Intron mutations of GABRG2 gene in patients with epilepsy combined with febrile seizures plus:an in vitro splicing assay / 中华神经医学杂志

Objective To screen the GABRG2 gene in Chinese patients diagnosed as having epilepsy with febrile seizures plus (EFS+) and analyze the in vitro splicing of intron mutations of GABRG2 gene. Methods After collecting the blood samples from patients with EFS+, all 9 coding exons and introns relevant to mRNA splice of GA BRG2 gene were sequenced by PCR. PCR products of exons 7, 8 and 9 and part of the introns of both ends of GABRG2 gene were cloned into the pTARGET vector to construct pTARGET-Exon-7-8-9 minigene vector and its Exon8+45C>T mutation vector.Wild-type and Exon8+45C>T mutation vector were transfected into HEK 293 cells and extracted RNA for RT-PCR. Results We did not detect mutation in GABRG2 gene coding region, but found 1 mutation in intron Exon8+45C>T. After splicing, the size of RT-PCR products of Wild-type and Exon8+45 OT mutation were both 522 bp. Conclusion Mutations in GABRG2 gene coding region are not likely to be substantially involved in the etiology of EFS+. Exon8+45C>T mutation does not affect the splicing of GABRG2 gene.

Mesenchymal stem cells transplanted in mdx mice differentiate into myocytes and express dystrophin/utrophin / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into myocytes and their expression of dystrophin/utrophin after transplantation in mdx mice.</p><p><b>METHODS</b>BrdU-labeled fifth-passage rat MSCs were transplanted in mdx mice with previous total body gamma irradiation (7 Gy). At 4, 8, 12 and 16 weeks after the transplantation, the mice were sacrificed to detect dystrophin/BrdU and utrophin expressions in the gastrocnemius muscle using immunofluorescence assay, RT-PCR and Western blotting. Five normal C57 BL/6 mice and 5 mdx mice served as the positive and negative controls, respectively.</p><p><b>RESULTS</b>Four weeks after MSC transplantation, less than 1% of the muscle fibers of the mdx mice expressed dystrophin, which increased to 15% at 16 weeks. Donor-derived nuclei were detected in both single and clusters of dystrophin-positive fibers. Some BrdU-positive nuclei were centrally located, and some peripherally within myofibers. Utrophin expression decreased over time after transplantation.</p><p><b>CONCLUSION</b>The myofibers of mdx mice with MSC transplantation express dystrophin, which is derived partially from the transplanted MSCs. Dystrophin expression from the transplanted MSCs partially inhibits the upregulation of utrophin in mdx mouse muscle, showing a complementary relation between them.</p>

Construction of a recombinant plasmid expressing microdystrophin gene fused with HSV-1 VP22 and its biological characterization / 中华神经医学杂志

Objective To construct a recombinant plasmid expressing human microdystrophin gene fused with VP22 ofhmnan herpes simplex virus 1(HSV-1),and investigate the expression of microdystrophin in vitro and the characteristics of VP22-mediated protein transduction.Methods Full length HSV-1 VP22 gene was amplified by PCR from the plasmid pSINrep5-VP22 and cloned into the eukatyotic expression vector pAVXl to conslruct recombinant plasmid pAVP22.Microdystrophin cDNA was obtained from the recombinant plasmid pBSK-MICRO digested with Not Ⅰ,and the product Was inserted into plasmid pAVP22 to construct the plasmid pAVP22-MICDYS. 3T3 cells were transfected with pAVP22-MICDYS,and the expression of microdystrophin was detected by RT-PCR,Western blotting and immtmocytochemislry.The supematant of 3T3 cells transfected with pAVP22-MICDYS were collected to infect human mesenchymal cells(MSCs),and the expression of the fusion protein VP22-microdystrophin in the cells was detected using immunohistochemistry to assess VP22-mediated protein transduction. Results The recombinant plasmid expressing microdystrophin-VP22 fusion gene capable of in vitro expression of the fusion protein was constructed successfully.VP22 was shown to enhance the expression efficiency of microdystrophin in 3T3 cells and transduce VP22-microdystrophin fusion protein from 3T3 cells to human MSCs. Conclusion The recombinant plasmid expressing microdystrophin-VP22 fusion gene with protein transduction capacity provides an important basis for further study of the fusion protein in treatment of Duchenne muscular dystrophy.

