CSNK2A1 Research Articles

Patient organization perspective: a research roadmap for Okur-Chung Neurodevelopmental Syndrome.

Okur-Chung neurodevelopmental syndrome (OCNDS) is an ultra-rare disorder caused by variants in the CSNK2A1 gene. CSNK2A1 encodes for the alpha subunit of casein kinase 2 (CK2), a serine/threonine kinase critical in neural development. CK2 is implicated in many human pathologies, including viral infections, cancer, inflammation, cardiovascular, neurodegenerative, and psychiatric diseases. However, the mechanism of action for the CSNK2A1 variants observed in OCNDS is not fully understood, although studies suggest a loss of function or altered substrate specificity. There are no approved treatments for OCNDS, and current treatments focus on symptom management. The CSNK2A1 Foundation was established in 2018 and aims to find a cure for OCNDS and provide support to affected individuals. OCNDS presents with symptoms at varying severity, including developmental delay/intellectual disabilities, autism, disrupted sleep, speech delays/inability to speak, short stature, and, in ~25% of cases, epilepsy. The foundation has developed a research toolbox that is readily available to researchers worldwide and has awarded ~$1 million in grant funding. These efforts have provided valuable insights into CK2 biology and the natural history of OCNDS. However, additional efforts are needed to fully characterize the disease mechanism and investigate potential treatment interventions. Continued investigation into CK2 and its role in neural development holds promise for a better understanding of OCNDS and related disorders in the future. To accelerate research, we have developed a research roadmap highlighting key focus areas of landscape analysis/toolbox expansion, biomarker development, and therapeutic testing through a series of steps that are nonlinear; we expect these efforts to guide decision-making for therapeutic exploration whether that be drug repurposing, gene therapy, novel drug discovery, or a combination. In this perspective article, we describe OCNDS and the CSNK2A1 gene, highlight gaps in OCNDS research, discuss the research roadmap, and offer the founder's perspective on our growth and future opportunities.

The chemical composition and the preservative, antimicrobial, and antioxidant effects of Thymus broussonetii Boiss. essential oil: an in vitro and in silico approach.

The aim of this study was to evaluate the antioxidant, antimicrobial, and preservative efficacy of Thymus broussonetii Boiss. essential oil (EO) in a topically applied formulation using a challenge test. The essential oil was extracted from the aerial part of T. broussonetii using hydrodistillation, and the obtained EO was further analyzed by gas chromatography/mass spectrometry (GC/MS). The antioxidant effect of the EO was evaluated using three methods: the inhibition of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene-linoleic acid, and the ferric reducing antioxidant power (FRAP) methods. The antimicrobial activity and the minimum inhibitory concentration (MIC) of this EO were assayed by the disk-diffusion method and the broth microdilution method, respectively. The preservative efficacy of T. broussonetii EO was assayed at 1% and 2% (v/w) in a topical cream formulation using a challenge test against standard-specific microorganisms recommended by the European Pharmacopoeia. Furthermore, the identified phytochemical compounds were docked for their effect on nicotinamide adenine dinucleotide phosphate oxidase, human casein kinase 1 alpha 1 (CSNK1A1), glycogen synthase kinase 3, Staphylococcus aureus nucleoside diphosphate kinase, Escherichia coli beta-ketoacyl-[acyl-carrier protein] synthase, Pseudomonas aeruginosa LasR ligand-binding domain, and sterol 14-alpha demethylase (CYP51) from Candida albicans. The ADME/toxicity was predicted by analyzing the absorption, distribution, metabolism, and excretion parameters. chemical composition of the EO revealed the presence of thymol (63.09%), p-cymene (11%), and γ-terpinene (8.99%) as the major components. The antioxidant assays revealed that the essential oil exhibited strong antioxidant activity, as indicated by the minimum inhibitory concentration IC50 (IC50 = 210 ± 0.3μg/mL for the DPPH assay, IC50 = 145 ± 0.1μg/mL for the β-carotene assay, and IC50 = 84 ± 0.21μg/mL for the FRAP assay) when compared to quercetin and butylated hydroxytoluene (BHT) as controls. The investigated essential oil exhibited important antimicrobial activity against all the tested microorganisms, and the MICs of the EO against bacteria and fungi were 0.02%-1%. Moreover, the EO of T. broussonetii evaluated at 2% (v/w) in a cream formulation succeeded in satisfying the A criteria for preservation efficacy against S. aureus, E. coli, and Aspergillus brasiliensis but exhibited less efficacy against P. aeruginosa (1.78 log reduction in the number of CFU/g after 7days of evaluation) and C. albicans (1.09 log reduction in the number of CFU/g after 14days of evaluation) when compared to the synthetic preservative phenoxyethanol 1% (v/w). In silico results showed that the antimicrobial activity of T. broussonetii EO is mostly attributed to thymol, terpinen-4-ol, and aromadendrene, while the antioxidant activity is attributed to thymol. These results indicate that the EO of T. broussonetii possesses important antimicrobial and antioxidant properties and can, therefore, be used as a natural preservative ingredient in the cosmetic industry.

