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We report a Japanese girl with Coffin‐Lowry syndrome phenotype such as hypertelorism, hypodontia, and tapering fingers and 46,XX,t(X;11)(p22;p15)dn. Whole genome sequencing revealed RPS6KA3 disruption by the translocation, and X‐inactivation analysis indicated preferential inactivation of the normal X chromosome. The results explain the development of an X‐linked disease in this girl.

Background: Although ribosomal protein S6 kinases, 90 kDa, polypeptide 3 (RSK2, RPS6KA3) has been reported to play an important role in cancer cell proliferation, invasion, and migration, including breast cancer, its clinical implication in primary breast cancer patients is not well understood, and there were not many studies to explore the relationship between RSK2 and breast cancer on a clinical level.Methods: A systematic series matrix file search uploaded from January 1, 2008 to November 31, 2017 was undertaken using ArrayExpress and Gene Expression Omnibus (GEO) databases. Search filters were breast cancer, RNA assay, and array assay. Files eligible for inclusion met the following criteria: a) sample capacity is over 100, b) tumor sample comes from unselected patient’s primary breast tumor tissue, and c) expression of RSK2 and any clinical parameters of patients were available from the files. We use median as the cutoff value to assess the association between the expression of RSK2 and the clinical indexes of breast cancer patients.Finding: The meta-analysis identified 13 series matrix files from GEO database involving 3,122 samples that come from patients’ primary breast cancer tissue or normal tissue. The expression of RSK2 in tumor tissues is lower than that in normal tissues [odds ratio (OR), 0.54; 95% credible interval (CI), 0.44–0.67; Cochran’s Q test p = 0.14; I2 = 41.7%]. Patients with a high expression of RSK2 showed more favorable overall survival [hazard ratio (HR), 0.71; 95% CI, 0.49–0.94; Cochran’s Q test p = 0.95; I2 = 0.0%] and less potential of distant metastasis (OR, 0.59; 95% CI, 0.41–0.87; Cochran’s Q test p = 0.88; I2 = 0.0%) and lymph node infiltration (OR, 0.81; 95% CI, 0.65–0.998; Cochran’s Q test p = 0.09; I2 = 42.8%). Besides, the expression of RSK2 in luminal breast cancer is lower than Cochran’s Q test p = 0.06; I2 = 63.5%). RSK2 overexpression corresponded with higher histological grade (OR, 1.329; 95% CI, 1.03–1.721; Cochran’s Q test p = 0.69; I2 = 0.0%). RSK2 expression is also associated with estrogen receptor (ER) and age.Conclusion: The meta-analysis provides evidence that RSK2 is a potential biomarker in breast cancer patients. The expression of RSK2 is distinctive in different intrinsic subtypes of breast cancer, indicating that it may play an important role in specific breast cancer. Further study is needed to uncover the mechanism of RSK2 in breast cancer.Systematic Review Registration: (website), identifier (registration number).

Coffin-Lowry syndrome (CLS) is a well-described syndrome characterized by intellectual disability, growth retardation, recognizable dysmorphic features, and skeletal changes. It is an X-linked syndrome where males are more severely affected and females have high variability in clinical presentations. This case series reports nine molecularly confirmed Chinese CLS patients from six unrelated families (three with familial variants and three with de novo variants). There is a wide genotypic spectrum with five novel variants in RPS6KA3 gene. Clinical phenotype and facial features of these Chinese CLS patients are comparable to what has been described in other ethnicities.

Background/Aims: Three rare MAPK7 variants that predispose individuals to adolescent idiopathic scoliosis have previously been identified. However, the mechanism underlying the effects of the mutations remain unknown. Methods: Human mesenchymal stem cells (hMSCs) were isolated from both patients and healthy volunteer donors, and MAPK7 expression was detected by western blotting and real-time quantitative PCR (RT-qPCR). Zebrafish embryos were injected with mapk7 morpholinos or co-injected with morpholinos and wild-type (WT) MAPK7 messenger RNA (mRNA) at the one-cell stage, followed by calcein staining to evaluate bone formation. hMSCs were transfected with MAPK7 small interfering RNAs and osteogenesis was induced for 14 days. Alizarin red staining was performed and osteoblast markers were detected by western blotting and RT-qPCR. Since RPS6KA3 is a downstream target of MAPK7 and plays an important role in the osteogenesis, zebrafish embryos were then injected with rps6ka3 morpholinos, or co-injected with rps6ka3 or mapk7 morpholinos and WT RPS6KA3 mRNA at the one-cell stage. Results: MAPK7 expression in the patient group was much lower than in the control group. Morpholino-induced mapk7 knockdown in zebrafish embryos led to body curvature, which was significantly reversed by WT MAPK7 mRNA. Calcein staining revealed that mapk7-knockdown delayed the ossification of the vertebrae. MAPK7 silencing in hMSCs impaired osteogenesis and downregulated osteoblast marker expression. Morpholino-induced rps6ka3-knockdown in zebrafish embryos led to body curvature, which was reversed by WT RPS6KA3 mRNA. Interestingly, RPS6KA3 mRNA also partially reversed the phenotype induced by mapk7 morpholinos. Conclusion: Impaired osteogenesis is linked to mutant MAPK7-induced idiopathic scoliosis , and RPS6KA3 may play an important role in this process.

