to progressive loss of vision and, in many instances, to legal blindness at the end stage. The RP28 locus was assigned in 1999 to the short arm of chromosome 2 by homozygosity mapping in a large Indian family segregating autosomal-recessive RP (arRP). Following a combined approach of chromatin immunoprecipitation and parallel sequencing of genomic DNA, we identified a gene, FAM161A, which was shown to carry a homozygous nonsense mutation (p.Arg229X) in patients from the original RP28 pedigree. Another homozygous FAM161A stop mutation (p.Arg437X) was detected in three subjects from a cohort of 118 apparently unrelated German RP patients. Age at disease onset in these patients was in the second to third decade, with severe visual handicap in the fifth decade and legal blindness in the sixth to seventh decades. FAM161A is a phylogenetically conserved gene, expressed in the retina at relatively high levels and encoding a putative 76 kDa protein of unknown function. In the mouse retina, Fam161a mRNA is developmentally regulated and controlled by the transcription factor Crx, as demonstrated by chromatin immunoprecipitation and organotypic reporter assays on explanted retinas. Fam161a protein localizes to photoreceptor cells during development, and in adult animals it is present in the inner segment as well as the outer plexiform layer of the retina, the synaptic interface between photoreceptors and their efferent neurons. Taken together, our data indicate that null mutations in FAM161A are responsible for the RP28-associated arRP.

Expression of neutral endopeptidase, endothelin-1, and nuclear factor kappa B in prostate cancer: interrelations and associations with prostate-specific antigen recurrence after radical prostatectomy.

Objective. To study the impact of the neutral endopeptidase (NEP)/neuropeptides (NPs) axis and nuclear factor kappa B (NFκB) as predictors of prostate-specific antigen (PSA) recurrence after radical prostatectomy (RP). Patients and Methods. 70 patients with early-stage PC were treated with RP and their tumor samples were evaluated for expression of NEP, endothelin-1 (ET-1) and NFκB (p65). Time to PSA recurrence was correlated with the examined parameters and combined with preoperative PSA level, Gleason score, pathological TNM (pT) stage, and surgical margin (SM) assessment. Results and Limitations. Membranous expression of NEP (P < 0.001), cytoplasmic ET-1 (P = 0.002), and cytoplasmic NFκB (P < 0.001) were correlated with time to PSA relapse. NEP was associated with ET-1 (P < 0.001) and NFκB (P < 0.001). ET-1 was also correlated with NFκB (P < 0.001). NEP expression (P = 0.017), pT stage (P = 0.013), and SMs (P = 0.036) were independent predictors of time to PSA recurrence. Conclusions. There seems to be a clinical model of NEP/NPs and NFκB pathways interconnection, with their constituents following inverse patterns of expression in accordance with their biological roles and molecular interrelations.

Retinal degeneration progression changes lentiviral vector cell targeting in the retina