Association between cutaneous adverse reactions to antiepileptie drugs and HLA-B*IS02 allele / 中华神经医学杂志

Objective To investigate the association between cutaneous adverse drug reactions (CADRs) caused by antiepileptic drugs and HLA-B*1502 allele. Methods In 31 epileptic patients presented to the Epilepsy Clinic of the Second Affiliated Hospital of Guangzhou Medical College between January 2007 and May 2008, 13 had CADR to carbanazepine (CBZ) including 6 with Stevens-Johnson syndrome (SJS) and 7 with mild maculopapular exanthona (MPE);15 were CBZ-tolerant, and 3 had lamotrigine (LTG)-indueed MPE. All the patients underwent examinations using polymerase chain reaction with sequence specific palmers to analyze HLA -B*1502 allele frequencies, with 30 healthy subjects without a history of using CBZ or LTG as the control. Results HLA-B*IS02 allele frequency was 100% (6/6) in patients with CBZ-SJS, 57% (4/7) in patients with CBZ-induced MPE, and 33% (1/3) in patients with LTG-induced MPE. The frequency was 7% (1/15) in CBZ-tolerant patients and 10% (3/30) in the control subjects. Compared with the CBZ-tolerant patients and the control subjects, the patients with CBZ-induced SJS and MPE had significantly increased HLA -B*1502 allele frequency (P<0.05). Conclusions HLA-B*1502 allele is associated with CADRs to CBZ in epileptic patients.

Clinical features and mutations of voltage-gated sodium channel subunit type 1 gene in myoclonic-astatic epilepsy in infancy / 中华神经医学杂志

Objective To study the clinical features and genetic mechanism of myoclonic-astafic epilepsy (MAE) in infancy. Methods This study was conducted among 10 infants with MAE (including 7 male and 3 female patients) diagnosed between 2006 and 2008 according to the criteria of International League Against Epilepsy (2001). The clinical data including onset age, seizure type, physical signs, EEG, brain maguetic resonance imaging (MRI), effects of anti-epileptic drugs and prognosis were analyzed. The mutations of voltage-gated sodium channel subunit type 1 gene (SCN1A gene) were screened by denaturing high performance liquid chromatography and direct sequencing. Results The 10 MAE cases included 8 sporadic cases and 2 with a family history of febrile seizure and epilepsy. The onset age ranged from 5 months to 39 months, and all the MAE patients had multiple generalized seizure types, including myoclonic-atonic, myoclonic, atonic, tonic-clonic and absence seizures. Two patients had myoclonic status epilepticus, and 7 showed mental retardation. All the patients showed normal findings in MRI. SCN1A gene was screened in 8 of the MAE patients, and no mutation was found. Valproate, clonazepam and levetiracetam were effective in these MAE cases. Conclusion MAE is a rare epilepsy syndrome, whose genetic mechanism is still unclear. Valproate, clonazepam and levetiracetam are effective for MAE, which is associated with poor prognosis.

Construction of recombinant adenovirus including microdystrophin and expression in the mesenchymal cells of mdx mice / 生物工程学报

