We demonstrate here the importance of interleukin signalling pathways in cognitive function and the normal physiology of the CNS. Thorough investigation of an MRX critical region in Xp22.1-21.3 enabled us to identify a new gene expressed in brain that is responsible for a non-specific form of X-linked mental retardation. This gene encodes a 696 amino acid protein that has homology to IL-1 receptor accessory proteins. Non-overlapping deletions and a nonsense mutation in this gene were identified in patients with cognitive impairment only. Its high level of expression in post-natal brain structures involved in the hippocampal memory system suggests a specialized role for this new gene in the physiological processes underlying memory and learning abilities.

X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.

X-linked mental retardation is a very common condition that affects approximately 1 in 600 males. Despite recent progress, in most cases the molecular defects underlying this disorder remain unknown. Recently, a study using the candidate gene approach demonstrated the presence of mutations in PAK3 (p21-activating kinase) associated with nonspecific mental retardation. PAK3 is a member of the larger family of PAK genes. PAK proteins have been implicated as critical downstream effectors that link Rho-GTPases to the actin cytoskeleton and to MAP kinase cascades, including the c-Jun amino-terminal kinase (JNK) and p38. We screened 12 MRX pedigrees that map to a large region overlying Xq21-q24. Mutation screening of the whole coding region of the PAK3 gene was performed by using a combination of denaturing gradient gel electro-phoresis and direct sequencing. We have identified a novel missense mutation in exon 2 of PAK3 gene (R67C) in MRX47. This confirms the involvement of PAK3 in MRX following the report of a nonsense mutation recently reported in MRX30. In the MRX47 family, all affected males show moderate to severe mental retardation. No seizures, statural growth deficiency, or minor facial or other abnormal physical features were observed. This mutation R67C is located in a conserved polybasic domain (AA 66-68) of the protein that is predicted to play a major role in the GTPases binding and stimulation of Pak activity. Am. J. Med. Genet. 93:294-298, 2000.

Fas ligand (CD95L) and tumor necrosis factor-α (TNF-α) are pivotal inducers of hepatocyte apoptosis. Uncontrolled activation of these two systems is involved in several forms of liver injury. Although the broad antiapoptotic action of Bcl-2 and Bcl-xL has been clearly established in various apoptotic pathways, their ability to inhibit the Fas/CD95- and TNF-α-mediated apoptotic signal has remained controversial. We have demonstrated that the expression of BCL-2 in hepatocytes protects them against Fas-induced fulminant hepatitis in transgenic mice. The present study shows that transgenic mice overexpressing[Formula: see text]in hepatocytes are also protected from Fas-induced apoptosis in a dose-dependent manner. Bcl-xL and Bcl-2 were protective without any change in the level of endogenous[Formula: see text]or Bax and inhibited hepatic caspase-3-like activity. In vivo injection of TNF-α caused massive apoptosis and death only when transcription was inhibited. Under these conditions,[Formula: see text]mice were partially protected from liver injury and death but PK-BCL-2 mice were not. A similar differential protective effect of Bcl-xL and Bcl-2 transgenes was observed when Fas/CD95 was activated and transcription blocked. These results suggest that apoptosis triggered by activation of both Fas/CD95 and TNF-α receptors is to some extent counteracted by the transcription-dependent protective effects, which are essential for the antiapoptotic activity of Bcl-2 but not of Bcl-xL. Therefore, Bcl-xL and Bcl-2 appear to have different antiapoptotic effects in the liver whose characterization could facilitate their use to prevent the uncontrolled apoptosis of hepatocytes.

To identify methylation-silenced genes in prostate cancers, a microarray analysis for genes up-regulated by treatment with a demethylating agent, 5-aza-2 ¶-deoxycytidine, was performed using three rat prostate cancer cell lines. Eight genes (Aebp1, Dysf, Gas6, LOC361288, Nnat, Ocm, RGD1308119, and Tgfbr2) were re-expressed at 16-fold or more, and their promoter CpG islands were shown to be densely methylated in the cancer cell lines. From the eight genes, Tgfbr2, a key mediator of transforming growth factor-B (TGF-B) signaling that has been strongly implicated in human and rat prostate carcinogenesis, was selected, and its silencing in primary samples was analyzed further. Tgfbr2 was methylated and markedly down-regulated in three of seven 3,2 ¶-dimethyl-4-aminobiphenyl-induced invasive adenocarcinomas in the dorsolateral lobe of the rat prostate. In humans, marked down-regulation of TGFBR2 protein was observed in 12 of 20 high-grade prostatic intraepithelial neoplasia and 36 of 60 prostate cancers. DNA methylation of the human TGFBR2 promoter CpG islands repressed transcription, if present, but neither methylation nor mutation were detected in 27 human prostate cancers analyzed. Methylation silencing of rat Tgfbr2 was associated with histone H3 lysine 9 trimethylation, whereas decreased expression of human TGFBR2 was mainly due to decreased transcription activity, sometimes in concert with histone deacetylation and H3 lysine 27 trimethylation. The identification of methylation silencing of Tgfbr2 in rat prostate cancers, in accordance with TGFBR2 down-regulation in human prostate cancers, will enable us to analyze how aberrant methylation is induced in vivo and identify factors that promote and suppress the induction of aberrant methylation. [Cancer Res 2008;68(7):2112-21]

