Multiple Endocrine Neoplasia Type I Syndrome (MEN 1) is a monogenic autosomal dominantly inherited cancer syndrome caused by a heterozygous loss of the MEN1 gene, predisposing for endocrine cell proliferation and tumor formation. MEN 1 carriers classically develop tumors in endocrine organs; the parathyroids, the endocrine pancreas, and the pituitary. Other organs, endocrine and non-endocrine, may also be affected. The most common cause of death in MEN 1 is pancreatic endocrine tumor (PNET), which exhibit inactivation of both MEN1 alleles. The increased proliferation prior to loss of the wild-type allele indicates haploinsufficiency, and little is known concerning the mechanisms behind MEN 1 PNET development. The MEN1 protein, menin, lacking homology with other known proteins, is involved in several aspects of transcriptional regulation and chromatin organization. We report differential expression and subcellular localization of transcription factors important in pancreatic development, in human and mouse MEN 1 pancreas, compared to non-MEN 1 pancreas. A predominantly cytoplasmic localization of Neurogenin3 and NeuroD1 was observed in tumors as well as in MEN 1 non-tumorous pancreas. Notch signaling factor expression and localization were examined in the pancreas of a heterozygous Men1 mouse model, and compared with that of wild-type littermates. Nuclear Hes1 was lost in tumors, concomitant to weaker Notch1 NICD expression, and further, analyzed using qPCR, it was shown that Notch1 was less expressed in heterozygous islets compared to wild-type islets. Performing a global gene expression array, we identified differential gene expression in five-week-old heterozygous Men1 mouse islets, compared to islets from wild-type littermates. The array results for a subset of the differentially regulated genes were corroborated using qPCR, western blotting and in situ PLA. We additionally observed significantly accelerated proliferation in islets from young heterozygous animals. It is often problematic to determine prognosis for individual patients with PNET. This is especially true in the group of patients with well differentiated endocrine carcinomas. In the absence of metastases, morphological signs of malignancy are frequently lacking. We evaluated the expression of nuclear and cytoplasmic survivin in a clinically characterized patient material (n=111), and a high nuclear survivin expression proved to be a significant negative prognostic factor for survival.

While there has been an opulence of data and studies surrounding the study of the developing pancreas in mammals and other vertebrates, the focus has largely been in mice. The paucity of research in the development of the human pancreas has led to diminished knowledge in the area, compared to other species. Recent discoveries provide growing evidence for discrepancies between mouse and human pancreatic development and diseases and highlight the fact that developmental studies of the pancreas in humans are imperative. The need to develop therapies for diabetes, a growing and one of the leading health problems worldwide, further compels more exploration in this area to deepen our understanding in the different aspects of diabetes in humans and its underlying causes. Research involving modelling human diseases in vitro enables the investigation of the cellular and molecular mechanisms underlying these diseases as well as the development of therapies for treating them. The availability of hPSCs brings with it the advantage of overcoming the limitations of animal models for certain disorders such as pancreatic agenesis, the focus of my project. The use of site-specific nucleases such as TALENs for such a purpose represents a paradigm shift in disease modelling, where TALENs are capable of directly correcting disease-causing mutations, therefore permanently eliminating the symptoms with precise genome modifications. Alternatively, TALENs can also be used to inactivate specific genes by inducing site-specific mutations. Using these tools, I found that GATA6 is required for the formation of the definitive endoderm (DE) and pancreas in humans; hPSCs harbouring homozygous GATA6 mutations fail to form the definitive endoderm, and consequently the pancreas, whereas hPSCs harbouring heterozygous GATA6 mutations exhibited impairment in definitive endoderm development, although it remains unclear if this is a protocol dependent defect. At the pancreatic stage, heterozygous GATA6 mutations consistently compromised pancreas formation regardless of protocol used. I also found that GATA6 transcriptionally activates the development of the definitive endoderm and pancreatic endoderm, and possibly represses the development of mesoderm. Furthermore, I also established that GATA6 directly interacts with key definitive endoderm markers CXCR4 and SOX17, and pancreatic marker PDX1. Taken together, the work herein demonstrates the successful use of hPSCs coupled with the TALEN genome editing technology as a unique in vitro system for disease modelling. These findings also establish two developmental windows, the DE and pancreatic progenitor stages, where GATA6 haploinsufficiency can result in the impairment of pancreatic development leading to pancreatic hypoplasia observed in human GATA6 heterozygous patients. Lastly, my work also provides the molecular mechanism by which GATA6 regulates pancreatic development. Overall, this study provided new insights in the role of GATA6 during development of the human pancreas. These results will be important in developing new methods of differentiation for hPSCs and understanding the interconnection between early organogenesis and late onset of diabetes.

