It is well-known that one of the main clinical features of the syndrome is the presence of a decreased expression of alpha-globin genes leading to a form of alpha thalassemia. Indeed, qRT-PCR analysis confirmed a decrease of the alpha-globin mRNA/beta-globin mRNA ratios, in agreement with the known molecular alterations of the disease. Moreover, the increase in beta-like chain gene transcription, observed in microarray analysis, could be considered a compensatory activity to alpha chain deficiency. However much less is known about the mechanisms responsible for the other dominant clinical feature, the mental retardation. Many lines of evidence support a role for ATRX protein during cerebral development and Berube et al.  proposed that transcription-dependent events regulated by ATRX play a critical role in mediating the survival of neurons in the developing cortex and hippocampus. To date, although the ATRX protein structure suggests a role in chromatin regulation, it is not clear whether ATRX imparts predominantly a blocking or a positive effect on gene expression.
The complexity of the syndrome suggests that ATRX protein could be involved in the regulation of various unidentified genes and the present work shows, indeed, that altered expression in specific genes can be found in this disease. As an exploratory tools to identify potential candidate genes we used a cDNA array analysis, that does not cover the entire range of cellular transcripts. Indeed, such analysis suggested some specific transcripts whose changes were confirmed by quantitative RT-PCR analysis (a putative lymphocyte G0/G1 switch or G0S2 gene [24, 25] and the human Keratin 23 gene, HAIK1 . On the other hand, the gene expression change for oligophrenin-1 was not confirmed by RT-PCR but drew our attention on an homologous gene, the GRAF1/OPHN-1-L gene, localized at chromosome 5q31 and encoding for a Rho-GTPase-activating protein (Rho-GAP). Indeed, GRAF1/OPHN-1-L expression is decreased both in blood mononuclear cells and in immortalized lymphoblastoid cells of ATRX patients. Although, our analysis has been performed in peripheral blood lymphomonocytes, it is interesting that the GRAF1/OPHN-1-L transcript is highly expressed in the human brain  and homologous to a gene, OPHN-1, previously associated with X-linked mental retardation . GRAF1/OPHN-1-L protein contains a centrally located GAP domain followed by a serine/proline-rich domain and a carboxyl-terminal SH3 domain. The SH3 domain was shown to specifically bind to a proline-rich region in the carboxy-terminus of FAK, protein-tyrosine kinase associated with focal adhesions. Hildebrand et al.  reported that, in vitro, GRAF1/OPHN-1-L has GAP activity for Cdc42 and RhoA. Taylor et al. [27, 30] showed that GRAF1/OPHN-1-L specifically regulates Rho activity in vivo, down-regulating Rho activity in Swiss 3T3 cells but enhancing Rho-dependent effects in PC12 cells. The latter cell line, used primarily as a neuron model, has been established from a rat pheochromocytoma, expressing high level of GRAF1/OPHN-1-L, while Swiss 3T3 cells (or other fibroblast cell lines) did not express detectable levels . Moreover, GRAF1/OPHN-1-L is enriched in the brain, where it could regulate Rho-mediated neurite retraction, and it has been suggested that GRAF1/OPHN-1-L might play an important role in neuronal cell morphology [27, 30]. In particular GRAF1/OPHN-1-L should function as mediator linking the extracellular guiding signals to the intracellular signal transduction pathways that are important for neuronal morphogenesis as well as for cytoskeletal dynamics within neuronal growth cones . Alteration of such pathways influencing growth and guidance of axon outgrowth at neuronal growth cones might lead to impaired formation of brain structures.
In the present paper, we report the tissue distribution of a novel alternative splicing transcript (variant-3), lacking the entire exon 21, and show that it represents the main transcript in the brain. Exon 21 encodes for a portion of GRAF1/OPHN-1-L protein (from aminoacid 664 to 755 in variant-1) localized between the serine/proline rich domain (from aminoacid 584 to 701) and a carboxy-terminal Src-homology 3 (SH3) domain (from aminoacid 756 to 814). The three isoforms represent tissue-specific alternative products. It is possible to suggest that occurrence of exon 21 might be of importance to regulate the distance between the serine/proline rich domain and SH3 domain and the composition of the serine/proline rich domain.
Another issue for future investigations is suggested by the observation that somatic mutations of ATRX are associated with alpha thalassaemia myelodysplastic syndrome, an acquired form of alpha-thalassaemia that most commonly arises in the context of myelodysplasia [31, 32]. Taking into consideration that GRAF1/OPHN-1-L inactivation has been detected in myelodysplasias and leukemias [21, 33], suggesting a role as tumor suppressor for this gene, the link between inactivating mutations of ATRX and decreased expression of GRAF1/OPHN-1-L, observed in the present work, might explain the selection of ATRX mutations in the course of leukemic progression. Analysis of GRAF1/OPHN-1-L expression and correlation with ATRX activity deserves further studies in such hematological malignancies.