Unusual splice-site mutations in the RSK2 gene and suggestion of genetic heterogeneity in Coffin-Lowry syndrome

Coffin-Lowry syndrome (CLS) is a syndromic form of X-linked mental retardation that is characterized, in male patients, by psychomotor and growth retardation and various skeletal anomalies. Typical facial changes and specific clinical and radiological hand aspects exhibited by patients are essential clues for the diagnosis. CLS is caused by mutations in a gene that is located in Xp22.2 and that encodes RSK2, a growth-factor-regulated protein kinase. RSK2 mutations are extremely heterogeneous and lead to premature termination of translation and/or loss of phosphotransferase activity. Surprisingly, among a series of 250 patients screened by single-strand conformation polymorphism (SSCP) analysis, in whom a clinical diagnosis of CLS was made, no mutations were detected in 66% (165) of the patients. To determine what proportion of these latter patients have a RSK2 mutation that has not been detected and what proportion have different disorders that are phenotypically similar to CLS, we have, in the present article, investigated, by western blot analysis and in vitro kinase assay, cell lines from 26 patients in whom no mutation was previously identified by SSCP analysis. This approach allowed us to identify seven novel RSK2 mutations: two changes in the coding sequence of RSK2, one intragenic deletion, and four unusual intronic nucleotide substitutions that do not affect the consensus GT or AG splice sites. We have also determined the nucleotide sequence of the promoter region of the RSK2 gene, and we have screened it for mutations. No disease-causing nucleotide change was identified, suggesting that mutations affecting the promoter region are unlikely to account for a large number of patients with CLS. Finally, our results provide evidence that some patients have a disease that is phenotypically very similar to CLS, which is not caused by RSK2 defects. This suggests that there are defects in either additional genes or combinations of genes that may result in a CLS-like phenotype.     

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.     

Delineation of the mechanisms of aberrant splicing caused by two unusual intronic mutations in the RSK2 gene involved in Coffin-Lowry syndrome

Coffin–Lowry syndrome (CLS) is caused by mutations in the RSK2 gene encoding a protein kinase of the Ras signalling pathway. We have studied two point mutations which cause aberrant splicing but do not concern the invariant GT or AG nucleotides of splice sites. The first, an A→G transition at position +3 of the 5′ splice site of exon 6, results in vivo and in vitro in exon skipping and premature translation termination. The natural 5′ splice site, although intrinsically weak, is not transactivated under normal conditions. Consequently, replacement of an A/U by a G/U base pairing with U1 snRNA reduces its strength below a critical threshold. The second mutation, an A→G transition 11 nt upstream of exon 5, creates a new AG near the natural 3′ splice site. In vitro this synthetic 3′ AG is used exclusively by the splicing machinery. In vivo this splicing event is also observed, but is underestimated because the resulting RSK2 mRNA contains premature stop codons which trigger the nonsense-mediated decay process. We show that a particular mechanism is involved in the aberrant splicing of exon 5, implying involvement of the natural 3′ AG during the first catalytic step and the new 3′ AG during the second step. Thus, our results explain how these mutations cause severe forms of CLS.     

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene

The aim of this study was to investigate associations of two candidate gene SNPs of the endocannabinoid receptor type 1 gene (CNR1) with overweight, obesity and obesity-related traits in Chinese retired women. The study subjects were a subsample of the Taizhou Retiree Women Cohort, consisting of 2812 retired women aged 50-64 years recruited from Taizhou, Jiangsu, China. Neither rs2023239 nor rs806381 polymorphism was significantly associated with body mass index-defined overweight and obesity or waist-to-hip-ratio-defined obesity. For obesity-related traits, rs2023239 was significantly associated with glutamate pyruvate transaminase (GPT) (median, 18.00 vs 17.00 for TT and TC genotypes, respectively, P=0.043). The rs806381 also showed significant association with triglyceride (TG) (mean±SD, 1.46±0.20 vs 1.53±0.20 for GA and GG+AA genotypes, respectively, P=0.013) under the dominant genetic model. In conclusion, the rs2023239 and rs806381 polymorphisms of CNR1 were not associated with increased overweight and obesity risk. But the rs2023239 polymorphism was significantly associated with GPT, and the rs806381 polymorphism was significantly associated with TG.     

