Somatic mosaicism in skin fragility disorders: mechanisms and therapeutic perspectives
Final Report Abstract
The term mosaicism defines the presence of at least two populations of cells within one individual that are derived from the same zygote, but are genotypically different. This results from a single mutation during the organism’s development that is propagated only in a limited number of cells. Revertant mosaicism (RM) is the phenomenon occurring in an individual with a genetic disorder where a subpopulation of its cells have been corrected spontaneously, resulting in a co-existence of mutated and spontaneously corrected, also called reverted, cells. This phenomenon was described for the first time in the skin in 1997, and was thought to be extremely rare. However, recent studies had showed that it occurred in patients with mutations in 5 different genes causing the genetic skin fragility disorder epidermolysis bullosa (EB). The long term goal of this project was to elucidate the mechanisms that underlie RM in EB and use that knowledge for the development of novel therapies making use of these patient-own, spontaneously-corrected cells. The specific objectives of this proposal were: a) identification of patients with EB and RM and elucidation of the underlying molecular mechanisms; b) definition of the natural history of RM in our large well-characterized EB-cohort; c) characterization of the reverted keratinocytes and d) assessment of the applicability of reverted cells for molecular therapies. The project has developed well, found answers to the questions posed in the application and – as an unexpected result – generated knowledge on postzygotic mechanism mutations driving disease in 2 cases of genodermatoses with extensive skin involvement. All patients visiting the EB-Center Freiburg during the funding period were specifically asked for the presence of clinically healthy-appearing skin areas. To validate whether these areas really represent reverted skin, we took a 4-6 mm punch biopsy from each possible reverted skin area for immunofluorescence microscopy. In total, we took biopsies from clinically healthy-appearing skin from 49 patients. From these 49 patients, in 30 cases (61%) the staining of the biopsies revealed the presence of RM. Specifically, we identified revertant skin patches in 8 patients with KS, 15 patients with DEB and 7 patients with JEB, of them 2 carrying mutations in LAMB3 and 5 in COL17A1 genes respectively. We established primary keratinocyte cultures from reverted skin, altogether from 30 different biopsies from proven reverted skin and created immortalized keratinocyte lines from mutant and reverted keratinocytes from 6 EB patients. The immortalized reverted cells of patients with recessive dystrophic EB and RM were characterized in respect of their migration, dissociation, proliferation and adhesion. The reverted cells behaved in a clearly different way than the mutant ones and comparably to normal control keratinocytes. We also assessed the longterm survival of the reverted keratinocytes versus mutant keratinocytes. In dystrophic EB, the reverted cells did not diminish in culture, unlike what has been published before in junctional EB. This result is compatible with the use of reverted cells for skin grafting on affected patient’s skin. Our research improved the general understanding on the role of the proteins that are important for the mechanical resistance of the skin and on the significance of the reverted cells as disease modifying and therapeutic factors. There were several articles published in the media reporting the success of the project and presentation of our work: http://www.spiegel.de/spiegel/print/d-129853808.html; https://www.pr.uni-freiburg.de/pm/personalia/kiritsi_dimitra?set_language=de; http://www.deutsche-stiftung-kinderdermatologie.de/newsevents/news/artikel/article/mittwoch.html.
