A familial case of Coffin-Lowry syndrome caused by RPS6KA3 C.898C>T mutation associated with multiple abnormal brain imaging findings.

Coffin-Lowry syndrome (CLS) is a rare X linked mental retardation syndrome characterised by severe psychomotor and growth retardation, distinct facial phenotype, and progressive skeletal malformations. It is caused by mutations in the RPS6KA3 gene located at Xp22.2. In this report we describe a family with CLS consists of three affected males, and two affected females, arising from c.898C>T mutation in RPS6KA3 gene. A 6 year-old, and a 3 year-old boy both had distinct clinical features of Coffin-Lowry syndrome; severe mental and motor retardation, microcephaly, prominent forehead, hypertelorism, large mouth, large ears, large soft hands, puffy tapered fingers, and pectus carinatum. In addition, they had multiple abnormal brain MRI findings. Other siblings presented with a mild and variable phenotype.

Deletion of the Coffin-Lowry syndrome gene Rsk2 in mice is associated with impaired spatial learning and reduced control of exploratory behavior.

Coffin-Lowry Syndrome (CLS) is an X-linked syndromic form of mental retardation associated with skeletal abnormalities. It is caused by mutations of the Rsk2 gene, which encodes a growth factor regulated kinase. Gene deletion studies in mice have shown an essential role for the Rsk2 gene in osteoblast differentiation and function, establishing a causal link between Rsk2 deficiency and skeletal abnormalities of CLS. Although analyses in mice have revealed prominent expression of Rsk2 in brain structures that are essential for learning and memory, evidence at the behavioral level for an involvement of Rsk2 in cognitive function is still lacking. Here, we have examined Rsk2-deficient mice in two extensive batteries of behavioral tests, which were conducted independently in two laboratories in Zurich (Switzerland) and Orsay (France). Despite the known reduction of bone mass, all parameters of motor function were normal, confirming the suitability of Rsk2-deficient mice for behavioral testing. Rsk2-deficient mice showed a mild impairment of spatial working memory, delayed acquisition of a spatial reference memory task and long-term spatial memory deficits. In contrast, associative and recognition memory, as well as the habituation of exploratory activity were normal. Our studies also revealed mild signs of disinhibition in exploratory activity, as well as a difficulty to adapt to new test environments, which likely contributed to the learning impairments displayed by Rsk2-deficient mice. The observed behavioral changes are in line with observations made in other mouse models of human mental retardation and support a role of Rsk2 in cognitive functions.

Identification of novel mutations in the RSK2 gene (RPS6KA3) in patients with Coffin-Lowry syndrome.

The Coffin-Lowry syndrome (CLS) is a rare X-linked semidominant syndrome characterized by severe psychomotor retardation, facial dysmorphism, digit abnormalities and progressive skeletal deformations. CLS is caused by mutations in a gene located in Xp22.2, RPS6KA3. This gene encodes for a growth factor-regulated serine/threonine protein kinase, RSK2 (ribosomal S6 kinase 2), acting in the Ras-mitogen-activated protein kinase signaling pathway. Mutations in the RPS6KA3 gene are extremely heterogeneous and lead to premature termination of translation and/or to loss of phosphotransferase activity of the RSK2 protein. Screening for RSK2 mutations is essential in most cases to confirm the diagnosis as well as for genetic counseling. Here we present 44 novel mutations in RSK2 causing CLS. The overall number of CLS mutations reported now is 128. Thirty-three percent of mutations are missense mutations, 15% nonsense mutations, 20% splicing errors and 29% short deletion or insertion events. Only four large deletions have so far been found. They are distributed throughout the RPS6KA3 gene, and the majority has been found in a single family. This study further confirms the high rate of new mutations at the RSK2 locus. It is important to consider the possibility of mosaicism when providing genetic counseling in CLS families.

Coffin-Lowry syndrome: clinical and molecular features.

The Coffin-Lowry syndrome (CLS) is a rare X linked disorder in which affected males show severe mental retardation with characteristic dysmorphism, most notably affecting the face and hands. The typical facial features consist of a prominent forehead, hypertelorism, a flat nasal bridge, downward sloping palpebral fissures, and a wide mouth with full lips. Mild progression in facial coarsening occurs during childhood and adult life. The hands are broad with soft, stubby, tapering fingers. Other clinical findings include short stature (95%), a pectus deformity (80%), a kyphosis and/or scoliosis (80%), mitral valve dysfunction, and sensorineural hearing loss. The causal gene, RSK2, was identified in 1996 and contains 22 exons which encode a protein of 740 amino acids. Over 75 distinct pathogenic mutations have been identified in 250 unrelated CLS patients.

