| Background and ObjectiveSpinal muscular atrophy(SMA)is an autosomal recessive disease caused by a deficiency of the survival motor neuron 1(SMN1)protein,which causes the loss of motor neurons in the anterior horn of the spinal cord.A genetically similar gene,SMN2,has a translationally silent C-to-T transition at Position 6 in its 7th exon that,via alternative splicing,causes only 10% correctly spliced full-length and functional SMN protein.However,in SMA-affected individuals,SMN2 is the sole source of SMN protein and defined to be a disease-modifying gene because of the relationship between its copy number(1 to 8)and disease severity.Previously,SMA symptoms could only be relieved by physical therapy,but gratifyingly,Nusinersen(Spinraza)obtained orphan drug qualification in Food and Drug Administration(FDA)and European Medicines Agency(EMA)successively,marking that SMA is no longer an incurable disease.Hence,definitive diagnosis,precise molecular evaluations,and effective treatment in early stage are important for SMA patients.In addition,there is an urgent need to develop a novel technique for rapid,simple,and inexpensive detection of SMA,as well as precise molecular evaluations of SMN2 copy number for analyzing correlation of SMA genotype and phenotype.Also,preemptive therapy are already clear.This study is divided into four parts.The first part is to develop a rapid,sensitive and specific method to identify the homozygous deletion of SMN.The second part is to develop a method that can specifically detect the copy number of SMN1.In the third part,the reproducibility of two methods of droplet digital PCR(dd PCR)and multiplex ligation-dependent probe amplification(MLPA)was compared for assessing copy numbers of SMN1 and SMN2.In the fourth part,we performed a preliminary study on the life span of male and female FVB mice used to construct the mouse model of spinal muscular atrophy.Method1.A total of 95 SMA samples and 89 normal samples from the First Affiliated Hospital of Fujian Medical University from January 1997 to December 2019 were randomly selected.The homozygous deletion of SMN1 and SMN2 was analyzed by comparing polymerase chain reaction-restricted fragment length polymorphism(PCR-RFLP)with the loop-mediated isothermal amplification(LAMP).2.A total of 77 samples were randomly selected,and the copy number of SMN1 was detected by using multiple quantitative fluorescent polymerase chain reaction(QF-PCR),and then verified by MLPA subsequently.3.The reproducibility of the two different methods was compared for assessing copy numbers of SMN1 and SMN2 in 21 samples.4.The differences in expression levels of longevity-related gene of IGF-1R in different tissues(muscle,brain,heart)at different ages(neonatal,preadolescent,adolescent,and senile)for wild-type FVB female and male mice.Result1.In direct comparison,there was close agreement between classifications of SMN1 homozygous deletion status by PCR-RFLP and LAMP,with a Cohen κ = 1 and no discordant cases.Additionally,the performance of SMN1 homozygous deletion testing was: sensitivity 100%(95%CI,95-100);specificity 100%(95%CI,95-100);positive likelihood ratio infinity(95%CI,NA-infinity);negative likelihood ratio 0(95%CI,0-NA).Besides,the performance of SMN2 homozygous deletion testing was as follows:sensitivity 100%(95%CI,52-100);specificity 99.42%(95%CI,96-100);positive likelihood ratio 173(95%CI,25-1221);negative likelihood ratio 0(95%CI,0-NA),Cohen κ 1(95%CI,0.76-1).2.The performance of copy number detection of SMN1 used QF-PCR was as follows:sensitivity 100%(95%CI,52-100);specificity 99.42%(95%CI,96-100);positive likelihood ratio 173(95%CI,25-1221);negative likelihood ratio 0(95%CI,0-NA),Cohen κ 1(95%CI,0.76-1).3.Reproducibility of SMN1 copy number assessment was 74.1%(20/27)and SMN2 copy number was 100%(27/27)for MLPA while both 100%(27/27)for dd PCR.4.Discordancy of SMN1 copy number was observed with SMN1 c.844C>T mutation by MLPA or dd PCR,as a result,MLPA assessed the true number.5.The life expectancy of severe SMA mice was only 11 days.Nevertheless,the average life span of female mice treated with ASO-11-27 was extended to 256 days,and that of male mice was extended to 147 days,with a statistically significant difference(P<0.0001).In addition,there was a significant decrease in testosterone in SMA male mice with castrated surgery(P=0.0273),but no significant change in estradiol in SMA female mice with castrated surgery(P=0.7744).Therefore,it was no significant difference in the life span between the female castrated surgery group and the sham operation group in SMA mice with ASO treatment(P=0.5826),while the life span of the male castrated surgery group was significantly longer than that of the sham operation group(P=0.0002).Via life monitoring,the average life span was 721 days of female mice for FVB,458 days of sham operation group,and 642 days of castrated surgery group,with a statistical difference in life span among these three groups(P=0.0004).However,it was no significant difference among female mice group,male mice sham operation group,and male mice operation group for C57BL/6(P=0.6944).6.There were significant differences in the expression of IGF-1R in muscle and heart tissues of male and female mice during FVB neonatal period(P<0.0001).The expression level of IGF-1R in the muscle tissue of newborn males was higher than that of females,and the expression level of IGF-1R in the heart tissue was lower than that in the brain tissue,while the expression level of IGF-1R in the brain tissue was similar between males and females.Significantly,from pre-puberty(day15)to puberty(day80)of FVB,the expression of IGF-1R in all tissues of male mice gradually tended to be low,and the expression of IGF-1R in all tissues of FVB male mice undergoing castration surgery was up-regulated,while the expression of IGF-1R in all tissues of C57BL/6male mice undergoing castration surgery was down-regulated.However,the expression level of IGF-1R in FVB aging period(day220)was highly expressed in male muscle tissue while low in male cardiac tissue(P<0.0001).Conclusion1.We successfully develop a visual LAMP assay for rapid detection of SMN homozygous deletion by optimizing the reagents and reaction conditions as well as reducing the reaction time to 30 minutes,which is in excellent accordance with the results of PCR-RFLP.2.QF-PCR can detect the copy number of SMN1 quickly and accurately using three pairs of internal reference genes compared with SMN1,and only PCR and fragment analysis are needed.3.By comparing methods of MLPA and dd PCR,the dd PCR is a robust platform for analysis of SMN1 and SMN2 copy number,so as to further provide the explanation for the phenotype-genotype correlation as well as the treatment of SMA.In addition,it is recommended that SMN1 compound heterozygous mutation needs joint analysis of multiple methods.4.Wild-type FVB male mice are affected by androgens,resulting in shorter average life span than females,and the level of testosterone is regulated by longevity related gene IGF-1R. |
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