A novel mutation in the RPS6KA3 gene in a patient with Coffin-Lowry syndrome

Coffin-Lowry syndrome is an X-linked disorder characterized by mental retardation, characteristic facial features, skeletal abnormalities, and tapering fingers. Herein we report a novel missense mutation in exon 7 at codon 180 in the RPS6KA3 gene in a boy with Coffin-Lowry syndrome.     

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene

In European populations, large rearrangements contribute to approximately 2% of CF mutations. Here, we reported a novel duplication, the CFTRdup2, identified in a patient heterozygous for Phe508del and suffering from a mild CF. Using a combination of functional tests, we studied the impact of duplication/deletion on CFTR expression. We showed that the copy number variations of exon 2, in addition to abolishing the rate of the mature CFTR protein, affect the CFTR mRNA levels. These data illustrate the importance to perform functional analysis to better understand the molecular basis responsible for cystic fibrosis. Determining the impact of deletions or duplications is relevant for a more comprehensive diagnosis and prognosis of patients.     

Characterization of the first FGFRL1 mutation identified in a craniosynostosis patient

Fibroblast growth factor receptor-like 1 (FGFRL1) is a recently discovered transmembrane protein whose functions remain unclear. Since mutations in the related receptors FGFR1-3 cause skeletal malformations, DNA samples from 55 patients suffering from congenital skeletal malformations and 109 controls were searched for mutations in FGFRL1. One patient was identified harboring a frameshift mutation in the intracellular domain of this novel receptor. The patient showed craniosynostosis, radio-ulnar synostosis and genital abnormalities and had previously been diagnosed with Antley–Bixler syndrome. The effect of the FGFRL1 mutation was studied in vitro. In a reporter gene assay, the wild-type as well as the mutant receptor inhibited FGF signaling. However, the mutant protein differed from the wild-type protein in its subcellular localization. Mutant FGFRL1 was mainly found at the plasma membrane where it interacted with FGF ligands, while the wild-type protein was preferentially located in vesicular structures and the Golgi complex. Two motifs from the intracellular domain of FGFRL1 appeared to be responsible for this differential distribution, a tandem tyrosine based motif and a histidine-rich sequence. Deletion of either one led to the preferential redistribution of FGFRL1 to the plasma membrane. It is therefore likely that mutant FGFRL1 contributes to the skeletal malformations of the patient.     

Molecular characterization of the first missense mutation in the fibrinogen Aalpha-chain gene identified in a compound heterozygous afibrinogenemic patient

Congenital afibrinogenemia is a rare coagulopathy characterized by extremely low levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait and is invariantly associated with mutations affecting 1 of the 3 fibrinogen genes (FGA, FGB, and FGG, coding for Aalpha, Bbeta, and gamma chain, respectively). Most genetic defects causing afibrinogenemia are truncating mutations, whereas only few missense mutations (6) have been identified so far, all located in FGB. In this study, the mutational screening of an afibrinogenemic Italian male identified the first missense mutation (Met51Arg) in FGA leading to afibrinogenemia. The patient was a compound heterozygote for a previously described frameshift mutation (1215delT) in the same gene. Met51Arg involves a residue located at the very beginning of the coiled-coil domain, in a region demonstrated to play a pivotal role in hexamer formation. In-vitro expression experiments showed that Met51Arg strongly reduces secretion of hexameric fibrinogen, whereas traces of not completely assembled trimeric intermediate were found in conditioned media. Western blot analysis on the proband's plasma confirmed the presence in vivo of the trimeric fibrinogen, supporting the hypothesis that Met51Arg prevents the final step of fibrinogen assembly.     