Ce travail de thèse a été réalisé au sein de l'Unité de Thérapie Génique et Biologie des Cellules Souches de l'Hôpital Jules- Gonin dans le Service d'Ophtalmologie de l'Université de Lausanne. Ce laboratoire recherche des solutions thérapeutiques pour des maladies dégénératives et incurables de la rétine comme les rétinites pigmentaires (RP). Ayant déjà montré certains résultats dans le domaine, la thérapie génique a été notre outil pour ce travail. Cette méthode se base sur le principe de remplacer un gène déficient par sa copie normale, en transportant celle-ci au coeur même du noyau par un vecteur. Il existe à l'heure actuelle différents vecteurs. Un des plus efficaces est un vecteur viral non-réplicatif : le lentivirus, dérivé de HIV-1. Celui-ci a la capacité d'intégrer le génome de la cellule cible, lui conférant ainsi un nouveau matériel génétique. Notre but a été d'établir le tropisme du lentivirus dans une rétine en dégénérescence. Ce lentivirus est connu pour transduire efficacement les cellules de l'épithélium pigmentaire rétinien dans l'oeil adulte sain, ainsi que celles de la neurorétine, mais ce, uniquement durant le développement. On sait aussi que le vecteur lentiviral présente un tropisme différent selon les enveloppes dont il est muni ; par exemple, le lentivirus avec une enveloppe Mokola est connu pour transduire les cellules gliales du système nerveux central. La rétine qui dégénère montre quant à elle des changements de sa structure qui pourraient influencer la diffusion du vecteur et/ou son tropisme. Le postulat de base a été le suivant : chez l'adulte, la transduction des neurones de la rétine via le lentivirus pourrait être facilitée par l'altération de la membrane limitante externe induite par la dégénérescence (meilleure pénétrance du virus). D'un point de vue technique, nous avons utilisé deux types distincts de modèles murins de dégénérescence rétinienne : des souris Balb/C soumises à une dose toxique de lumière et les souris Rhodopsin knockout, animaux génétiquement modifiés. Comme vecteur viral, nous avons employé deux différents pseudotypes de lentivirus (caractérisés par les enveloppes virales) avec différents promoteurs (séquence d'ADN qui initie la transduction et confère la spécificité d'expression d'un gène). En changeant l'enveloppe et le promoteur, nous avons essayé de trouver la meilleure combinaison pour augmenter l'affinité du vecteur vis-à-vis des photorécepteurs d'abord, puis vis-à-vis d'autres cellules de la rétine. Nos résultats ont montré que la membrane limitante externe est effectivement altérée chez les deux modèles de dégénérescence, mais que cette modification ne favorise pas la transduction des photorécepteurs lorsqu'on utilise un vecteur lentiviral contenant une enveloppe VSVG et un promoteur photorécepteur-spécifique ou ubiquitaire. En effet, une forte réaction gliale a été observée. Par contre, en utilisant le lentivirus avec une enveloppe Mokola et un promoteur ubiquitaire, nous avons constaté une très bonne transduction au niveau des cellules de Millier dans la rétine en dégénérescence, phénomène non observé chez les souris sauvages. Ce travail a donc permis de trouver un vecteur viral efficace pour atteindre et transduire les cellules de Miiller, ceci seulement pendant la dégénérescence de la rétine. Ces cellules, une fois transduites, pourraient être utilisées pour sécréter dans la rétine des agents thérapeutiques tels que des facteurs neurotrophiques pour soutenir la survie des photorécepteurs ou des facteurs anti-angiogéniques pour prévenir la néo-vascularisation lors de diabète ou de dégénérescence maculaire liée à l'âge. - In normal mice, the lentiviral vector (LV) is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the iuter limiting membrane (OLM). Two different LV pseudotypes were tested using the VSVG arid the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/-) mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an altération of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptór-specific promoter activity change during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the tfokola envelope allows a wide dispersion of the ctor into the retina (corresponding to the injection bleb) with preferential targeting of Muller cells, a situation Mc\ does ot occur in the wild- type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina.

FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies.

Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the photoreceptor connecting cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathies.

Establishment of models to study mouse and human retinogenesis "in vitro" : isolation and characterization of retinal stem cells

