Identification of new genes involved in hereditary steroid-resistant nephrotic syndrome using next generation sequencing and in vivo functional characterization in drosophila melanogaster Sara De Almeida Gonçalves To cite this version: Sara De Almeida Gonçalves. Identification of new genes involved in hereditary steroid-resistant nephrotic syndrome using next generation sequencing and in vivo functional characterization in drosophila melanogaster. Urology and Nephrology. Université Sorbonne Paris Cité, 2017. English. NNT : 2017USPCB030. tel-02180575 HAL Id: tel-02180575 https://tel.archives-ouvertes.fr/tel-02180575 Submitted on 11 Jul 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Doctoral Thesis of PARIS DESCARTES University Doctoral School of Bio Sorbonne Paris Cité Sara DE ALMEIDA GONÇALVES A dissertation submitted in accordance with the requirements for the award of the degree of PhD in Science Identification of new genes involved in hereditary steroid-resistant nephrotic syndrome using next generation sequencing and in vivo functional characterization in Drosophila melanogaster Defense date - 28th September, 2017 Jury panel Pr. Alain FISCHER Dr. Clément CARRÉ Pr. Nine KNOERS Pr. Moin SALEEM Pr. Corinne ANTIGNAC To my father 1 Acknowledgements 3 Acknowledgements At the end of this important chapter of my life I would like to acknowledge all the people who have, directly and/or indirectly, allowed me to grow in this passionate and always partially mysterious world of science. First and foremost I would like to thank my mentor Corinne Antignac. Thank you for guiding me through the world of genetics, methods of research, in developing my passion for science and for all the exciting discussions. Thank you for always being there, for demanding a lot but only because you cared. Thank you for your patience and your kindness with me. Thank you for your example of strength, perseverance and passion, even when obstacles come. Thank you for your kind attitude in difficult or joyful moments of my personal life. These are things that I will always be grateful of, much more so than I can express. To Matias Simons, thank you for accepting me into your lab whilst you were still in Freiburg, where I first met the fruit fly under a stereomicroscope. Thanks for introducing me to the world of Drosophila and for guiding me in the Drosophila experiments. Thanks for all the exciting lab meetings and also for your help in the conclusion and writing of the ADD3 and KAT2B paper. To the reviewers of my thesis, Clément Carré and Nine Knoers, as well as to the other members of the jury, Alain Fischer and Moin Saleem, who have agreed to evaluate my work. To Julie Patat, who helped me in my experiments and was brave enough to sequence the KAT2B gene in 73 patients and to start dangerous and new experimental procedures such as the heat-fixation (I hope you will go ahead with your patent!). To Noëlle Lachaussée, the M2 student who worked with me in the initial phases of the ADD3 and KAT2B project. Thanks for your enthusiasm, speed and energy. To Christelle Arrondel, with whom I learned the ABC of experimental techniques at the bench! Thank you for all of your teaching and patience. To the remaining podocyte team, both present and past members: Géraldine Mollet (for your scientific advice and joyful character), Olivier Gribouval (for all your help in and outside the lab), Evelyne Huynh Cong (the queen of podocytes in culture, for all the help with protocols and experiments), Olivia Boyer (for your encouragement and help with patient data), Guillaume Doal the )eafish opetito… Dosophila is ette!, Maia “eao o ee lose to e, Lauie Busaa, Faes Tille ou ko I do ot at to kill ou, dot ou? ad Giulia Menara. To Kalman Tory and his endless good humor! May all of your work reveal more and more of this octopus cell! To Daiel Poul fo the stiosis tea, fo his Audi stlé, aotues, é-fot ad othe aazig Caaa-like jokes. To Fred Bernard and Maria Rujano, the top Drosophilists that guided me in the passionate world of Drosophila. To the rest of the Simons team, including those who have already left, Simon Gerber (hope to see you soon again), Clara Guida (who still worked with me on the other side of the Atlantic, breaking the hearts of adult flies), Virginie Hauser and Zvoni Marelja (now you are the only one to keep the good old huo i the la!!! …ad to the oe eet ees of the la: Valetia 5 Marchesin and Magda Cannata Serio (the Italian team) Mathilda Bedin, Gwenn Le Meur, Laure Villoing-Gaudè, Albert Pérez. Thanks to all of you for making my days