Coffin-Lowry syndrome (CLS) is a rare X-linked disorder characterized by moderate to severe intellectual disability, hypotonia, craniofacial features, tapering digits, short stature, and skeletal deformities. Using whole exome sequencing and high-resolution targeted comparative genomic hybridization array analysis, we identified a novel microduplication encompassing exons five through nine of RPS6KA3 in three full brothers. Each brother presented with intellectual disability and clinical and radiographic features consistent with CLS. qRT-PCR analyses performed on mRNA from the peripheral blood of the three siblings revealed a marked reduction of RPS6KA3 levels suggesting a loss-of-function mechanism. PCR analysis of the patients' cDNA detected a band greater than expected for an exon 4-10 amplicon, suggesting this was likely a direct duplication that lies between exons 4 through 10, which was later confirmed by Sanger sequencing. This microduplication is only the third intragenic duplication of RPS6KA3, and the second and smallest reported to date thought to cause CLS. Our study further supports the clinical utility of methods such as next-generation sequencing and high-resolution genomic arrays to detect small intragenic duplications. These methods, coupled with expression studies and cDNA structural analysis have the capacity to confirm the diagnosis of CLS in these rare cases.

Mutation of RPS6KA3 can induce Coffin–Lowry syndrome, an X-linked syndrome. The case here reported manifests its signature characteristic of short stature, facial dysmorphism, development retardation, hearing defect. The mutation of RPS6KA3 we detected by NGS analysis is c.2185 C > T. The short stature is a noteworthy problem we discuss here to improve the patient's growth and development. The efficacy and safety of application of growth hormone analogs on patients with CLS are not confirmed and need to be carefully considered.

Coffin-Lowry syndrome (CLS) is an X-linked semi-dominant disorder characterized by diverse phenotypes including intellectual disability, facial and digital anomalies. Loss-of-function mutations in the Ribosomal Protein S6 Kinase Polypeptide 3 (RPS6KA3) gene have been shown to be responsible for CLS. Among the large number of mutations, however, no exonic mutation causing exon skipping has been described. Here, we report a male patient with CLS having a novel mutation at the 3' end of an exon at a splice donor junction. Interestingly, this nucleotide change causes both a novel missense mutation and partial exon skipping leading to a truncated transcript. These two transcripts were identified by cDNA sequencing of RT-PCR products. In the carrier mother, we found only wildtype transcripts suggesting skewed X-inactivation. Methylation studies confirmed X-inactivation was skewed moderately, but not completely, which is consistent with her mild phenotype. Western blot showed that the mutant RSK2 protein in the patient is expressed at similar levels relative to his mother. Protein modeling demonstrated that the missense mutation is damaging and may alter binding to ATP molecules. This is the first report of exon skipping from an exonic mutation of RPS6KA3, demonstrating that a missense mutation and concomitant disruption of normal splicing contribute to the manifestation of CLS.

