Genomic sequencing of a dyslexia susceptibility haplotype encompassing ROBO1

Satu Massinen,Krista Laivuori,Isabel Tapia Paez,Andrea Bieder,Hong Jiao,Jingwen Wang,Juha Kere

doi:10.1186/s11689-016-9136-y

Abstract

BackgroundThe 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.MethodsWe 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.ResultsWe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s11689-016-9136-y) contains supplementary material, which is available to authorized users.

Highlights

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
We combined individual- and pooled-sample sequencing strategies; we sequenced the whole genomes of two affected individuals at Complete Genomics Inc. (CGI) and used Illumina sequencing on a pooled sample including an equal amount of DNA from all the 19 affected members carrying the susceptibility haplotype
In the whole genome sequencing of two individuals performed at CGI, 97 % of the genome was covered in both individuals and over 80 % of the genome contained at least 30-fold reads

Summary

Introduction

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. The largest family so far reported to show simple autosomal dominant segregation for DD is a Finnish family in which a genetic linkage study implicated a locus on chromosome 3, named DYX5 [7] (Fig. 1), carrying a 33-Mb susceptibility haplotype for dyslexia. Gene expression study of ROBO1 in lymphoblasts from members of the large linkage family suggested suppressed expression from the rare haplotype segregating with DD. No such allelic suppression was observed for neighboring genes. Consistent with an important developmental role of the ROBO1 locus, a 15Mb deletion involving ROBO1 and a few neighboring genes was found in a child with developmental delay [14]

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