SUMMARY IN FRENCH Les cellules souches sont des cellules indifférenciées capables a) de proliférer, b) de s'auto¬renouveller, c) de produire des cellules différenciées, postmitotiques et fonctionnelles (multipotencialité), et d) de régénérer le tissu après des lésions. Par exemple, les cellules de souches hematopoiétiques, situées dans la moelle osseuse, peuvent s'amplifier, se diviser et produire diverses cellules différenciées au cours de la vie, les cellules souches restant dans la moelle osseuse et consentant leur propriété. Les cellules souches intestinales, situées dans la crypte des microvillosités peuvent également régénérer tout l'intestin au cours de la vie. La rétine se compose de six classes de neurones et d'un type de cellule gliale. Tous ces types de cellules sont produits par un progéniteur rétinien. Le pic de production des photorécepteurs se situe autour des premiers jours postnatals chez la souris. A cette période la rétine contient les cellules hautement prolifératives. Dans cette étude, nous avons voulu analyser le phénotype de ces cellules et leur potentiel en tant que cellules souches ou progénitrices. Nous nous sommes également concentrés sur l'effet de certains facteurs épigéniques sur leur destin cellulaire. Nous avons observé que toutes les cellules prolifératives isolées à partir de neurorétines postnatales de souris expriment le marqueur de glie radiaire RC2, ainsi que des facteurs de transcription habituellement trouvés dans la glie radiaire (Mash1, Pax6), et répondent aux critères des cellules souches : une capacité élevée d'expansion, un état indifférencié, la multipotencialité (démontrée par analyse clonale). Nous avons étudié la différentiation des cellules dans différents milieux de culture. En l'absence de sérum, l'EGF induit l'expression de la β-tubulin-III, un marqueur neuronal, et l'acquisition d'une morphologie neuronale, ceci dans 15% des cellules présentes. Nous avons également analysé la prolifération de cellules. Seulement 20% des cellules incorporent le bromodéoxyuridine (BrdU) qui est un marqueur de division cellulaire. Ceci démontre que l'EGF induit la formation des neurones sans une progression massive du cycle cellulaire. Par ailleurs, une stimulation de 2h d'EGF est suffisante pour induire la différentiation neuronale. Certains des neurones formés sont des cellules ganglionnaires rétiniennes (GR), comme l'indique l'expression de marqueurs de cellules ganglionnaires (Ath5, Brn3b et mélanopsine), et dans de rare cas d'autres neurones rétiniens ont été observés (photorécepteurs (PR) et cellules bipolaires). Nous avons confirmé que les cellules souches rétiniennes tardives n'étaient pas restreintes au cours du temps et qu'elles conservent leur multipotencialité en étant capables de générer des neurones dits précoces (GR) ou tardifs (PR). Nos résultats prouvent que l'EGF est non seulement un facteur contrôlant le développement glial, comme précédemment démontré, mais également un facteur efficace de différentiation pour les neurones rétiniens, du moins in vitro. D'autre part, nous avons voulu établir si l'oeil adulte humain contient des cellules souches rétiniennes (CSRs). L'oeil de certains poissons ou amphibiens continue de croître pendant l'âge adulte du fait de l'activité persistante des cellules souches rétiniennes. Chez les poissons, le CSRs se situe dans la marge ciliaire (CM) à la périphérie de la rétine. Bien que l'oeil des mammifères ne se développe plus pendant la vie d'adulte, plusieurs groupes ont prouvé que l'oeil de mammifères adultes contient des cellules souches rétiniennes également dans la marge ciliaire plus précisément dans l'épithélium pigmenté et non dans la neurorétine. Ces CSRs répondent à certains critères des cellules souches. Nous avons identifié et caractérisé les cellules souches rétiniennes résidant dans l'oeil adulte humain. Nous avons prouvé qu'elles partagent les mêmes propriétés que leurs homologues chez les rongeurs c.-à-d. auto-renouvellement, amplification, et différenciation en neurones rétiniens in vitro et in vivo (démontré par immunocoloration et microarray). D'autre part, ces cellules peuvent être considérablement amplifiées, tout en conservant leur potentiel de cellules souches, comme indiqué par l'analyse de leur profil d'expression génique (microarray). Elles expriment également des gènes communs à diverses cellules souches: nucleostemin, nestin, Brni1, Notch2, ABCG2, c-kit et son ligand, aussi bien que cyclin D3 qui agit en aval de c-kit. Nous avons pu montré que Bmi1et Oct4 sont nécessaires pour la prolifération des CSRs confortant leur propriété de cellules souches. Nos données indiquent que la neurorétine postnatale chez la souris et l'épithélium pigmenté de la marge ciliaire chez l'humain adulte contiennent les cellules souches rétiniennes. En outre, nous avons développé un système qui permet d'amplifier et de cultiver facilement les CSRs. Ce modèle permet de disséquer les mécanismes impliqués lors de la retinogenèse. Par exemple, ce système peut être employé pour l'étude des substances ou des facteurs impliqués, par exemple, dans la survie ou dans la génération des cellules rétiniennes. Il peut également aider à