Background Increased expression of RNA polymerase 1 (POL1) molecular pathway was reported to be associated with increased disease activity in patients with multiple sclerosis (MS). However, the operating molecular mechanisms that characterize the pattern of acute MS relapse activity has not been thoroughly studied. Objective To assess POL1 pathway expression during acute MS relapse. Methods We studied POL1 pathway associated biomarkers during the first acute optic neuritis attack of MS, and in relapsing-remitting MS patients treated with disease-modifying drugs (DMDs) experiencing an acute MS relapse or a radiological relapse using gene expression microarrays and quantitative RT-PCR. Results In MS patients (N = 6) during the first acute optic neuritis attack POL1 pathway activation was evident by over-expression of POL1 related network including transcription factor UBTF and downstream components of Assembly of RNA POL1 complex (p=1.92E-03). POL1 related biomarkers RRN3, POLR1D and LRPPRC were over-expressed x1.6 (p = .002), ×1.7 (p = .01) and x2.0 (p = .001) times higher respectively, in MS patients (N = 30) during acute clinical relapse as compared with remission. Similarly, in MS patients (N = 21) that presented with a radiological relapse, we observed significant activation of POL1 related biomarkers including RRN3 (p = .01), POLR1D (p = .002), POLR1E (p = .0001) and LRPPRC (p = .006), as compared with remission, as well as overexpression of a large group of genes encoding ribosomal proteins like RPS6KA3 (p = 7.2E-6), RRP8 (p = .0002) and RPCS9 (p = .0008). Conclusions Our findings demonstrated increased POL1 pathway activity in acute MS relapse and suggest that targeted inactivation of POL1 pathway represent a novel strategy for a better treatment of acute MS relapse.

Coffin-Lowry syndrome (CLS) is an X-linked syndromic form of mental retardation characterized by various skeletal dysmorphisms, moderate to severe mental retardation, and in some cases, psychosis. CLS is caused by loss-of-function mutations of the p90 ribosomal S6 kinase 2 (RPS6KA3) gene encoding a growth factor-regulated serine/threonine kinase, ribosomal S6 kinase 2 (RSK2). We previously identified RSK2 as a novel interacting protein that tonically inhibits 5-HT2A receptor signaling by phosphorylating Ser-314 within the third intracellular loop. To determine if RSK2 inhibits 5-HT2A receptor signaling in vivo and whether disruption of RSK2 could lead to schizophrenia-like behaviors - as is seen in some CLS patients - we genetically disrupted the function of RSK2 either globally or selectively in forebrain pyramidal neurons in mice. Both global and forebrain-selective RSK2 deletion augmented the locomotor responses to the psychotomimetic drugs phencyclidine (PCP) and amphetamine (AMPH). Significantly, forebrain-selective deletion of RSK2 augmented 5-HT2A receptor signaling as exemplified by enhanced 5-HT2A-mediated c-fos activation and head-twitch response without altering the levels or distribution of 5-HT2A receptor protein. Thus, RSK2 modulates 5HT2A receptor function in vivo, and disruption of RSK2 leads to augmented psychostimulant-induced responses reminiscent of those seen in many animal models of schizophrenia. These findings strengthen the association between 5-HT2A receptor dysfunction and psychosis, and provide a potential mechanism underlying the schizophrenia-like symptoms present in some CLS patients.

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20–30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.

Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70-80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20-30% of cases is unknown. We performed next-generation sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gain-of-function alleles in Ras-like without CAAX 1 (RIT1) and mitogen-activated protein kinase kinase 1 (MAP2K1) and previously unseen loss-of-function variants in RAS p21 protein activator 2 (RASA2) that are likely to cause NS in these patients. Expression of the mutant RASA2, MAP2K1, or RIT1 alleles in heterologous cells increased RAS-ERK pathway activation, supporting a causative role in NS pathogenesis. Two patients had more than one disease-associated variant. Moreover, the diagnosis of an individual initially thought to have NS was revised to neurofibromatosis type 1 based on an NF1 nonsense mutation detected in this patient. Another patient harbored a missense mutation in NF1 that resulted in decreased protein stability and impaired ability to suppress RAS-ERK activation; however, this patient continues to exhibit a NS-like phenotype. In addition, a nonsense mutation in RPS6KA3 was found in one patient initially diagnosed with NS whose diagnosis was later revised to Coffin-Lowry syndrome. Finally, we identified other potential candidates for new NS genes, as well as potential carrier alleles for unrelated syndromes. Taken together, our data suggest that next-generation sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients.

An elevated odds ratio for low IQ has been found for cryptorchid boys. Furthermore, poor school performance has been observed in cryptorchid boys with impaired mini-puberty. Gene expression analysis, qPCR and immunohistology were performed on testicular biopsies from 7 boys who underwent orchiopexy and had testicular histology typical of a high risk of infertility (HIR). The results were compared with 12 biopsies from cryptorchid boys with a low risk for developing infertility. The following genes associated with mental retardation were identically expressed: GDI1, OPHN1, PAK3, ARHGEF6, IL1RAPL, ACSL4, MECP2, RPS6KA3, ARX, and ATRX. However, boys in the HIR group had low or no expression of EGR4, FMR2 (AFF2) and VCX3A. In conclusion, impaired expression of genes known to encode proteins involved in signaling pathways that regulate cytoskeletal organization, synaptic vesicle transport and the establishment of connections between neuronal cells may contribute to reduced intellectual and cognitive functioning in infertile cryptorchid males.