Non-specific X-linked mental retardation (MRX) is a very common disorder which affects approximately 1 in 600 males. Despite this high frequency, little is known about the molecular defects underlying this disorder, mainly because of the clinical and genetic heterogeneity which is evident from linkage studies. Recently, a collaborative study using the candidate gene approach demonstrated the presence of mutations in GDIalpha, a Rab GDP-dissociation inhibitor encoded by a gene localized in Xq28, associated with non-specific mental retardation. GDIalpha is mainly a brain-specific protein that plays a critical role in the recycling of Rab GTPases involved in membrane vesicular transport. The study presented here was designed to assess the prevalence of mutations in the GDIalpha in mentally retarded patients and to discuss the clinical phenotypes observed in affected individuals. Mutation screening of the whole coding region of the GDIalpha gene, using a combination of denaturing gradient gel electrophoresis and direct sequencing, was carried out in 164 patients found negative for expansions across the FRAXA GCC repeat. In addition to the nonsense mutation recently reported in MRX48, we have identified a novel missense mutation in exon 11 of the GDIalpha gene in one familial form of non-specific mental retardation. In this family (family R), all affected males show moderate to severe mental retardation, and the X-linked semidominant inheritance is strongly suggested by the severe phenotypes in males with respect to mildly affected females or unaffected obligatory carriers. This study showed that the prevalence of GDIalpha mutations in non-specific mental retardation could be estimated to be 0.5-1%, and molecular diagnosis and genetic counselling in some cases of non-specific mental handicap can now be provided.

Background/Aims: CpG island (CGI) methylator phenotype (CIMP) is strongly associated with poor prognosis in neuroblastomas (NBLs; hazard ratios 7–22). Methylation of nonpromoter CGIs is useful to detect the presence of the CIMP, while the poor prognosis is considered to be caused by gene silencing due to promoter methylation. Here, promoter CGIs targeted by the CIMP were searched for. Methods: A genome-wide screening was performed by methylation-sensitive representational difference analysis of CIMP(+) and CIMP(–) NBLs. Results: Promoter CGIs of 9 genes were methylated in CIMP(+) NBL cell lines and caused silencing of their downstream genes. On analysis of 90 clinical specimens, CYP26C1,FERD3L (N-TWIST), CRYBA2 and PCDHGC4 were methylated at significantly higher incidences in CIMP(+) NBLs than CIMP(–) NBLs, while the difference was unclear for NPY, SPAG6, DDIT4L, CHR3SYT and C6Orf141. Methylation of CYP26C1 and FERD3L was significantly associated with poor prognosis, but weaker than the presence of the CIMP. Treatment of an NBL cell line with a demethylating agent caused demethylation of multiple promoter CGIs, and enhanced 13-cis-retinoic acid-induced neuronal differentiation. Conclusion: Our results indicate that the CIMP causes poor prognosis of NBLs by inducing methylation of multiple promoter CGIs with various incidences.

The severe neurodegenerative disorder, Tays-Sachs disease, is caused by a beta-hexosaminidase alpha-subunit deficiency which prevents the formation of lysosomal heterodimeric alpha-beta enzyme, hexosaminidase A (HexA). No treatment is available for this fatal disease; however, gene therapy could represent a therapeutic approach. We previously have constructed and characterized, in vitro, adenoviral and retroviral vectors coding for alpha- and beta-subunits of the human beta-hexosaminidases. Here, we have determined the in vivo strategy which leads to the highest HexA activity in the maximum number of tissues in hexA -deficient knock-out mice. We demonstrated that intravenous co-administration of adenoviral vectors coding for both alpha- and beta-subunits, resulting in preferential liver transduction, was essential to obtain the most successful results. Only the supply of both subunits allowed for HexA overexpression leading to massive secretion of the enzyme in serum, and full or partial enzymatic activity restoration in all peripheral tissues tested. The enzymatic correction was likely to be due to direct cellular transduction by adenoviral vectors and/or uptake of secreted HexA by different organs. These results confirmed that the liver was the preferential target organ to deliver a large amount of secreted proteins. In addition, the need to overexpress both subunits of heterodimeric proteins in order to obtain a high level of secretion in animals defective in only one subunit is emphasized. The endogenous non-defective subunit is otherwise limiting.