Molecular Genetic Analysis of Survival Motor Neuron Gene in 460 Turkish Cases with Suspicious Spinal Muscular Atrophy Disease

How to Cite This Article: Rashnonejad A, Onay H, Atik T, Atan Sahin O, Gokben S, Tekgul H, Ozkinay F. Molecular Genetic Analysis of Survival Motor Neuron Gene in 460 Turkish Cases with Suspicious Spinal Muscular Atrophy Disease. Iran J Child Neurol. Autumn 2016; 10(4):30-35.AbstractObjectiveTo describe 12 yr experience of molecular genetic diagnosis of Spinal Muscular Atrophy (SMA) in 460 cases of Turkish patients. Materials & MethodsA retrospective analysis was performed on data from 460 cases, referred to Medical Genetics Laboratory, Ege University’s Hospital, Izmir, Turkey, prediagnosed as SMA or with family history of SMA between 2003 and 2014.The PCR-restriction fragment length polymorphism (RFLP) and the Multiplex ligation–dependent probe amplification (MLPA) analysis were performed to detect the survival motor neuron (SMN)1 deletions and to estimate SMN1 and SMN2 gene copy numbers. ResultsUsing PCR-RFLP test, 159 of 324 postnatal and 18 of 77 prenatal cases were detected to have SMN1 deletions. From positive samples, 88.13% had a homozygous deletion in both exon 7 and exon 8 of SMN1. Using MLPA, 54.5% of families revealed heterozygous deletions of SMN1, and 2 or 3 copies of SMN2, suggesting a healthy SMA carrier. Among patients referred for SMA testing, the annual percentage of patients diagnosed as SMA has decreased gradually from 90.62% (2003) down to 20.83% (2014). ConclusionAlthough PCR-RFLP method is a reliable test for SMA screening, MLPA is a necessary additional test and provide relevant data for genetic counseling of families having previously affected child. The gradual decrease in the percentage of patients molecularly diagnosed as SMA shows that clinicians have begun to use genetic tests in the differential diagnosis of muscular atrophies. Cost and availability of these genetic tests has greatly attributed to their use.   References1. Brichta L, Holker I, Haug K, Klockgether T, Wirth B. In vivo activation of SMN in spinal muscular atrophy carriers and patients treated with valprotae. Ann Neurol 2006;59:970-5.2. Prior TW, Krainer AR, Hua Y, Swoboda KJ, Snyder PC, Bridgeman SJ, et al. A positive modifier of spinal muscular atrophy in the SMN2 gene. Am J Hum Genet 2009;85:408-13.3. Striano P, Boccella P, Sarappa C, Striano S. Spinal muscular atrophy and progressive myoclonic epilepsy: one case report and characteristics of the epileptic syndrome. Seizure 2004;13:582-6.4. Wirth B. An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mutat 2000;15:228-37.5. Van der Steege G, Grootscholten PM, Van der Vlies P, Draaijers TG, Osinga J, Cobben JM, et al. PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 1995;345:985-6.6. Rekik I, Boukhris A, Ketata S, Amri M, Essid N, Feki I, et al. Deletion analysis of SMN and NAIP genes in Tunisian patients with spinal muscular atrophy. Ann Indian Acad Neurol 2013;16:57-61.7. de Souza Godinho FM, Bock H, Gheno TC, Saraiva-Pereira ML. Molecular Analysis of Spinal Muscular Atrophy: A genotyping protocol based on TaqMan realtime PCR. Genet Mol Biol 2012;35:955-9.8. Burghes AH. 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Prenatal prediction of spinal muscular atrophy. Experience with linkage studies and consequences of present SMN deletion analysis. Eur J Hum Genet 1996;4:231-6.15. Miskovic M, Lalic T, Radivojevic D, Cirkovic S, Ostojic S, Guc-Scekic M. Ten years of experience in molecular prenatal diagnosis and carrier testing for spinal muscular atrophy among families from Serbia. Int J Gynaecol Obstet 2014;124:55-8.16. Mailman MD, Heinz JW, Papp AC, Snyder PJ, Sedra MS, Burghes AHM, Wirth B, Prior TW. Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2. Genet Med 2002;4:20–26.17. Ogino S, Leonard DG, Rennert H, Ewens WJ, Wilson RB. Genetic risk assessment in carrier testing for spinal muscular atrophy. Am J Med Genet 2002;110:301-7.18. Wirth B. An update on the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mutat 2000;15:228–37

Spinal musküler atrofi'li farelerde, adeno ilişkili virüs serotipi 9 vektörü kullanarak prenatal gen tedavisi

