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.

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.

Genetic susceptibility to non-necrotizing erysipelas/cellulitis.

BACKGROUND: Bacterial non-necrotizing erysipelas and cellulitis are often recurring, diffusely spreading infections of the skin and subcutaneous tissues caused most commonly by streptococci. Host genetic factors influence infection susceptibility but no extensive studies on the genetic determinants of human erysipelas exist. METHODS: We performed genome-wide linkage with the 10,000 variant Human Mapping Array (HMA10K) array on 52 Finnish families with multiple erysipelas cases followed by microsatellite fine mapping of suggestive linkage peaks. A scan with the HMA250K array was subsequently performed with a subset of cases and controls. RESULTS: Significant linkage was found at 9q34 (nonparametric multipoint linkage score (NPL(all)) 3.84, p=0.026), which is syntenic to a quantitative trait locus for susceptibility to group A streptococci infections on chromosome 2 in mouse. Sequencing of candidate genes in the 9q34 region did not conclusively associate any to erysipelas/cellulitis susceptibility. Suggestive linkage (NPL(all)>3.0) was found at three loci: 3q22-24, 21q22, and 22q13. A subsequent denser genome scan with the HMA250K array supported the 3q22 locus, in which several SNPs in the promoter of AGTR1 (Angiotensin II receptor type I) suggestively associated with erysipelas/cellulitis susceptibility. CONCLUSIONS: Specific host genetic factors may cause erysipelas/cellulitis susceptibility in humans.

Human ROBO1 regulates interaural interaction in auditory pathways.

In rodents, the Robo1 gene regulates midline crossing of major nerve tracts, a fundamental property of the mammalian CNS. However, the neurodevelopmental function of the human ROBO1 gene remains unknown, apart from a suggested role in dyslexia. We therefore studied axonal crossing with a functional approach, based on magnetoencephalography, in 10 dyslexic individuals who all share the same rare, weakly expressing haplotype of the ROBO1 gene. Auditory-cortex responses were recorded separately to left- and right-ear sounds that were amplitude modulated at different frequencies. We found impaired interaural interaction that depended on the ROBO1 in a dose-dependent manner. Our results indicate that normal crossing of the auditory pathways requires an adequate ROBO1 expression level.

Acquisition of complement factor H is important for pathogenesis of Streptococcus pyogenes infections: evidence from bacterial in vitro survival and human genetic association.

Streptococcus pyogenes (or group A streptococcus [GAS]) is a major human pathogen causing infections, such as tonsillitis, erysipelas, and sepsis. Several GAS strains bind host complement regulator factor H (CFH) via its domain 7 and, thereby, evade complement attack and C3b-mediated opsonophagocytosis. Importance of CFH binding for survival of GAS has been poorly studied because removal of CFH from plasma or blood causes vigorous complement activation, and specific inhibitors of the interaction have not been available. In this study, we found that activation of human complement by different GAS strains (n = 38) correlated negatively with binding of CFH via its domains 5-7. The importance of acquisition of host CFH for survival of GAS in vitro was studied next by blocking the binding with recombinant CFH5-7 lacking the regulatory domains 1-4. Using this fragment in full human blood resulted in death or radically reduced multiplication of all of the studied CFH-binding GAS strains. To study the importance of CFH binding in vivo (i.e., for pathogenesis of streptococcal infections), we used our recent finding that GAS binding to CFH is diminished in vitro by polymorphism 402H, which is also associated with age-related macular degeneration. We showed that allele 402H is suggested to be associated with protection from erysipelas (n = 278) and streptococcal tonsillitis (n = 209) compared with controls (n = 455) (p < 0.05). Taken together, the bacterial in vitro survival data and human genetic association revealed that binding of CFH is important for pathogenesis of GAS infections and suggested that inhibition of CFH binding can be a novel therapeutic approach in GAS infections.

Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons.

DCDC2 is one of the candidate susceptibility genes for dyslexia. It belongs to the superfamily of doublecortin domain containing proteins that bind to microtubules, and it has been shown to be involved in neuronal migration. We show that the Dcdc2 protein localizes to the primary cilium in primary rat hippocampal neurons and that it can be found within close proximity to the ciliary kinesin-2 subunit Kif3a. Overexpression of DCDC2 increases ciliary length and activates Shh signaling, whereas downregulation of Dcdc2 expression enhances Wnt signaling, consistent with a functional role in ciliary signaling. Moreover, DCDC2 overexpression in C. elegans causes an abnormal neuronal phenotype that can only be seen in ciliated neurons. Together our results suggest a potential role for DCDC2 in the structure and function of primary cilia.

Functional interaction of DYX1C1 with estrogen receptors suggests involvement of hormonal pathways in dyslexia.

Dyslexia, or specific reading disability, is the unexpected failure in learning to read and write when intelligence and senses are normal. One of the susceptibility genes, DYX1C1, has been implicated in neuronal migration, but little is known about its interactions and functions. As DYX1C1 was suggested to interact with the U-box protein CHIP (carboxy terminus of Hsc70-interacting protein), which also participates in the degradation of estrogen receptors alpha (ERalpha) and beta (ERbeta), we hypothesized that the effects of DYX1C1 might be at least in part mediated through the regulation of ERs. ERs have shown to be important in brain development and cognitive functions. Indeed, we show that DYX1C1 interacts with both ERs in the presence of 17beta-estradiol, as determined by co-localization, co-immunoprecipitation and proximity ligation assays. Protein levels of endogenous ERalpha or exogenous ERbeta were reduced upon over-expression of DYX1C1, resulting in decreased transcriptional responses to 17beta-estradiol. Furthermore, we detected in vivo complexes of DYX1C1 with ERalpha or ERbeta at endogenous levels along neurites of primary rat hippocampal neurons. Taken together, our data suggest that DYX1C1 is involved in the regulation of ERalpha and ERbeta, and may thus affect the brain development and regulate cognitive functions. These findings provide novel insights into the function of DYX1C1 and link neuronal migration and developmental dyslexia to the estrogen-signaling effects in the brain.

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.

A locus on 2p12 containing the co-regulated MRPL19 and C2ORF3 genes is associated to dyslexia.

DYX3, a locus for dyslexia, resides on chromosome 2p11-p15. We have refined its location on 2p12 to a 157 kb region in two rounds of linkage disequilibrium (LD) mapping in a set of Finnish families. The observed association was replicated in an independent set of 251 German families. Two overlapping risk haplotypes spanning 16 kb were identified in both sample sets separately as well as in a joint analysis. In the German sample set, the odds ratio for the most significantly associated haplotype increased with dyslexia severity from 2.2 to 5.2. The risk haplotypes are located in an intergenic region between FLJ13391 and MRPL19/C2ORF3. As no novel genes could be cloned from this region, we hypothesized that the risk haplotypes might affect long-distance regulatory elements and characterized the three known genes. MRPL19 and C2ORF3 are in strong LD and were highly co-expressed across a panel of tissues from regions of adult human brain. The expression of MRPL19 and C2ORF3, but not FLJ13391, were also correlated with the four dyslexia candidate genes identified so far (DYX1C1, ROBO1, DCDC2 and KIAA0319). Although several non-synonymous changes were identified in MRPL19 and C2ORF3, none of them significantly associated with dyslexia. However, heterozygous carriers of the risk haplotype showed significantly attenuated expression of both MRPL19 and C2ORF3, as compared with non-carriers. Analysis of C2ORF3 orthologues in four non-human primates suggested different evolutionary rates for primates when compared with the out-group. In conclusion, our data support MRPL19 and C2ORF3 as candidate susceptibility genes for DYX3.