The axon guidance receptor gene ROBO1 is a candidate gene for developmental dyslexia

Abstract

Dyslexia, or specific reading disability, is the most common learning disorder with a complex, partially genetic basis, but its biochemical mechanisms remain poorly understood. A locus on Chromosome 3, DYX5, has been linked to dyslexia in one large family and speech-sound disorder in a subset of small families. We found that the axon guidance receptor gene ROBO1, orthologous to the Drosophila roundabout gene, is disrupted by a chromosome translocation in a dyslexic individual. In a large pedigree with 21 dyslexic individuals genetically linked to a specific haplotype of ROBO1 (not found in any other chromosomes in our samples), the expression of ROBO1 from this haplotype was absent or attenuated in affected individuals. Sequencing of ROBO1 in apes revealed multiple coding differences, and the selection pressure was significantly different between the human, chimpanzee, and gorilla branch as compared to orangutan. We also identified novel exons and splice variants of ROBO1 that may explain the apparent phenotypic differences between human and mouse in heterozygous loss of ROBO1. We conclude that dyslexia may be caused by partial haplo-insufficiency for ROBO1 in rare families. Thus, our data suggest that a slight disturbance in neuronal axon crossing across the midline between brain hemispheres, dendrite guidance, or another function of ROBO1 may manifest as a specific reading disability in humans.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1. Delineation of Translocation Breakpoint Region and ROBO1 Structure

(A) Fluorescence in situ hybridization with BAC clone RP11-143B12 as a probe, showing hybridization signals in Chromosome 3 (upward arrow), der(3), and der(8) (horizontal arrows). (B) Southern hybridization with a probe derived from RP11-143B12 shows genomic rearrangements (arrowheads) with five restriction enzymes in translocation patient (P) compared to the control sample. (C) A gene map of Chromosome 3p13-3q11.1 showing the cytogenetic localization of the translocation breakpoint (black bar). An arrow indicates the direction of ROBO1 transcription. Localization of the translocation breakpoint (square bracket) to BACs AC117479 and AC117461. (D) Splice variants and exon structure of ROBO1 (exons numbered from 1–30). Novel exons a and 7b and additional sequence to exon 1 are indicated in solid black. Exons unique to ROBO1 (hatched black) and DUTT1 (hatched grey) and common to both ROBO1 and DUTT1 (solid grey) are indicated. Corresponding BACs to exons are shown below. The translocation disrupting ROBO1 between exons 1 and 2 in AC117479 is shown by vertical grey bar. Dotted lines indicate DUTT1 variants. Novel splice variants are shown by grey lines and numbered (1), exclusion of exon 2 (89–169 of AF040990) (2), exclusion of DUTT1 exon 2 (1019–1345 of Z95705) (3), exclusion of exon 19 (2813–2829 of AF040990) (4), initial 165 bp of exon 22 (3037–3201 of AF040990) (5), 905 bp from exons 24–28 (3603–4508 of AF040990) (6), 878 bp from exons 25–28 (3641–4528 of AF040990) and (7), exclusion of exon 29 (4745–4939 of AF040990).

Figure 2. Analysis of ROBO1 in the Large Family Linked to DYX5

(A) An abridged pedigree of the family linked to DYX5 [17,19]. Numbers refer to samples studied for ROBO1 expression (C and D). A dot indicates carriers of the dyslexia-linked haplotype [17] and circled dots indicate individuals genotyped for all markers (B). Diamonds denote individuals genotyped for all markers, but not sharing the haplotype. Affected individuals are shaded black and unverified dyslectics are shaded gray. (B) Markers (right) and alleles (left) that define the haplotype linked to dyslexia (A). The bar indicates the extent of the ROBO1 haplotype carried by patients marked with a dot (A). (C) Sequencing of cDNA reveals absent or attenuated expression (p < 0.017 for all measurements) of the ROBO1 allele (SNP 6483A > T indicated by arrows) encoded by the dyslexia-linked haplotype as compared to genomic sequence. In the control, both alleles show equal allelic ratios in genomic and cDNA. Patient numbers refer to (A). (D) Attenuation of ROBO1 mRNA expression from the dyslexia-associated allele. Allelic expression of ROBO1 was assessed by sequencing the SNP 6483 (A/T) as in (C). Allelic ratios were assessed by five to six replicated sequencing tracings in four controls (21 data points) and four dyslexic individuals (24 data points). The results are expressed as the mRNA level of the dyslexia-associated allele as compared to the corresponding allele mRNA level in controls. Data are shown as mean ± 1 standard error of the mean (bars). Significance was assessed by two-tailed t test.

Figure 3. Coding Changes of ROBO1 during Primate Evolution

Phylogenetic tree of ROBO1 protein evolution in hominoids. Rat was used as the out-group in sequence comparisons. dN/dS ratios of the branches were calculated with the Codeml program, assuming a freely varying ratio. A model in which omega value was higher in lineages leading to humans, chimpanzees, and gorillas was significantly better than a free-ratio model (p < 0.001).