The New Gene Mutation Function And Neuron Apoptosis Mechanism Research Of Methylmalonic Acidemia

Part 1 A study on the function of MUT missense mutationsObjective: Investigate the effects of eight missens mutations of MUT gene detected from eight methylmalonic acidemia patients on the levels of MCM protein expression and enzymatic activities, to confirm their pathogenicity.Methods:（1） Predict the effects of 8 missense mutations（L140P, A141 T, G161 V, W309 G, I505 T, Q514 K, I597 R, and G723D）on MCM protein structure and function by bioinformatics softwares. The 8 mutations have not been reported and detected from 8 methylmalonic acidemia patients.（2） Construct the expression vector with wild type and eight missense mutation types of MUT c DNA, and transfected them into 293 T cells.（3） Detect the proteins expression of widetype and 8 kinds of mutant in 293 T cells by western bolt.（4） MCM activity of wide type and mutant types expressed in 293 T cells were assayed by UPLC.Results: The recombinant expression vectors containing MUT c DNA and 8 missense mutations（L140P, A141 T, G161 V, W309 G, I505 T, Q514 K, I597 R, and G723D） were constructed subsequently. Real time PCR results showed that 8 missense mutations plasmid had no impact on the MUT m RNA levels in transfected 293 T cells.Western blot results showed the MCM protein levels in 5 missense mutations（L140P, A141 T, G161 V, W309 G, and Q514K）were lower than that in wild type（P<0.05）, while the expressions amount of I505 T, I597 R, and G723 D had no difference with wild type（P>0.05）. 8 kinds of missense mutation contributed to markedly reduced enzymatic activities of MCM（0.31±0.21, 1.66 ±0.01, 0.76 ±0.04, 0.19 ±0.13, 1.37±0.24, 0.46 ±0.09, 0.86±0.27, and 1.37 ±0.13 nmol/min/mg protein）than control（3.11±0.13 nmol/min/mg protein）（all P<0.05）. Combined analyze with the clinical phenotype, we confirmed that the 8 MUT missense mutations are pathogenic mutations.Conclusions: Eight kinds of MUT missense mutations（G161V, W309 G, Q514 K, G723 D, L140 P, A141 T and I505T） the mutations did not affect m RNA expression level, but 7 of them impaired the expression of MCM protein content, and all of them resulted in the decrease of enzyme activity. So they are pathogenic mutations.Part II Observation of apoptosis changes and analysis of the relationship of Lnc RNA expression with m RNA in apoptosis signaling pathway in mice neurons induced by MMAObjective: In this study, we will detect the mice cortex neurons induced by methylmalonic acid（MMA） through expression profile chip to screen the differential expressing Long noncoding RNA（Lnc RNA） and m RNA,, and to explore the neuronal apoptosis signaling pathways involved of the Lnc RNA. Besides we will analysis the relationship between Lnc RNA and m RNA in apoptosis signaling pathways, so as to discuss the MMA brain damage mechanism.Methods: Using different concentration methylmalonic acid to induce mice primary cortical neurons for 6h, 12 h and 24 h, then we observed the neuronal apoptosis;（2） Detecting the differential expression of Lnc RNA and m RNA by expression profile chip;（3） Analysis of apoptosis related Lnc RNA and m RNA against differential expressing ones through gene ontology enrichment（GO） and Pathway analysis;（4） Analyzing the possible pathways of Lnc RNA involved in the neuronal apoptosis induced by methyl malonic acid.（5）Analyzing the relationship of Lnc RNA with m RNA involved in apoptosis signaling pathways.Results: The mice neuron showed concentration and time dependent apoptosis phenomenon after induced by methyl malonic acid.（2） In the chip results, there were 113 Lnc RNA and 248 m RNA showed a trend of increasing expression, while 61 Lnc RNA and 191 m RNA showed a trend of decline expression. These genes mainly related to transcription regulation, cell proliferation, apoptosis, and so on functions.（3） Pathway analysis showed that 22 m RNA contained in MAPK signal pathway, and 8 m RNA in P53 pathway.（4） Atf4, Dusp8, Hspa2 etc, total 7 m RNA included in MAPK signaling pathways correlated closely to Lnc RNA.（5） Six Lnc RNA（NONMMUT041857, Know TID₀0005724, NONMMUT001282 、NONMMUT044301, NONMMUT006249, NONMMUT062897） had significant correlation with Atf4.（6） Lnc RNA may regulate the expression of Atf4, activate a series of apoptosis related gene（p- JNK, c- Jun and p- Akt） expression in MAPK and P53 signaling pathways, and then result in neurons apoptosis.Conclusions:（1） Our study confirmed that brain neurons apoptosis is one of MMA brain damage mechanism;（2） The MMA induced neuronal apoptosis maybe through MAPK and P53 signaling pathway;（3） The Lnc RNA may regulate Atf4 differentially expression, then effect the MAPK signal pathway, thereby be involved in the MMA induced neuronal apoptosis. The conclusion might serve as a theory basis of mechanism of MMA brain injury.