Background More than 100 X-linked intellectual disability (X-LID) genes have been identified to be involved in 10–15% of intellectual disability (ID). Method To identify novel possible candidates, we selected 18 families with a male proband affected by isolated or syndromic ID. Pedigree and/or clinical presentation suggested an X-LID disorder. After exclusion of known genetic diseases, we identified seven cases whose mother showed a skewed X-inactivation (>80%) that underwent whole exome sequencing (WES, 50X average depth). Results WES allowed to solve the genetic basis in four cases, two of which (Coffin-Lowry syndrome, RPS6K3 gene; ATRX syndrome, ATRX gene) had been missed by previous clinical/genetics tests. One further ATRX case showed a complex phenotype including pontocerebellar atrophy (PCA), possibly associated to an unidentified PCA gene mutation. In a case with suspected Lujan-Fryns syndrome, a c.649C>T (p.Pro217Ser) MECP2 missense change was identified, likely explaining the neurological impairment, but not the marfanoid features, which were possibly associated to the p.Thr1020Ala variant in fibrillin 1. Finally, a c.707T>G variant (p.Phe236Cys) in the DMD gene was identified in a patient retrospectively recognized to be affected by Becker muscular dystrophy (BMD, OMIM 300376). Conclusion Overall, our data show that WES may give hints to solve complex ID phenotypes with a likely X-linked transmission, and that a significant proportion of these orphan conditions might result from concomitant mutations affecting different clinically associated genes.

Multiplex ligation-dependent probe amplification (MLPA) and array- comparative genomic hybridization analysis have been proven to be useful in the identification of submicroscopic copy-number imbalances in families with nonsyndromic X-linked intellectual disability (NS-XLID). Here we report the first description of a child with mild intellectual disability and a submicroscopic duplication at Xp22.12 identified by MLPA with a P106 MRX kit (MRC-Holland, Amsterdam, Netherlands) and further confirmed and characterized with a custom 244-k oligo-array, fluorescence in situ hybridization, quantitative polymerase chain reaction (qPCR), and immunoblotting. This 1.05-megabase duplication encompasses 7 genes, RPS6KA3 being the only of these genes known to be related to ID. The proband was an 8-year-old boy referred to the genetics unit for psychomotor retardation and learning disabilities. Both maternal brothers also showed learning difficulties and delayed language during childhood in a similar way to the proband. These boys also carried the duplication, as did the healthy mother and grandmother of the proband. The same duplication was also observed in the 5-year-old younger brother who presented with features of developmental delay and learning disabilities during the previous year. Increased RPS6KA3/RSK2 levels were demonstrated in the proband by qPCR and immunoblotting. To our knowledge, this is the first family identified with a submicroscopic duplication including the entire RPS6KA3/RSK2 gene, and our findings suggest that an increased dose of this gene is responsible for a mild form of NS-XLID.

Background Coffin-Lowry syndrome is a semi-dominant condition characterized by severe psychomotor retardation with facial, hand and skeletal malformations resulting from mutations in RSK2 gene, encoding for a serine/threonine kinase. More than 100 different mutations have been identified to date; however, about 50% of clinically diagnosed patients apparently do not have mutations. In order to exclude that these patients have RSK2 mutations missed by standard mutation detection techniques, a rapid and sensitive assay allowing evaluation of RSK2 activity was needed. Methods RSK2 capacity to phosphorylate a synthetic CREB-peptide in basal and PMA-stimulated conditions was evaluated in lymphoblasts from 3 patients with RSK2 mutations and normal controls. Results Patients RSK2 activity is normal in nonstimulated conditions but fails to grow following stimulation. The evaluation of the stimulated/non-stimulated activity ratio demonstrated a statistically significant impairment in patients. Conclusions We have set up an assay which allows the identification of even partial alterations of RSK2 activity and seems to give good results also in females with a balanced X-chromosome inactivation and thus with a presumably normal enzymatic activity in about 50% of cells. Moreover, our data seem to confirm previous reports of a potential direct correlation between the level of RSK2 activity and the severity of cognitive impairment.