Diploid organisms including yeast, most animals, and humans, typically carry two copies of each gene. Variation above or below two copies can however sometimes occur. When gene copy number reduction from two to one causes a disadvantage, that gene is considered haploinsufficient (HI). In the first part of my work, I identified associations between Saccharomyces cerevisiae gene properties and genome-scale HI phenotypes from earlier work. I compared HI profiles against 23 gene properties and found that genes with (i) greater numbers of protein interactions, (ii) greater numbers of genetic interactions, (iii) greater gene sequence conservation, and (iv) higher protein expression were significantly more likely to be HI. Additionally, HI showed negative relationships with (v) cell cycle regulation and (vi) promoter sequence conservation. I exploited the aforementioned associations using Linear Discriminant Analysis (LDA) to predict HI in existing data and guide experimental identification of 6 novel HI phenotypes, previously undetected in genome-scale screenings. I also found significant relationships between HI and two gene properties in Schizosaccharomyces pombe, relationships that hold despite the lack of conserved HI between S. cerevisiae and Sz. pombe orthologue gene pairs. These data suggest associations between HI and gene properties may be conserved in other organisms. The relationships and model presented here are a step towards understanding HI and its underlying mechanisms. Increases in copy number can occur through gene duplication. When duplication produces two functional gene copies, both experience relaxed selection and rapid mutation. This sometimes leads to interesting evolutionary events such as gain of novel function (neofunctionalisation). Previous work shows an ancient ancestor of S. cerevisiae underwent whole genome duplication (WGD) followed by massive redundant gene loss. Interestingly some duplicate pairs show retention of both copies, including the pair TUB1 and TUB3. Existing sequence data shows that TUB3 has experienced a very high rate of evolution post-WGD, suggesting neofunctionalisation. To characterise TUB3, I have carried out experiments measuring fitness effects of varying TUB1, TUB2 and TUB3 copy number across many environments. In ethanol media, some TUB1 and TUB3 null mutants interestingly show severe defects. Other data suggest stress response, ethanol tolerance, protein degradation and/or regulatory roles, which may involve the regulatory Snf1p protein kinase complex.