Construction of recombinant adenovirus, which contain human microdystrophin, and then transfection into mesenchymal cells( MSCs) of mdx mice were done, and genetically-corrected isogenic MSCs were acquired; the MSCs transplantation into the mdx mice was then done to treat the Duchenne muscular dystrophy( DMD). Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I ; the production was inserted directionally into pShuttle-CMV. The plasmid of pShuttle-CMV-MICRO was digested by Pme I , the fragment containing microdystrophin was reclaimed and transfected into E. coli BJ5183 with plasmid pAdeasy-1. After screening by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the CPE of cells and by the PCR method. Finally, MSCs of mdx mice were infected with the culture media containing recombinant adenovirus, and the expression of microdystrophin was detected by RT-PCR and immunocytochemistry. Recombinant adenovirus including microdystrophin was constructed successfully and the titer of recombinant adenovirus was about 5.58 x 10(12) vp/mL. The recombinant adenovirus could infect MSC of mdx mice and microdystrophin could be expressed in the MSC of mdx mice. Recombinant adenovirus including microdystrophin was constructed successfully, and the microdystrophin was expressed in the MSC of mdx mice. This lays the foundation for the further study of microdystrophin as a target gene to correct the dystrophin-defected MSC for stem cell transplantation to cure DMD.

Sibling brother and sister both with Duchenne muscular dystrophy / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the clinical and lab features of sibling brother and sister both with Duchenne muscular dystrophy (DMD).</p><p><b>METHODS</b>We conducted comprehensive clinical and lab investigations including the test of serum enzymes, electromyography (EMG), electrocardiography, color Doppler echocardiography, HE staining of skeletal muscles, immunohistochemical study of dystrophin and utrophin, multiple ligation probe amplification (MLPA) on exon 1-79 of dystrophin gene, and short tandem repeat-poly- merase chain reaction of CA repeats located in dystrophin gene.</p><p><b>RESULTS</b>These two patients were confirmed to suffer from DMD. They were characterized by typical features of DMD including typical clinical manifestations, increased serum enzymes, EMG presenting myogenic impairment, HE staining presentation belonging to DMD, negative dystrophin in brother, and inconstantly positive on the sarcolemma of sister. Furthermore, no deletion or duplication was found in the 1-79 exons of dystrophin gene. The suffering brother and sister carried the same maternal X chromosome.</p><p><b>CONCLUSIONS</b>Carriers of DMD gene show typical clinical and laboratory manifestations of DMD. Comprehensive examinations should be performed for such carriers.</p>

Expression of human micro-dystrophin gene after retrovirus infection in mdx mice bone marrow-derived mesenchymal stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct the retroviral vector containing human micro-dystrophin gene and detect the expression of human micro-dystrophin in mdx mice bone marrow-derived mesenchymal stem cells (MSCs) after retrovirus infection.</p><p><b>METHODS</b>Retroviral vector for micro-dystrophin gene was constructed and transferred into the packing cell PA317 mediated by Lipofectamine 2000. The retroviral supernatant containing the target genes were subsequently used to infect mdx mice MSCs. Micro-dystrophin expression was examined by methods of immunofluorescence staining and reverse transcriptase-polymerase chain reaction.</p><p><b>RESULTS</b>Micro-dystrophin retroviral vector was successfully constructed and transferred into PA317 cells, and 48 h after infection with the recombinant retrovirus in mdx mice MSCs, 319 bp fragment could be detected by electrophoresis in the RT-PCR products. The red particles could be detected in some infected mdx mice MSCs with immunofluorescence staining. CONCLUSION mdx mice MSCs infected with retrovirus containing micro-dystrophin gene can express micro-dystrophin protein.</p>

Transfection and in vitro expression of human microdystrophin gene in rat mesenchymal stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the eukaryotic expression vector of human microdystrophin gene and observe its expression in rat mesenchymal stem cells (rMSCs) in vitro.</p><p><b>METHODS</b>The plasmid PBSK-MICRO containing human microdystrophin cDNA was digested by restriction endonuclease, and the resultant microdystrophin fragment was inserted into the NotI site of pcDNA3.1(+) to prepare the eukaryotic expression vector-pcDNA3.1(+)/ microdystrophin, which was identified by endonuclease digestion and sequencing. The recombinant plasmid was transfected into rMSCs via lipofectamine, and after G418 selection, the expression of microdystrophin was detected by RT-PCR and indirect immunofluorescence assay.</p><p><b>RESULTS</b>Microdystrophin gene fragment was correctly inserted into the plasmid pcDNA3.1(+), as conformed by sequencing and digestion with Not I and Hind III. The total mRNA of the transfected rMSCs was extracted and microdystrophin mRNA expression was found in the cells by RT-PCR. Indirect immunofluorescence assay for the protein expression of microdystrophin showed bright red fluorescence in the transfected rMSCs.</p><p><b>CONCLUSION</b>Eukaryotic expression plasmid pcDNA3.1(+)/microdystrophin has been constructed successfully and microdystrophin can be expressed in transfected rMSCs in vitro, which may facilitate further research of Duchenne muscular dystrophy treatment by genetically modified allogeneic stem cell transplantation.</p>

