Impaired muscle morphology in a Drosophila model of myosin storage myopathy was supressed by overexpression of an E3 ubiquitin ligase

Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/β-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases of myosin storage myopathy. We have developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of the R1845W mutation. This study shows that a higher expression level of the mutated allele is concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was suppressed by expression of Thin (herein referred to as Abba), an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities, including extensive loss of thick filaments with selective A-band loss, and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Furthermore, the impaired muscle morphology associated with the mutant allele was suppressed by expression of Abba. These findings suggest that modification of the ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients

A cluster of genes located in 1p36 are down-regulated in neuroblastomas with poor prognosis, but not due to CpG island methylation

BACKGROUND: A common feature of neuroblastoma tumours are partial deletions of the short arm of chromosome 1 (1p-deletions). This is indicative of a neuroblastoma tumour suppressor gene being located in the region. Several groups including our have been studying candidate neuroblastoma genes in the region, but no gene/genes have yet been found that fulfil the criteria for being a neuroblastoma tumour suppressor. Since frequent mutations have not been detected, we have now analyzed the expression and promoter CpG island methylation status of the genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 in the 1p36.22 region in order to find an explanation for a possible down-regulation of this region. RESULTS: The current study shows that gene transcripts in high stage neuroblastoma tumours are significantly down-regulated compared to those in low stage tumours in the 1p36.22 region. CpG island methylation does not seem to be the mechanism of down-regulation for most of the genes tested, since no methylation was detected in the fragments analyzed. One exception is the CpG island of APITD1. Methylation of this gene is also seen in blood from control individuals and is therefore not believed to participate in tumour development. CONCLUSION: The genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 are down-regulated in high stage NB tumours, a feature that can not be explained by CpG island methylation

Genetic alterations in Scandinavian neuroblastoma tumors

The results presented here derive from our efforts to find genes and chromosomal regions of interest in the formation and progression of the childhood tumor neuroblastoma.Neuroblastoma is the most common extracranial solid tumor of childhood. It is a tumor of the postganglionic sympathetic nervous system, and clinically, the hallmark of neuroblastoma is its heterogeneity.In order to characterize chromosomal regions of interest the methods loss of heterozygozity (LOH) studies using PCR-based polymorphic markers, representational difference analysis (RDA), somatic cell hybrid mapping, fluorescent in situ hybridization (FISH), radiation hybrid mapping and physical mapping by construction of a BAC-contig have been used. For mutation analysis of genes the methods single strand conformation polymorphism (SSCP), heteroduplex (HD) and DNA-sequencing have been used, and the expression studies of candidate genes have been performed using RT-PCR.In the Scandinavian neuroblastoma tumor material we have been able to show that deletions of chromosome arm 3p is the second most common deleted chromosomal region (16%) next to 1p (22%). We could also see a difference in clinical outcome between patients with tumors displaying deletion of the entire chromosome 3 versus the ones with tumors displaying deletions of region 3p only. The ones with 3p-deletion have all died of disease whereas the ones with entire chromosome 3 loss are alive and well. We have also characterized a region on chromosome 1, 1p36, shown, by others and us, to be commonly deleted in neuroblastoma tumors. A critical region of app. 25 cM between genetic markers D1S80 and D1S244 could be defined. We have also by using combined LOH-data from both neuroblastoma and germ cell tumors defined a region of common deletion for both tumor types, this region could be defined by markers D1S508 and D1S244 and is app. 5 cM.Also some 1p36 putative tumor suppressor genes, i.e. CDC2L1, TP73 and CORT, have been fine mapped and tested for expression and mutations in neuroblastomas. No evidence for any of them to be the 1p36 neuroblastoma tumor suppressor gene has however been discovered

Digitoxin Affects Metabolism, ROS Production and Proliferation in Pancreatic Cancer Cells Differently Depending on the Cell Phenotype

Digitoxin has repeatedly shown to have negative effects on cancer cell viability; however, the actual mechanism is still unknown. In this study, we investigated the effects of digitoxin (1-100 nM) in four pancreatic cancer cell lines, BxPC-3, CFPAC-1, Panc-1, and AsPC-1. The cell lines differ in their KRAS/BRAF mutational status and primary tumor or metastasis origin. We could detect differences in the basal rates of cell proliferation, glycolysis, and ROS production, giving the cell lines different phenotypes. Digitoxin treatment induced apoptosis in all four cell lines, but to different degrees. Cells derived from primary tumors (Panc-1 and BxPC-3) were highly proliferating with a high proportion of cells in the S/G2 phase, and were more sensitive to digitoxin treatment than the cell lines derived from metastases (CFPAC-1 and AsPC-1), with a high proportion of cells in G0/G1. In addition, the effects of digitoxin on the rate of glycolysis, ROS production, and proliferation were dependent on the basal metabolism and origin of the cells. The KRAS downstream signaling pathways were not altered by digitoxin treatment, thus the effects exerted by digitoxin were probably disconnected from these signaling pathways. We conclude that digitoxin is a promising treatment in highly proliferating pancreatic tumors.CC BY 4.0Correspondence: [email protected]: This research was funded by Assar Gabrielsson Foundation, grant FB19-80.</p

