Regulation and function of ciliary dyslexia candidate genes

Dyslexia is defined as an unexpected difficulty in reading despite normal intelligence, senses and instruction. It is the most common learning disability with estimated 5-10% of the population affected. Its heredity is estimated to about 40-60%. Despite the established heredity of the condition, it has been very challenging to pinpoint the underlying genes. In the past 15 years, a number of dyslexia candidate genes have been suggested. A handful of them have been replicated in several studies, including DYX1C1, DCDC2 and KIAA0319. More recently, the very same genes have been independently associated to functions of the cilium. Cilia are microtubule-based organelles present on the surface of most eukaryotic cells. The aim of this thesis was to investigate the molecular functions of ciliary dyslexia candidate genes and their role at the cilium. In paper I, we found X-box motifs in the promoter regions of DYX1C1, DCDC2 and KIAA0319 and showed that they are functional and able to bind ciliogenic RFX transcription factors. Knockdown of certain RFX transcription factors altered the expression of DYX1C1 and DCDC2, but not KIAA0319. Overall, we strengthened the evidence for DYX1C1 and DCDC2 as ciliary genes. In paper II, we identified DCDC2 as a causative gene for nephronophthisis-related ciliopathy (NPHP-RC) with loss-of function mutations present in two affected families. We observed localization of DCDC2 to the ciliary axoneme of affected organs and demonstrated a crucial role of the Wnt pathway in the pathogenesis of NPHP-RC. 3D modeling in spheroids and in vivo modeling in zebrafish confirmed these observations. In paper III, we identified CPAP as an interacting partner of both DYX1C1 and DCDC2. In addition, we observed genetic pathway synergy between DYX1C1 and DCDC2 using zebrafish and a human ciliated cell model. In paper IV, we performed transcriptomics on differentiating human neuroepithelial stem cells and characterized the expression of dyslexia candidate genes. We found that some dyslexia candidate genes are upregulated during human neuronal differentiation. Remarkably, we identified the group of ciliary genes as the major group of upregulated genes. In addition, we showed that cilia are present on the surface of neuronal cells throughout differentiation. In paper V, we asked whether dyslexia and ciliopathies might have a common genetic origin by investigating the genome of two individuals with situs inversus and dyslexia. We identified rare variants in dynein heavy chain genes likely causing their situs inversus phenotype. Their involvement in dyslexia remains to be determined. In conclusion, the work conducted within this thesis strengthened and expanded on the role of DYX1C1 and DCDC2 at the cilium and in ciliopathies and identified the group of ciliary genes as a major gene class in human neuronal differentiation. A link between cilia and dyslexia remains elusive

Genomic sequencing of a dyslexia susceptibility haplotype encompassing ROBO1

Background: The DYX5 locus for developmental dyslexia was mapped to chromosome 3 by linkage study of a large Finnish family, and later, roundabout guidance receptor 1 (ROBO1) was implicated as a candidate gene at DYX5 with suppressed expression from the segregating rare haplotype. A functional magnetoencephalographic study of several family members revealed abnormal auditory processing of interaural interaction, supporting a defect in midline crossing of auditory pathways. In the current study, we have characterized genetic variation in the broad ROBO1 gene region in the DYX5-linked family, aiming to identify variants that would increase our understanding of the altered expression of ROBO1. Methods: We have used a whole genome sequencing strategy on a pooled sample of 19 individuals in combination with two individually sequenced genomes. The discovered genetic variants were annotated and filtered. Subsequently, the most interesting variants were functionally tested using relevant methods, including electrophoretic mobility shift assay (EMSA), luciferase assay, and gene knockdown by lentiviral small hairpin RNA (shRNA) in lymphoblasts. Results: We found one novel intronic single nucleotide variant (SNV) and three novel intergenic SNVs in the broad region of ROBO1 that were specific to the dyslexia susceptibility haplotype. Functional testing by EMSA did not support the binding of transcription factors to three of the SNVs, but one of the SNVs was bound by the LIM homeobox 2 (LHX2) protein, with increased binding affinity for the non-reference allele. Knockdown of LHX2 in lymphoblast cell lines extracted from subjects from the DYX5-linked family showed decreasing expression of ROBO1, supporting the idea that LHX2 regulates ROBO1 also in human. Conclusions: The discovered variants may explain the segregation of dyslexia in this family, but the effect appears subtle in the experimental settings. Their impact on the developing human brain remains suggestive based on the association and subtle experimental support.Peer reviewe

