Dyslexia and language impairment associated genetic markers influence cortical thickness and white matter in typically developing children

Dyslexia and language impairment (LI) are complex traits with substantial genetic components. We recently completed an association scan of the DYX2 locus, where we observed associations of markers in DCDC2, KIAA0319, ACOT13, and FAM65B with reading-, language-, and IQ-related traits. Additionally, the effects of reading-associated DYX3 markers were recently characterized using structural neuroimaging techniques. Here, we assessed the neuroimaging implications of associated DYX2 and DYX3 markers, using cortical volume, cortical thickness, and fractional anisotropy. To accomplish this, we examined eight DYX2 and three DYX3 markers in 332 subjects in the Pediatrics Imaging Neurocognition Genetics study. Imaging-genetic associations were examined by multiple linear regression, testing for influence of genotype on neuroimaging. Markers in DYX2 genes KIAA0319 and FAM65B were associated with cortical thickness in the left orbitofrontal region and global fractional anisotropy, respectively. KIAA0319 and ACOT13 were suggestively associated with overall fractional anisotropy and left pars opercularis cortical thickness, respectively. DYX3 markers showed suggestive associations with cortical thickness and volume measures in temporal regions. Notably, we did not replicate association of DYX3 markers with hippocampal measures. In summary, we performed a neuroimaging follow-up of reading-, language-, and IQ-associated DYX2 and DYX3 markers. DYX2 associations with cortical thickness may reflect variations in their role in neuronal migration. Furthermore, our findings complement gene expression and imaging studies implicating DYX3 markers in temporal regions. These studies offer insight into where and how DYX2 and DYX3 risk variants may influence neuroimaging traits. Future studies should further connect the pathways to risk variants associated with neuroimaging/neurocognitive outcomes

Genetics of developmental dyslexia

Developmental dyslexia is a highly heritable disorder with a prevalence of at least 5% in school-aged children. Linkage studies have identified numerous loci throughout the genome that are likely to harbour candidate dyslexia susceptibility genes. Association studies and the refinement of chromosomal translocation break points in individuals with dyslexia have resulted in the discovery of candidate genes at some of these loci. A key function of many of these genes is their involvement in neuronal migration. This complements anatomical abnormalities discovered in dyslexic brains, such as ectopias, that may be the result of irregular neuronal migration

Approach to epigenetic analysis in language disorders

Language and learning disorders such as reading disability and language impairment are recognized to be subject to substantial genetic influences, but few causal mutations have been identified in the coding regions of candidate genes. Association analyses of single nucleotide polymorphisms have suggested the involvement of regulatory regions of these genes, and a few mutations affecting gene expression levels have been identified, indicating that the quantity rather than the quality of the gene product may be most relevant for these disorders. In addition, several of the candidate genes appear to be involved in neuronal migration, confirming the importance of early developmental processes. Accordingly, alterations in epigenetic processes such as DNA methylation and histone modification are likely to be important in the causes of language and learning disorders based on their functions in gene regulation. Epigenetic processes direct the differentiation of cells in early development when neurological pathways are set down, and mutations in genes involved in epigenetic regulation are known to cause cognitive disorders in humans. Epigenetic processes also regulate the changes in gene expression in response to learning, and alterations in histone modification are associated with learning and memory deficits in animals. Genetic defects in histone modification have been reversed in animals through therapeutic interventions resulting in rescue of these deficits, making it particularly important to investigate their potential contribution to learning disorders in humans

Genome-wide association study of shared components of reading disability and language impairment

Written and verbal languages are neurobehavioral traits vital to the development of communication skills. Unfortunately, disorders involving these traits-specifically reading disability (RD) and language impairment (LI)-are common and prevent affected individuals from developing adequate communication skills, leaving them at risk for adverse academic, socioeconomic and psychiatric outcomes. Both RD and LI are complex traits that frequently co-occur, leading us to hypothesize that these disorders share genetic etiologies. To test this, we performed a genome-wide association study on individuals affected with both RD and LI in the Avon Longitudinal Study of Parents and Children. The strongest associations were seen with markers in ZNF385D (OR = 1.81, P = 5.45 × 10(-7) ) and COL4A2 (OR = 1.71, P = 7.59 × 10(-7) ). Markers within NDST4 showed the strongest associations with LI individually (OR = 1.827, P = 1.40 × 10(-7) ). We replicated association of ZNF385D using receptive vocabulary measures in the Pediatric Imaging Neurocognitive Genetics study (P = 0.00245). We then used diffusion tensor imaging fiber tract volume data on 16 fiber tracts to examine the implications of replicated markers. ZNF385D was a predictor of overall fiber tract volumes in both hemispheres, as well as global brain volume. Here, we present evidence for ZNF385D as a candidate gene for RD and LI. The implication of transcription factor ZNF385D in RD and LI underscores the importance of transcriptional regulation in the development of higher order neurocognitive traits. Further study is necessary to discern target genes of ZNF385D and how it functions within neural development of fluent language