Na+/K+‑ATPase subunit α3 expression is associated with the efficacy of digitoxin treatment in pancreatic cancer cells

The alpha subunits (ATP1A1-3) of Na+/K+-ATPase binds digitoxin with varying affinity. The expression levels of these subunits dictate the anticancer effects of digitoxin. In the present study, three pancreatic cancer cell lines, AsPC-1, Panc-1 and CFPAC-1, were used to investigate the effects of digitoxin in relation to the expression of the subunits ATP1A1 and ATP1A3. Cell viability and intracellular calcium concentrations was measured in relation to the gene and protein expression of ATP1A1 and ATP1A3. Digitoxin was used to treat the cells at concentrations of 1-100 nM, and the intracellular calcium concentrations increased in a concentration-dependent manner in the Panc-1 and in the CFPAC-1 cells with treatment at 100 nM. In the AsPC-1 cells only the supraphysiological concentration of digitoxin (100 nM) resulted in a decrease in the number of viable cells (unviable cells increased to 22%), whereas it had no effect on intracellular calcium levels. The number of viable Panc-1 and CFPAC-1 cells decreased after digitoxin treatment at 25-100 nM (unviable Panc-1 cells increased to 33-59%; unviable CFPAC-1 cells increased to 22-56%). Digitoxin treatment also affected the transcriptional expression of the ATP1A1 and ATP1A3 subunits. In Panc-1 cells, ATP1A3 gene expression was negatively associated with the digitoxin concentration (25-100 nM). In the AsPC-1 and CFPAC-1 cells, the expression of the ATP1A1 gene increased in the cells treated with the 100 nM digitoxin concentration. The protein expression of ATP1A1 and ATP1A3 was not altered with digitoxin treatment. The basal protein expression of ATP1A1 was high in the AsPC-1 and CFPAC-1 cells, compared to the Panc-1 cells, in contrast to the basal expression of ATP1A3, which was higher in the Panc-1 cells, compared to the other pancreatic cancer cells used. On the whole, the present study demonstrates that the high expression of ATP1A3 renders pancreatic cancer cells more susceptible to digitoxin-induced cell death. The findings suggest that the expression of ATP1A3 may be used as a marker for tumor sensitivity to digitoxin treatment, where a high expression of ATP1A3 is favorable for the anticancer effects of digitoxin.CC BY-NC-ND 4.0Correspondence to: Dr Ferenc Szekeres, Department of Biomedicine, Translational Medicine, School of Health Sciences, University of Skövde, Högskolevägen 1, 54145 Skövde, Sweden E‑mail: [email protected] funding was received.</p

Biallelic MED27 variants lead to variable ponto-cerebello-lental degeneration with movement disorders

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants. Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 ± 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed. MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed. This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'. CC BY 4.0 DEED© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.Correspondence to: Mariasavina Severino Neuroradiology Unit, IRCCS Istituto Giannina Gaslini Via Gerolamo Gaslini 5, 16147 Genova, GE, Italy E-mail: [email protected] may also be addressed to: Reza Maroofian Department of Neuromuscular Diseases, University College London Queen Square Institute of Neurology Queen Square, London WC1N 3BG, UK E-mail: [email protected] of this research was possible thanks to the Deciphering Developmental Disorders (DDD) study. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003). R.K. was supported by the European Academy of Neurology Research Training Fellowship and Rosetrees Trust PhD Plus award (PhD2022\100042). J.R.L. was supported by the National Institute for Neurological Disorders and Stroke Research Program Award R35 NS105078 and the Baylor College of Medicine-GREGoR Program (NHGRI U01 HG001758). H.T. was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 608473. M.A.K. and R.S. were supported by a National Institute for Health Research professorship, Sir Jules Thorn Charitable Trust Award for Biomedical Research and the Rosetrees Trust. This study was also supported by the Wellcome Trust (WT093205MA and WT104033AIA), the Medical Research Council (H.H.), European Union’s Seventh Framework Programme (FP7/2007-2013, under grant agreement no. 2012-305121), the National Institute for Health Research (NIHR), University College London Hospitals (UCLH) and UCLH Biomedical Research Centre (BRC). For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. The NIHR Oxford Biomedical Research Centre Programme and a Wellcome Trust Core Award (203141/Z/16/Z). P.I. was supported by Foundation for Pediatric Research.</p

The loss of DLG2 isoform 7/8, but not isoform 2, is critical in advanced staged neuroblastoma