disséquer la fonction de gènes ou les facteurs impliqués dans la restriction ou la spécification du destin cellulaire. En outre, dans les pays occidentaux, la rétinite pigmentaire (RP) touche 1 individu sur 3500 et la dégénérescence maculaire liée à l'âge (DMLA) affecte 1 % à 3% de la population âgée de plus de 60 ans. La génération in vitro de cellules rétiniennes est aussi un outil prometteur pour fournir une source illimitée de cellules pour l'étude de transplantation cellulaire pour la rétine. SUMMARY IN ENGLISH Stem cells are defined as undifferentiated cells capable of a) proliferation, b) self maintenance (self-renewability), c) production of many differentiated functional postmitotic cells (multipotency), and d) regenerating tissue after injury. For instance, hematopoietic stem cells, located in bone marrow, can expand, divide and generate differentiated cells into the diverse lineages throughout life, the stem cells conserving their status. In the villi crypt, the intestinal stem cells are also able to regenerate the intestine during their life time. The retina is composed of six classes of neurons and one glial cell. All these cell types are produced by the retinal progenitor cell. The peak of photoreceptor production is reached around the first postnatal days in rodents. Thus, at this stage the retina contains highly proliferative cells. In our research, we analyzed the phenotype of these cells and their potential as possible progenitor or stem cells. We also focused on the effect of epigenic factor(s) and cell fate determination. All the proliferating cells isolated from mice postnatal neuroretina harbored the radial glia marker RC2, expressed transcription factors usually found in radial glia (Mash 1, Pax6), and met the criteria of stem cells: high capacity of expansion, maintenance of an undifferentiated state, and multipotency demonstrated by clonal analysis. We analyzed the differentiation seven days after the transfer of the cells in different culture media. In the absence of serum, EGF led to the expression of the neuronal marker β-tubulin-III, and the acquisition of neuronal morphology in 15% of the cells. Analysis of cell proliferation by bromodeoxyuridine incorporation revealed that EGF mainly induced the formation of neurons without stimulating massively cell cycle progression. Moreover, a pulse of 2h EGF stimulation was sufficient to induce neuronal differentiation. Some neurons were committed to the retinal ganglion cell (RGC) phenotype, as revealed by the expression of retinal ganglion markers (Ath5, Brn3b and melanopsin), and in few cases to other retinal phenotypes (photoreceptors (PRs) and bipolar cells). We confirmed that the late RSCs were not restricted over-time and conserved multipotentcy characteristics by generating retinal phenotypes that usually appear at early (RGC) or late (PRs) developmental stages. Our results show that EGF is not only a factor controlling glial development, as previously shown, but also a potent differentiation factor for retinal neurons, at least in vitro. On the other hand, we wanted to find out if the adult human eye contains retina stem cells. The eye of some fishes and amphibians continues to grow during adulthood due to the persistent activity of retinal stem cells (RSCs). In fish, the RSCs are located in the ciliary margin zone (CMZ) at the periphery of the retina. Although, the adult mammalian eye does not grow during adult life, several groups have shown that the adult mouse eye contains retinal stem cells in the homologous zone (i.e. the ciliary margin), in the pigmented epithelium and not in the neuroretina. These RSCs meet some criteria of stem cells. We identified and characterized the human retinal stem cells. We showed that they posses the same features as their rodent counterpart i.e. they self-renew, expand and differentiate into retinal neurons in vitro and in vivo (indicated by immunostaining and microarray analysis). Moreover, they can be greatly expanded while conserving their sternness potential as revealed by the gene expression profile analysis (microarray approach). They also expressed genes common to various stem cells: nucleostemin, nestin, Bmil , Notch2, ABCG2, c-kit and its ligand, as well as cyclin D3 which acts downstream of c-kit. Furthermore, Bmil and Oct-4 were required for RSC proliferation reinforcing their stem cell identity. Our data indicate that the mice postnatal neuroretina and the adult pigmented epithelium of adult human ciliary margin contain retinal stem cells. We developed a system to easily expand and culture RSCs that can be used to investigate the retinogenesis. For example, it can help to screen drugs or factors involved, for instance, in the survival or generation of retinal cells. This could help to dissect genes or factors involved in the restriction or specification of retinal cell fate. In Western countries, retinitis pigmentosa (RP) affects 1 out of 3'500 individuals and age-related macula degeneration (AMD) strikes 1 % to 3% of the population over 60. In vitro generation of retinal cells is thus a promising tool to provide an unlimited cell source for cellular transplantation studies in the retina.