Purpose: Routine protocols for the study of mental retardation include karyotype, analysis for fragile X syndrome, and subtelomeric rearrangements. Nevertheless, detection of cryptic rearrangements requires more sensitive techniques. Mutation screening in all known genes responsible for X-linked mental retardation is not feasible, and linkage analysis is sometimes limited. Multiplex ligation probe amplification is a recently developed technique based on the amplification of specific probes that allows relative quantification of 40 to 46 different target DNA sequences in a single reaction. Methods: In the present study, we assessed multiplex ligation probe amplification for the detection of microduplications/microdeletions in 80 male patients with suspicion of X-linked mental retardation. Results: We detected four copy number aberrations (5%): three duplications (GDI1, RPS6KA3, and ARHGEF6) and one deletion (OPHN1). All these changes were confirmed by other molecular techniques, and patients were clinically re-evaluated. Conclusions: We strongly recommend the use of multiplex ligation probe amplification as a first screening method for the detection of copy number aberrations in patients with mental retardation because of its cost-effectiveness.

The ribosomal protein S6 kinase, 90 kb, polypeptide 3 gene (RPS6KA3) is responsible for Coffin-Lowry syndrome (CLS), which is characterized by intellectual disability (ID) and facial and bony abnormalities. This gene also affects nonsyndromic X-linked ID and nonsyndromic X-linked ID without bony abnormalities. Two families have been previously reported to have genetic microduplication including RPS6KA3. In the present study, we used array-comparative genomic hybridization (CGH) analysis with Agilent Human genome CGH 180K and detected a 584-kb microduplication spanning 19.92-20.50 Mb of Xp22.12 (including RPS6KA3) in the members of one family, including three brothers, two sisters, and their mother. The 15-year-old male proband and one of his brothers had mild ID and localization-related epilepsy, whereas his other brother presented borderline intelligence quotient (IQ) and attention-deficit-hyperactivity disorder (ADHD). One sister presented pervasive development disorder (PDD). Analysis of this family suggests that RPS6KA3 duplication is responsible for mild ID, ADHD, and localization-related epilepsy, and possibly for PDD.

Two novel mutations of the ribosomal S6 kinase 2 gene (also known as RSK2) have been identified in two unrelated patients with Coffin–Lowry syndrome. The first mutation consists of a de novo insertion of a 5′-truncated LINE-1 element at position −8 of intron 3, which leads to a skipping of exon 4, leading to a shift of the reading frame and a premature stop codon. The L1 fragment (2800 bp) showed a rearrangement with a small deletion, a partial inversion of the ORF 2, flanked by short direct repeats which duplicate the acceptor splice site. However, cDNA analysis of the patient shows that both sites are apparently not functional. The second family showed the nucleotide change 803T>C in exon 10, resulting in the F268S mutation. This mutation was detected in two monozygotic twin patients and in their mother, who was mildly affected. The patients fulfill the clinical criteria of the syndrome, and therefore the mutation provides further support for the importance of phenylalanine at position 268, which is highly conserved in the protein kinase domain of many serine–threonine protein kinases.

We report an African-American family that was identified after the proposita was referred for diagnostic evaluation at 4½ months with a history of Hirschsprung and dysmorphic features typical of Waardenburg syndrome (WS). Family evaluation revealed that the father had heterochromidia irides and hypertelorism supporting the clinical diagnosis of WS; however, examination of the mother revealed characteristic facial and digital features of Coffin-Lowry syndrome (CLS). Molecular testing of the mother identified a novel 2 bp deletion (c.865_866delCA) in codon 289 of RPS6KA3 leading to a frame-shift and premature termination of translation 5 codons downstream (NM_004586.2:p.Gln289ValfsX5). This deletion also was identified in the proposita and her three sisters with a clinical suspicion of CLS, all of whom as carriers for this X-linked disorder had very subtle manifestations. The molecular confirmation of WS type 4 (Shah-Waardenburg; WS4) was not as straightforward. To evaluate WS types 1-4, multiple sequential molecular tests were requested, including Sanger sequencing of all exons, and deletion/duplication analysis using MLPA for PAX3, MITF, SOX10, EDN3 and EDNRB. Although sequencing did not identify any disease causing variants, MLPA identified a heterozygous deletion of the entire EDNRB in the father. This deletion was also found in the proposita and the oldest child. Since the heterozygous deletion was the only change identified in EDNRB, this family represents one of the few cases of an autosomal dominant inheritance of WS4 involving the endothelin pathway. Altogether, clinical evaluation of the family revealed one child to be positive for WS4 and two positive for CLS, while two children were positive for both diseases simultaneously (including the proposita) while another pair test negative for either disease. This kinship is an example of the coincidence of two conditions co-segregating in one family, with variable phenotypes requiring molecular testing to confirm the clinical diagnoses.