Background: Neuroblastoma is a childhood neural crest tumor showing large clinical and genetic heterogeneity, one form displaying 11q-deletion is very aggressive. It has been shown that 11q-deletion results in decreased expression of DLG2, a gene residing in the deleted region. DLG2 has a number of different isoforms with the main difference is the presence or absence of a L27 domain. The L27 domain containing DLG proteins can form complexes with CASK/MPP and LIN7 protein family members, which will control cell polarity and signaling. Methods: We evaluated the DLG gene family and the LIN7 gene family for their expression in differently INSS staged neuroblastoma from publically available data and primary tumors, we included two distinct DLG1 and DLG2 N-terminal transcript isoforms encoding L27 domains for their expression. Functionality of DLG2 isoforms and of LIN7A were evaluated in the 11q-deleted neuroblastoma cell line SKNAS. Results: In neuroblastoma only two DLG2 isoforms were expressed: isoform 2 and isoform 7/8. Using the array data we could determine that higher expression of DLG members that contain L27 domains correlated to better survival and prognosis. Whilst DLG1 showed a decrease in both isoforms with increased INSS stage, only the full length L27 containing DLG2 transcripts DLG2-isoform 7/8 showed a decrease in expression in high stage neuroblastoma. We could show that the protein encoded by DLG2-isoform 7 could bind to LIN7A, and increased DLG2-isoform 7 gene expression increased the expression of LIN7A, this reduced neuroblastoma cell proliferation and viability, with increased BAX/BCL2 ratio indicating increased apoptosis. Conclusion: We have provided evidence that gene expression of the L27 domain containing DLG2-isoform 7/8 but not L27 domain lacking DLG2-isoform 2 is disrupted in neuroblastoma, in particular in the aggressive subsets of tumors. The presence of the complete L27 domain allows for the binding to LIN7A, which will control cell polarity and signaling, thus affecting cancer cell viability. CC BY 4.0The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.© 2021, The Author(s).© 2021 BioMed Central Ltd unless otherwise stated. Part of Springer Nature.Correspondence: [email protected]</p

DLG2 impairs dsDNA break repair and maintains genome integrity in neuroblastoma

Background In primary neuroblastoma, deletions on chromosome 11q are known to result in an increase in the total number of chromosomal breaks. The DNA double-strand break repair pathways mediated by NHEJ are often upregulated in cancer. DLG2, a candidate tumor suppressor gene on chromosome 11q, has previously been implicated in DNA repair. Methods We evaluated an association between gene expression and neuroblastoma patient outcome, risk categorization, and 11q status using publicly available microarray data from independent neuroblastoma patient datasets. Functional studies were conducted using comet assay and H2AX phosphorylation in neuroblastoma cell lines and in the fruit fly with UVC-induced DNA breaks. Results We show that the NHEJ genes PARP1 and FEN1 are over expressed in neuroblastoma and restoration of DLG2 impairs their gene and protein expression. When exposed to UVC radiation, cells with DLG2 over expression show less DNA fragmentation and induce apoptosis in a p53 S46 dependent manner. We could also confirm that DLG2 over expression results in CHK1 phosphorylation consistent with previous reports of G2/M maintenance. Conclusions Taken together, we show that DLG2 over expression increases p53 mediated apoptosis in response to etoposide and UVC mediated genotoxicity and reduced DNA replication machinery.CC BY 4.0Corresponding author: E-mail address: [email protected] (S. Keane).We thank the Swedish Childhood Cancer Fund [PR2016–0060], Jane and Dan Olsson Foundation [2020–29], Assar Gabrielssons Foundation [FB20–13], Nilsson-Ehle Endowments, Kungliga Fysiografiska sällskapet i Lund and University of Skövde for financial support.</p

Low DLG2 gene expression, a link between 11q-deleted and MYCN-amplified neuroblastoma, causes forced cell cycle progression, and predicts poor patient survival

BACKGROUND: Neuroblastoma (NB) is a childhood neural crest tumor. There are two groups of aggressive NBs, one with MYCN amplification, and another with 11q chromosomal deletion; these chromosomal aberrations are generally mutually exclusive. The DLG2 gene resides in the 11q-deleted region, thus makes it an interesting NB candidate tumor suppressor gene. METHODS: We evaluated the association of DLG2 gene expression in NB with patient outcomes, stage and MYCN status, using online microarray data combining independent NB patient data sets. Functional studies were also conducted using NB cell models and the fruit fly. RESULTS: Using the array data we concluded that higher DLG2 expression was positively correlated to patient survival. We could also see that expression of DLG2 was inversely correlated with MYCN status and tumor stage. Cell proliferation was lowered in both 11q-normal and 11q-deleted NB cells after DLG2 over expression, and increased in 11q-normal NB cells after DLG2 silencing. Higher level of DLG2 increased the percentage of cells in the G2/M phase and decreased the percentage of cells in the G1 phase. We detected increased protein levels of Cyclin A and Cyclin B in fruit fly models either over expressing dMyc or with RNAi-silenced dmDLG, indicating that both events resulted in enhanced cell cycling. Induced MYCN expression in NB cells lowered DLG2 gene expression, which was confirmed in the fly; when dMyc was over expressed, the dmDLG protein level was lowered, indicating a link between Myc over expression and low dmDLG level. CONCLUSION: We conclude that low DLG2 expression level forces cell cycle progression, and that it predicts poor NB patient survival. The low DLG2 expression level could be caused by either MYCN-amplification or 11q-deletion. Video Abstract.CC BY 4.0</p