Coffin-Lowry Syndrome Patients Research Articles

The natural history of spinal deformity in patients with Coffin-Lowry syndrome

PurposeLittle is known about the natural history of spinal deformities in Coffin-Lowry syndrome (CLS). Our goal was to evaluate the spinal deformity progression and clinical impact.MethodsIn this institutional review board-approved study, we performed a multinational retrospective review of six male CLS patients, aged 13 to 22 years at final follow-up, for a mean of 7.25 years (3 to 13).ResultsAll showed delayed skeletal maturity. Three had calcifications of their lower cervical ligamentum flavum, all experienced neural axis abnormalities, including lower extremity weakness, numbness and tingling and in one, quadriparesis. Only two were ambulatory at final follow-up.All had significant spinal abnormalities, including severe progressive thoracic lordosis, thoracolumbar kyphosis and scoliosis. All had undergone spinal fusion or were being evaluated for surgery.ConclusionCLS is a rare X-linked mutation in the RSK2 gene, affecting between 1/50 000 to 100 000 people. There are two reports in the literature of patients with calcifications of their ligamentum flavum. Both had neural axis abnormalities and one had acute onset quadriplegia. Analysis of their ligamentum flavum found abundant central calcifications. Despite our small cohort we found 50% had calcifications and 100% had neurologic consequences associated with those calcifications. There was a 100% rate of deformity progression.They all exhibited delay in skeletal maturity, which mandates longer follow-up and has implications for surgical planning.From our cohort and literature review, the natural history of CLS supports frequent patient evaluation and a lower threshold for correction of spinal deformities. Aiming to avoid spinal cord compression and improve or avoid neurological deterioration.Level of EvidenceIV - retrospective study

High diagnostic yield of clinically unidentifiable syndromic growth disorders by targeted exome sequencing.

As syndromic short stature and overgrowth are heterogeneous and the list of causative genes is rapidly expanding, there is an unmet need for identifying genetic causes based on conventional gene testing or karyotyping. Early diagnosis leads to the proper management of the patient and providing genetic counseling for family members at risk in a timely manner. We conducted targeted exome sequencing to identify the genetic causes of undiagnosed syndromic short stature or overgrowth in 15 pediatric patients from 13 families in Korea. We applied targeted exome sequencing using the Next Seq platform and a TruSight One panel. Among the 13 families, 6 different disorders in 8 patients with short stature or overgrowth were identified, and the diagnostic yield was 46.2%. One boy with overgrowth had a TGFB3 gene mutation. In the short stature group, Coffin-Lowry syndrome (CLS), trichorhinophalangeal syndrome, DYRK1A haploinsufficiency syndrome, short stature with optic atrophy and Pelger-Huët anomaly syndrome with recurrent hepatitis, and type 4 Meier-Gorlin syndrome were identified. One CLS patient had a co-existing monogenic disease, congenital glaucoma, caused by the compound heterozygote mutations of the CYP1B1 gene. Targeted exome sequencing is a powerful method for diagnosing syndromic growth disorders. It enables us to understand molecular pathophysiology and investigate new treatments for growth disorders.

Rsk2, the Kinase Mutated in Coffin-Lowry Syndrome, Controls Cementum Formation

The ribosomal S6 kinase RSK2 is essential for osteoblast function, and inactivating mutations of RSK2 cause osteopenia in humans with Coffin-Lowry syndrome (CLS). Alveolar bone loss and premature tooth exfoliation are also consistently reported symptoms in CLS patients; however, the pathophysiologic mechanisms are unclear. Therefore, aiming to identify the functional relevance of Rsk2 for tooth development, we analyzed Rsk2-deficient mice. Here, we show that Rsk2 is a critical regulator of cementoblast function. Immunohistochemistry, histology, micro–computed tomography imaging, quantitative backscattered electron imaging, and in vitro assays revealed that Rsk2 is activated in cementoblasts and is necessary for proper acellular cementum formation. Cementum hypoplasia that is observed in Rsk2-deficient mice causes detachment and disorganization of the periodontal ligament and was associated with significant alveolar bone loss with age. Moreover, Rsk2-deficient mice display hypomineralization of cellular cementum with accumulation of nonmineralized cementoid. In agreement, treatment of the cementoblast cell line OCCM-30 with a Rsk inhibitor reduces formation of mineralization nodules and decreases the expression of cementum markers. Western blot analyses based on antibodies against Rsk1, Rsk2, and an activated form of the 2 kinases confirmed that Rsk2 is expressed and activated in differentiating OCCM-30 cells. To discriminate between periodontal bone loss and systemic bone loss, we additionally crossed Rsk2-deficient mice with transgenic mice overexpressing the osteoanabolic transcription factor Fra1. Fra1 overexpression clearly increases systemic bone volume in Rsk2-deficient mice but does not protect from alveolar bone loss. Our results indicate that cell autonomous cementum defects are causing early tooth loss in CLS patients. Moreover, we identify Rsk2 as a nonredundant regulator of cementum homeostasis, alveolar bone maintenance, and periodontal health, with all these features being independent of Rsk2 function in systemic bone formation.

