(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).

(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.

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).