Specific Reading Disorder : Cellular and Neurodevelopmental Functions of Susceptibility Genes

Specific reading disorder (SRD), or developmental dyslexia, is defined as an unexpected difficulty in learning to read and write when intelligence and senses are normal. Hereditary factors are estimated to play a substantial role in the etiology of SRD, although the exact neurobiological mechanisms involved are rather poorly understood. In this thesis we have investigated the function of three SRD susceptibility candidate genes, DYX1C1, DCDC2 and ROBO1, with the aim of finding neurodevelopmental and molecular pathways that might shed light on the etiology of SRD. When research for this thesis began, knockdown of the rodent orthologs of DYX1C1 and DCDC2 had been shown to disturb radial neuronal migration in the developing cerebral cortex, but the function of human DYX1C1 and DCDC2 at the cellular level was still unclear. We discovered that both DYX1C1 and DCDC2 are involved in signalling pathways that are important in brain development; DYX1C1 is involved in estrogen signalling and DCDC2 is involved in ciliary signalling. We found that the effect of DYX1C1 on estrogen signalling was concerted through its interaction with estrogen receptors (ERs) in in the presence of the endogenous ligand, 17β-estradiol. We observed that DYX1C1 regulates the degradation of ERs, resulting in decreased transcriptional responses to 17β-estradiol. Our findings suggest that the effects of DYX1C1 on brain development may be at least partially mediated by ERs and that hormonal factors may play a role in SRD. We also observed DYX1C1 and ERα complexes in the neurites of primary rat hippocampal neurons, which suggests a role for DYX1C1 in rapid non-genomic ER signalling. The effect of DCDC2 on the ciliary signalling was such that the overexpression of DCDC2 was found to activate SHH signalling, whereas the downregulation of DCDC2 expression was found to enhance WNT signalling. We also observed that the DCDC2 protein localizes to the primary cilium in primary rat hippocampal neurons and is involved in regulating the length of the cilium through its role in stabilizing microtubules. DCDC2 was also found to interact with the ciliary kinesin-2 subunit KIF3A, a key molecule in function and maintenance of cilia. Consistent with a role in ciliary function, the overexpression of DCDC2 in C. elegans resulted in an abnormal neuronal phenotype that could only be observed in ciliated neurons. Our results were the first to suggest a role for DCDC2 in the structure and function of primary cilia. Later, others have reported more links between ciliary function and SRD candidate genes, most notably the putative role of DYX1C1 as a cytoplasmic assembly factor for ciliary dynein. ROBO1 has been discovered as a SRD susceptibility gene in a large multi-generation family, in whom a rare haplotype in the broad genomic area of ROBO1 is co-segregated with SRD. The expression of ROBO1 has been shown to be reduced from the SRD-associated haplotype, but the causal factor for the reduced expression was not known. In this thesis we have characterized genetic variation within the SRD-susceptibility haplotype by whole genome sequencing, aiming to identify variants that would increase our understanding of the altered expression of ROBO1. We found several novel variants in the SRD susceptibility haplotype and tested transcription factor binding to four of the variants by EMSA. We did not detect transcription factor binding to three of the variants. However, one of the variants was bound by the LIM homeobox 2 (LHX2) transcription factor with increased binding affinity to the non-reference allele. Knockdown of LHX2 in lymphoblast cell lines extracted from subjects of the DYX5-linked family showed decreased expression of ROBO1 supporting the idea that LHX2 regulates ROBO1. Because the regulation of ROBO1 is likely to be complex and the effect of the novel variants was at the most very subtle in our experiments, it remains unknown if any of them are causal factors for the SRD susceptibility. The mouse ortholog of ROBO1 has been shown to have many functions in brain development: it is involved in neuronal migration of interneurons and pyramidal cells and in axonal guidance of major nerve tracts. The role of ROBO1 in mouse brain led us to test two hypotheses on two human populations: 1) We tested whether ROBO1 controls midline crossing of auditory pathways in the family with reduced expression of ROBO1 and 2) we tested whether in the normal population ROBO1 is involved in the development of the corpus callosum, the major axon tract connecting the cerebral hemispheres. The axonal crossing of the auditory pathways was studied using a functional approach, based on magnetoencephalography and frequency tagging. We found impaired interaural interaction in the subjects