Ribosomal S6 Kinase 2 (RSK2) Maintains Genomic Stability by Activating the Atm/p53-Dependent DNA Damage Pathway

Ribosomal S6 Kinase 2 (RSK2) is a member of the p90RSK family of serine/threonine kinases, which are widely expressed and respond to many growth factors, peptide hormones, and neurotransmitters. Loss-of function mutations in the RPS6KA3 gene, which encodes the RSK2 protein, have been implicated in Coffin-Lowry Syndrome (CLS), an X-linked mental retardation disorder associated with cognitive deficits and behavioral impairments. However, the cellular and molecular mechanisms underlying this neurological disorder are not known. Recent evidence suggests that defective DNA damage signaling might be associated with neurological disorders, but the role of RSK2 in the DNA damage pathway remains to be elucidated. Here, we show that Adriamycin-induced DNA damage leads to the phosphorylation of RSK2 at Ser227 and Thr577 in the chromatin fraction, promotes RSK2 nuclear translocation, and enhances RSK2 and Atm interactions in the nuclear fraction. Furthermore, using RSK2 knockout mouse fibroblasts and RSK2-deficient cells from CLS patients, we demonstrate that ablation of RSK2 impairs the phosphorylation of Atm at Ser1981 and the phosphorylation of p53 at Ser18 (mouse) or Ser15 (human) in response to genotoxic stress. We also show that RSK2 affects p53-mediated downstream cellular events in response to DNA damage, that RSK2 knockout relieves cell cycle arrest at the G2/M phase, and that an increased number of γH2AX foci, which are associated with defects in DNA repair, are present in RSK2-deficient cells. Taken together, our findings demonstrated that RSK2 plays an important role in the DNA damage pathway that maintains genomic stability by mediating cell cycle progression and DNA repair.

Transcriptome profile reveals AMPA receptor dysfunction in the hippocampus of the Rsk2-knockout mice, an animal model of Coffin–Lowry syndrome

Coffin-Lowry syndrome (CLS) is a syndromic form of mental retardation caused by loss of function mutations in the X-linked RPS6KA3 gene, which encodes RSK2, a serine/threonine kinase acting in the MAPK/ERK pathway. The mouse invalidated for the Rps6ka3 (Rsk2-KO) gene displays learning and long-term spatial memory deficits. In the current study, we compared hippocampal gene expression profiles from Rsk2-KO and normal littermate mice to identify changes in molecular pathways. Differential expression was observed for 100 genes encoding proteins acting in various biological pathways, including cell growth and proliferation, cell death and higher brain function. The twofold up-regulated gene (Gria2) was of particular interest because it encodes the subunit GLUR2 of the AMPA glutamate receptor. AMPA receptors mediate most fast excitatory synaptic transmission in the central nervous system. We provide evidence that in the hippocampus of Rsk2-KO mice, expression of GLUR2 at the mRNA and at the protein levels is significantly increased, whereas basal AMPA receptor-mediated transmission in the hippocampus of Rsk2-KO mice is significantly decreased. This is the first time that such deregulations have been demonstrated in the mouse model of the Coffin-Lowry syndrome. Our findings suggest that a defect in AMPA neurotransmission and plasticity contribute to mental retardation in CLS patients.

The Coffin-Lowry Syndrome-Associated Protein rsk2 and Neurosecretion

Coffin-Lowry syndrome (CLS) is a syndromic form of X-linked mental retardation, characterized in male patients by psychomotor and growth retardation and various skeletal anomalies. CLS is caused by mutations in the RPS6KA3 gene, which encodes RSK2, a growth factor-regulated protein kinase. Cognitive deficiencies in CLS patients are prominent, but markedly variable in severity, even between siblings. However, the vast majority of patients are severely affected, with mental retardation ranging from moderate to profound. We used a RSK2-KO mouse model that shows no obvious brain abnormalities at the anatomical and histological levels to study the function of RSK2 in neurosecretion. Behavioral studies revealed normal motor coordination, but a profound retardation in spatial learning and a deficit in long-term spatial memory, providing evidence that RSK2 plays similar roles in mental functioning both in mice and human. We found that associative LTP at cortical inputs to the lateral amygdala was blocked in Rsk2 KO mice. Using an RNA interference rescue strategy in PC12 cells, we were able to demonstrate that RSK2 regulates catecholamine release through the phosphorylation of PLD. These results provide the first molecular evidence that RSK2 could regulate neurotransmitter release by activating PLD production of lipids required for exocytosis.

