The Gene Diagnosis Research Of Spinal Muscular Atrophy Using PCR-RFLP And MLPA

ObjectiveSpinal muscular atophy (SMA) is a common autosomal recessive neuromuscular disorder caused by degeneration of neurons in the anterior horn of the spinal cord. The degeneration process leads to progressive, symmetric muscle weakness and muscle atrophy. And it is more serious proximal than distal. The frequency of the disease is calculated to be approximately1:10000with a carrier frequency of1/50-1/34. SMA represents the second most frequent autosomal-recessive discorder after cystic fiborosis. Currently there is no treatment for the disease. So, the probands group of genetic tests and the carriers screening for genetic counselling, guidance of growth have important roles.The primary SMA disease-determation gene is the survival mortor neuron gene (SMN), located in5q11.2-13.3. It has two homologous gene SMN1and SMN2, the two gene are too homologous that only5base pair differences between them. Generally, the SMN1gene is primary responsible for the SMA, and the SMN2gene can modify the phenotype of the disease. About95%SMA patients are homologous deletion of exon7and/or8in SMN1or the SMN1conversation to the SMN2. The other5%SMA patients are due to the gene mutations. So the detection of exon7and8of the SMN1is the main direction for the SMA.The PCR-restriction fragment length polymorphism (PCR-RFLP) which the most widely used method and the new multiplex ligation dependent probe amplification (MLPA) method were applied to perform gene diagnosis for30suspected patients with spinal muscular atrophy (SMA) in this study. The consistency of two methods was compared in the various types of the SMN gene for proband’s diagnosis. The evaluation of MLPA contribution in screening the carriers were also observed.MethodGenomic DNA was isolated from peripheral blood of all the research subjects using salting-out method. DNA concentration and purity was determined by nucleic acid quantitative instrument (NADO DROP2000)). Exon7and8of SMN gene was amplified by allele specific PCR-restriction fragment length polymorphism.The PCR products were digested with Dra I and Dde I.The PCR products and the enzyme digested products analyzed by2%agarose gel electrophoresis. Simultaneously, the DNA samples were performed by SALSA MLPA KIT P021, and the SMN copies were caculated. The dataes were analyzed by SPSS16.0software.ResultsIn this study twenty-four SMA patients were detected from30suspected patients by above two methods, including22patients with exon7and8homozygous deletion, and2patients with only exon7homozygous deletion of SMN1. The results of the two method agreed with eath orther. Reviewing the clinical,age of onset and disease progress, the24cases were ranked with9cases of type I,9type II and6type Ⅲ. The other6cases and parents presented no homozygous deletion by PCR-RFLP, but one child and two morthers of them were detected heterozygous by MLPA. Also MLPA analysis found three "2+0"carriers from3families. The MLPA method detected52carriers and18normal individuals. The results also showed that the SMN2copy numbers were mainly4or5in SMA patients, while the carriers and the normal individuals were2or3and1or2copies respectively. There were clear statistical significance in the groups of patient-carrier and patient-normal individuals (p<0.001). The carrier-normal group appeared no statistical significance (p>0.05).ConclutionCompared with PCR-RFLP, MLPA is more convenient, precise and high-effective. And the MLPA can be fast and accurate quantitate SMN1and SMN2, needing only a small amount of DNA template. It also helps to distinguish SMA phenotype and screen the carriers. In short, it is a kind of technique of gene diagnosis for common genetic disease, and may provide reliable information for genetic consulting. It is belived that it will be widely used in the clinical in the future.