that had reduced ROBO1 expression supporting a defect in midline crossing of auditory pathways. Moreover, the deficit in interaural interaction depended on the ROBO1 in a dose-dependent manner. Our results suggest that ROBO1 controls midline crossing of the auditory pathways and were the first evidence of a SRD susceptibility gene being linked to a specific sensory function in the human brain. The role of ROBO1 in callosal development was assessed by studying whether polymorphisms in ROBO1 correlate with variation in the white matter structure in the corpus callosum. By using data acquired by both structural magnetic resonance imaging and diffusion tensor imaging we found that five polymorphisms in the regulatory region of ROBO1 were associated with white matter density in the posterior part of the corpus callosum. One of the polymorphisms, rs7631357, was also significantly associated with the probability of connections from the body of the corpus callosum to the parietal cortical regions. Our results suggest that the human ROBO1 may be involved in the regulation of the structure and connectivity of the posterior part of the corpus callosum. Overall, our results support the idea that similarly as in mice, the human ROBO1 is likely to play many different roles in brain development. In conclusion, the results of this study have advanced the field of SRD research by suggesting new functions for SRD candidate susceptibility genes in cellular and developmental pathways that are highly relevant in the context of brain development. More studies will be needed to clarify the role of genes in the etiology of SRD and in the neurobiology of reading, but our results have provided clues that may be worthwhile to be investigated.Lukivaikeus (eli lukemisen erityisvaikeus tai dysleksia) ilmenee odottamattomana vaikeutena lukemaan oppimisessa henkilöillä, joiden aistit ja älykkyys ovat normaaleja. Perinnöllisillä tekijöillä on arvioitu olevan merkittävä rooli lukivaikeudessa, vaikkakin neurobiologisia mekanismeja lukivaikeuden taustalla tunnetaan vielä melko huonosti. Tässä väitöskirjatyössä tutkimuksen kohteena oli kolme mahdollista lukivaikeuden alttiusgeeniä: DYX1C1, DCDC2 ja ROBO1. Tutkimuksen tavoitteena oli selvittää geenien toimintaa sekä solutasolla että aivojen kehityksessä. Havaitsimme, että kaksi alttiusgeeniä DYX1C1 ja DCDC2 vaikuttavat solunsisäisiin viestintäreitteihin, jotka ovat tärkeitä aivojen kehityksessä. Tuloksemme osoittivat että DYX1C1 säätelee estrogeenireseptorien hajotusta soluissa ja vaikuttaa sitä kautta estrogeenireseptorien viestintään. On mahdollista että hormonaalisten tekijöiden osuutta lukivaikeudessa kannattaisi tutkia tarkemmin. Havaitsimme että DCDC2 osallistuu erityisen antennimaisen soluelimen cilian (eli värekarvan) kautta tapahtuviin viestintäreitteihin. Lisäksi DCDC2 säätelee cilian pituutta. Tuloksemme olivat ensimmäinen tutkimus joissa lukihäiriön alttiusgeeni yhdistettiin cilian toimintaan. Myöhemmin muut tutkimukset ovat löytäneet lisää yhteyksiä cilian ja lukivaikeuden alttiusgeenien välille. ROBO1 geenin harvinainen muoto periytyy yhdessä lukivaikeuden kanssa suuressa suomalaisessa perheessä. ROBO1:n ilmentyminen on vähentynyt harvinaisen geenimuodon kantajilla, mutta tarkkaa syytä tähän ei tiedetä. Tässä väitöskirjatutkimuksessa käytimme uuden sukupolven sekvensointitekniikoita etsiäksemme ROBO1 geenin harvinaisen muodon kantajilta uusia geneettisiä variantteja ROBO1 geenin alueelta ja sen ympäristöstä. Löysimme useita variantteja, jotka saattavat vaikuttaa ROBO1:n ilmentymiseen. Lisäksi tutkimme magnetoenkefalografian avulla aivoissa tapahtuvaa vasemmasta ja oikeasta korvasta tulevien viestien vuorovaikutusta (binauraalinen vuorovaikutus) ja huomasimme että harvinaisen ROBO1 geenimuodon kantajilla oli vähemmän binauraalista vuorovaikutusta kuin kontrollihenkilöillä. Binauraalinen vuorovaikutus oli sitä heikompaa mitä vähemmän ROBO1 geeni ilmentyi. Tuloksemme osoittavat että todennäköisesti ROBO1 säätelee kuuloratojen risteämistä aivoissa, sillä binauraalinen vuorovaikutus on riippuvaista kuuloratojen osittaisesta risteämisestä. Tutkimme myös ROBO1 geenin toimintaa aivokurkiaisen kehityksessä diffuusiotensorikuvantamisen ja rakenteellisen magneettiresonanssikuvantamisen avulla. Tässä tutkimuksessa koehenkilöt olivat otos normaaliväestöstä. Havaitsimme, että viisi ROBO1 geenin alueella olevaa yhden emäksen muutosta korreloivat aivokurkiaisen takaosan vakoisen aineen tiheyden kanssa. Tuloksemme viittaavat siihen että ROBO1 voi osallistua aivokurkiaisen rakenteen säätelyyn. Kaiken kaikkiaan tuloksemme tukevat ajatusta että ROBO1 geeni osallistuu monien aivojen kehityksen kannalta oleellisiin toimintoihin. Tämän väitöskirjatutkimuksen tulokset ovat edistäneet lukivaikeuden ymmärtämistä ehdottamalla uusia mahdollisia toimintoja lukivaikeuden alttiusgeeneille

