GLI3 variants causing isolated polysyndactyly are not restricted to the protein's C-terminal third

Loss of function variants of GLI3 are associated with a variety of forms of polysyndactyly: Pallister-Hall syndrome (PHS), Greig-Cephalopolysyndactyly syndrome (GCPS), and isolated polysyndactyly (IPD). Variants affecting the N-terminal and C-terminal thirds of the GLI3 protein have been associated with GCPS, those within the central third with PHS. Cases of IPD have been attributed to variants affecting the C-terminal third of the GLI3 protein. In this study, we further investigate these genotype-phenotype correlations. Sequencing of GLI3 was performed in patients with clinical findings suggestive of a GLI3-associated syndrome. Additionally, we searched the literature for reported cases of either manifestation with mutations in the GLI3 gene. Here, we report 48 novel cases from 16 families with polysyndactyly in whom we found causative variants in GLI3 and a review on 314 previously reported GLI3 variants. No differences in location of variants causing either GCPS or IPD were found. Review of published data confirmed the association of PHS and variants affecting the GLI3 protein's central third. We conclude that the observed manifestations of GLI3 variants as GCPS or IPD display different phenotypic severities of the same disorder and propose a binary division of GLI3-associated disorders in either PHS or GCPS/polysyndactyly

Identifizierung Pathogenese-relevanter Kandidatengene im Hodgkin-Lymphom durch CRISPR/Cas9-basiertes knockout-Screening

Das klassische Hodgkin-Lymphom (classical Hodgkin lymphoma, cHL) ist eine häufige lymphatische Neoplasie der B-Zell-Reihe, deren molekulare Pathogenese in den letzten Jahrzehnten Gegenstand intensiver Forschung war. Trotz zahlreicher Erkenntnisse, wie u.a. der Beschreibung der Rolle des NF-κB- und JAK/STAT-Signalwegs sowie von AP-1- und IRF-Transkriptionsfaktoren, ist die Frage nach den letztendlich tumorbiologisch entscheidenden molekularen Treibern des cHL bis heute nur unzureichend beantwortet. Um in einem unvoreingenommenen Ansatz nach diesen Treibern zu suchen, wurde in dieser Arbeit ein genomweiter loss-of-function Screen in Hodgkin- und Kontrollzelllinien durchgeführt. Hierfür wurde das CRISPR/Cas9-System etabliert und verwendet, mit dem ein gezielter knockout von Zielgenen erreicht werden kann. Nach zunächst genomweitem knockout-Screening erfolgte ein unabhängiger Bestätigungs-Screen anhand einer individuellen, selbst erstellten sgRNA-Bibliothek der ermittelten potentiellen Kandidatengene. Durch diese beiden sequentiellen CRISPR/Cas9-basierten knockout-Screens konnten zahlreiche bereits beschriebene Gene für die Pathogenese des cHL unabhängig identifiziert und bestätigt werden, was gleichzeitig die Validität des gewählten experimentellen Ansatzes sowie der CRISPR-Screens selbst demonstriert. Unter den so bestätigten Genen befinden sich zahlreiche Faktoren des NF-κB- und JAK/STAT-Signalwegs, die AP-1-Transkriptionsfaktoren JUNB und BATF3, der IRF-Transkriptionsfaktor IRF4 sowie u.a. der Zellzyklusregulator Cyclin D2. Darüber hinaus konnten mehrere neue Kandidatengene identifiziert werden, darunter bspw. weitere Transkriptionsfaktoren und Zellzyklusregulatoren, Faktoren der DNA-Reparatur und epigenetische Modifikatoren. Im Anschluss an die beiden CRISPR-Screens erfolgte die weitere Validierung ausgewählter Kandidatengene anhand von Kompetitions-Assays, in denen die Mehrzahl dieser Kandidatengene bestätigt werden konnte. Weitere funktionelle Untersuchungen unter Einsatz molekularer Inhibitoren konnten die Rolle von Cyclin D2 und weiterer Zellzyklusregulatoren für die Proliferation von Hodgkin-Zellen demonstrieren. Schließlich konnten ausgewählte neue Kandidatengene für die Pathogenese des cHL verifiziert werden; darunter KEAP1, das als ein Substratadapterprotein eines E3-Ubiquitin-Ligase-Komplexes fungiert und eine zentrale Rolle in der Regulation der zellulären Antwort auf oxidativen Stress einnimmt.Classical Hodgkin lymphoma (cHL) is a common lymphoid malignancy that is derived from B cells. The pathogenesis of cHL has been studied intensively over the past decades and a number of deregulated signalling cascades (NF-κB, JAK/STAT) as well as transcription factor networks (AP-1, IRFs) could be identified. However, the question of the central and specific molecular mechanisms that drive the disease and their relative contribution still remains unanswered. To address this question by an unbiased approach, we performed genome-wide loss of function screening in Hodgkin and control cell lines. To this end, we applied a CRISPR/Cas9-based knockout approach which allows for a targeted knockout of genes on a genomic scale. Our genome-wide CRISPR/Cas9 knockout screen identified most of the so far known key factors of cHL pathogenesis, such as numerous members of the NF-κB signaling pathway and JAK/STAT signaling pathways, the AP-1 family transcription factors JUNB and BATF3 and the IRF transcription factor IRF4 as well as cell cycle regulators such as Cyclin D2; i.e. our screen provides the first unbiased and genomic view of functionally relevant factors in cHL tumorbiology. The identification of these previously described factors also served as a strong validation for the chosen approach and our screening results. Moreover, several new potential candidate genes for the pathogenesis of cHL could be discovered, including additional transcription factors and cell cycle regulators, epigentic modifiers as well as genes involved in DNA repair. To confirm our results obtained by genome-wide CRISPR/Cas9 screening, we designed and generated a targeted re-screen library and performed a second independent CRISPR/Cas9 screen. By these means, most of the known candidate genes as well as several new candidate genes could be validated. Following their identification by CRISPR/Cas9 screening, selected candidate genes were analyzed on a single candidate level by competition assays. Here, most of the selected candidate genes could be validated independently. Further functional studies applying molecular inhibitors of cell cycle regulators demonstrated the important role of these regulators for the proliferation of Hodgkin lymphoma cells. Finally, also selected novel candidate genes for the pathogenesis of cHL could be verified. Among those is KEAP1 that serves as a substrate adaptor protein of an E3 ubiquitin ligase complex and is a key regulator of the cellular response to oxidative stress