Abstract
BackgroundATRX is a severe X-linked disorder characterized by mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassemia. The disease is caused by mutations in ATRX gene, which encodes a protein belonging to the SWI/SNF DNA helicase family, a group of proteins involved in the regulation of gene transcription at the chromatin level. In order to identify specific genes involved in the pathogenesis of the disease, we compared, by cDNA microarray, the expression levels of approximately 8500 transcripts between ATRX and normal males of comparable age.MethodscDNA microarray was performed using total RNA from peripheral blood mononuclear cells of ATRX and normal males. Microarray results were validated by quantitative real-time polymerase chain reaction.ResultscDNA microarray analysis showed that 35 genes had a lower expression (30-35% of controls) while 25 transcripts had a two-fold higher expression in comparison to controls. In the microarray results the probe for oligophrenin-1, a gene known for its involvement in mental retardation, showed a decreased hybridization signal. However, such gene was poorly expressed in blood mononuclear cells and its decrease was not confirmed in the quantitative real-time RT-PCR assay. On the other hand, the expression of an homologous gene, the GTPase regulator associated with the focal adhesion kinase 1/Oligophrenin-1-like (GRAF1/OPHN-1-L), was relatively high in blood mononuclear cells and significantly decreased in ATRX patients. The analysis of the expression pattern of the GRAF1/OPHN-1-L gene in human tissues and organs revealed the predominant brain expression of a novel splicing isoform, called variant-3.ConclusionsOur data support the hypothesis of a primary role for altered gene expression in ATRX syndrome and suggest that the GRAF1/OPHN-1-L gene might be involved in the pathogenesis of the mental retardation. Moreover a novel alternative splicing transcript of such gene, predominantly expressed in brain tissues, was identified.
Highlights
ATRX is a severe X-linked disorder characterized by mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassemia
Several lines of evidence suggest that XNP/ATRX is involved in gene expression regulation via chromatin remodelling through the following mechanisms: 1) its association with the human EZH2 protein [5,13], a human homologue of the Enhancer of Zeste Drosophila gene involved in the regulation of homeotic gene expression through chromatin remodelling; 2) its interaction with the heterochromatin protein HP1 [14]; 3) the abnormalities of DNA methylation profiles of repetitive elements induced by ATRX mutations [12]; 4) the nuclear localization and the close association with pericentromeric heterochromatin during mitosis [13,15]; 5) the documented DNA-binding activity of the zinc finger domain [6]; 6) the co-localization with the transcription co-activator Daxx in promyelocytic leukaemia nuclear bodies [16]
GTPase regulator associated with the focal adhesion kinase 1/Oligophrenin-1-like (GRAF1/OPHN1-L), one of the gene showing altered expression level in ATRX, is potentially involved in Central Nervous System (CNS) function and in mental retardation and we report the identification of a novel alternative transcript and its tissue and brain distribution
Summary
ATRX is a severe X-linked disorder characterized by mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassemia. Several lines of evidence suggest that XNP/ATRX is involved in gene expression regulation via chromatin remodelling through the following mechanisms: 1) its association with the human EZH2 protein [5,13], a human homologue of the Enhancer of Zeste Drosophila gene involved in the regulation of homeotic gene expression through chromatin remodelling; 2) its interaction with the heterochromatin protein HP1 [14]; 3) the abnormalities of DNA methylation profiles of repetitive elements induced by ATRX mutations [12]; 4) the nuclear localization and the close association with pericentromeric heterochromatin during mitosis [13,15]; 5) the documented DNA-binding activity of the zinc finger domain [6]; 6) the co-localization with the transcription co-activator Daxx in promyelocytic leukaemia nuclear bodies [16]. Despite the rarity of this syndrome the identification of involved genes can supply useful data for the general understanding of the molecular mechanisms responsible for mental retardation
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