The Studies Of The Detection And Expression Of SMN Gene Of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease that characterized by the degeneration of the anterior cells of the spinal cord, leading to progressive symmetry muscle weakness, atrophy and paralysis in the limbs and trunk. The incidence of a disease is near 1/10000. According to the onset of the disease and the clinical course, SMA is classified into the following 3 types: type I ( Werdning - Hoffmann disease) , the most severe form with onset before the age of 6 months, never able to sit without support, death u-sually ensues by 2 years of age from respiratory failure or infection; type II (intermediate) , is characterized by onset before age 18 months, these patients are able to sit but not walk, and survival is usually more than2 years; type III (Kugelberg - Welander disease) is the mild form with onset after the age of 18 months. Up to now, pathological and physiological of SMA is not very clear. The changes of clinical phenotype maybe associated with the regulation of SMN2 gene. All three forms of SMA were mapped to the long arm of chromosome 5, at 5qll. 2 ~5q 13.3. The survival motor neuron (SMN) is the primary SMA - determining gene . There are two highly homologous copies in this region: SMN1 (the telomeric copy) and SMN2( the centromeric copy). The SMN 1 gene exon 7 is homozygous deleted or mutation in more than 96% of SMA patients. So, it is very valuable to detect the homozygous deletion of SMN 1 gene exon 7 for the diagnosis of SMA. In the past, on the basis of nucleotide mismatch between SMN1 fP SMN2 and homozygous deleted of SMN1 exon 7 and 8 of SMA patients, thegenetic diagnosis is used by single strand conformation polymorphism (SSCP) , and restriction fragment length polymorphism ( RFLP). But, it was expensive and time consuming for these methods as well as technique was trivial and complicated. Therefore, it is necessary to probe a method that is more conveniently and quickly than that of past for SMA gene diagnosis. So, the SMN gene of 57 SMA patients has been detected by using AS - PCR ( AUele - Specific Amplification ) method that can detect single base mutation. The incidence of carriers of SMA is approximately 1: 50, so it is very important to screen carriers of SMNl deletion in genetic counseling. However because the reproducibility of SMN site, it is difficult to detect it. So, in part II of our study we detect the SMN gene copy number of different types of SMA, SMA parents and normal by using fluorescent quantitative PCR with TaqMan technique and MGB ( minor groove binder, MGB) probe. By quantitative analysis of the amount of SMN gene, we can get the change of genotype in normal, carriers of SMA, and distinguish the carriers and no - carriers of SMA. Now, the genetic basis of clinical phenotype of SMA is not clear, But many studies show that up - regulating SMN2 gene expression maybe lessen the severity of SMA. Therefore, to detect the change of SMN2 gene copy number in different types SMA can investigate the relationship between them, as well as to provide index for treating the SMA. Although the studies show that in general the SMN2 copy number is correlated with the severity of SMA, the most of SMN2 gene can product 20% - 30% containing exon 7 full -length transcripts. Therefore, we presumed that the different types of SMA could have different full - length transcripts of SMN2 . In part III of our study, we detected the SMN gene mRNA expression of different types of SMA by using RT - PCR, to probe the relationship between the the expression of SMN gene mRN A and the clinical classification of SMA.Materials and MethodsSamples:1. Patients Selection:Fifty — seven SMA patients who were final diagnosed by genetic diagnosis(SMN1 exon 7 homozygous deletion) (type I 21, type H 25, type 111). A total of 114 patients'parents and 40 controls who were employee of laboratory in the hospital. There was no genetic relationship among them. 2. Isolated cells(1) Leucocytes: venous blood of subject 5ml + 1.5% EDTA 0. 5 - 1. 0ml anticoagulation, 3000rpm/min, lOmin, abandon plasma, RBC + double volume hypotonic RBC lysate, quake lightly, keep 40min, centrifugation, remove supernatant , once more, WBC was kept at - 20c€.(2)Peripheral blood lymphocyte; Venous blood of subjects 5ml + 1. 5% EDTA 0.5 -1.0ml anticoagulation + normal saline dilution, plus it on the liquid of lymphocyte isolation, centrifugation , 2200rpm 13min. taking white flocc buffy coat and placing in EP, keep at -80X1.Materials1. Reagents:(l)Extract genome DNA: 1. 5% EDTA, hypotonic RBC lysate (0. 7% NH4CL,7. 12% NH4HCO3) .extract DNA buffer(lOmmol/L EDTA,lOmmol/L naCL,10mmol/L TrisCL.O. 5% SDS) ,20mg/ml protease K ( TaKaRa Company) , saturate phenol .(2) Electrophoresis: agarose , 100 ladder DNA marker (TaKaRa) , loading buffer, polyacrylamide, methylene bisacrylamid, ultrapure urea (Sigma)(3) PCR: MGB probe x fluorescent quantitative PCR Kit, Taq DNAase (TaKaRa).(4) RNA: TRIzol (Invitrogen) , liquid of lymphocyte isolation, AMV reverse transciption Kit2. Primer designed according to references;(1) Microsatellite polymorphism C212 marker (TaKaRa)(2) Fluorescent quantitative primer and probe of SMNland SMN2(3) AS-PCR primer of SMN(4) primer of SMN gene mRNA (TaKaRa) 2. Methods1. AS-PCR method (l)Phenol/chloroform extract DNA;(2)PCR: Detecting the deletion of SMNl and SMN2 gene exon 7 by using allele -specific primers of SMNl, SMN2 and SMN7R, respectively. The fragment is 404bp. In order to avoid false negative results, we amplified NAT2 as internal standard at the same time. The fragment is 500bpo The products were separated by 2% agarose gel electrophoresis, dyed by EB.2. SMNl and SMN2 copy numbers were detected by real - time fluorescent quantitative PCR method.(l)Phenol/chloroform extracted DNA:(2)Detecting the OD of DNA(3)Microsatellite polymorphism C212 marker genotyping0The objects of experiement: 2 healthy persons who have homozygous deletion of SMN2 and 57 S>MA patients who have homozygous deletion of SMNl.(DPCR amplification; 25|xl, DNA 50ng, dNTP 200mmol/L, primer 0. 2u,mol/L, 10 x Buffer, Taqase 1U. Amplification condition:941 3min, 94t 40s, 65*0408, 72