The Tissue Expression Of NMNAT1 And SIRT1 In LCA Mice With Compound Heterozygote With NMNAT1 Mutation And A Novel Frameshift Mutation Of SMN1 Gene Cause Spinal Muscular Atrophy

PartⅠ The tissue expression of NMNAT1 and SIRT1 in LCA mice with compound heterozygote with NMNAT1 mutationBackground:Leber’s congenital amurosis（LCA）is a disorder characterized by retinal dystrophy and the child will be low visual at birth or infancy.Autosomal recessive inheritance is major type of LCA.Currently,among 18 genes are associate with LCA and our lab provided the evidence that NMNAT1 gene mutation can cause LCA by using whole-exome Sequencing.NMNAT1 gene encodes nicotinamide mononucleotide adenylyltransferase 1 which is an important enzyme to catalyze the biosynthesis of NAD+ and play a role in the metabolism involve NAD+.SIRT1 is an NAD+-dependent deacetylase and perform to deacetylation of histone,which participate in multiple regulating mechanism of the disease such as metabolic diseases,malignant tumor,cardiovascular disease and neurological diseases.Previous study demonstrate that the interection between NMNAT1 and SIRT1 can transfer the NAD+ to SIRT1 to play a vital funtction in deacetylation.Thus,NMNAT1 is closely related to SIRT1 protein.Objective:The NMNAT1 proteins express in multiple tissues,especially in retina,liver,testis and skeletal muscle etc.In our study,the knock-in mice with mutant NMNAT1 gene were generated to test if there is any difference of expression status of NMNAT1 and SIRT1 in mice with NMNAT1 compound heterozygote mutations and the status of tissues which the main purpose was study the damage status because of NMNAT1 mutation.Method:Mouse models of NMNAT1 gene compound heterozygote mutation were created.Genomic DNA were extracted and Sanger sequenced to validate the geneotypes.Total RNAs were isolated from multiple tissues samples with the TRIzol Reagent and used for detecting the expression of NMNAT1 and SIRT1 by qPCR.Immunohistochemistry were used to examine the protein level and the cellular location.H&E staining was used to observe the pathological and morphological changes in mouse tissues.Result:The expression of NMNAT1 and SIRT1 genes of NMNAT1 compound heterozygous mutant mice were statistically significantly decreased in liver and testis,but not in eyes,muscle,heart and kidney.The immunohistochemistry results also showed that the expression levels of NMNAT1 and SIRT1 were significantly down-regulated in mutant mice.H&E staining indicated that the testis and liver tissues of the mutant mice were severely damage.Conclusion:NMNAT1 compound heterozygous mutation result in not only severe retinal dystrophy,but also the damage for liver and testis.In addition,we hypothesize that the pathological changes were associate with the improper expression of SIRT1 caused by NMNAT1 mutations.PartⅡ A novel framshift mutation of SMN1 gene causing Spinal muscular atrophyBackground:Spinal muscular atrophy（SMA）is an autosomal recessive disease characterised with degeneration of spinal cord motor neurons,atrophy of skeletal muscles,and generalised weakness.The absence or defect of survival motor neuron（SMN1）result in SMAObjective:Molecular diagnosis and carrier screening were performed for three SMA families to help proper genetic counseling and prenatal diagnosis.Method:Genomic DNA and RNA were extracted from peripheral blood samples.SMN gene dosage analysis were conducted by qPCR and results were confirmed by STR.Sanger sequencing was performed to screen the mutations.Bioinformation analysis was used to predict the change of mutant protein function and structure.In addition,the aberrant function of mutant SMN1 were presented by cloning constructs and cell transfection experiment.Result:A novel frameshift mutation c.728₇29delTT（p.Ile243Thrfsl2）at SMN1 gene was identified that let to a truncated protein in one family.The cellular localization of mutant proteins is altered.The bioinformatics result suggested this novel mutation severely destroyed the structure and function of SMN protein.In addition,a rare "2+0"pattern（one chromosome with two copies of the SMN1 gene,one chromosome with no copies the SMN1 gene deletion）of SMN1 gene was founded in carriers of the second SMA family.The last patient with exon7 homozygous deletion was diagnosed.Conclusion:Our study provided the new pathogenic mutant information for future molecular diagnosis of SMA.