Discovery and Exploration of Protein Kinase CK2 Binding Sites Using CK2α′Cys336Ser as an Exquisite Crystallographic Tool

The structural knowledge about protein kinase CK2 is dominated by crystal structures of human CK2α, the catalytic subunit of human CK2, and the product of the CSNK2A1 gene. In contrast, far fewer structures of CK2α′, its paralogous isoform and the product of the CSNK2A2 gene, have been published. However, according to a PDB survey, CK2α′ is the superior alternative for crystallographic studies because of the inherent potential of the single mutant CK2α′Cys336Ser to provide crystal structures with atomic resolution. In particular, a triclinic crystal form of CK2α′Cys336Ser is a robust tool to determine high-quality enzyme-ligand complex structures via soaking. In this work, further high-resolution CK2α′Cys336Ser structures in complex with selected ligands emphasizing this trend are described. In one of these structures, the “N-terminal segment site”, a small-molecule binding region never found in any eukaryotic protein kinase and holding the potential for the development of highly selective substrate-competitive CK2 inhibitors, was discovered. In order to also address the binding site for the non-catalytic subunit CK2β, which is inaccessible in these triclinic CK2α′Cys336Ser crystals for small molecules, a reliable path to a promising monoclinic crystal form of CK2α′Cys336Ser is presented. In summary, the quality of CK2α′Cys336Ser as an exquisite crystallographic tool is solidified.

Casein kinase 1 and 2 phosphorylate Argonaute proteins to regulate miRNA-mediated gene silencing.

MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs post-transcriptionally. Argonaute proteins are subjected to intensive regulation via various post-translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in Caenorhabditis elegans, two conserved serine/threonine kinases - casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2) - regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA-specific AGO protein ALG-1. We show that CK1A1 phosphorylates ALG-1 at sites S992 and S995, while CK2 phosphorylates ALG-1 at sites S988 and S998. Furthermore, we demonstrate that phospho-mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC-target RNA binding and silencing.

Knockdown of Csnk1a1 results in preimplantation developmental arrest in mice

Casein kinase 1, alpha 1 (CSNK1A1), is a member of the highly conserved serine/threonine protein kinase family. This study was established to analyze the expression and localization of CSNK1A1 and its function in early embryonic development in mice. Csnk1a1 mRNA and protein are expressed in multiple mouse tissues including the ovary. After ovulation and fertilization, Csnk1a1 mRNA and protein were detected in preimplantation embryos and their expression was highest in two-cell-stage embryos. CSNK1A1 protein was also mainly localized in the cytoplasm of preimplantation embryos. Moreover, knockdown of Csnk1a1 in zygotes led to a significant decrease in the rate of blastocyst formation. Furthermore, treatment of zygotes with the CSNK1A1-specific inhibitor D4476 also resulted in embryonic developmental arrest. These results provide the first evidence for a novel function of CSNK1A1 in early embryonic development in mice.

