The Association And Functional Study Of The 5-methylcytosine Meththyltransferase NSUN5 Mutations In The Tratralogy Of Fallot

Background:Congenital heart disease(CHD)is characterized by structural and conduction abnormalities,with a most common congenital anomaly in live birth worldwide.It is reported that approximately 28%of all major congenital anomalies consist of heart defects,which is one of the leading causes of infants and children death with about 1%incidence.According to the Birth defect Prevention and Treatment Documentation published by Ministry of Health of China in 2012,CHD has become the most common congenital anomaly in live births and there are about 180,000-220,000 infants born with congenital heart diseaseTetralogy of Fallot(TOF)is the most common form of cyanotic congenital heart disease and nearly 5%of all infants born with CHD have tetralogy of Fallot,corresponding to one in 3,000 live births.TOF is defined as the combination of an over-riding of the aorta,ventricular septal defect,pulmonary artery stenosis,and right ventricular hypertrophy.Compared with other subtypes of CHDs,the infants with TOF often develop symptom s of severe hypoxia and pneumonnia.TOF patients who fail to diagnose and treat in time will die and lead to almost 90%mortality in adulthood.Despite onging improvements in the initial surgical repair,there are lingering concerns regarding the long-term outcomes that may be complicated by right ventricular dyssynchrony,and sudden cardiac death,which gives both economic and mental burden to patients' families as well as the whole societyCardiac development is an extremely complicated process,and the entire development process involves a series of complex and specific signaling pathways and transcriptional regulation.Epigentic modification enzymes are reported to be necessary for development of CHD.Epigenetics,mainly including DNA methylation,RNA methylation,histone modification and chromatin remodeling,play an important role not only in transcriptional level,also in post-transcriptional and translation modification.A recent study compared the incidence of mutations in 362 severe CHD cases and 264 controls by analysing exome sequencing and it reveled that a marked excess of mutations in genes involved in H3K4 and H3K27 modification and regulated expression of the genes in cardiac development,which led to heart defects.In recent years,epigenetic modification has been widely researched and it is mainly involved in regulating genes mRNA post-transcriptional modification.There are currently about 112 RNA modifications that have been reported and most RNA modifications occur on tRNA,mRNA,rRNA,and a small portion occur on small RNAs such as snRNA,miRNA.RNA methylation accounts for more than 60%of RNA modifications.Of which,5-methylcytidine(m5C)is one of the most prevalent mRNA modification.It has been confirmed that m5C modification affects the formation of aminoacylation and the recognition of codons in many archaea and eukaryotic tRNAs.The m5C modification also exists in the region where rRNA binds tRNA to play translation activity.Moreover,the m5C modified genes are highly enriched in mRNA,mainly in GC regions.The m5C modified genes are also specifically expressed in different tissues.The modification of m5C may regulate the selective cleavage of mRNA;and the modification level of m5C may affect the retention level of exon and the assembly form of transcripts.In addition,the enrichment of m5C modification suggests that it may be closely related to protein translation.m5C may be related to the stability of mRNA,the stability of mRNA modified by m5C is strong and the decrease of m5C level can lead to the decrease of mRNA stability.m5C modification may also affect the RNA degradation pathways involved in microRNA.At present,multiple studies focus on RNA m5C modification which involves in various biological functions.These researches are mainly on m5C modification enzymes,and the NSUN gene family is mostly explored.The human NSUN family has 9 genes totally,which are closely related to the human embryonic development.Studies have identified that NSUN2 is an mRNA m5C methyltransferase and Nsun2 deletion mice are small in size with specific tissues,such as skin,and development delay.In the process of testis development,Nsun2 mediated m5C modification is involved in reproductive regulation.In addition,besides NSUN family,it is reported that Ybx1 mediated m5C modification facilitates the maternal-to-zygotic transition in early development.Ybx1 deletion causes embryonic development delay in the blastocyst and gastrula stage.These studies provide evidence for the correlation between RNA methylase genes and embryonic development.It has been researched that 7q11.23 locus deletion has