Publications
- Targeting epidermal lipids for treatment of Mendelian disorders of cornification. Orphanet J Rare Dis. 9:33, 2014
Kiritsi D, Schauer F, Wölfle U, Valari M, Bruckner-Tuderman L, Has C, Happle R
- Novel and recurrent AAGAB mutations: clinical variability and molecular consequences. J Invest Dermatol. 133(10):2483-6, 2013
Kiritsi D, Chmel N, Arnold AW, Jakob T, Bruckner-Tuderman L, Has C
- A band-like balding disorder. Lancet. 383(9917):e14, 2014
Kiritsi D, Diaz-Cascajo C, Hoffmann R, Happle R, Jakob T, Kern JS
(See online at https://doi.org/10.1016/S0140-6736(13)60804-1) - Extensive postzygotic mosaicism for a novel keratin 10 mutation in epidermolytic ichthyosis. Acta Derm Venereol. 94(3):346-8, 2014
Kiritsi D, Nanda A, Kohlhase J, Bernhard C, Bruckner-Tuderman L, Happle R, Has C
(See online at https://doi.org/10.2340/00015555-1695) - Mechanisms of natural gene therapy in dystrophic epidermolysis bullosa. J Invest Dermatol. 134(8):2097-104, 2014
Kiritsi D, Garcia M, Brander R, Has C, Meijer R, Jose Escámez M, Kohlhase J, van den Akker PC, Scheffer H, Jonkman MF, del Rio M, Bruckner-Tuderman L, Pasmooij AM
(See online at https://doi.org/10.1038/jid.2014.118) - 'Double trouble': diagnostic challenges in genetic skin disorders. Br J Dermatol. 172(1):276-8, 2015
Kiritsi D, Valari M, Mileounis K, Bruckner-Tuderman L, Has C
(See online at https://doi.org/10.1111/bjd.13159) - Blaschko line acne on pre-existent hypomelanosis reflecting a mosaic FGFR2 mutation. Br J Dermatol. 172(4):1125-7, 2015
Kiritsi D, Lorente AI, Happle R, Bernabeu Wittel J, Has C
(See online at https://doi.org/10.1111/bjd.13491) - Junctional epidermolysis bullosa with LAMB3 splicesite mutations. Acta Derm Venereol. 95(7):849-51, 2015
Kiritsi D, Huilaja L, Franzke CW, Kokkonen N, Pazzagli C, Schwieger-Briel A, Larmas M, Bruckner-Tuderman L, Has C, Tasanen K
(See online at https://doi.org/10.2340/00015555-2073) - Losartan ameliorates dystrophic epidermolysis bullosa and uncovers new disease mechanisms. EMBO Mol Med. 7(9):1211-28, 2015
Nyström A, Thriene K, Mittapalli V, Kern JS, Kiritsi D, Dengjel J, Bruckner-Tuderman L
(See online at https://doi.org/10.15252/emmm.201505061) - Whole-exome sequencing in patients with ichthyosis reveals modifiers associated with increased IgE levels and allergic sensitizations. J Allergy Clin Immunol. 135(1):280-3, 2015
Kiritsi D, Valari M, Fortugno P, Hausser I, Lykopoulou L, Zambruno G, Fischer J, Bruckner- Tuderman L, Jakob T, Has C
(See online at https://doi.org/10.1016/j.jaci.2014.09.042) - Genotype, Clinical Course, and Therapeutic Decision Making in 76 Infants with Severe Generalized Junctional Epidermolysis Bullosa. J Invest Dermatol. 136(11):2150-2157, 2016
Hammersen J, Has C, Naumann-Bartsch N, Stachel D, Kiritsi D, Söder S, Tardieu M, Metzler M, Bruckner-Tuderman L, Schneider H
(See online at https://doi.org/10.1016/j.jid.2016.06.609) - Injury-Driven Stiffening of the Dermis Expedites Skin Carcinoma Progression. Cancer Res. 76(4):940-51, 2016
Mittapalli VR, Madl J, Löffek S, Kiritsi D, Kern JS, Römer W, Nyström A, Bruckner-Tuderman L
(See online at https://doi.org/10.1158/0008-5472.CAN-15-1348) - Keratin-dependent thymic stromal lymphopoietin expression suggests a link between skin blistering and atopic disease. J Allergy Clin Immunol. 138(5):1461- 1464.e6, 2016
Kumar V, Behr M, Kiritsi D, Scheffschick A, Grahnert A, Homberg M, Schwieger-Briel A, Jakob T, Bruckner-Tuderman L, Magin TM
(See online at https://doi.org/10.1016/j.jaci.2016.04.046) - The mysteries of mosaicism: phenotypic variability in a family with incontinentia pigmenti. Eur J Dermatol. 26(5):504-506, 2016
Kiritsi D, Valari M, Michos A, Karakosta V, Has C
(See online at https://doi.org/10.1684/ejd.2016.2829)