The Okur-Chung Neurodevelopmental Syndrome Mutation CK2K198R Leads to a Rewiring of Kinase Specificity.

Okur-Chung Neurodevelopmental Syndrome (OCNDS) is caused by heterozygous mutations to the CSNK2A1 gene, which encodes the alpha subunit of protein kinase CK2. The most frequently occurring mutation is lysine 198 to arginine (K198R). To investigate the impact of this mutation, we first generated a high-resolution phosphorylation motif of CK2WT, including the first characterization of specificity for tyrosine phosphorylation activity. A second high resolution motif representing CK2K198R substrate specificity was also generated. Here we report the impact of the OCNDS associated CK2K198R mutation. Contrary to prior speculation, the mutation does not result in a complete loss of function, but rather shifts the substrate specificity of the kinase. Broadly speaking the mutation leads to 1) a decreased preference for acidic residues in the +1 position, 2) a decreased preference for threonine phosphorylation, 3) an increased preference for tyrosine phosphorylation, and 4) an alteration of the tyrosine phosphorylation specificity motif. To further investigate the result of this mutation we have developed a probability-based scoring method, allowing us to predict shifts in phosphorylation in the K198R mutant relative to the wild type kinase. As an initial step we have applied the methodology to the set of axonally localized ion channels in an effort to uncover potential alterations of the phosphoproteome associated with the OCNDS disease condition.

Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2αLys198Arg.

Specific de novo mutations in the CSNK2A1 gene, which encodes CK2α, the catalytic subunit of protein kinase CK2, are considered as causative for the Okur-Chung neurodevelopmental syndrome (OCNDS). OCNDS is a rare congenital disease with a high phenotypic diversity ranging from neurodevelopmental disabilities to multi-systemic problems and characteristic facial features. A frequent OCNDS mutation is the exchange of Lys198 to Arg at the center of CK2α′s P+1 loop, a key element of substrate recognition. According to preliminary data recently made available, this mutation causes a significant shift of the substrate specificity of the enzyme. We expressed the CK2αLys198Arg recombinantly and characterized it biophysically and structurally. Using isothermal titration calorimetry (ITC), fluorescence quenching and differential scanning fluorimetry (Thermofluor), we found that the mutation does not affect the interaction with CK2β, the non-catalytic CK2 subunit, and that the thermal stability of the protein is even slightly increased. However, a CK2αLys198Arg crystal structure and its comparison with wild-type structures revealed a significant shift of the anion binding site harboured by the P+1 loop. This observation supports the notion that the Lys198Arg mutation causes an alteration of substrate specificity which we underpinned here with enzymological data.

Clinical Features of Okur-Chung Neurodevelopmental Syndrome: Case Report and Literature Review

Introduction: Autosomal dominant pathogenic variations in the CSNK2A1 gene cause Okur-Chung neurodevelopmental syndrome (OCNDS). Methods: The proband and her parents were examined thoroughly and observed for any issues related to OCNDS. Furthermore, peripheral blood samples were collected from each subject for further investigations. Whole-exome sequencing identified a pathogenic variant in CSNK2A1 (NM_001895: c.62G>A, p.R21Q; rs1402734448). Results: The proband has global developmental delay, speech disorders, epilepsy, and behavioral issues. Despite the previously reported cases, she manifested both atonic and myoclonic seizures simultaneously. Lastly, we provide a review of the reported cases with OCNDS. Discussion: p.R21Q causes OCNDS. Further studies are highly recommended concerning this mutation to validate the results of this study and expand the knowledge regarding CSNK2A1 and the phenotypic spectrum of OCNDS.