SMN gen ekspresyonunun intrauterin (IU) şeklinde düzeltilmesi SMA hastalığın tedavisinde kritik bir yaklaşım olabilir. Bu çalışmada, fare embriyolarına rAAV9-SMN vektörün intraserebroventriküler (ICV) enjeksiyonu ile hastalığın semptomlarının yok edilmesi araştırılımıştır. Gen tedavisi çalışması için, 4 x 10^10 vgk ss ya da scAAV9-SMN vektörlerden ICV enjeksiyonu ile sırasıyla, 44 ve 39 adet SMA fetüsüne aktarılmıştır. Doğumdan sonra, Enjekte edilmiş embriyoların SMN protein ekspresyonu, sağ kalım süreleri ve hastalık semptomlarının iyileşmesi araştırılmıştır. IU şeklinde enjekte edilmiş SMA'lı fetüslerin canlı doğum oranı yaklaşık %43.85'dir ki total hayatta kalma oranından (%69.41) daha düşüktür. Ss ve scAAV9-SMN vektörlerinin prenatal aktarılması SMA'lı fetüslerinin yaşam sürelerini artırarak sırasıyla 63±30 ve 105±50 gün hayatta kalmalarını sağlamıştır. Her iki çalışma grubunda motor nöron sayısı ve kas patolojisinde iyileştirmeler saptanmıştır, ancak scAAV9-SMN vektörünün etkinliği ss-vektörden daha fazla belirlenmiştir. Sonuç olarak, rAAV9-SMN vektörlerin intrauterin ICV enjeksiyonu SMA hastalığın tedavisi için yeni bir terapötik yaklaşım sunabilir. Ancak, bu yöntemin anne ve embriyo için güvenirliğini incelemesi konusunda fazla araştırma yapılmalıdır.Intrauterine (IU) correction of SMN gene expression seems can be critical in the treatment of SMA disease. In this study, using Intracerebroventricular (ICV) delivery of rAAV9-SMN into mice embryos correction of disease related symptoms was investigated. For gene therapy study, 44 and 39 SMA fetuses received 4 x 10^10 vgc ss and scAAV9-SMN via ICV injection, respectively. The SMN protein expression, survival rate, and improving disease symptoms of injected mice was investigated after birth. The survival rate of IU injected SMA fetuses was around 43.85% that was lower than total survival rate (69.41%). Prenatally delivery of both ss and scAAV9-SMN vectors cause to increased lifespan of injected SMA fetuses, respectively, 63±30 ve 105±50 days. The muscle pathology and number of the motor neurons have been improved in both study groups, however, the efficiency of the scAAV9-SMN vector was determined more than ss-vector. Thus, intrauterine administration of rAAV9-SMN via ICV injection may provide a new therapeutic approach for treating SMA disease. However, further analysis should be done for investigating the safety of this method for mother and embryo

Pyocyanine Biosynthetic Genes in Clinical and Environmental Isolates of Pseudomonas aeruginosa and Detection of Pyocyanine’s Antimicrobial Effects with or without Colloidal Silver Nanoparticles

Objective: Pyocyanine plays an important role in the pathogenesis of Pseudomonas aeruginosa, (P. aeruginosa) and is known to have inhibitory and bactericidal effects. This study has aimed to detect the phenazine biosynthetic operon (phz ABCDEFG) and two phenazine modifying genes (phzM and phzS) by polymerase chain reaction (PCR) and detection of its possible protein bands by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). The antimicrobial effects of pyocyanine alone and mixed with colloidal silver nanoparticles were studied.Materials and Methods: In this descriptive study, clinical and environmental species of P. aeruginosa were isolated by thioglycollate medium culture and cetrimide agar, respectively. The existence of a phenazine biosynthetic operon and two phenazine modifying genes as well as their protein products were confirmed by PCR and SDS-PAGE, respectively. Pyocyanine was extracted with chloroform and its antimicrobial effects against bacteria such as; Escherichia coli (E. coli), P. aeruginosaand Staphylococcus aureus (S. aureus) bacteria and yeast Candida albicans (C. albicans) were tested using well, spot and disk diffusion methods.Results: In this study, 3 out of 48 clinical strains were unable to produce pyocyanine on cetrimide and Mueller Hinton (MH) agar. Two strains did not have phenazine modifying gene bands. Another strain did not have the possible protein band of the phzM gene. Pyocyanine had antimicrobial effects against the microbial strains, which increased in the presence of silver nanoparticles.Conclusion: According to the results of the present study, some P. aeruginosa strains are unable to produce pyocyanine due to the absence of the phzM or phzS genes. Therefore, these genes have an important role in pyocyanine production in P. aeruginosa. Pyocyanine shows synergistic antimicrobial effects in the presence of silver nanoparticles against microbial strains