Here, we report on a patient with a 625 kb duplication in Xp22.12, detected by array comparative genomic hybridization (CGH). The duplicated region contains only one gene, RPS6KA3, that results in partial duplication. The same duplication was present in his mother and his maternal uncle. This partial duplication inhibits the RPS6KA3 expression, mimicking the effect of loss-of-function mutations associated with Coffin-Lowry syndrome (CLS). The phenotype of the patient here presented is not fully evocative of this syndrome because he does not present some of the facial, digital and skeletal abnormalities that are considered the main diagnostic features of CLS. This case is one of the few examples where RPS6KA3 mutations are associated with a non-specific X-linked mental retardation.

Coffin-Lowry syndrome (CLS) is a rare X-linked dominant disorder characterized by intellectual disability, craniofacial abnormalities, short stature, tapering fingers, hypotonia, and skeletal malformations. CLS is caused by mutations in the Ribosomal Protein S6 Kinase, 90 kDa, Polypeptide 3 (RPS6KA3) gene located at Xp22.12, which encodes Ribosomal S6 Kinase 2 (RSK2). Here we analyzed RPS6KA3 in three unrelated CLS patients including one from the historical Coffin-Lowry syndrome family and found two novel mutations. To date, over 140 mutations in RPS6KA3 have been reported. However, the etiology of the very first familial case, which was described in 1971 by Lowry with detailed phenotype and coined the term CLS, has remained unknown. More than 40 years after the report, we succeeded in identifying deposited fibroblast cells from one patient of this historic family and found a novel heterozygous 216 bp in-frame deletion, encompassing exons 15 and 16 of RPS6KA3. Drop episodes in CLS patients were reported to be associated with truncating mutations deleting the C-terminal kinase domain (KD), and only one missense mutation and one single basepair duplication involving the C-terminal KD of RSK2 in the patients with drop episode have been reported thus far. Here we report the first in-frame deletion in C-terminal KD of RPS6KA3 in a CLS patient with drop episodes.

An adolescent female presented with intellectual disability, stimulus-induced drop episodes (SIDEs), facial characteristics that include wide set eyes, short nose with wide columella, full and everted lips with wide mouth and progressive skeletal changes: scoliosis, spondylolisthesis and pectus excavatum. These findings were suggestive of Coffin-Lowry syndrome (CLS), and this was confirmed by the identification of a novel mutation in RPS6KA3, a heterozygous one basepair duplication at nucleotide 1570 (c.1570dupA). This mutation occurs within the C-terminal kinase domain of the protein, and, therefore contradicts the previous report that SIDEs is only associated with premature truncation of the protein in the N-terminal kinase domain or upstream of this domain. As CLS is X-linked, it is unusual for a female to have such a classic phenotype.

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.

RPS6KA3 encodes the p90 ribosomal S6 kinase 2 (p90RSK2 or RSK2), a protein kinase to relay the upstream transduction signal of the mitogen-activated protein kinase (MAPK) pathway. Currently, no characterization on RSK2 in human HCC has been performed. We determined the RSK2 mRNA expression in paired human HCC samples and their corresponding non-tumorous livers (n = 22) by real time PCR. RSK2 over-expression (≥2 folds) was found in 38% of the human HCCs and this over-expression was associated with tumor microsatellite formation, a feature of HCC metastasis. The oncogenic and pro-metastatic roles of RSK2 were further assessed and demonstrated in vivo by an orthotopic liver injection model in nude mice with stable shRSK2 knockdown HCC cells. Stable shRSK2 knockdown cells showed a significant reduction in tumor incidence in liver and lung metastasis. By exome-sequencing and targeted sequencing in a cohort of HBV-associated HCC patient samples (n = 111), we found that RSK2 was recurrently mutated in 6.3% of the samples being screened (n = 7). All the RSK2 mutants were confirmed by independent Sanger sequencing. Intriguingly, among the identified somatic RSK2 mutants, half of them introduced pre-mature stop gain to the transcript, which leads to the loss-of function of RSK2 through various mechanisms. Taken together, our data suggested that RSK2 may play differential roles in different subsets of HCC patients. Further characterization will definitely derive novel insights on the functional role of mutant RSK2 and its potential use to stratify HCC patients into different molecular subgroups. (This study was supported by a RGC GRF fund (17116414), SK Yee Medical Research Fund 2011 and University Development Fund of The University of Hong Kong. Ng is Loke Yew Professor in Pathology) Citation Format: Lo-Kong Chan, Daniel Wai-Hung Ho, Yung-Tuen Chiu, Alan Ka-Lun Kai, Chun-Ming Wong, Irene Oi-Lin Ng. Functional characterization of RPS6KA3 (RSK2) and identification of its naturally occurring mutants in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 690.