Abstract 2868: A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro

Abstract Introduction: Ovarian cancer recurrence and survival rates have remained stagnant over time with current therapeutics displaying low efficacy and often rendered ineffective by degradation. This work presents novel fusogenic peptides that will electrostatically complex with siRNAs and cause a pH-responsive insertion into the endosomal membrane, resulting in destabilization and breakdown allowing for release of siRNAs into the cytoplasm of ovarian cancer cells. Delivery of bioactive siRNAs targeting the CSNK2A1 gene in ovarian cancer cells is hypothesized to reduce cell viability, migration, and invasion. Experimental Procedures: Peptide formulations were complexed with non-targeting siRNAs (siNT) and CSNK2A1 siRNAs (siCSNK2A1) at various N:P ratios for all procedures. Analysis of complex formation was completed through gel-shift assays and dynamic light scattering. After characterization of the complexes, each formulation without siRNA was delivered to OVCAR-3 and CAOV-3 cells for cytotoxicity analysis measured via MTS assay. Observation of cellular uptake and endosomal escape was completed through fluorescent imaging after delivering each peptide formulation complexed with Cy3-labeled siNT (Cy3-siNT) and performing early endosome antigen-1 staining. To confirm bioactivity of targeted siRNAs, complexes with siCSNK2A1 were delivered and gene silencing was analyzed via western blotting. Results: Complexation of siRNAs with each formulation was achieved at N:P ratios of 40:1 and greater. Each of the three peptide formulations were able to form uniform nanocomplexes, at 195 nm, 111 nm, and 59 nm with 8.25 mV, -14.56 mV, and -18.42 mV surface charges respectively, and did not display vehicle cytotoxicity at an 80:1 ratio. Each peptide also mediated siRNA uptake into ovarian cancer cells, with fluorescent siRNA signal increasing with increasing N:P ratios. Of note, we observed that Cy3-siRNA appeared separate from the endosome, indicative of endosomal escape, which was seen in several peptide formulations with both CAOV-3 and OVCAR-3 cell lines. Confirmation of bioactive delivery of siCSNK2A1 is shown through western blot imaging, demonstrating knockdown of CSNK2A1 in comparison to cells treated with siNT alone or left untreated. Conclusion: Our novel fusogenic peptides have shown the ability to complex with and deliver siRNAs into ovarian cancer cells. Each peptide mediates endosomal escape activity, with varying degrees of effectiveness, and silencing of CSNK2A1 as measured by western blot. Further study into downstream effects of CSNK2A1 is ongoing. Future work includes optimization of a single fusogenic sequence for optimal delivery of siRNAs and development of a novel targeting peptide to increase cancer targeting and uptake efficiency. Acknowledgements: This research was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740. Citation Format: Timothy Samec, Jessica Boulos, Serena Gilmore, Angela Alexander-Bryant. A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2868.

Dual molecular diagnosis of tricho-rhino-phalangeal syndrome type I and Okur-Chung neurodevelopmental syndrome in one Chinese patient: a case report

BackgroundOkur-Chung neurodevelopmental syndrome (OCNDS) and tricho-rhino-phalangeal syndrome type I (TRPSI) are rare Mendelian diseases. OCNDS is caused by CSNK2A1 gene variants and TRPSI is caused by the TRPS1gene. However, to have two Mendelian diseases in one patient is even rarer.Case presentationA 6-year-10-month-old boy characterized by special facial features, short stature and mental retardation was referred to our pediatric endocrinology department. Whole-exome sequencing (WES) was done to detect the molecular basis of his disease. This patient was confirmed to carry two variants in the CSNK2A1 gene and one in the TRPS1 gene. The variant in the CSNK2A1 gene was vertically transmitted from his father, and the variant in TRPS1 gene from his mother. These two variants are classified as pathogenic and the causes of the presentation in this child. This patient’s father and mother have subsequently been diagnosed as having OCNDS and TRPSI respectively.ConclusionThis is the first reported case of a dual molecular diagnosis of tricho-rhino-phalangeal syndrome type I and Okur-Chung neurodevelopmental syndrome in the same patient. This patient is the first published example of vertical transmission of this recurrent CSN2A1 variant from parent to child. A novel variant in the TRPS1 gene that is pathogenic was also identified. In conclusion, identification of the variants in this patient expands the phenotypes and molecular basis of dual Mendelian diseases.