Rare variants in dynein heavy chain genes in two individuals with situs inversus and developmental dyslexia : a case report

Background Developmental dyslexia (DD) is a neurodevelopmental learning disorder with high heritability. A number of candidate susceptibility genes have been identified, some of which are linked to the function of the cilium, an organelle regulating left-right asymmetry development in the embryo. Furthermore, it has been suggested that disrupted left-right asymmetry of the brain may play a role in neurodevelopmental disorders such as DD. However, it is unknown whether there is a common genetic cause to DD and laterality defects or ciliopathies. Case presentation Here, we studied two individuals with co-occurring situs inversus (SI) and DD using whole genome sequencing to identify genetic variants of importance for DD and SI. Individual 1 had primary ciliary dyskinesia (PCD), a rare, autosomal recessive disorder with oto-sino-pulmonary phenotype and SI. We identified two rare nonsynonymous variants in the dynein axonemal heavy chain 5 gene (DNAH5): a previously reported variant c.7502G > C; p.(R2501P), and a novel variant c.12043 T > G; p.(Y4015D). Both variants are predicted to be damaging. Ultrastructural analysis of the cilia revealed a lack of outer dynein arms and normal inner dynein arms. MRI of the brain revealed no significant abnormalities. Individual 2 had non-syndromic SI and DD. In individual 2, one rare variant (c.9110A > G;p.(H3037R)) in the dynein axonemal heavy chain 11 gene (DNAH11), coding for another component of the outer dynein arm, was identified. Conclusions We identified the likely genetic cause of SI and PCD in one individual, and a possibly significant heterozygosity in the other, both involving dynein genes. Given the present evidence, it is unclear if the identified variants also predispose to DD and further studies into the association between laterality, ciliopathies and DD are needed.Peer reviewe

Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

DYX1C1, DCDC2, and KIAA0319 are three of the most replicated dyslexia candidate genes (DCGs). Recently, these DCGs were implicated in functions at the cilium. Here, we investigate the regulation of these DCGs by Regulatory Factor X transcription factors (RFX TFs), a gene family known for transcriptionally regulating ciliary genes. We identify conserved X-box motifs in the promoter regions of DYX1C1, DCDC2, and KIAA0319 and demonstrate their functionality, as well as the ability to recruit RFX TFs using reporter gene and electrophoretic mobility shift assays. Furthermore, we uncover a complex regulation pattern between RFX1, RFX2, and RFX3 and their significant effect on modifying the endogenous expression of DYX1C1 and DCDC2 in a human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1). In addition, induction of ciliogenesis increases the expression of RFX TFs and DCGs. At the protein level, we show that endogenous DYX1C1 localizes to the base of the cilium, whereas DCDC2 localizes along the entire axoneme of the cilium, thereby validating earlier localization studies using overexpression models. Our results corroborate the emerging role of DCGs in ciliary function and characterize functional noncoding elements, X-box promoter motifs, in DCG promoter regions, which thus can be targeted for mutation screening in dyslexia and ciliopathies associated with these genes.Peer reviewe

Ablation of cdk4 and cdk6 affects proliferation of basal progenitor cells in the developing dorsal and ventral forebrain