Establishing a simple and effective mutation screening method is one of the most compelling problems with applying genetic diagnosis to clinical use. Because there is no reliable and inexpensive screening system, amplifying by PCR and performing direct sequencing of every coding exon is the gold standard strategy even today. However, this approach is expensive and time consuming, especially when gene size or sample number is large. Previously, we developed CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) as an ideal simple mutation screening system constructed with only conventional apparatuses and commercially available reagents. In this study, we evaluated the utility of CHIPS technology for genetic diagnosis in clinical practice by applying this system to screening for the COL2A1 , WRN and RPS6KA3 mutations in newly diagnosed patients with Stickler syndrome (autosomal dominant inheritance), Werner syndrome (autosomal recessive inheritance) and Coffin–Lowry syndrome (X-linked inheritance), respectively. In all three genes, CHIPS detected all DNA variations including disease causative mutations within a day. Direct sequencing of all coding exons of these genes confirmed 100% sensitivity and specificity. We demonstrate high sensitivity, high cost performance and reliability of this simple system, with compatibility to all inheritance modes. Because of its low technology, CHIPS is ready to use and potentially disseminate to any laboratories in the world.

Coffin–Lowry syndrome (CLS) is a syndromic form of X-linked mental retardation, which is characterized in male patients by psychomotor and growth retardation and various skeletal anomalies. Typical facial changes and specific clinical and radiological signs in the hand are useful aids in the diagnosis. CLS is caused by mutations in the RPS6KA3 gene located at Xp22.2, which encodes RSK2, a growth-factor-regulated protein kinase. RPS6KA3 mutations are extremely heterogeneous and lead to loss of phosphotransferase activity in the RSK2 kinase, most often because of premature termination of translation.

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.

We describe three families with X-linked mental retardation, two with a deletion of a single amino acid and one with a missense mutation in the proximal domain of the RSK2(RPS6KA3) (ribosomal protein S6 kinase, 90 kDa, polypeptide 3) protein similar to mutations found in Coffin-Lowry syndrome (CLS). In two families, the clinical diagnosis had been nonsyndromic X-linked mental retardation. In the third family, although CLS had been suspected, the clinical features were atypical and the degree of intellectual disability much less than expected. These families show that strict reliance on classical clinical criteria for mutation testing may result in a missed diagnosis. A less targeted screening approach to mutation testing is advocated.

International audience; 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 (CLS; MIM #303600) is characterized by learning difficulties and dysmorphic traits in male patients and in some female carriers of the X-chromosome–linked gene mutation. The dysmorphic traits, skeletal abnormalities, and other clinical findings have been described (1). Mutations of the RSK2 gene (also called RPS6KA3 , MIM *300075), mapping to Xp22.2, are found in the disease (2). The gene encodes a 740-amino acid protein member of the 90-kDa ribosomal S6 serine/threonine kinase family (3). In humans, the RSK family includes 4 growth factor–regulated members (RSK1 to -4) produced in all examined tissues and regions of the brain (4). The highly conserved feature of all these proteins is the presence of 2 nonidentical kinase catalytic domains. The N-terminal kinase domain (amino acids 68–323 in RSK2) is responsible for phosphotransferase activity toward substrates, whereas the C-terminal kinase domain (amino acids 422–675 in RSK2) is necessary for enzymatic activation of the N-terminal domain. RSKs are activated through direct phosphorylation by the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) in response to insulin and growth factors, oncogenic events, and ultraviolet irradiation. RSKs have been implicated in the stimulation of cell proliferation and differentiation, in the cellular stress response, and in apoptosis (5). To date, only a few RSK2-specific physiologic substrates have been described: the transcription factor CREB, histone H3, STAT3 (6), ATF4(7), and p53(8). According to the most recent data (http://alsace.u-strasbg.fr/chimbio/diag/coffin/index.html), mutations in the RSK2 gene occur within all 22 exons, except exon 2. Approximately one third of the RSK2 gene mutations are missense changes, the other two thirds lead to premature translation termination. The proportion of de novo mutations is unusually high compared with other X-linked disorders, with most cases (80%) being sporadic (1). The 2 largest studies on the molecular diagnosis of CLS used single-strand conformational polymorphism …