Expression of the RSK2 gene during early human development

The 90 kDa ribosomal S6 serine/threonine kinase 2 gene (RSK2, U08316) has been recently identified as a disease-causing gene in an X-linked disorder, the Coffin-Lowry Syndrome (MIM 303600) characterized by severe mental retardation, facial dysmorphisms and progressive skeletal malformations. To investigate its possible role in cerebral cortex development, we performed RNA in situ hybridization at three stages of human development: day 32 (Carnegie 15), 9 weeks (Carnegie 23) and 13 weeks. RSK2 expression is detected in the embryonic anterior and posterior telencephalon (hippocampus anlagen), mesencephalon, rhombencephalon and cerebellum. RSK2 gene expression is also observed in dorsal root ganglia, cranial nerve ganglia, and sensory epithelium of the inner ear, liver, lung and jaw anlagen. This pattern of expression may be involved in cognitive impairment and facial dysmorphisms found in Coffin-Lowry Syndrome.     

Alu-Alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ehlers-Danlos syndrome.

The type VI variant of the Ehlers-Danlos syndrome (EDS) is a recessively inherited connective-tissue disorder. The characteristic features of the variant are muscular hypotonia, kyphoscoliosis, ocular manifestations, joint hypermobility, skin fragility and hyperextensibility, and other signs of connective-tissue involvement. The biochemical defect in most but not all patients is a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase is an enzyme that catalyzes the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. We have recently reported an apparently homozygous large-duplication rearrangement in the gene for lysyl hydroxylase, leading to the type VI variant of EDS in two siblings. We now report an identical, apparently homozygous large duplication in an unrelated 49-year-old female originally analyzed by Sussman et al. Our simple-sequence-repeat-polymorphism analysis does not support uniparental isodisomy inheritance for either of the two duplications. Furthermore, we indicate in this study that the duplication in the lysyl hydroxylase gene is caused by an Alu-Alu recombination in both families. Cloning of the junction fragment of the duplication has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with the type VI variant of EDS.     

The first nuclear-encoded complex I mutation in a patient with Leigh syndrome.

Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain. The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2. The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids. Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome. The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H). Both mutations were absent in 70 control alleles and cosegregated within the family. A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient. In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA. This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.     

Detection of a large duplication mutation in the myosin-binding protein C3 gene in a case of hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease with autosomal dominant inheritance caused by mutations in genes coding for sarcomeric and/or regulatory proteins expressed in cardiomyocytes. In a small cohort of HCM patients (n = 8), we searched for mutations in the two most common genes responsible for HCM and found four missense mutations in the MYH7 gene encoding cardiac β-myosin heavy chain (R204H, M493V, R719W, and R870H) and three mutations in the myosin-binding protein C3 gene (MYBPC3) including one missense (A848V) and two frameshift mutations (c.3713delTG and c.702ins26bp). The c.702ins26bp insertion resulted from the duplication of a 26-bp fragment in a 54-year-old female HCM patient presenting with clinical signs of heart failure due to diastolic dysfunction. Although such large duplications (> 10 bp) in the MYBPC3 gene are very rare and have been identified only in 4 families reported so far, the identical duplication mutation was found earlier in a Dutch patient, demonstrating that it may constitute a hitherto unknown founder mutation in central European populations. This observation underscores the significance of insertions into the coding sequence of the MYBPC3 gene for the development and pathogenesis of HCM.     

Dopaminergic system dysregulation in the mrsk2_KO mouse, an animal model of the Coffin-Lowry syndrome

The Coffin-Lowry syndrome, a rare syndromic form of X-linked mental retardation, is caused by loss-of-function mutations in the hRSK2 (RPS6KA3) gene. To further investigate RSK2 (90-kDa ribosomal S6 kinase) implication in cognitive processes, a mrsk2_KO mouse has previously been generated as an animal model of Coffin-Lowry syndrome. The aim of the present study was to identify possible neurochemical dysregulation associated with the behavioral and morphological abnormalities exhibited by mrsk2_KO mice. A cortical dopamine level increase was found in mrsk2_KO mice that was accompanied by an over-expression of dopamine receptor of type 2 and the dopamine transporter. We also detected an increase of total and phosphorylated extracellular regulated kinase that may be responsible for the increased level of tyrosine hydroxylase phosphorylation also observed. By taking into consideration previously reported data, our results strongly suggest that the dopaminergic dysregulation in mrsk2_KO mice may be caused, at least in part, by tyrosine hydroxylase hyperactivity. This cortical hyperdopaminergia may explain some non-cognitive but also cognitive alterations exhibited by mrsk2_KO mice.     

