Neural Tube Defects Affected Subject Carrying Rare Variant TCOF1 Gene A491g Disrupts Ribosome Biosynthesis

Objective:Functional destructive rare variant, which refers to a base changes in this gene, may cause functional changes. While the frequency of rare variant in individuals is very low.Rare variation plays an important role in the study of complex diseases. Neural tube defects is a multifactorial polygenic complex congenital disease, its manifestations include anencephalus, spina bifida, encephalocele, etc.Treacle, as a nucleolar protein, involved in ribosomal DNA transcription and ribosome biogenesis. In addition, it’s has found craniofacial malformations and anencephalus in a Tcof1+/-mouse model.The purpose of this study is whether there were a rare functional destructive TCOF1 gene variants in the human neural tube defects.Methods:We studied 354 cases with a neural tube defect and 238 control subjects during the same period, they were from Shanxi Province, China. We analyzed the genotypic distributions and allele frequencies of TCOF1 in DNA samples from the case and control groups. We extracted genomic DNA from Han Chinese NTDs brain and did TCOF1 gene sequencing. We found rare variants in TCOF1 gene via blasting with Genome1000, db SNP,NHLBI Exome Sequencing Project(ESP). To screen for new rare variants in TCOF1 gene,we adopted bioinformatic analysis, such as conservative analysis of amino acids and physical and chemical characteristics of amino acids. We transfected the plasmid of TCOF1, which constructed by Origene, into Human embryonic kidney 293(HEK293T)cells. We detected the transfection efficiency and the level of the m RNA andprotein of wide-type and rare variants in TCOF1 gene. We detected Nucleolus pressure by immunofluorescence. We used real-time PCR method for analysing the level of the RNA45S5 m RNA. Differences between wide-type and rare variants in TCOF1 gene was analyzed using chi-square tests. We used ALLPrep DNA / RNA / Protein Mini Kit and Coomassie blue method for detecting protein changes in total.Results:(1)A rare functional destructive variants in TCOF1 gene were up to21.2% of NTDs,non-synonymous mutation to 2.5%.(2)The clinical phenotype of NTDs which carried rare variants in TCOF1 gene enriched in anencephalus.(3)A491G affected the expression of protein and m RNA, which led to the differences phenomenon of nucleolar splitting between wide type and A491 G.(4)A491G affected the synthesis of RNA45S5, which led to the synthesis of total protein in the end.Conclusions:It has found 21.2% of NTDs carried a rare functional destructive variants in TCOF1 gene. The clinical phenotype of rare variants focued on anencephalus. A491 G is the pathogenic mutations which led to patients who sufferde from anencephalus binding with Treacher Collins syndrome.