always more joyful! To Marion Delous, with whom I shared my office and my jokes day after day (imagine how tough that can be for one person alone!). Thank you for not despairing! Thank you for our scientific discussions and for checking each of my scientific projects, publications, posters and oral presentations as if they were your own! To Rebecca Ryan, thank you for reading my thesis, for your scientific and English-native-speaker advice. Thanks for encouraging me in the dark days, especially during the writing of the sphingolipids chapter, and for your great creativity (if you ever doubt it you can just look at your knitted creations!). To the other teams of the lab and their PIs: Sophie Saunier, Alexandre Benmerah, Cécile Jeanpierre. Thank you for your kindness and nice discussions. To the old ones, starting with Albane Bizet (who is at least 1000 years old), who looked after me from the very beginning, gave me a place to stay when I was homeless and always helped me with tough experimental procedures; to Flora Legendre alas ith a ie sile to e; to Geltas Ode, ee though e fight eah othe ou ko I hae ee loed ou! I ould ot e ithout ou hat alade ad ou déiles jokes; ad to the young ones in the lab, Hugo Garcia (may the force be with you on your new leader position in the S.F. group), Louise Reilly (may the force be with you to defeat Hugo Garcia in the S.F. group and continue making cakes), petite Marie Dupont (viva le bigeminism!), Lara De Tomasi (after all your laugh is not awkward), Charline Henry (bon courage!), Esther Porée (if you want to reconsider you can still enter the S.F. group). To the secret friends group (you know who you are!). Thank you for your unconditional friendship and amazing cakes! To the ones that were in the lab when I started but have already left - Maxence Macia (the sweetest friend), Valentina Grampa deaest Italia fied, house ate ad the oe I as téouie of), Zuza Andrzejewska, Lucie Thomas, Fabien Nevo, Nathalie Nevo. For most of you we continue linked by the outside-lab life (yes, there is one!). To Meriem Garfa-Traoré and Nicolas Goudin, for their help with confocal microscopy. Without you (and Rebecca Ryan) there would have been no prize for the best funny picture of the YRII congress. To Antonio Gomes da Costa, who understood my passion for research and allowed me to start this PhD. Lastly, my gratefulness to all my family and friends: Thomas, thank you for enduring the thesis- stress with me, for the endless thesis formatting and for all your kindness to me; Mariana and mum, thank you for your infinite patience and support; Teresa, Cristina, Ines, Cristininhas, Fili, Mariana, Eunice, Filipas, Mordi, and all the others that I do not mention here, thank you for your everlasting friendship. 6 Abstract 7 Abstract Nephrotic syndrome (NS) is a kidney disease characterized by disruption of the glomerular filtration barrier and the massive loss of proteins into the urine. Although in the majority of cases treatment with steroids leads to remission of the disease, in 15-20% of cases the disease is not responsive to this therapy and is classified as steroid-resistant nephrotic syndrome (SRNS). SRNS is a clinical condition with high morbidity leading to progressive renal failure as well as multiple metabolic and cardiovascular complications. Extensive research over the last 20 years has identified more than 40 SRNS causing genes that are crucial for function of the podocyte, a highly specialized kidney epithelial cell. However, the mutated gene is still unknown in about half of the familial cases. We have used exome sequencing to identify new genes mutated in SRNS. In order to prove the pathogenicity of the identified mutations we used the Drosophila model, assessing defects of fly viability and the structure and function of nephrocytes, podocyte like-cells. My thesis work consists of two projects. Firstly, we identified biallelic mutations in a new candidate gene, SGPL1, encoding the sphingosine 1- phosphate lyase, in individuals presenting SRNS with facultative adrenal insufficiency, ichthyosis, neurological defects and immunodeficiency. SGPL1 is the main catabolic enzyme of sphingolipids, irreversibly degrading sphingosine 1-phosphate into phosphoethanolamine and hexadecenal. In flies, these mutations were shown to decrease viability, induce nephrocyte defects and lead to the accumulation of sphingoid bases due to the loss of SGPL1 catabolic activity. Together, these results indicate that the identified SGPL1 mutations are pathogenic and cause a new syndromic form of SRNS.