The genetic causes of intellectual disability (ID) and autism spectrum disorder (ASD) are frequently associated with mutations in genes that encode synaptic proteins. A recent screen of ID patients has revealed that approximately 4% of individuals carry spontaneous autosomal-dominant de novo mutations in the SYNGAP1 gene. This gene encodes the synaptic GTPase activating protein (SYNGAP) a known regulator of Ras signalling. Investigations into the pathological consequences of Syngap1 haploinsufficiency (Syngap+/−) in mice have reported abnormalities in behaviour, synaptic plasticity and dendritic spine development. These are analogous to findings from the mouse model of fragile X syndrome (FXS; Fmr1-/y), the most common inherited form of ID. One of the prominent phenotypes reported in the mouse model of FXS is that a form of hippocampal long-term depression (LTD) mediated by the activation of Group 1 (Gp1) metabotropic glutamate (mGlu) receptors is enhanced and independent of new protein synthesis (Huber et al. 2002; Nosyreva et al. 2006). The cause of these synaptic plasticity deficits together with other cognitive abnormalities observed in FXS are thought to arise, in part, from excessive protein synthesis, the consequence of altered mGlu5 receptor signalling via the Ras-ERK1/2 signalling pathway. Enhanced protein synthesis rates in Fmr1-/y mice can be corrected by either inhibiting mGlu5 receptors or reducing Ras and subsequent ERK1/2 activity (Osterweil et al. 2013). In this thesis mGluR-dependent LTD was examined at Schaffer collateral/commissural inputs to CA1 pyramidal neurones in hippocampal slices obtained from Fmr1-/y, Syngap+/− and Fmr1-/ySyngap+/− double mutant mice. Extracellular field recordings reveal that acute application of the Gp1 mGluR agonist dihydroxyphenylglycine (DHPG) induces a form of mGluR-dependent LTD that is enhanced and independent of new protein synthesis in CA1 of Fmr1-/y mice. In Syngap+/− mice, the magnitude of mGluR-dependent LTD is also significantly increased relative to WT littermates and insensitive to protein synthesis inhibitors. Furthermore, in the Fmr1-/ySyngap+/− double mutant, Syngap haploinsufficiency occludes the increase in mGluR-dependent LTD caused by the loss of FMRP. In addition, metabolic labelling studies reveal basal protein synthesis rates to be modestly enhanced in the hippocampus of Fmr1-/y mice compared to WT mice. Importantly this phenotype translates to the rat model of FXS. In Syngap+/- hippocampal slices, basal protein synthesis rates are also significantly elevated compared to WT counterparts. Interestingly, elevated basal protein synthesis rates in Syngap+/- mice could be corrected in the hippocampus by similarly pharmacological strategies employed in Fmr1-/y mice. The comparable neuropathophysiology we observe between Syngap+/− and Fmr1-/y mice suggests that SYNGAP and fragile X mental retardation protein (FMRP) may converge on similar biochemical pathways raising the intriguing possibility that therapeutic strategies used in the treatment of FXS may also be of benefit in treating individuals with ID caused by mutations in SYNGAP1.

More than 300 human conditions, ranging from cancer predisposition to developmental and neurological mendelian disorders, are caused by haploinsufficiency (HI), a genetic condition by which mutational inactivation of a single allele leads to reduced protein levels and is enough to produce the disease phenotype. Therefore, translational enhancement of the spare allele could exert a therapeutic effect. Here we propose a novel approach for the potential rescue of haploinsufficiency disease loci based on the insertion of specific single nucleotide changes in the Kozak sequence. Since this sequence controls translation by regulating start codon recognition, we aimed at identifying and introducing specific nucleotide variations to enhance translation and rescue haploinsufficiency. To do so, we used CRISPR-Cas base editors, able to generate single nucleotide changes in genomic DNA without the need of a donor DNA and without creating double-strand breaks. We performed a high-throughput screening to evaluate the strength of the Kozak sequences of 231 haploinsufficient genes. We compared the translational efficiency of each wild-type sequence to that of several variants using FACS-seq, which combines fluorescence-activated cell sorting and high-throughput DNA sequencing. We thus selected 5 candidate genes (PPARGC1B, FKBP6, GALR1, NRXN1, and NCF1) and several nucleotide variations able to up-regulate translation. Finally, we used CRISPR-Cas base editors to reproduce the most efficient variants of NCF1 in a cell model relevant for the associated haploinsufficient disease and verified the increase of protein levels. This study proposes a novel therapeutic strategy to rescue haploinsufficiency and sheds new insights into the regulatory mechanisms underlying the translational process. On a broader level, the possibility of modulating gene expression by acting exclusively on translation expands the CRISPR-Cas genome editing applications.