Okur-Chung neurodevelopmental syndrome: Eight additional cases with implications on phenotype and genotype expansion.

Okur-Chung syndrome is a neurodevelopmental condition attributed to germline CSNK2A1 pathogenic missense variants. We present 8 unreported subjects with the above syndrome, who have recognizable dysmorphism, varying degrees of developmental delay and multisystem involvement. Together with 6 previously reported cases, we present a case series of 7 female and 7 male subjects, highlighting the recognizable facial features of the syndrome (microcephaly, hypertelorism, epicanthic fold, ptosis, arched eyebrows, low set ears, ear fold abnormality, broad nasal bridge and round face) as well as frequently occurring clinical features including neurodevelopmental delay (93%), gastrointestinal (57%), musculoskeletal (57%) and immunological (43%) abnormalities. The variants reported in this study are evolutionary conserved and absent in the normal population. We observed that the CSNK2A1 gene is relatively intolerant to missense genetic changes, and most variants are within the protein kinase domain. All except 1 variant reported in this cohort are spatially located on the binding pocket of the holoenzyme. We further provide key recommendations on the management of Okur-Chung syndrome. To conclude, this is the second case series on Okur-Chung syndrome, and an in-depth review of the phenotypic features and genomic findings of the condition with suggestions on clinical management.

Extending the phenotype associated with the CSNK2A1-related Okur-Chung syndrome-A clinical study of 11 individuals.

Variants in the Protein Kinase CK2 alpha subunit, encoding the CSNK2A1 gene, have previously been reported in children with an intellectual disability and dysmorphic facial features syndrome: now termed the Okur-Chung neurodevelopmental syndrome. More recently, through trio-based exome sequencing undertaken by the Deciphering Developmental Disorders Study (DDD study), a further 11 children with de novo CSNK2A1 variants have been identified. We have undertaken detailed phenotyping of these patients. Consistent with previously reported patients, patients in this series had apparent intellectual disability, swallowing difficulties, and hypotonia. While there are some shared facial characteristics, the gestalt is neither consistent nor readily recognized. Congenital heart abnormalities were identified in nearly 30% of the patients, representing a newly recognized CSNK2A1 clinical association. Based upon the clinical findings from this study and the previously reported patients, we suggest an initial approach to the management of patients with this recently described intellectual disability syndrome.

Current diagnosis and treatment for myelodysplastc syndromes.

Genetic analysis of myelodysplastic syndrome (MDS) using next-generation sequencing yields medcially important information, showing gene mutations in 90% of MDS cases. The World Health Organization (WHO) classification was revised in 2016 to incorporate SF3B1 gene mutations, frequently seen in MDS with ringed sideroblasts, into the diagnostic criteria. Unlike the poor prognosis seen in cases with ASXL1, EZH2, RUNX1 and in particular, TP53 MDS-related mutations, SF3B1 gene mutations show a favorable prognosis. In low-risk patients such as these, darbepoetin treatment is an option. Moreover, the CSNK1A1 gene is known to play a role in the mechanism of action of lenalidomide. Hematopoietic stem cell transplantation is the only curable treatment for MDS, but azacitidine (AZA) is administered to high-risk patients who are not candidates for transplantation, a situation that remains unchanged. Even with allogeneic hematopoietic cell transplantation, the prognosis is poor with TET2, DNMT3A, ASXL1, RUNX1, and TP53 mutations, with survival time being significantly shorter with TP53 and PTPN11 mutations, regardless of the responsiveness to AZA. In the case of TP53 mutations, prognosis is poor for both hematopoietic stem cell transplantation and AZA treatment, although, patients with TP53 mutations have been shown to respond favorably to decitabine administration for 10 days. It is thought that the importance of genetic screening and its role in treatment decisions for MDS will further increase with time.

Gene mutations in myelodysplastic syndromes.