p53 and cyclooxygenase-2 expression are directly associated with cyclin D1 expression in radical prostatectomy specimens of patients with hormone-naïve prostate cancer.

Prostate cancer (PCa) is a potentially curable disease when diagnosed in early stages and subsequently treated with radical prostatectomy (RP). However, a significant proportion of patients tend to relapse early, with the emergence of biochemical failure (BF) as an established precursor of progression to metastatic disease. Several candidate molecular markers have been studied in an effort to enhance the accuracy of existing predictive tools regarding the risk of BF after RP. We studied the immunohistochemical expression of p53, cyclooxygenase-2 (COX-2) and cyclin D1 in a cohort of 70 patients that underwent RP for early stage, hormone naïve PCa, with the aim of prospectively identifying any possible interrelations as well as correlations with known prognostic parameters such as Gleason score, pathological stage and time to prostate-specific antigen (PSA) relapse. We observed a significant (p = 0.003) prognostic role of p53, with high protein expression correlating with shorter time to BF (TTBF) in univariate analysis. Both p53 and COX-2 expression were directly associated with cyclin D1 expression (p = 0.055 and p = 0.050 respectively). High p53 expression was also found to be an independent prognostic factor (p = 0.023). Based on previous data and results provided by this study, p53 expression exerts an independent negative prognostic role in localized prostate cancer and could therefore be evaluated as a useful new molecular marker to be added in the set of known prognostic indicators of the disease. With respect to COX-2 and cyclin D1, further studies are required to elucidate their role in early prediction of PCa relapse after RP.

FAM161A, associated with autosomal recessive retinitis pigmentosa,localizes at the level of the photoreceptor cilium and interacts with proteins involved in ciliopathies

Purpose: We have previously demonstrated that mutations in the FAM161A gene, encoding a protein with unknown function and no similarities with other characterized sequences, cause autosomal recessive retinitis pigmentosa (RP). The purpose of this work is to investigate the functional role of FAM161A within the retina and its relationship with other proteins involved in RP. Methods: The subcellular localization of FAM161A in the retina was assessed by immunohistochemistry of retinal sections and dissociated photoreceptors from mice, which were stained using antibodies against FAM161A and antibodies against cilium markers. The function of FAM161A was further assessed in ciliated mammalian cell lines by expression of recombinant FAM161A with various fusion tags. The binary interaction between FAM161A and a collection of ciliary and ciliopathy-associated proteins was analyzed using a yeast two-hybrid assay. The results obtained with this technique were validated using independent protein-protein interaction assays (GST-pull downs, co-transfection and co-immunoprecipitation). Results: Native FAM161A localized at the connecting cilium of photoreceptor cells, as demonstrated by immunofluorescence in both dissociated photoreceptors and retinal sections of mice. More specifically, co-staining with markers for ciliary sub-structures (RPGRIP1L, Centrin, RP1, GT335) demonstrated that FAM161A decorated the basal body and the very apical part of the connecting cilium. Upon overexpression in ciliated cultured mammalian cells, FAM161A localized to the ciliary basal body. Yeast two-hybrid analysis of the binary interaction of FAM161A and an array of ciliary proteins revealed the direct interaction of FAM161A with three proteins of which the cognate genes are mutated in retinal ciliopathies. The confirmation of these interactions using different biochemical assays is currently in progress. Conclusions: FAM161A is a ciliary basal body protein of the photoreceptor connecting cilium, rendering the associated RP as a novel retinal ciliopathy. The confined expression of FAM161A in the retina and the direct interaction of FAM161A with other retinal ciliopathy-associated proteins may explain the retinal phenotype of this specific subset of mechanistically and phenotypically connected retinal disorders.

