Investigation On The Mechanism Of U1snRNA And Morpholino Correcting The Aberrant Splicing Of SCN1A Mutations

PurposeTo investigate the aberrant splicing of three SCN1A gene mutations from thepatients with febrile seizure-related epilepsy syndrome and the potential therapeuticstrategies for them, we predicted their possible aberrant splicing using bioinformaticssoftware and analyzed their expression and splicing in vitro mini-gene. Furthermore,to explore the mechanism of the specific U1snRNA and morpholino repair aberrantsplicing.MethodsWe have identified three SCN1A gene mutations (c.473+5G>A, c.909A>G, andc.3705+1G>T) in our previous study, using PCR and genomic DNA extraction fromthe whole blood of the patients with febrile seizures related-epilepsy syndrome. Weused Human Splicing Finder2.4software to predict the aberrant splicing ofc.473+5G>A, and c.909A>G (in addition, the predictive aberrant splicing ofc.3705+1G>T has been reported in our previous study). The mini-gene plasmids foreach mutation were constructed and transfected into HEK293cells.Using the in vitrocellular expression system, The total RNA was extracted and RT-PCR was followed.We analyzed the final transcripts based on the fragment length and the ratio of theindividual fragments. The specific U1snRNA plasmids were designed complementary to the splice donorsite or its vicinity intron for three mutations respectively.They were delivered intoHEK293cells with co-transfection of the corresponding mutation mini-gene.ThemRNA transcript were analysed and the therapeutic effect of specific U1snRNA wasevaluated with the ratio of normal transcription.Morpholino oligonucleotidescomplementary to the newly activated splice donor site of c.909A>G was applied andthe transcript results were compared with those of specific U1snRNA. Eachexperiment was repeated three times to obtain the mean, the measurement data ispresented as mean±standard deviation(X±S). We use SPSS16.0software forstatistical analysis. Two samples are compared using t test. P<0.05was consideredstatistically significant.Results:1. The results of splcing in vitroCompared with the normal transcripts, the abnormal transcript of c.473+5G>Amutation of SCN1A gene lacks98bp,the deletion which contained8nucleotide basepairs of the5’–end of exon2and the entire exon3.The aberrant transcripts ofc.909A>G mutation misses the60bp of exon6. The length of two aberrant transcriptsrevealed by mini-gene expression system was consistent with that of the predictivesplicing by software.2. The efficiency of Specific U1snRNA repairing the aberrant splcing(1)The ratio of the normal transcript expression of c.473+5G>A mini-gene was35.37%±3.29%. After the specific U1snRNA(E3Mu, E3+1, E3+9, and E3+16) wasco-transfected into the mini-gene expressing cells,the ratio of the normal transcriptwas increased to59.88%±4.46%，54.94%±1.97%，100%，and100%respectively.Statistically, each of them was significantly different from that of none co-transfectedmini-gene (p<0.05).(2) The ratio of the normal transcript expression of c.909A>G mini-genewas30.34%±2.71%. After the co-transfection with specific U1snRNA(E6+1, E6+9, E6+16, E6-9, E6-18),the cells expressed the normal transcripts with the increasedratio,92.85%±2.08%,100%,100%,27.47%±1.80%,29.83%±3.54%respectively. Theratio of E6+1, E6+9, and E6+16were significantly increased (p<0.05), while that ofE6-9, E6-18are not significantly increased (p>0.05).(3)The c.3705+1G>T mini-gene did not express normal transcript, instead, twoaberrant ones, the shorter fragment skipping the49bp in5’-end of exon18and thelonger one including the20bp in5’end of intron18.The ratio of the longer abnormaltranscript expression of c.3705+1G>T was37.53%±2.27%. After the co-transfectionwith specific U1snRNA(E18Mu, E18+1, E18+9, E18+16),the ratio of the largerabnormal transcript was increased to55.49%±1.38%，64.66%±3.64%，46.39%±2.14％，80.29%±3.51%respectively, which were significantly increased(p<0.05).However, specific U1snRNAsdid neither make c.3705+1G>T mini-gene toexpress the normal transcript.3. The efficiency of Morpholino oligonuclwotides repairing the aberrant splcingFor c.909A>G mini-gene, the ratio of the normal transcript expression wasincreased by co-transfection with morpholino(10uM,50uM), to41.64%±1.94%，64.54%±2.47%respectively, which were both significantly increased, compared tothat of non-morpholino treated mini-gene (p<0.05).Conclusion1. The SCN1A gene mutationsc.473+5G>A and c.909A>G cause aberrant splicingwhich may have been relevant to the clinical phenotype.2. The mutation changing splice site+1causes none-repairable aberrant splicing.3. The binding U1snRNA could fix the aberrant splicing more efficiently than themorpholino.