The regulation of DCDC2, a candidate gene for dyslexia

Within the human genome, genetic mapping studies have identified ten regions of different chromosomes, known as DYX loci, in genetic linkage with dyslexia. The gene DCDC2, located within the DYX2 region on chromosome 6p22, has been shown to have genetic association with dyslexia in several independent studies. Functional assays of DCDC2 indicate that it may help guide the migration of neurons during early brain development. DCDC2 polymorphisms that display the strongest association with dyslexia are located in a highly GC-rich region in intron 2 known as BV677278. These polymorphisms contain several transcription factor binding sites, including the canonical 8-base recognition site for PEA3, a transcription factor known to modulate neuronal migration in mice. We hypothesized that 1) BV677278 is an enhancer element for DCDC2 that regulates its expression level, location, or timing, and that 2) PEA3 regulates DCDC2 expression by binding BV677278. To test these hypotheses we showed that PEA3 binds to regions within BV677278, and that siRNA knockdown of PEA3 appears to delay the expression of DCDC2 during neuronal differentiation of mouse cells. We concluded that PEA3 was a viable candidate transcription factor for DCDC2, with the ability to bind BV677278. Taken together, these data suggest a possible mechanism by which BV677278 polymorphisms alter PEA3 binding and DCDC2 expression, which in turn may modulate neuronal migration and affect the risk of dyslexia

Genome-wide association study of shared components of reading disability and language impairment

Written and verbal languages are neurobehavioral traits vital to the development of communication skills. Unfortunately, disorders involving these traits—specifically reading disability (RD) and language impairment (LI)—are common and prevent affected individuals from developing adequate communication skills, leaving them at risk for adverse academic, socioeconomic and psychiatric outcomes. Both RD and LI are complex traits that frequently co-occur, leading us to hypothesize that these disorders share genetic etiologies. To test this, we performed a genome-wide association study on individuals affected with both RD and LI in the Avon Longitudinal Study of Parents and Children. The strongest associations were seen with markers in ZNF385D (OR = 1.81, P = 5.45 × 10−7) and COL4A2 (OR = 1.71, P = 7.59 × 10−7). Markers within NDST4 showed the strongest associations with LI individually (OR = 1.827, P = 1.40 × 10−7). We replicated association of ZNF385D using receptive vocabulary measures in the Pediatric Imaging Neurocognitive Genetics study (P = 0.00245). We then used diffusion tensor imaging fiber tract volume data on 16 fiber tracts to examine the implications of replicated markers. ZNF385D was a predictor of overall fiber tract volumes in both hemispheres, as well as global brain volume. Here, we present evidence for ZNF385D as a candidate gene for RD and LI. The implication of transcription factor ZNF385D in RD and LI underscores the importance of transcriptional regulation in the development of higher order neurocognitive traits. Further study is necessary to discern target genes of ZNF385D and how it functions within neural development of fluent language

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

White matter connections : developmental neuroimaging studies of the associations between genes, brain and behavior

Development of cognitive abilities across childhood and adulthood parallels brain maturation in typically developing samples. Cognitive abilities such as reading and working memory have been linked to neuroimaging measures in relevant brain regions. Though the correlations between inter-individual brain differences and their related cognitive abilities are well established, the cause of this inter-individual variability is still not fully known. This thesis aims to understand the neural bases of the inter-individual variability in reading ability by studying the associations between dyslexia susceptibility genes and white and gray matter brain structures, and determine whether the measures of associated regions correlate with variability in reading ability. Moreover, it aims to identify the brain measures that correlate with concurrent measures of working memory and those that are predictive of future working memory, using a longitudinal cohort of typically developing children and young adults. Studies I and II: Three genes, DYX1C1, DCDC2 and KIAA0319, have been previously associated with dyslexia, neuronal migration, and ciliary function. We investigated whether the polymorphisms within these genes would affect variability in white and gray matter brain structures. Rs3743204 (DYX1C1), rs793842 (DCDC2), and rs6935076 (KIAA0319) were associated with left temporo-parietal white matter volume connecting middle temporal cortex to angular and supramarginal gyri as well as lateral occipital cortex. Rs793842 was significantly associated with thickness of left parietal areas and the lateral occipital cortex. Both white and gray matter measures correlated with current reading ability, but only white matter predicted future reading. Study III: We aimed to investigate whether MRPL19/C2ORF3 dyslexia genes, found to be correlated with verbal and non-verbal IQ, have a significant influence on white matter brain structures. Rs917235 showed a significant association with white matter volume in bilateral posterior parts of the corpus callosum and the cingulum, with connections to parietal, occipital and temporal cortices that are involved in both language and general cognitive abilities. Study IV: ROBO1 is a dyslexia gene that has been associated with axonal guidance and midline crossing. We assessed whether the polymorphisms within this gene have an influence on structure of the corpus callosum. Rs7631357 was associated with probability of connections within the fibers extending through the body of corpus callosum to parietal brain regions. The results fit well with previous reports on the role of Robo1 in axonal path finding in mice. Study V: Working memory has been associated with greater brain activity, thinner cortex, and white matter maturation in cross-sectional studies of children and young adults. Here, we aimed to investigate the role of differences in brain structure and function in the development of working memory. We assessed the concurrent and predictive relationships between working memory performance and neuroimaging measures in the fronto-parietal and fronto-striatal networks important for working memory. Working memory performance correlated with brain activity in frontal and parietal regions, cortical thickness in parietal cortex, and white matter volume of fronto-parietal and fronto-striatal tracts. White matter microstructure and brain activity in the caudate predicted future working memory. This work highlights the impact of imaging genetics research, revealing important associations between genes, brain and behavior. The results identify the neural mechanism underlying two cognitive abilities, reading and working memory. Specifically, the findings identify the important role of white matter in driving the development of working memory and reading ability, connecting the related cortical areas, as well as bridging the gap between genes and behavior