The ribosomal S6 kinases, cAMP-responsive element-binding, and STAT3 proteins are regulated by different leukemia inhibitory factor signaling pathways in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

Mutations in the X-linked RSK2 gene (RPS6KA3) in patients with Coffin-Lowry syndrome.

RSK2 is a growth factor-regulated serine-threonine protein kinase, acting in the Ras-Mitogen-Activated Protein Kinase (MAPK) signaling pathway. Mutations in the RSK2 gene (RPS6KA3) on chromosome Xp22.2, have been found to cause Coffin-Lowry syndrome (CLS), an X-linked disorder characterized by psychomotor retardation, characteristic facial and digital abnormalities, and progressive skeletal deformations. By screening of 250 patients with clinical features suggestive of Coffin-Lowry syndrome, 71 distinct disease-associated RSK2 mutations have been identified in 86 unrelated families. Thirty-eight percent of mutations are missense mutations, 20% are nonsense mutations, 18% are splicing errors, and 21% are short deletion or insertion events. About 57% of mutations result in premature translation termination, and the vast majority are predicted to cause loss of function of the mutant allele. These changes are distributed throughout the RSK2 gene and show no obvious clustering or phenotypic association. However, some missense mutations are associated with milder phenotypes. In one family, one such mutation was associated solely with mild mental retardation. It is noteworthy that nine mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms.

Activation of RSK by UV-light: phosphorylation dynamics and involvement of the MAPK pathway.

Ribosomal S6 kinases (RSKs) are serine/threonine kinases activated by mitogenic signals through the Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinases (MAPK/ERK). RSKs contain two heterologous complete protein kinase domains. Phosphorylation by ERK of the C-terminal kinase domain allows activation of the N-terminal kinase domain, which mediates substrate phosphorylation. In human, there are three isoforms of RSK (RSK1, RSK2, RSK3), whose functional specificity remains undefined. Importantly, we have shown that mutations in the RSK2 gene lead to the Coffin-Lowry syndrome (CLS). In this study, we characterize two monoclonal antibodies raised against phosphorylated forms of the N- and C-terminal domain of RSK2 (P-S227 and P-T577, respectively). Using these two antibodies, we show that stress signals, such as UV light, induce phosphorylation and activation of the three RSKs to an extent which is comparable to Epidermal Growth Factor (EGF)-mediated activation. The use of specific kinase inhibitors indicates that UV-induced phosphorylation and activation of RSK2 is mediated by the MAPK/ERK pathway, but that the Stress-Activated Protein Kinase 2 (SAPK2)/p38 pathway is also involved. These results modify the view of RSKs as kinases restricted to the mitogenic response and reveal a previously unappreciated role of MAPKs in stress induced signaling. Oncogene (2000) 19, 4221 - 4229

Unreported RSK2 missense mutation in two male sibs with an unusually mild form of Coffin-Lowry syndrome.

An unreported missense mutation of the ribosomal S6 kinase 2 (RSK2) gene has been identified in two male sibs with a mild form of Coffin-Lowry syndrome (CLS) inherited from their healthy mother. They exhibit transient severe hypotonia, macrocephaly, delay in closure of the fontanelles, normal gait, and mild mental retardation, associated in the first sib with transient autistic behaviour. Some dysmorphic features of CLS (in particular forearm fullness and tapering fingers) and many atypical findings (some of which were reminiscent of FG syndrome) were observed as well. The moderate phenotypic expression of this mutation extends the CLS phenotype to include less severe mental retardation and minor, hitherto unreported signs. The missense mutation identified may be less deleterious than those previously described. As this mutation occurs in a protein domain with no predicted function, it could be responsible for a conformational change affecting the protein catalytic function, since a non-polar amino acid is replaced by a charged residue.

Requirement of Rsk-2 for epidermal growth factor-activated phosphorylation of histone H3.