Coffin–Lowry syndrome: A role for RSK2 in mammalian neurogenesis

Coffin–Lowry Syndrome (CLS) is an X-linked genetic disorder associated with cognitive and behavioural impairments. CLS patients present with loss-of-function mutations in the RPS6KA3 gene encoding the mitogen-activated protein kinase (MAPK)–activated kinase p90 ribosomal S6 kinase 2 (Rsk2). Although Rsk2 is expressed in the embryonic brain, its function remains largely uncharacterized. To this end, we isolated murine cortical precursors at embryonic day 12 (E12), a timepoint when neuronal differentiation is initiated, and knocked-down Rsk2 expression levels using shRNA. We performed similar experiments in vivo using in utero electroporations to express shRNA against Rsk2. Rsk2 knockdown resulted in a significant decrease in neurogenesis and an increase in the proportion of proliferating Pax6-positive radial precursor cells, indicating that Rsk2 is essential for cortical radial precursors to differentiate into neurons. In contrast, reducing Rsk2 levels in vitro or in vivo had no effect on the generation of astrocytes. Thus, Rsk2 loss-of-function, as seen in CLS, perturbs the differentiation of neural precursors into neurons, and maintains them instead as proliferating radial precursor cells, a defect that may underlie the cognitive dysfunction seen in CLS.

Four novel RSK2 mutations in females with Coffin–Lowry syndrome

Coffin–Lowry syndrome (CLS) is an X-linked semi-dominant disorder caused by mutations in the RSK2 gene and characterized by moderate to severe mental retardation, characteristic facial features, skeletal deformities, and tapering fingers in males. Females are usually much more mildly and variably affected thus more difficult to diagnose. In this study, molecular genetic analysis was carried out in four female patients presenting features of Coffin–Lowry syndrome. The probands were sporadic cases with no affected males in their families. The molecular analysis of the RSK2 gene revealed four novel mutations, including two frameshift and one missense mutation identified by sequencing, and one large deletion detected by multiplex ligation-dependent probe amplification (MLPA) analysis. Females exhibited a random X-chromosome inactivation pattern. To our knowledge, this is the first report of applying MLPA in the diagnostics of CLS and the first description of a large deletion in a CLS female. These results support including screening for large rearragements in the genetic analysis of female CLS patients.

P90 Ribosomal s6 kinase 2 negatively regulates axon growth in motoneurons

Mutations in Ribosomal s6 kinase 2 ( Rsk2) are associated with severe neuronal dysfunction in Coffin–Lowry syndrome (CLS) patients, flies and mice. So far, the mechanisms of how Rsk2 regulates development, maintenance and activity of neurons are not understood. We have investigated the consequences of Rsk2 deficiency in mouse spinal motoneurons. Survival of isolated Rsk2 deficient motoneurons is not reduced, but these cells grow significantly longer neurites. Conversely, overexpression of a constitutively active form of Rsk2 leads to reduced axon growth. Increased axon growth in Rsk2 deficient neurons was accompanied by higher Erk 1/2 phosphorylation, and the knockout phenotype could be rescued by pharmacological inhibition of MAPK/Erk kinase (Mek). These data indicate that Rsk2 negatively regulates axon elongation via the MAPK pathway. Thus, the functional defects observed in the nervous system of CLS patients and animal models with Rsk2 deficiency might be caused by dysregulated neurite growth rather than primary neurodegeneration.

The first large duplication of the RSK2 gene identified in a Coffin-Lowry syndrome patient

Heterogeneous mutations in the X-linked gene RPS6KA3, encoding the protein kinase RSK2, are responsible for Coffin-Lowry Syndrome. Here we have further studied a male patient with a highly suggestive clinical diagnosis of CLS but in whom no mutation was found by exon sequencing. Western blot analysis revealed a protein much larger than the normal expected size. Sequencing of the RSK2 cDNA, showed the presence of an in-frame tandem duplication of exons 17-20. The mutated RSK2 protein was found to be inactive in an in-vitro kinase assay. This event, which was the result of a homologous unequal recombination between Alu sequences, is the first reported large duplication of the RPS6KA3 gene. Our finding provides further evidence that immunoblot analysis, or a molecular assay capable to detect large genomic mutational events, is essential for patients with a highly suggestive CLS clinical diagnosis but remaining without mutation after exon sequencing.