The aim of this thesis was to identify genetic factors involved in frontotemporal lobar degeneration (FTLD), a neurodegenerative disorder clinically characterised by a progressive change in personality, behaviour and language. FTLD is a genetically complex disorder and a positive family history is found in up to 40% of the cases. In 10-20% of the familial cases the disease can be explained by mutations in the gene encoding the microtubule associated protein tau (MAPT). In the first study we describe the clinical and neuropathological features of a Finnish family with FTLD caused by a mutation in MAPT. We also provide evidence that the pathogenic mechanism of this mutation is through altered splicing of MAPT transcripts. Recently, mutations in the gene encoding progranulin (PGRN) were identified as a major cause of FTLD. In the second study we describe a Swedish family with FTLD caused by a frameshift mutation in PGRN. We provide a clinical and neuropathological description of the family, as well as evidence that the pathogenicity of this mutation is through nonsense-mediated decay of the mutant mRNA transcripts and PGRN haploinsufficiency. In the third study we describe a novel PGRN splice site mutation and a previously described PGRN frameshift mutation, found in a mutation screen of 51 FTLD patients. We describe the clinical and neuropathological characteristics of the mutation carriers and demonstrate that haploinsufficiency is the pathogenic mechanism of the two mutations. In the fourth study we investigate the prevalence of PGRN and MAPT gene dosage alterations in 39 patients with FTLD. No gene dosage alterations were identified, indicating that variations in copy number of the PGRN and MAPT genes are not a common cause of disease, at least not in this FTLD patient collection.

La maladie de Whipple (MW) est une maladie infectieuse rare, sévère et chronique qui ne touche qu’une minorité d’individus infectés par Tropheryma whipplei (T. whipplei). Le portage chronique et asymptomatique de T. whipplei est moins rare. La pathogénie de la MW reste largement inconnue. Par une approche génétique combinant une analyse de liaison du génome entier et le séquençage de l’ensemble des exons, nous avons testé l’hypothèse d’une prédisposition génétique à la MW. Nous avons étudié une famille multiplexe comprenant quatre patients atteints de la MW mais n’ayant pas d’autres pathologies associées et cinq porteurs asymptomatiques de T. whipplei, pour laquelle nous avons envisagé un modèle autosomique dominant (AD) avec une pénétrance incomplète liée à l’âge. Nous avons montré que la mutation c.292 C>T (p.R98W) dans le gène IRF4 était le seul variant hétérozygote très rare et non-synonyme commun aux quatre patients. Chez la souris, le gène Irf4 est un facteur de transcription qui joue un rôle pléiotrope dans l’immunité. La caractérisation moléculaire de l’allèle muté a montré un effet délétère par un mécanisme d’haplo-insuffisance pour la fonction de facteur de transcription de la protéine. Une augmentation de la localisation cytoplasmique de la protéine a également été observée. De plus, le défaut IRF4 étudié confère une réponse transcriptomique distincte dans les leucocytes stimulés par le BCG ou T. whipplei. En conclusion, nous avons identifié la première étiologie génétique associée à la MW. Le mode d’hérédité est AD avec une pénétrance incomplète, le portage chronique précédant probablement la MW de plusieurs décennies chez les individus hétérozygotes infectés par T. whipplei. Ces travaux permettront de mieux comprendre la pathogénie de la MW, de mieux définir les mécanismes de l’immunité contre T. whipplei et de pouvoir offrir un diagnostic moléculaire et génétique adapté aux familles. / Whipple's disease (WD) is a rare, severe and chronic infectious disease that affects only a small minority of individuals infected with Tropheryma whipplei (T. whipplei). The chronic and asymptomatic carriage of T. whipplei is less rare. The pathogenesis of WD remains largely unknown. Using a genetic approach combining genome-wide linkage and whole exon sequencing, we tested the hypothesis of a genetic predisposition to WD. We studied a multiplex family containing four otherwise healthy WD patients and five asymptomatic T. whipplei carriers. We tested the hypothesis that WD follows autosomal dominant (AD) inheritance with age-dependent incomplete penetrance. We showed that the c.292 C> T mutation (p.R98W) of IRF4 gene was the only heterozygote variant that was very rare and non-synonymous for all four patients. In mice, the Irf4 gene is a transcription factor that plays pleiotropic roles in immunity. Molecular characterization of the mutated allele showed a deleterious effect by a haplo-insufficiency mechanism for the transcription factor function of the protein. Increase localization of the protein in the cytoplasmic has also been observed. In addition, the defect IRF4 studied confers a distinct transcriptomic response in leukocytes stimulated by BCG or T. whipplei. In conclusion, we identified the first genetic etiology associated with WD. The mode of inheritance is AD with incomplete penetrance, chronic carriage probably preceding WD for several decades in heterozygous individuals infected with T. whipplei. This work will help to better understand the pathogenesis of WD, to better define the mechanisms of immunity against T. whipplei and to be able to offer a molecular and genetic adapted diagnosis to families.

