title: Genomic sequencing of a dyslexia susceptibility haplotype encompassing ROBO1.
abstract: 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.
authors.affiliation: Molecular Neurology Research Program, University of Helsinki, Helsinki, Finland ; Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland.
authors.forename: Satu
authors.initials: S
authors.lastname: Massinen
authors.affiliation: Department of Biosciences and Nutrition, and Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Wang
authors.affiliation: Molecular Neurology Research Program, University of Helsinki, Helsinki, Finland ; Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland.
authors.forename: Krista
authors.initials: K
authors.lastname: Laivuori
authors.affiliation: Department of Biosciences and Nutrition, and Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bieder
authors.affiliation: Department of Biosciences and Nutrition, and Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Isabel
authors.initials: I
authors.lastname: Tapia Paez
authors.affiliation: Department of Biosciences and Nutrition, and Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Hong
authors.initials: H
authors.lastname: Jiao
authors.affiliation: Molecular Neurology Research Program, University of Helsinki, Helsinki, Finland ; Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland ; Department of Biosciences and Nutrition, and Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Juha
authors.initials: J
authors.lastname: Kere
keywords: Dyslexia; ROBO1; Whole genome sequencing