Title: Causal genomic copy number gains in patients with X-linked intellectual disability: gene overexpression and cognition
Other Titles: Causale genomische duplicaties in patiënten met X-gekoppelde verstandelijke beperking: overexpressie van genen en cognitie
Authors: Vandewalle, Joke
Issue Date: 12-Jun-2012
Abstract: Intellectual disability (ID), previously referred to as mental retardation (MR), is a heterogeneous group of disorders with non-progressive cognitive impairment as a common characteristic, and affects ~2% of the population in developed countries. Patients lack the necessary mental capabilities and adaptive skills required to live independently, and rely on family members and other caretakers for help in daily life. ID therefore constitutes an important medical and socio-economical issue. Genetic causes are thought to account for 25-35% of the patients. These genetic causes do not only comprise mutations altering the coding sequence or large chromosomal aberrations, but also submicroscopic genomic gains and losses (copy number variations or CNVs). In the past, these microdeletions and microduplications could not be detected since they fall below the detection limit of karyotyping. The more recently developed array-CGH technique has now demonstrated that these CNVs constitute a very important disease mechanism. Much of the ID research has focused on the X-chromosome, in part because there are many large families with an X-linked inheritance pattern of the ID phenotype (XLID). In these families, only males are affected whereas the female carriers are usually asymptomatic due to preferential inactivation of the aberrant X-chromosome. In order to screen these XLID families for pathogenic CNVs, our lab developed a full-tiling X-chromosome-specific BAC array. Data from our lab and other groups indicate that pathogenic CNVs are present in 5-10% of the families with XLID. Interestingly, a high rate of causal copy number gains compared to deletions has been noted for the X-chromosome, in contrast to the autosomes. Despite this finding, copy number gains have received far less attention compared to deletions and loss-of-function mutations. This project aims to gain more insight in the role of gene overexpression in cognition by the identification of new candidate gene-dosage-sensitive ID genes followed by their functional characterization. In the first part of this project, a screen was performed to identify new pathogenic CNVs in XLID patients by means of our X-chromosome-specific BAC array. This led to the identification of two new duplication hotspots, at Xp11.22 and Xq28. At Xp11.22 we identified non-recurrent overlapping duplications in six unrelated XLID families, varying in size from 0.4 to 0.8 Mb. For the Xq28 region we detected a 0.3 Mb recurrent copy number gain in four unrelated XLID families. Interestingly, we clearly saw a dosage-dependent severity of the phenotype in these patients, as one family with five copies had a severe form of syndromic ID in comparison to the other families with two or three copies that had mild-to-moderate ID with minor additional features. For both regions, a qPCR screen in 100 sporadic ID patients did not reveal additional duplications.The BAC array that was used in this screen has a detection limit of ~80 kb. In order to detect even smaller aberrations, we developed an X-chromosome-specific oligo array. With this array, we detected five potentially pathogenic aberrations in a screen of 54 idiopathic ID patients. Three of these aberrations were, or could not have been identified with our BAC array. In addition, the array also proved useful to delineate previously identified aberrations in more detail. In the second part of this project, we performed overexpression studies on the candidate gene-dosage-sensitive ID genes of the two frequent copy number gains that were detected in part one. Owing to the identification of seven additional XLID families with duplications at Xp11.22, we could narrow down the smallest region of overlap to one gene; HUWE1. In addition, we also detected point mutations in HUWE1 in three ID patients. For the Xq28 copy number gains, we selected GDI1 because its copy number correlates with the severity of the phenotype, and mutations in this gene had already been found in patients with non-syndromic XLID. For both candidate genes, we demonstrated a highly stable expression in mouse brain as well as in EBV-transformed blood lymphocytes (EBV-PBL) of control individuals, indicating a tightly regulated expression of these genes. Additionally, we detected increased expression levels in EBV-PBL of the patients. For GDI1, the increased expression levels were also correlated with the genomic copy number of this gene in the different patients. Overexpression of HUWE1 in Drosophila did not lead to major structural abnormalities in the fly brain. We also investigated the dorsal cluster neurons (DCNs), which are often used as a model to investigate axonal pathfinding, outgrowth and branching due to their typical growth pattern and grid-like branching structure. We found that overexpression of HUWE1 led to an increase in axonal branching. In addition, our data suggested that this phenotype was mediated via a negative regulatory effect of HUWE1 on the Wnt/ß-catenin pathway. To this point, no effect of HUWE1 overexpression on fly behavior has been found.Overexpression of rat Gdi1 in rat primary hippocampal neurons significantly increased the dendritic length. This finding is in agreement with earlier studies that showed an inhibition of axonal outgrowth after downregulation of Gdi1 in cultured hippocampal neurons. Our Drosophila model for GDI1 overexpression did not reveal morphological or behavioral abnormalities. However, more detailed studies will be needed to examine the possibility of more subtle aberrations that were not yet detected in our studies.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Department of Human Genetics - miscellaneous
Human Genome Laboratory

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