During the immediate-early response of mammalian cells to mitogens, histone H3 is rapidly and transiently phosphorylated by one or more unidentified kinases. Rsk-2, a member of the pp90rsk family of kinases implicated in growth control, was required for epidermal growth factor (EGF)-stimulated phosphorylation of H3. RSK-2 mutations in humans are linked to Coffin-Lowry syndrome (CLS). Fibroblasts derived from a CLS patient failed to exhibit EGF-stimulated phosphorylation of H3, although H3 was phosphorylated during mitosis. Introduction of the wild-type RSK-2 gene restored EGF-stimulated phosphorylation of H3 in CLS cells. In addition, disruption of the RSK-2 gene by homologous recombination in murine embryonic stem cells abolished EGF-stimulated phosphorylation of H3. H3 appears to be a direct or indirect target of Rsk-2, suggesting that chromatin remodeling might contribute to mitogen-activated protein kinase-regulated gene expression.

Non-random distribution of mutations in the PHEX gene, and under-detected missense mutations at non-conserved residues.

Thirty newly detected mutations in the PHEX gene are reported, and pooled with all the previously published mutations. The spectrum of mutations displayed 16% deletions, 8% insertions, 34% missense, 27% nonsense, and 15% splice site mutations, with two peaks in exon 15, and 17. Since 32.8% of PHEX amino acids were conserved in the endopeptidases family, the number of missense mutations detected at non-conserved residues was smaller than expected, whereas the number of nonsense mutations observed at non-conserved residues was very close to the expected number. Compared with conserved amino acids, the changes in non-conserved amino acids may result in benign polymorphisms or possibly mild disease that may go undiagnosed.

Evaluation of a mutation screening strategy for sporadic cases of ATR-X syndrome.

We report on the evaluation of a strategy for screening for XNP/ATR-X mutations in males with mental retardation and associated dysmorphology. Because nearly half of the mutations in this gene reported to date fall into a short 300 bp region of the transcript, we decided to focus in this region and to extend the mutation analysis to cases with a negative family history. This study includes 21 mentally retarded male patients selected because they had severe mental retardation and a typical facial appearance. The presence of haemoglobin H or urogenital abnormalities was not considered critical for inclusion in this study. We have identified six mutations which represents a mutation detection rate of 28%. This figure is high enough for us to propose this strategy as a valid first level of screening in a selected subset of males with mental retardation. This approach is simple, does not require RNA preparation, does not involve time consuming mutation detection methods, and can thus be applied to a large number of patients at a low cost in any given laboratory.

Novel mutations in Rsk-2, the gene for Coffin-Lowry syndrome (CLS).

Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by facial dysmorphism, digit abnormalities and severe psychomotor retardation. CLS had previously been mapped to Xp22.2. Recently, mutations in the ribosomal S6 kinase (Rsk-2) gene were shown to be associated with CLS. We have tested five unrelated individuals with CLS for mutations in nine exons of Rsk-2 using Single Strand Conformation Polymorphism (SSCP) analysis. Two patients had the same missense mutation (C340T), which causes an arginine to tryptophan change (R114W). This mutation falls just outside the N-terminal ATP-binding site in a highly conserved region of the protein and may lead to structural changes since tryptophan has an aromatic side chain whereas arginine is a 5 carbon basic amino acid. The third patient also had a missense mutation (G2186A) resulting in an arginine to glutamine change (R729Q). The fourth patient had a 2bp deletion (AG) of bases 451 and 452. This creates a frameshift that results in a stop codon 25 amino acids downstream, thereby producing a truncated protein. This deletion also falls within the highly conserved amino-catalytic domain of the protein. The fifth patient has a nonsense mutation (C2065T) which results in a premature stop codon, thereby producing a truncated protein. These mutations further confirm Rsk-2 as the gene involved in CLS and may help in understanding the structure and function of the protein.

Rapid immunoblot and kinase assay tests for a syndromal form of X linked mental retardation: Coffin-Lowry syndrome.