Little is known about the molecular players driving proliferation of neural progenitor cells (NPCs) during embryonic mouse development. Here, we demonstrate that proliferation of NPCs in the developing forebrain depends on a particular combination of cell cycle regulators. We have analyzed the requirements for members of the cyclin-dependent kinase (cdk) family using cdk-deficient mice. In the absence of either cdk4 or cdk6, which are both regulators of the G1 phase of the cell cycle, we found no significant effects on the proliferation rate of cortical progenitor cells. However, concomitant loss of cdk4 and cdk6 led to a drastic decrease in the proliferation rate of NPCs, specifically the basal progenitor cells of both the dorsal and ventral forebrain at embryonic day 13.5 (E13.5). Moreover, basal progenitors in the forebrain of Cdk4;Cdk6 double mutant mice exhibited altered cell cycle characteristics. Cdk4;cdk6 deficiency led to an increase in cell cycle length and cell cycle exit of mutant basal progenitor cells in comparison to controls. In contrast, concomitant ablation of cdk2 and cdk6 had no effect on the proliferation of NCPs. Together, our data demonstrate that the expansion of the basal progenitor pool in the developing telencephalon is dependent on the presence of distinct combinations of cdk molecules. Our results provide further evidence for differences in the regulation of proliferation between apical and basal progenitors during cortical development

Genetic and protein interaction studies between the ciliary dyslexia candidate genes DYX1C1 and DCDC2

BackgroundDYX1C1 (DNAAF4) and DCDC2 are two of the most replicated dyslexia candidate genes in genetic studies. They both have demonstrated roles in neuronal migration, in cilia growth and function and they both are cytoskeletal interactors. In addition, they both have been characterized as ciliopathy genes. However, their exact molecular functions are still incompletely described. Based on these known roles, we asked whether DYX1C1 and DCDC2 interact on the genetic and the protein level.ResultsHere, we report the physical protein-protein interaction of DYX1C1 and DCDC2 as well as their respective interactions with the centrosomal protein CPAP (CENPJ) on exogenous and endogenous levels in different cell models including brain organoids. In addition, we show a synergistic genetic interaction between dyx1c1 and dcdc2b in zebrafish exacerbating the ciliary phenotype. Finally, we show a mutual effect on transcriptional regulation among DYX1C1 and DCDC2 in a cellular model.ConclusionsIn summary, we describe the physical and functional interaction between the two genes DYX1C1 and DCDC2. These results contribute to the growing understanding of the molecular roles of DYX1C1 and DCDC2 and set the stage for future functional studies.Peer reviewe

Acute doses of caffeine shift nervous system cell expression profiles toward promotion of neuronal projection growth

Caffeine is a widely consumed psychoactive substance, but little is known about the effects of caffeine stimulation on global gene expression changes in neurons. Here, we conducted gene expression profiling of human neuroepithelial stem cell-derived neurons, stimulated with normal consumption levels of caffeine (3 mu M and 10 mu M), over a period of 9 h. We found dosage-dependent activation of immediate early genes after 1 h. Neuronal projection development processes were up-regulated and negative regulation of axon extension processes were down-regulated at 3 h. In addition, genes involved in extracellular matrix organization, response for wound healing, and regulation of immune system processes were down-regulated by caffeine at 3 h. This study identified novel genes within the neuronal projection guidance pathways that respond to acute caffeine stimulation and suggests potential mechanisms for the effects of caffeine on neuronal cells.Peer reviewe

Taxation of corporates in the Czech Republic and Austria

This bachelor thesis aims to determine and compare the tax burden of corporates in terms of tax on corporate profits in the Czech Republic and Austria. It consists of two parts. The first one is focused on introduction of the corporate tax structure in both countries. The second part compares the tax burden using real and fictitious indicators. The analysis of all obtained values shows that Austria is subject of higher corporate tax burden. Despite that, for some companies, other aspects, such as the possibility of group taxation in Austria, may be relevant

Additional file 3: Table S3. of Genomic sequencing of a dyslexia susceptibility haplotype encompassing ROBO1

Rare coding heterozygous SNVs detected by both platforms in the linkage region. (DOC 28 kb