Human ROBO1 regulates white matter structure in corpus callosum

The axon guidance receptor, Robo1, controls the pathfinding of callosal axons in mice. To determine whether the orthologous ROBO1 gene is involved in callosal development also in humans, we studied polymorphisms in the ROBO1 gene and variation in the white matter structure in the corpus callosum using both structural magnetic resonance imaging and diffusion tensor magnetic resonance imaging. We found that five polymorphisms in the regulatory region of ROBO1 were associated with white matter density in the posterior part of the corpus callosum pathways. One of the polymorphisms, rs7631357, was also significantly associated with the probability of connections to the parietal cortical regions. Our results demonstrate that human ROBO1 may be involved in the regulation of the structure and connectivity of posterior part of corpus callosum.Peer reviewe

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

Genetic Susceptibility to Non-Necrotizing Erysipelas/Cellulitis

Peer reviewe

Increased Expression of the Dyslexia Candidate Gene DCDC2 Affects Length and Signaling of Primary Cilia in Neurons

Peer reviewe

The complex of TFII-I, PARP1, and SFPQ proteins regulates the DYX1C1 gene implicated in neuronal migration and dyslexia

DYX1C1 was first identified as a candidate gene for dyslexia susceptibility, and its role in controlling neuronal migration during embryogenesis and effect on learning in rodents have been verified. In contrast, genetic association studies have been ambiguous in replicating its effects on dyslexia. To better understand the regulation of DYX1C1 and the possible functional role of genetic variation in the promoter of DYX1C1, we selected three single-nucleotide polymorphisms (SNPs) with predicted functional consequences or suggested associations to dyslexia for detailed study. Electrophoretic mobility shift assays suggested the allele-specific binding of the transcription factors TFII-I (to rs3743205) and Sp1 (to rs16787 and rs12899331) that could be verified by competition assays. In addition, we purified a complex of protein factors binding to the previously suggested dyslexia-related SNP, −3G/A (rs3743205). Three proteins, TFII-I, PARP1, and SFPQ, were unambiguously identified by mass spectrometry and protein sequencing. Two SNPs, rs16787 and rs3743205, showed significant allelic differences in luciferase assays. Our results show that TFII-I, PARP1, and SFPQ proteins, each previously implicated in gene regulation, form a complex controlling transcription of DYX1C1. Furthermore, allelic differences in the promoter or 5′ untranslated region of DYX1C1 may affect factor binding and thus regulation of the gene.—Tapia-Páez, I., Tammimies, K., Massinen, S., Roy. A. L., Kere, J. The complex of TFII-I, PARP1, and SFPQ proteins regulates the DYX1C1 gene implicated in neuronal migration and dyslexia

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