Dystrophin expression and pathology of diaphragm muscles of mdx mice after xenogenic bone marrow stem cell transplantation / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of bone marrow stem cell transplantation (BMT) on the diaphragm muscles of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD).</p><p><b>METHODS</b>The bone marrow-derived stem cells form male SD rats was transplanted through the tail vein into 18 female 8-week-old mdx mice, which were sacrificed at 4, 8 and 12 weeks after BMT (6 at each time point), respectively. The diaphragm muscles of the mice were subjected to HE staining, immunofluorescence detection of dystrophin, reverse transcription (RT)-PCR analysis of dystrophin mRNA transcripts and PCR analysis of Sry (sex-determining region on the Y chromosome) gene, with age-matched female C57 mice and untreated mdx mice as the controls.</p><p><b>RESULTS</b>The proportion of centrally nucleated fibers (CNF) in the diaphragm muscle of the recipient mdx mice was (15.58+/-0.91) %, (12.50+/-1.87) % and (10.17+/-1.17) % at 4, 8 and 12 weeks after BMT, respectively, significantly smaller than that of untreated mdx mice [(19.5+/-1.87) %], and the fibers after BMT showed less inflammatory infiltration. Compared with the untreated mice, the recipient mdx mice showed green fluorescence on significantly more diaphragm muscle cell membranes [with the proportion of dystrophin-positive fibers of (1.00+/-0.32) %, (6.00+/-1.05) % and (11.92+/-1.11) % at 4, 8, and 12 weeks after BMT]. RT-PCR of dystrophin mRNA also demonstrated significantly higher relative levels of dystrophin in the recipient mdx mice (0.19+/-0.05, 0.26+/-0.06 and 0.36+/-0.04 at 4, 8 and 12 weeks after BMT) than in untreated mdx mice, and Sry gene was present in the recipient mice.</p><p><b>CONCLUSION</b>BMT can partially restore dystrophin expression and ameliorate the pathology in the diaphragm muscles of mdx mice, and has great potential to produce general therapeutic effect in patients with DMD.</p>

Dystrophin expression in mdx mice after bone marrow stem cells transplantation / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the dynamic changes of dystrophin expression in mdx mice after bone marrow stem cells transplantation.</p><p><b>METHODS</b>The bone marrow stem cells of C57 BL/6 mice (aged 6 to 8 weeks) were injected intravenously into the mdx mice (aged 7 to 9 weeks), which were preconditioned with 7Gy gamma ray. The amount of dystrophin;expression in gastrocnemius was detected by immunofluorescence, reverse transcription-polymerase chain reaction and Western blot at week 5, 8, 12 and 16 after transplantation.</p><p><b>RESULTS</b>At week 5 after bone marrow stem cells transplantation, the dystrophin expression detected in mdx mice were very low; however, its expression increased along with time. At week 16 week, about 12% muscle cells of all transplanted mice expressed dystrophin. There were less centrally placed myonuclei than the control mdx mice, whereas peripheral myonuclei increased.</p><p><b>CONCLUSIONS</b>After having been injected into mdx mice, the allogenic bone marrow stem cells have a trend to reach the injured muscle tissues and differentiate to fibers that can express dystrophin and the expression increased with time. The bone marrow stem cells participates in the repair and regeneration of the injured tissues permanently and constantly.</p>