Coffin-Lowry syndrome (CLS) is a syndromal form of X linked mental retardation, in which some associated facial, hand, and skeletal abnormalities are diagnostic features. Accurate diagnosis, critical for genetic counselling, is often difficult, especially in early childhood. We have recently shown that Coffin-Lowry syndrome is caused by mutations in the gene encoding RSK2, a growth factor regulated protein kinase. RSK2 mutations are very heterogeneous and most of them lead to premature termination of translation or to loss of phosphotransferase activity or both. In the present study, we have evaluated immunoblot and RSK2 kinase assays as a rapid and simple diagnostic test for CLS, using cultured lymphoblastoid or fibroblast cell lines. Western blot analysis failed to detect RSK2 in six patients, suggesting the presence of truncated proteins in these patients. This conclusion was confirmed in four patients, in whom the causative mutations, all leading to premature termination of translation, were identified. Of four patients showing a normal amount of RSK2 protein on western blot and tested for RSK2 phosphotransferase activity, one had a dramatically impaired activity. Analysis of the RSK2 cDNA sequence in this patient showed a mutation of a putative phosphorylation site that would be critical for RSK2 activity. Preliminary results show that, at least, the western blot protocol can be successfully applied to lymphocyte protein extracts prepared directly from blood samples. These assays promise to become important diagnostic tools for CLS, particularly with regard to very young patients with no family history of the condition.

Mutation analysis of the RSK2 gene in Coffin-Lowry patients: extensive allelic heterogeneity and a high rate of de novo mutations.

Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by severe psychomotor retardation, facial and digital dysmorphisms, and progressive skeletal deformations. By using a positional cloning approach, we have recently shown that mutations in the gene coding for the RSK2 serine-threonine protein kinase are responsible for this syndrome. To facilitate mutational analysis, we have now determined the genomic structure of the human RSK2 gene. The open reading frame of the RSK2 coding region is split into 22 exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single-strand conformation polymorphism analysis. We screened 37 patients with clinical features suggestive of CLS. Twenty-five nucleotide changes predicted to be disease-causing mutations were identified, including eight splice-site alterations, seven nonsense mutations, five frameshift mutations, and five missense mutations. Twenty-three of them were novel mutations. Coupled with previously reported mutations, these findings bring the total of different RSK2 mutations to 34. These are distributed throughout the RSK2 gene, with no clustering, and all but two, which have been found in two independent patients, are unique. A very high (68%) rate of de novo mutations was observed. It is noteworthy also that three mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms. No obvious correlation was observed between the position or type of the RSK2 mutations and the severity or particular clinical features of CLS.

Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of CREB protein and transcription of c-fos gene.

Activation by growth factors of the Ras-dependent signaling cascade results in the induction of p90 ribosomal S6 kinases (p90(rsk)). These are translocated into the nucleus upon phosphorylation by mitogen-activated protein kinases, with which p90(rsk) are physically associated in the cytoplasm. In humans there are three isoforms of the p90(rsk) family, Rsk-1, Rsk-2, and Rsk-3, which are products of distinct genes. Although these isoforms are structurally very similar, little is known about their functional specificity. Recently, mutations in the Rsk-2 gene have been associated with the Coffin-Lowry syndrome (CLS). We have studied a fibroblast cell line established from a CLS patient that bears a nonfunctional Rsk-2. Here we document that in CLS fibroblasts there is a drastic attenuation in the induced Ser-133 phosphorylation of transcription factor CREB (cAMP response element-binding protein) in response to epidermal growth factor stimulation. The effect is specific, since response to serum, cAMP, and UV light is unaltered. Furthermore, epidermal growth factor-induced expression of c-fos is severely impaired in CLS fibroblasts despite normal phosphorylation of serum response factor and Elk-1. Finally, coexpression of Rsk-2 in transfected cells results in the activation of the c-fos promoter via the cAMP-responsive element. Thus, we establish a link in the transduction of a specific growth factor signal to changes in gene expression via the phosphorylation of CREB by Rsk-2.

Germline mosaicism in Coffin-Lowry syndrome.

We have identified a Coffin-Lowry syndrome pedigree where the disorder is associated with a novel splice site mutation in the RSK2 gene, leading to in-phase skipping of exon 5. Western blot analysis, using an antibody directed against the C-terminus of RSK2, failed to reveal RSK2 in this patient, suggesting strongly that the resulting internally deleted protein is unstable. The mutation was present in the DNA of one affected son and one manifesting daughter but was absent in two asymptomatic daughters, who carry the at-risk haplotype, and in the mother's somatic cell (lymphocyte) DNA. The results are consistent with the mutation arising as a postzygotic event in the mother, who therefore is a germinal mosaic. The application of linked markers to identify the disease allele for conventional genetic counselling would have been misleading in this family. This observation again highlights the importance of precise identification of the disease-causing mutation.