The PROM1 mutation p.R373C causes an autosomal dominant bull's eye maculopathy associated with rod, rod-cone, and macular dystrophy.

PURPOSE: To characterize in detail the phenotype of five unrelated families with autosomal dominant bull's eye maculopathy (BEM) due to the R373C mutation in the PROM1 gene. METHODS: Forty-one individuals of five families of Caribbean (family A), British (families B, D, E), and Italian (family C) origin, segregating the R373C mutation in PROM1, were ascertained. Electrophysiological assessment, fundus autofluorescence (FAF) imaging, fundus fluorescein angiography (FFA), and optical coherence tomography (OCT) were performed in available subjects. Mutation screening of PROM1 was performed. RESULTS: The R373C mutant was present heterozygously in all affected patients. The age at onset was variable and ranged between 9 and 58 years, with most of the individuals presenting with reading difficulties. Subjects commonly had a mild to moderate reduction in visual acuity except for members of family C who experienced markedly reduced central vision. The retinal phenotype was characterized by macular dystrophy, with retinal pigment epithelial mottling in younger subjects, progressing to typical BEM over time, with the development of macular atrophy in older patients. In addition, all members of family C had typical features of RP. The electrophysiological findings were variable both within and between families. CONCLUSIONS: Mutations in PROM1 have been described to cause a severe form of autosomal recessive RP in two families of Indian and Pakistani descent. The results of this study have demonstrated that a distinct redundant PROM1 mutation (R373C) can also produce an autosomal dominant, fully penetrant retinopathy, characterized by BEM with little inter- and intrafamilial variability, and retinal dystrophy with variable rod or rod-cone dysfunction and marked intra- and interfamilial variability, ranging from isolated maculopathy without generalized photoreceptor dysfunction to maculopathy associated with very severe rod-cone dysfunction.

CD10 is inversely associated with nuclear factor-kappa B and predicts biochemical recurrence after radical prostatectomy.

INTRODUCTION: The cell surface endopeptidase CD10 (neutral endopeptidase) and nuclear factor-κB (NF-κB) have been independently associated with prostate cancer (PC) progression. We investigated the correlations between these two factors and their prognostic relevance in terms of biochemical (prostate-specific antigen, PSA) relapse after radical prostatectomy (RP) for localized PC. PATIENTS AND METHODS: The immunohistochemical expression of CD10 and NF-κB in samples from 70 patients who underwent RP for localized PC was correlated with the preoperative PSA level, Gleason score, pathological stage and time to PSA failure. RESULTS: CD10 expression was inversely associated with NF-κB expression (p < 0.001), stage (p = 0.03) and grade (p = 0.003), whereas NF-κB was directly related with stage (p = 0.006) and grade (p = 0.002). The median time to PSA failure was 56 months. CD10 and NF-κB were directly (p < 0.001) and inversely (p < 0.001) correlated with biochemical recurrence-free survival, respectively. CD10 expression (p = 0.022) and stage (p = 0.018) were independently associated with time to biochemical recurrence. CONCLUSION: Low CD10 expression is an adverse prognostic factor for biochemical relapse after RP in localized PC, which is also associated with high NF-κB expression. Decreased CD10 expression which would lead to increased neuropeptide signaling and NF-κB activity may be present in a subset of early PCs.