A novel EFNB1 mutation in a patient with craniofrontonasal syndrome and right hallux duplication

Craniofrontonasal syndrome (CFNS, MIM #304110) is a rare X-linked dominant developmental disorder that shows paradoxically greater severity in affected females than in affected males. Our female patient with frontonasal dysplasia, craniosynostosis and additional malformations was consistent with CFNS. EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases, is the only gene in which mutation is known to cause CFNS. Here, we describe 402 T > C, a novel de novo mutation on EFNB1. This mutation results in substitution of highly conserved isoleucine at 134th residue to threonine.     

Generation of mice with functional inactivation of talpid3, a gene first identified in chicken

Specification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid(3) chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid(3) chicken has a mutation in KIAA0586, which encodes a centrosomal protein required for the formation of primary cilia, which are sites of vertebrate Hh signalling. The highly conserved exons 11 and 12 of KIAA0586 are essential to rescue cilia in talpid(3) chicken mutants. We constitutively deleted these two exons to make a talpid3(-/-) mouse. Mutant mouse embryos lack primary cilia and, like talpid(3) chicken embryos, have face and neural tube defects but also defects in left/right asymmetry. Conditional deletion in mouse limb mesenchyme results in polydactyly and in brachydactyly and a failure of subperisoteal bone formation, defects that are attributable to abnormal sonic hedgehog and Indian hedgehog signalling, respectively. Like talpid(3) chicken limbs, the mutant mouse limbs are syndactylous with uneven digit spacing as reflected in altered Raldh2 expression, which is normally associated with interdigital mesenchyme. Both mouse and chicken mutant limb buds are broad and short. talpid3(-/-) mouse cells migrate more slowly than wild-type mouse cells, a change in cell behaviour that possibly contributes to altered limb bud morphogenesis. This genetic mouse model will facilitate further conditional approaches, epistatic experiments and open up investigation into the function of the novel talpid3 gene using the many resources available for mice.     

The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification.

The amplified DNA of HC50474, a Chinese hamster fibroblast cell line selected in three steps for high resistance to coformycin, consists chiefly of 150 copies of a large inverted duplication including the adenylate deaminase gene. Most if not all of these units are more than 2 x 120 kb long. The inverted duplication was first detected in the cells recovered from the second selection step, at the same chromosomal location as the first step amplified units. Its formation and amplification appear to be coupled since the second step cell line already contained 40 copies of this novel structure. Reamplification of the inverted duplication occurred at the third step of selection concomitant with the loss of amplified DNA acquired during the first step. The head-to-head junction has been formed by recombination within a recombinational hotspot described previously [Hyrien, O., Debatisse, M., Buttin, G. and Robert de Saint Vincent, B. (1987) EMBO J., 6, 2401-2408]. Sequences at the joint and in the corresponding wild-type region reveal that the crossover sites, one of which occurs in the putative promoter region of B2 repeat, are located at the top of significant stem-loop structures and that patchy homologies between the parental molecules on one side of the breakpoints allow alignment of these crossover sites. We present a model which explains the formation and amplification of this and other large inverted duplications by errors in DNA replication.     

Structure of an inverted duplication formed as a first step in a gene amplification event: implications for a model of gene amplification.

Inverted duplications have been observed to be a common feature of gene amplification in mammalian cells and appear to be generated as a primary event in the amplification process (Ford et al., 1985; Ford and Fried, 1986). The structural features of the amplified inverted duplication, containing the polyoma virus oncogene middle T-antigen, were analysed in transformed 3B rat cells. No unusual sequences such as transposition elements were detected at the site of the inversion. The inversion was generated by a simple illegitimate recombination event in which only a single nucleotide directly at the point of the inversion cannot be accounted for from the sequence of the two parental strands. Possible structural (hairpin formation) and sequence (rich AT) features may have been involved in the illegitimate recombination event at the inversion join. In the cellular DNA near one of its joins with polyoma virus DNA an unusual sequence of 198 bp composed of 99 consecutive purine-pyrimidine pairs has been detected. A model for the generation of amplified DNA containing inverted duplications is proposed.