multiple clinical features and circulatory system complications are the most common which include outflow tract(OFT)disorders,such as TOF.A typical 7q11.23 deletion contains 25 genes encoding transcriptional regulators,signaling molecules and others that function in various cellular processes.Among them,four genes,BAZ1B,EIF4H,ELN and NSUN5,are specifically expressed in both human and mouse embryonic heart,which indicate that these genes may exert important functions in heart development.Recent studies revealed that BAZ1B and EIF4H were responsible for hypothyroidism and amyotrophic lateral sclerosis respectively in the 7q11.23 deletion patients.And the ELN gene was identified to be the contributor to supravalvular aortic stenosis.NSUN5,encoding a cytosine-5 RNA methyltransferase,is required for embryonic development.By generating Nsun5 knockout mouse model,it is researched that Nsun5 deletion suppressed N-methyl-D-aspartic receptor(NMDAr)activity in neuronal cells probably through the disrupted development and function of oligodendrocyte precursor cells(OPCs),leading to deficits in spatial cognitive abilities in mice.Also,the deletion of Nsun5 caused a decrease in the length of the sagittal corpus callosum(CC)and the area of the coronal CC in mice,which was associated with fewer myelinated axons and the loose myelin sheath.In addition,Nsun5 played a critical role in development of cerebral cortex in mice through regulating radial glial scaffolds of radial glial cells(RCGs)to control migration of neocortical neurons.All above findings provide reasonable evidence that Nsun5 is probably involved in early development process.The above results indicate that NSUN5 is specific expressed in both human and mouse embryonic heart,which may exert important functions in heart development.Moreover,the deleted gene in 7q11.23 region that causes OFT defects is still unknown.Therefore,we infer that the NSUN5 gene may be related to the OFT defects in 7q11.23 regionTo dissect the role of NSUN5 in TOF,we study association and functional role of NSUN5 mutations in the occurrence of TOF by systematic epidemiological method,including epidemiology,cell biology,molecular biology and other methodsMethods:We enrolled 132 TOF cases and 2,000 in-house controls without birth defects and heart disease from the same area to detect the potenial pathogenenic mutations located in NSUN5.We downloaded the sequence of NSUN5 coding region from human reference genome(GRCh37/hg19)and used online software Primer 3(v.0.4.0)to design primers.Sanger sequencing was used to detect the mutations in NSUN5 and predicted functional mutations were annotated by Combined Annotation Dependent Depletion(CADD)tool.Fisher's exact test was used to detect the mutations frequency and enrichment of NSUN5 mutation in TOF patientsThe CRISPR/Cas9 mediated genome editing was conducted to get Nsun5 knockout mouse and explore the regulatory effects on Nsun5 deletion during OFT development.We performed immunostaining to confirm the effect of Nsun5 deletion on cell proliferation in heart.By RNA bisulfite sequencing(BS-seq)and mass spectrometry(LC-MS/MS)assay,we figured out the differentially methylated sites genes and differentially expressed proteins to detect the regulation mechanism by Nsun5 during heart OFT development processResults:We identified four mutations which bioinformatic annotations predicted to be deleterious.Collectively,these mutations conferred an increased risk for TOF(P=1.44×10-5).The result implied that these NSUN5 mutations were significantly associated with TOF in patients.By further analysis,c.219₂21delAAG(p.K65del)located in a conserved position and the highly conserved sequence region will play an important role in biological evolution.This mutation can cause NSUN5 open reading frame(ORF)shift and damage the sequence of the conservative region;c.324G>T(p.A100S)is located in C-5 methylase,RsmB/RsmF domain,which is mainly responsible for ribosomal RNA methylation,so that mRNA molecules can be expand on it.The missense mutation is predicted to be deleterious,which can destroy the functional domain of the structure,leading to the ribosomal RNA unfold and protein synthesis obstacle;c.1236₁240delTGCCT(p.CL404fs*5)is located in the S-adenosylmethionine-dependent methyltransferase domain,which is an important region for m5C modification,to ensure that m5C can be modified on all kinds of RNA The mutation may cause the destruction of this functional domain and lead to the inactivated NSUN5 function;c.1370₁373delAGAA(p.KE448fs*17)is located at the end of NSUN5 gene,and the frameshift mutation may lead to the abnormal termination of the translation and cause NSUN5 protein degradationTo further verify the causative effect on Nsun5 deletion during OFT development