Recent progress in next generation sequencing technologies has enabled us to investigate somatic mutations throughout the coding regions of myelodysplastic syndromes (MDS), and have likely provided us with almost the entire spectrum of driver mutations responsible for this disease. As shown by numerous recent studies, such frequent mutations play an important role in the leukemogenesis of MDS. In the first part of this review, we comprehensively describe the characteristics and frequencies of the most prevalent mutations already confirmed as drivers in MDS. Second, we focus on the associations between karyotypic abnormalities and somatic mutations involving chromosomes 5 and 7. In particular, CSNK1A1 mutations are the most recently identified among the 5q- syndromes. Finally, we summarize recently reported findings on the functional effects of frequent splice factor mutations on splicing defects, which have been clarified by multiple institutions.

Targeting Protein Kinase CK2 Suppresses Prosurvival Signaling Pathways and Growth of Glioblastoma

Gliomas are the most frequently occurring primary malignancies in the brain, and glioblastoma is the most aggressive of these tumors. Protein kinase CK2 is composed of two catalytic subunits (α and/or α') and two β regulatory subunits. CK2 suppresses apoptosis, promotes neoangiogenesis, and enhances activation of the JAK/STAT, NF-κB, PI3K/AKT, Hsp90, Wnt, and Hedgehog pathways. Aberrant activation of the NF-κB, PI3K/AKT, and JAK/STAT-3 pathways is implicated in glioblastoma progression. As CK2 is involved in their activation, the expression and function of CK2 in glioblastoma was evaluated. Analysis of 537 glioblastomas from The Cancer Genome Atlas Project demonstrates the CSNK2A1 gene, encoding CK2α, has gene dosage gains in glioblastoma (33.7%), and is significantly associated with the classical glioblastoma subtype. Inhibition of CK2 activity by CX-4945, a selective CK2 inhibitor, or CK2 knockdown by siRNA suppresses activation of the JAK/STAT, NF-κB, and AKT pathways and downstream gene expression in human glioblastoma xenografts. On a functional level, CX-4945 treatment decreases the adhesion and migration of glioblastoma cells, in part through inhibition of integrin β1 and α4 expression. In vivo, CX-4945 inhibits activation of STAT-3, NF-κB p65, and AKT, and promotes survival of mice with intracranial human glioblastoma xenografts. CK2 inhibitors may be considered for treatment of patients with glioblastoma.

CSNK1A1 (casein kinase 1, alpha 1)

Review on CSNK1A1 (casein kinase 1, alpha 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Influences of Genetic Variation on Fetal Hemoglobin

Fetal hemoglobin (HbF) plays a dominant role in ameliorating morbidity and mortality of hemoglobinopathies. The authors performed a replicated study following the genome-wide association study (GWAS) guidelines to identify the genetic mechanics that influence HbF. The authors recruited and phenotyped 312 unrelated β-thalassemia subjects. Single-nucleotide polymorphism (SNP) analysis was performed by using polymerase chain reaction (PCR)/restriction enzymes. Four independent regions of interest were identified: HBS1L-MYB intergenic region, BCL11A locus, β-globin gene cluster, and the CSNK2A1 gene. There were 10 SNPs associated with HbF levels. In addition, haplotypes of HBS1L-MYB and BCL11A were identified and showed association with HbF production. Three independent regions, including HBS1L-MYB intergenic region, BCL11A locus, and β-globin gene cluster, were associated with HbF levels. This study can significantly improve the GWAS findings in Chinese cohorts and is useful for further research in the field of common predictors of the erythropoiesis.

Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma

Background. Previous knowledge of cervical lymph node compromise may be crucial to choose the best treatment strategy in oral squamous cell carcinoma (OSCC). Here we propose a set four genes, whose mRNA expression in the primary tumor predicts nodal status in OSCC, excluding tongue. Material and methods. We identified differentially expressed genes in OSCC with and without compromised lymph nodes using Differential Display RT-PCR. Known genes were chosen to be validated by means of Northern blotting or real time RT-PCR (qRT-PCR). Thereafter we constructed a Nodal Index (NI) using discriminant analysis in a learning set of 35 patients, which was further validated in a second independent group of 20 patients. Results. Of the 63 differentially expressed known genes identified comparing three lymph node positive (pN +) and three negative (pN0) primary tumors, 23 were analyzed by Northern analysis or RT-PCR in 49 primary tumors. Six genes confirmed as differentially expressed were used to construct a NI, as the best set predictive of lymph nodal status, with the final result including four genes. The NI was able to correctly classify 32 of 35 patients comprising the learning group (88.6%; p = 0.009). Casein kinase 1alpha1 and scavenger receptor class B, member 2 were found to be up regulated in pN + group in contrast to small proline-rich protein 2B and Ras-GTPase activating protein SH3 domain-binding protein 2 which were upregulated in the pN0 group. We validated further our NI in an independent set of 20 primary tumors, 11 of them pN0 and nine pN + with an accuracy of 80.0% (p = 0.012). Conclusions. The NI was an independent predictor of compromised lymph nodes, taking into the consideration tumor size and histological grade. The genes identified here that integrate our “Nodal Index” model are predictive of lymph node metastasis in OSCC.

The catalytic subunit α′ gene of human protein kinase CK2 (CSNK2A2): Genomic organization, promoter identification and determination of Ets1 as a key regulator

The human genome contains four protein kinase CK2 loci, enclosing three active genes coding for the catalytic subunits alpha and alpha' and the regulatory subunit beta, and a processed alpha subunit pseudogene. Extensive structure and transcriptional control data of the genes are available, except for the CK2alpha' gene (CSNK2A2). Using in silico and experimental approaches, we find CSNK2A2 to be located on the long arm of chromosome 16 (in contrast to published data), to span 40kb and to consist of 12 exons, with the translational start in Exon 1 and the stop in Exon 11. Exon/intron boundaries conform to the gt/ag rule, and various potential polyadenylation signals determine transcript species with lengths of 1.7-5.7 kb. The upstream region of the gene displays housekeeping characteristics, lacking a TATA box and possessing several GC boxes as well as a CpG island around Exon 1. According to reporter gene assay results, the promoter activity ranges from -1308 to 197 with the highest activity in region -396 to -129. This region contains binding motifs for various transcription factors, including NFkappaB, Sp and Ets family members. Site-directed mutagenesis indicates that the Ets motifs play, in cooperation with Sp motif clusters, a central role in regulating CK2alpha' gene transcription. A similar control has been described for the transcription of the CK2alpha and CK2beta genes so that the presented data are compatible with the assumption of a coordinate transcriptional regulation of all three active human CK2 genes decisively determined by Ets family members.

Search for mutations involved in human globozoospermia*

Globozoospermia is a severe form of teratozoospermia characterized by round-headed sperm with an absence of acrosomes. Family cases of globozoopermia suggest that this pathology has genetic origins, but the mode of inheritance remains unknown. So far, no responsible genes have been identified. Recently, a mouse lacking the casein kinase IIalpha' (encoded by the Csnk2a2 gene) was described. This mutant mouse presents a single phenotype reminiscent of that seen in human globozoospermia. Interestingly, the fission yeast orthologue (orb5) exhibits, when mutated, a spherical phenotype. Casein kinase II is a heterotetramer, composed of two catalytic subunits alpha or alpha' and two regulatory beta subunits (encoded by the Csnk2b gene). Based on the evolution conservation, phenotypes observed in mouse and yeast mutant and the structure of casein kinase II, we analysed Csnk2a2 and Csnk2b genes in six patients with globozoospermia and 10 fertile controls. Genomic DNA was extracted from peripheral blood and PCR was performed to amplify Csnk2a2 and Csnk2b genes before sequencing. No mutation was identified among these six patients. Further work is needed, with a larger patient data set, to identify putative genes involved in this form of male infertility.