Molecular genetic analyses in developmental dyslexia & related endophenotypes

Developmental dyslexia is one of the most common neurodevelopmental disorders, with a prevalence of 5% to 12% among school-aged children. It is a severe and specific impairment in the acquisition of reading and spelling skills, which is unexpected in relation to other cognitive abilities. It is widely accepted that genetic factors play an essential role in dyslexia susceptibility. In this thesis, genetic analyses have been conducted (i) to investigate known susceptibility variants and (ii) to identify new genetic variants that would explain the heritability observed for both, dyslexia as qualitative trait and dyslexia-related quantitative endophenotypes. Causal susceptibility variants have been reported for two genes on chromosome 6p22, namely DCDC2 and KIAA0319. In our German dyslexia (DYS-) sample, we were not able to replicate prior findings on an intronic deletion or a compound STR marker in intron 2 of DCDC2 to be causative for dyslexia. Also, the analysis of further common variants in KIAA0319 did not provide evidence for a genetic effect in the German DYS-sample. However, we were able to confirm previous findings on epistatic gene-gene interactions between an intronic DCDC2 two-marker risk haplotype and SNPs within KIAA0319. This suggests that genetic variation within KIAA0319 might confer small modifying effects on dyslexia susceptibility in the presence of the DCDC2 risk haplotype. We next analyzed quantitative measures using candidate-gene approaches. Dyslexic children often perform poorly in verbal short-term memory tasks, and GRIN2B has been reported to play an important role in human memory and cognition. Our genetic results provide evidence that variation within intron 3 of GRIN2B contributes to the weakness of dyslexic children in short-term memory. A non-synonymous variant within a second gene, LRRTM1, was found to be associated with the measure of relative hand skill, which represents a correlate for asymmetrical brain function, and has been suggested to partly underlie the neural signature of dyslexia. We also attempted to identify new variants contributing to the development of dyslexia using a genome-wide association approach. To increase power, we combined individual genotyping with large pooling efforts. Our study revealed four new susceptibility loci on chr. 5p13, 9q21, 21q21 and 22q13. Promising candidate genes within these regions include GRIK1, TMC1 and FBLN1. As each of the identified variants only confers a small effect size, replication in large, independent samples will be required to confirm our findings. The application of the genome-wide approach on quantitative dimensions of dyslexia led to the identification of new susceptibility variants for two dyslexia-related endophenotypes. First, an intergenic marker on chromosome 4q32.1 was found to be highly associated with mismatch negativity, a neural correlate of speech perception. Our data indicate that the locus mediates its function via trans-regulational effects on the expression level of SLC2A3, a facilitative glucose transporter expressed in brain. It can be suggested that the effect is functionally relevant during childhood, when an increased amount of glucose substrate is required for the formation of synaptic connections in the human brain. In a second approach, we found that arithmetical skills are associated with alleles of rs133885, a non-synonymous marker in the long isoform of MYO18B. Structural MRI data provided evidence that in contrast to the non-risk group, carriers of the risk allele show a lower depth and volume of the right intraparietal sulcus, a structural entity involved in numerical processing. The new findings presented within this thesis might contribute to a better understanding of dyslexia susceptibility, genetic effects on related endophenotypes and the functional mechanisms that underlie human reading and writing skills