Expression analysis of RSK gene family members: the RSK2 gene, mutated in Coffin-Lowry syndrome, is prominently expressed in brain structures essential for cognitive function and learning.

Coffin-Lowry syndrome (CLS) is characterized by cognitive impairment, characteristic facial and digital findings and skeletal anomalies. The gene implicated in CLS encodes RSK2, a serine/threonine kinase acting in the Ras/MAPK signalling pathway. In humans, RSK2 belongs to a family of four highly homologous proteins (RSK1-RSK4), encoded by distinct genes. RSK2 mutations in CLS patients are extremely heterogeneous. No consistent relationship between specific mutations and the severity of the disease or the expression of uncommon features has been established. Together, the data suggest an influence of environmental and/or other genetic components on the presentation of the disease. Obvious modifying genes include those encoding other RSK family members. In this study we have determined the expression of RSK1, 2 and 3 genes in various human tissues, during mouse embryogenesis and in mouse brain. The three RSK mRNAs were expressed in all human tissues and brain regions tested, supporting functional redundancy. However, tissue specific variations in levels suggest that they may also serve specific roles. The mouse Rsk3 gene was prominently expressed in the developing neural and sensory tissues, whereas Rsk1 gene expression was the strongest in various other tissues with high proliferative activity, suggesting distinct roles during development. In adult mouse brain, the highest levels of Rsk2 expression were observed in regions with high synaptic activity, including the neocortex, the hippocampus and Purkinje cells. These structures are essential components in cognitive function and learning. Based on the expression levels, our results suggest that in these areas, the Rsk1 and Rsk3 genes may not be able to fully compensate for a lack of Rsk2 function.

Activation of JNK1, RSK2, and MSK1 Is Involved in Serine 112 Phosphorylation of Bad by Ultraviolet B Radiation

The Bcl-2 family member Bad is a pro-apoptotic protein, and phosphorylation of Bad by cytokines and growth factors promotes cell survival in many cell types. Induction of apoptosis by UV radiation is well documented. However, little is known about UV activation of cell survival pathways. Here, we demonstrate that UVB induces Bad phosphorylation at serine 112 in JNK1, RSK2, and MSK1-dependent pathways. Inhibition of mitogen-activated protein (MAP) kinases including ERKs, JNKs, and p38 kinase by the use of their respective dominant negative mutant or a specific inhibitor for MEK1 or p38 kinase, PD98059 or SB202190, resulted in abrogation of UVB-induced phosphorylation of Bad at serine 112. Incubation of active MAP kinase members with Bad protein showed serine 112 phosphorylation of Bad by JNK1 only. However, activated RSK2 and MSK1, downstream kinases of ERKs and p38 kinase, respectively, also phosphorylated Bad at serine 112 in vitro. Cells from a Coffin-Lowry syndrome patient (deficient in RSK2) or expressing an N-terminal or C-terminal kinase-dead mutant of MSK1 were defective for UVB-induced serine 112 phosphorylation of Bad. Furthermore, MAP kinase pathway-dependent serine 112 phosphorylation was shown to be required for dissociation of Bad from Bcl-X(L). These data illustrated that UVB-induced phosphorylation of Bad at serine 112 was mediated through MAP kinase signaling pathways in which JNK1, RSK2, and MSK1 served as direct mediators.

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.

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.

Mutations in the kinase Rsk-2 associated with Coffin-Lowry syndrome.

The Coffin-Lowry syndrome (CLS), an X-linked disorder, is characterized by severe psychomotor retardation, facial and digital dysmorphisms, and progressive skeletal deformations. Genetic linkage analysis mapped the CLS locus to an interval of 2-3 megabases at Xp22.2. The gene coding for Rsk-2, a member of the growth-factor-regulated protein kinases, maps within the candidate interval, and was tested as a candidate gene for CLS. Initial screening for mutations in the gene for Rsk-2 in 76 unrelated CLS patients revealed one intragenic deletion, a nonsense, two splice site, and two missense mutations. The two missenses affect sites critical for the function of Rsk-2. The mutated Rsk-2 proteins were found to be inactive in a S6 kinase assay. These findings provide direct evidence that abnormalities in the MAPK/RSK signalling pathway cause Coffin-Lowry syndrome.