The development of the central nervous system is dysregulated in neurodevelopmental disorders such as intellectual disability, autism spectrum disorder, and epilepsy. These three disorders have different clinical features, yet there is high comorbidity between them. They can be difficult to study due to their highly complex aetiologies, however there are various monogenic diseases that can cause all of them, including SYNGAP1 haploinsufficiency where the synaptic guanosine triphosphatase (GTPase)-activating protein (SYNGAP) protein levels are highly reduced; Fragile X syndrome where the fragile X mental retardation protein (FMRP) is no longer translated; and DNM1 epileptic encephalopathy where mutations in the Dynamin1 gene alter the protein function. These monogenic conditions are synaptopathies as the proteins affected play important roles in synapse stability and neurotransmission. Because of the high comorbidity between these disorders, it is hypothesised that there may be a common mechanism underlying them. We hypothesise that a deficit in presynaptic vesicle recycling may be part of a common mechanism underlying intellectual disability, autism spectrum disorder, and epilepsy especially in SYNGAP1 haploinsufficiency, Fragile X syndrome, and DNM1 epileptic encephalopathy. Using various fluorescent presynaptic activity reporters including synaptic pHluorins, tetramethylrhodamine dextran and calcium dyes to compare presynaptic activity in in vitro models of these monogenic conditions, we found differences in synaptic vesicle (SV) endocytosis in the genetically altered conditions compared to wildtype controls. We observed various SV endocytosis defects in clathrin-mediated endocytosis (CME) or activity-dependent bulk endocytosis (ADBE) in our models. We observed enhanced CME in SynGAP1 KO mouse hippocampal neurons. This enhanced SV endocytosis was accompanied by decreased SV cargo on the plasma membrane. Rat SynGAP1 KO hippocampal neurons did not display enhanced SV endocytosis, nor did neurons with the GTPase-activating (GAP) domain of SynGAP deleted. This was perhaps due to the altered time course of development between these rodent species. In mouse and rat models of Fragile X syndrome, CME was not altered compared to wildtype controls. However, in a rat model, we observed fewer nerve terminals undergoing ADBE which is the dominant SV endocytosis mode during elevated neuronal activity. De novo epileptic encephalopathy-associated mutations in DNM1 had differential effects on SV recycling through both CME and ADBE. Mouse hippocampal neurons overexpressing Dyn1R237W, Dyn1I289F and Dyn1H396D all showed less CME compared to overexpression of Dyn1WT. Moreover, fewer nerve terminals overexpressing Dyn1H396D were found to undergo ADBE. We also found that a large-conductance potassium (BK) channel opener can accelerate clathrin-mediated endocytosis and thus may be able to rescue the impaired SV endocytosis caused by these mutants. Although there is not yet a common underlying pathway at the presynaptic level between these conditions, SV recycling dysfunction is present across all of these models. Furthermore, we propose an axis of pathophysiology model where optimal SV endocytosis is required for optimised neural performance. We propose that either decreased or increased SV endocytosis can lead to the synaptic dysfunction observed in these models.