The Effect of Pre-Postnatal Nicotine Exposure on Types and Levels of Plasma Sialic Acids in Rats

Nicotine, is an alkaloid compound consisting of pyridine and pyrolidine ring. Its closed formula is C10H4N2. During smoking peak plasma concentration changes from 25 to 50 ng/mL. Its half-life is 1–2 h. Nicotine is metabolized primarily in the liver and excreted by the kidneys. The plasma concentration of the nicotine metabolite cotinine is 10 times more than nicotine and its half-life is longer around 15 to 20 h. Cotinine can be found in both amniotic fluid and umbilical cord blood given that it passes across the placental barrier. Adverse effects of nicotine and its metabolites on the fetus are suggested but have not been proven by scientific explanations till now. Sialic acid (Sia) is a modified nine-carbon sugar. They are located on the last end of the glycan chains located in the glycoconjugate structures. They organize a wide range of relationships between cells and their environment such as cellular recognition, adhesion, transmission, differentiation and aging. The aim of this study is to determine the possible changes in the sialic acid levels and types in the plasma after different levels of nicotine applied to Swiss Albino rats in order to assess the effect of long-term per oral nicotine administration

Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene

WOS: 000368064600004PubMed ID: 26607476Recombinant AAV (rAAV) vectors are a suitable vector for gene therapy studies because of desired characteristics such as low immunogenicity, transfection of non-dividing and dividing cells, and long-term expression of the transgene. In this study, the large-scale production of single stranded (ss) and self-complementary (sc) AAV9 carrying the human survival motor neuron (SMN) gene (AAV9-SMN) suitable for in vivo gene therapy studies of SMA was described. SMN cDNA has been cloned into pAAV-CB6-PI and pAAVsc-CB6-PI with and without its specific UTRs, respectively. Both plasmids bear CMV enhancer/beta-actin (CB) promoter, CMV IE enhancer, and polyadenylation signal sequences. 2.5 mu g of constructed pAAV-CB6-PI-SMN and pAAVsc-CB6-PI-SMN cause to, respectively, 4.853- and 2.321-fold increases in SMN protein levels in transfected cells compared to untransfected cells. Ss and scAAV9-SMN vectors were also produced from these plasmids by transient transfection of HEK293 cells using CaCl2 solution. The silver staining and electron microscopy analysis demonstrated good quality of both isolated vectors, ssAAV9-SMN and scAAV9-SMN, with the titers of 2.00E+13 and 1.00E+13 GC/ml. The results of this study show that, the plasmid containing UTR elements causes to twice more SMN gene expression in transfected cells. The quality control results show that both produced ss and scAAV9-SMN are suitable for in vivo studies.University of Massachusetts Medical School; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01NS076991-01, 1R21DA031952-01A, 2P01HL059407, 1P01AI100263-01]; Will Foundation; Jacob's Cure; NTSAD Foundation; Canavan Foundation; National High Technology Research and Development Program ("863" Program) of ChinaNational High Technology Research and Development Program of China [2012AA020810]This work is supported by University of Massachusetts Medical School (an internal grant), National Institutes of Health (R01NS076991-01, 1R21DA031952-01A, 2P01HL059407, 1P01AI100263-01), the Will Foundation, Jacob's Cure, NTSAD Foundation, Canavan Foundation, and partial support from a grant from the National High Technology Research and Development Program ("863" Program) of China (2012AA020810). G.G. is a co-founder of Voyager Therapeutics and holds equity in the company. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics and other pharmaceutical companies

Hematologic Malignancies in Children with Down Syndrome: Transient Myeloproliferative Disease and Acute Megakaryoblastic Leukemia

WOS: 000219054200011Down Syndrome (DS) is an important genetic disease resulting from partial or total trisomy of chromosome 21 and characterized by dysmorphic facial features, intellectual disabilities and multiple congenital anomalies. Children with DS are at increased risk of developing leukemia. Specifically, 3-10% of newborns with DS are diagnosed with transient myeloproliferative disease, and children with DS are 500 times more likely to develop acute megakaryoblastic leukemia (AMKL), and 20 times more likely to develop acute lymphoblastic leukemia (ALL) than children without DS. In this study, we report two children with DS presented with transient myeloproliferative disease