An X-linked recessive syndromic form of mental retardation is described in a family in which 10 males in four generations were affected. The main manifestations were severe to profound intellectual disability, muscular hypotonia in childhood, delayed walking, and difficult, aggressive behavior. There was a moderate reduction both in occipitofrontal circumference (OFC) and height and a similar facial appearance, triangular in shape with a high forehead, prominent ears, and a small pointed chin. Linkage analysis located the gene at Xp22 with maximum lod scores of 4.8 at theta = 0.0 for markers mapping between the closest recombination points at DXS7104 and DXS418. The physical length of this region is approximately 6 Mb. Mutations in the GRPR gene and M6b genes were excluded by sequence analysis. Nearby genes in which mutations are known to be associated with mental retardation (RPS6KA3, STK9, and VCXA, B and C), were excluded by position.

SummaryCoffin-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.

AIM: To identify the differentially expressed miRNAs and their targets in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). METHODS: Six hundred and sixty seven human miRNAs were quantitatively analyzed by Taqman low-density miRNA array (TLDA) in HBV-HCC tissues. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to analyze the significant function and pathway of the differentially expressed miRNAs in HBV-HCC. TargetScan software was used to predict the targets of deregulated miRNAs. Western blotting and luciferase assay were performed to verify the targets of these miRNAs. RESULTS: Ten up-regulated miRNAs (miR-217, miR-518b, miR-517c, miR-520g, miR-519a, miR-522, miR-518e, miR-525-3p, miR-512-3p, and miR-518a-3p) and 11 down-regulated miRNAs (miR-138, miR-214, miR-214#, miR-199a-5p, miR-433, miR-511, miR-592, miR-483-3p, miR-483-5p, miR-708 and miR-1275) were identified by Taqman miRNAs array and confirmed quantitatively by reverse transcription polymerase chain reaction in HCC and adjacent non-tumor tissues. GO and KEGG pathway analysis revealed that “regulation of actin cytoskeleton” and “pathway in cancer” are most likely to play critical roles in HCC tumorigenesis. MiR-519a and ribosomal protein S6 kinase polypeptide 3 (RPS6KA3) were predicted as the most significant candidates by miRNA-mRNA network. In addition, cyclin D3 (CCND3) and clathrin heavy chain (CHC), usually up-regulated in HCC tissues, were validated as the direct target of miR-138 and miR-199a-5p, respectively. CONCLUSION: Our data suggest an importance of miR-138 and miR-199a-5p as well as their targets CCND3 and CHC in HCC tumorigenesis, and may provide more evidence for reliability of integrative bioinformatics analysis.

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. Hum Mutat 17:103–116, 2001. © 2001 Wiley-Liss, Inc.

Background Drug resistance hampers the efficient treatment of malignancies, including advanced stage ovarian cancer, which has a 5-year survival rate of only 30 %. The molecular processes underlying resistance have been extensively studied, however, not much is known about the involvement of microRNAs. Methods Differentially expressed microRNAs between cisplatin sensitive and resistant cancer cell line pairs were determined using microarrays. Mimics were used to study the role of microRNAs in drug sensitivity of ovarian cancer cell lines and patient derived tumor cells. Luciferase reporter constructs were used to establish regulation of target genes by microRNAs. Results MiR-634 downregulation was associated with cisplatin resistance. Overexpression of miR-634 affected cell cycle progression and enhanced apoptosis in ovarian cancer cells. miR-634 resensitized resistant ovarian cancer cell lines and patient derived drug resistant tumor cells to cisplatin. Similarly, miR-634 enhanced the response to carboplatin and doxorubicin, but not to paclitaxel. The cell cycle regulator CCND1, and Ras-MAPK pathway components GRB2, ERK2 and RSK2 were directly repressed by miR-634 overexpression. Repression of the Ras-MAPK pathway using a MEK inhibitor phenocopied the miR-634 effects on viability and chemosensitivity. Conclusion miR-634 levels determine chemosensitivity in ovarian cancer cells. We identify miR-634 as a therapeutic candidate to resensitize chemotherapy resistant ovarian tumors. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0464-4) contains supplementary material, which is available to authorized users.