in vivo,we generated Nsun5 knockout mouse model using CRISPR/Cas9 system.From Nsun5 heterozygosity crossed mice,we observed that a decreased amount of Nsun5 knockout mice were identified with an imbalanced Mendelian ratio among postnatal day one(P1)newborns.The number of Nsun5 wild-type,heterzygous knockout and homozygous knockout mice was 41,87 and 25 respectively.The proportion of homozygous knockouts decreased significantly,which may be caused by embryonic death.To determine the developmental stage of lethality,embryos were genotyped at embryonic day 14.5(E14.5)which OFT septation finished around this stage.The genotype ratio from E14.5 was in accordance with the expected Mendelian ratio.The result suggested that OFT septation development delay could be one of the factors which led to the lethality of the Nsun5 knockout embryos occurred between E14.5 and P1.During gestation,the OFT aorta separates such that it becomes distinct vessel arising from left ventricular In Nsun5 knockout embryos,we found that OFT aorta did not separate properly and observed ventricular septum defects by Hematoxylin-eostin(H&E)staining,which led to form a common trunk vessel originating from right ventricle.These data demonstrated that loss of Nsun5 function may confer high risk of OFT developmental malformation.In addition,42.2%(27/64)of knockouts exhibited such a mark as aorta arising from right ventricle,which conferred an significant increased occurrence compared to wild-types(13.8%,8/58).Notably,marked as a part of OFT components,Nsun5 knockout E14.5 hearts had a thinner ventricular septum,suggesting severe defects in OFT cell growth.Moreover,the immunofluorescence staining of Ki67 and EdU revealed that there was significantly reduced cell proliferation of Nsun5 knockout mice in both OFT aorta and ventricular septum at E14.5.These findings indicated that OFT septation development delay seen in Nsun5 knockout E14.5 hearts were likely caused by cell undergrowth during embryonic developmentTo elucidate whether the NsuN5-mediated m5C modification was involved in OFT development,we first detected the m5C level in E14.5 hearts by dot blot assay and the result revealed that Nsun5 knockout hearts had a significantly decreased RNA m5C level at E14.5.By BS-seq and LC-MS/MS assays,the result of pathway enrichment analysis indicated that differentially methylated sites genes were associated with striated muscle contraction,cardiac muscle tissue development,and cardiac muscle tissue morphogenesis pathways and differentiated expressed genes on protein level showed a correlation with striated muscle cell differentiation and cardiac muscle tissue development pathways.All these pathways are involved in embryonic heart development.Intriguingly,we figured out the overlap in the BS-seq and LC-MS/MS data,and we found Tpml could be as potential downstream gene.Tpml is highly expressed in cardiac cells and also acts as a regulator involved in cell proliferation.Moreover,we detected that the m5C abundance of four differentially mehtylated sites in Tpml were significantly reduced in Nsun5 knockout E14.5 hearts.The immunofluorescence staining of Tpml revealed that there were significantly reduced intensity of Nsun5 knockout hearts in OFT area at E14.5.The result indicated that Nsun5-mediated m5C modification regualted Tpml expression.Finally,we carried out RNA sequencing in Nsun5 wild-type and knockout E14.5 hearts and found that the differentiated expressed genes on mRNA level were enriched in developmental cell growth pathway.The result indicated that these differentiated expressed genes may be associated with cell proliferation.Together,our findings indicated that Nsun5 deficient caused Tpml inactivation via m5C modification,which reduced Tpml translation efficiency followed by a decrease of cell proliferation.Conclusion:In the present study,we used epidemiological methods to systematically identify the enrichment of NSUN5 functional mutations in TOF patients.The potential functional mutations are located in the important NSUN5 gene position or functional domain,resulting in the inactivated NSUN5 function,the abnormal modification of m5C on various RNA or the disorder of protein synthesis which cause the OFT development delay.Mechanistically,the deletion of Nsun5 may lead to OFT development delay.Loss of Nsun5 function inactivated m5C modification of Tpml mRNA,which reduced Tpml translation efficiency followed by a decrease of cell proliferation.These observations indicated the importance of NSUN5/TPM1 axis in the progress of OFT development.The results of the study indicate that m5C modification and NSUN5 gene play an important role in heart development,which provides a new idea for genetic etiology and pathogenesis of tetralogy of Fallot.