The Association Analysis Between Spinal Muscular Atrophy 1 And Mitochondrial Genome

BackgroundSpinal muscular atrophy (SMA) is the second most common autosomal recessive neuromuscular disorder after cystic fibrosis (CF), affecting approximately one in 6,000 live births and with a carrier frequency of approximately one in 50. It is characterized by degeneration of the anterior horn cells of the spinal cord resulting in symmetric, progressive proximal muscle weakness and atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved and survivorship: SMA1 (OMIM:253300) , SMA2 (OMIM:253550 ) , SMA3 (OMIM:253400). SMA 1, the most severe form, is situated in the No. 1 leading to the childhood mortality in the world. Patients with SMA1 usually begin to severe, generalized muscle weakness at birth or within the first 6 months. They never achieve ability to sit without support. Feeding and breathing difficulties are usually responsible for death within two years of age. So far, no relieved or curative treatment is known for this devasting disease.The Survival of Motor Neuron (SMV) gene has been identified as a SMA-determining gene. In humans, SMN is contained in a 500-kb sequence on chromosome5ql2.2 - q13.3 which consists of nine exons. It is present in two copies: a telomeric one (SMN1) and a centromeric one (SMN2) . Perhaps it is a recent evolutionary event which they form each other the inverted repeat through gene transition. In rare conditions, an individual could have SMN1 only owing to the conversion from SMN2.SMN1 shows homozygous deletions of exon 7 in 90%～98% of the SMA patients, and it is the essential genetic diagnosis responsible for the SMA phenotype.It is reported that mitochondrial DNA deletion is a secondary factor for muscle atrophy caused by degeneration of the anterior horn cells of the spinal cord. In patients with clinical features of spinal muscular atrophy and no deletions in the survival of motor neuron gene, functional investigation of muscle tissue revealed diminished respiration rates and decreased enzyme activities of the respiratory chain including all respiratory chain enzyme complexes I-IV, cytochrome c oxidase (COX) or thymidine kinase 2 (TK2) may mutate. And mutation in the synthesis of cytochrome oxidase 2 (SCO2) can present as a SMA1 phenotype. The relation of SMA1 and mitochondrial DNA depletion was rarely reported and of great controversial.ObjectivesTo investigate the mitochondrial factor related to SMA1 through sequencing and analysis the mitochondrial genome of Han Chinese patients with SMA1.Patients32 unrelated Chinese Han patients with suspected SMA1 and 50 matched healthy controls.Methods(1) The patients and matched healthy controls were collected, genomic DNA fromperipheral venous blood was extracted using the phenol-chloroform method. (2) Genetic diagnosis was carried out through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) . (3) Ten SMA1 patients were selected randomly, amplifying their whole sequence of mtDNA, and bidirection sequencing with the universal M13 primer. (4) DNA sequence analysis and variation identification by DNAssist software.Results(1) 96.9% of the patients (31/32) had the deletion of SMN1 exon 7.(2) There were 5 consistency mutations: 73A>G, 263A>G, 10398A>G(Thr-Ala), 10400C＞'T and 16223C＞T in the mitochondrial genome of 9 patients with SMN1 exon 7 deletion and 1 patient without SMN1 exon 7 deletion. 10398A>G (Thr-Ala) lies in ND3, 10400OT is a synonymous mutation. Three mutations: 73A>G, 263A>G and 16223OT belong to the polymorphic nucleotide positions from the mtDNA control region.Conclusions(1) 10398A>G (Thr-Ala) / ND3 mutation likely associated with SMA1 pathogenesis.(2) ND3 assists electron transmission and oxidative phosphorylation to a great extent in the respiratory chain. It is one of nicotinamide adenine dinucleotide (NADH) subunit. NAD~+ accept 2H of most metabolite and form NADH, then these 2H were transmited to ubiquinone by mitochondrial respiratory complex I . We suggested that this mutation: 10398A>G (Thr-Ala) influence the mitochondrial respiratory complexI , lead to the mitochondrial respiratory chain dysfunction resulting in symmetric, progressive proximal muscle weakness and atrophy of the patients. And 73A>G,263A>G, 16223C＞T belong to the polymorphic nucleotide positions from the mtDNAcontrol region and were considered as no functional single nucleotide polymorphism(SNP) . The 4 consistency mutations coexist in 10 unrelated Chinese Han patients with SMA1 of our study. It indicates that they may influence the mitochondrial respiratory chain and the energy production of muscular cell, resulting in patients with SMA1 begin to progressive motor difficulties, proximal limb weakness and so on. Further research on the molecular consequence of these mutations are needed to confirm the mutation.