The aim of this thesis was to identify genetic factors involved in frontotemporal lobar degeneration (FTLD), a neurodegenerative disorder clinically characterised by a progressive change in personality, behaviour and language. FTLD is a genetically complex disorder and a positive family history is found in up to 40% of the cases. In 10-20% of the familial cases the disease can be explained by mutations in the gene encoding the microtubule associated protein tau (MAPT). In the first study we describe the clinical and neuropathological features of a Finnish family with FTLD caused by a mutation in MAPT. We also provide evidence that the pathogenic mechanism of this mutation is through altered splicing of MAPT transcripts. Recently, mutations in the gene encoding progranulin (PGRN) were identified as a major cause of FTLD. In the second study we describe a Swedish family with FTLD caused by a frameshift mutation in PGRN. We provide a clinical and neuropathological description of the family, as well as evidence that the pathogenicity of this mutation is through nonsense-mediated decay of the mutant mRNA transcripts and PGRN haploinsufficiency. In the third study we describe a novel PGRN splice site mutation and a previously described PGRN frameshift mutation, found in a mutation screen of 51 FTLD patients. We describe the clinical and neuropathological characteristics of the mutation carriers and demonstrate that haploinsufficiency is the pathogenic mechanism of the two mutations. In the fourth study we investigate the prevalence of PGRN and MAPT gene dosage alterations in 39 patients with FTLD. No gene dosage alterations were identified, indicating that variations in copy number of the PGRN and MAPT genes are not a common cause of disease, at least not in this FTLD patient collection.

Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a defect in red blood cell production. The disease is associated with growth retardation, malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. In a cellular model for DBA, siRNA knock-down of RPS19 results in a relative decrease of other ribosomal (r) proteins belonging to the small subunit (RPS20, RPS21, RPS24) when compared to r-proteins from the large ribosomal subunit (RPL3, RPL9, RPL30, RPL38). RPS19 mutant cells from DBA patients show a similar and coordinated down-regulation of small subunit proteins. The mRNA levels of the small subunit r-proteins remain relatively unchanged. We also show that RPS19 has an extensive number of transcriptional start sites resulting in mRNAs of variable 5’UTR length. The short variants are translated more efficiently. Structural sequence variations in the 5’UTR of RPS19 found in DBA patients show a 20%-30% reduced translational activity when compared to normal transcripts. Primary fibroblast from DBA patients with truncating mutations in RPS19 or RPS24 showed specific cell cycle defects. RPS19 mutant fibroblasts accumulate in the G1 phase whereas the RPS24 mutant cells show a defect in G2/M phase. The G1 phase arrest is associated with a reduced level of phosphorylated retinoblastoma (Rb) protein, cyclin E and cdk2 whereas the G2/M phase defect is associated with increased levels of p21, cyclin E, cdk4 and cdk6. RPS19 interacts with PIM-1 kinase. We investigated the effects of targeted disruptions of both Rps19 and Pim-1 in mice. Double mutant (Rps19+/-, Pim-1-/-) mice have increased peripheral white- and red blood cell counts when compared to the wild-type mice (Rps19+/+, Pim-1+/+). Bone marrow cells in Rps19+/-, Pim-1-/- mice showed up-regulated levels of c-Myc and the anti-apoptotic factors Bcl2, Bcl-xl and Mcl-1 and reduced levels of the apoptotic factors Bak and Caspase 3 as well as the cell cycle regulator p21. In summary, this thesis clarifies several mechanisms in the pathogenesis of DBA. Mutations in RPS19 results in coordinated down-regulation of several small subunit r-proteins causing haploinsufficiency for the small ribosomal subunit. RPS19 have multiple transcriptional start sites and mutations in the RPS19 5’UTR found in DBA patients result in reduced translational activity. At the cellular level, mutations in RPS19 and RPS24 cause distinct cell cycle defects and reduced cell proliferation. Finally, PIM-1 kinase and RPS19 cooperates in the proliferation of myeloid cells.