Constructing LCA-Nmnat1 Mice Models Using CRISPR-Cas9 Technology And Its Pathogenesis Research And A De Novo Mosaic Mutation Of PHEX Gene Causing Hypophosphatemic Rickets

Part I Constructing LCA-Nmnatl mice models using CRISPR-Cas9 technology and its pathogenesis researchBackground:Hereditary disease is a major cause of birth defects, among which there are nearly 300 kinds of simple eye genetic disorders, including Leber’s congenital amaurosis (LCA) —an earliest and the most serious genetic retinopathy. Up to now, People have found 18 types of pathogenic genes associated with LCA. Using Whole-exome Sequencing, Our team has identified that NMNAT1 gene mutations can lead to the 9th Leber’s congenital amaurosis (LCA9). But the specific pathogenic mechanism remains unknown.Methods:We explore the possible pathogenesis of the mutations in aspects of bioinformatics analysis, cytology level analysis and CRISPR-Cas9-constructed mice models. Bioinformatics analysis including homology analysis of mice and humans of NMNAT1 protein to ensure the credibility of constructing animal models using mice; Protein crystal structure analysis can help to find the candidate research mutation site which most likely to affect the structure of the protein; Protein interaction analysis with the String database can help to find the potential proteins interacting with NMNAT1. In terms of Cytological analysis, we have constructed a wild type and eight forms of mutant recombinant plasmids. Aiming to explore if the mutation can affect the subcellular localization and influence the quantity of RNA, confocal fluorescence and Real-time PCR experiments were performed. We will perform Western Blot experiments to detect the quantity of protein further. In refer to the construction of animal models of mice, we have acquired the gRNA, Cas9 mRNA and DNA templates for microinjection, bought and raised the donor mice (C57) and the receptor mice (ICR) at the same time. Once obtaining the mice animal models, we will explore the pathogenic mechanism from the phenotype, biochemical analysis, cell and molecular levels comprehensively, and explore feasible treatment further.Results:NMNAT1 protein is highly homologous in mice and humans. Protein crystal structure analysis indicates that valine 151 (V151) is located in hydrophobic core site of the protein, valine 98 (V98) is close to the ligand binding site, Trp85* (M255) and Trp 169* (M507) nonsense mutant proteins truncated and have a decentralized structure. Protein interaction prediction shows its possible interactions with a variety of proteins. Confocal fluorescence experiment indicates that the subcellular localization of Trp85* is obviously out of the nucleus while the subcellular localization of the other mutations remain normal. Real-time PCR experiments show that Trp85* (M255), Trp169* (M507) nonsense mutant, Met35Thr (M104) and Leu153Val (M457) missense mutant RNA quantities do not have distinct difference with wild type while Va1151Phe (M451) missense mutant RNA quantities decreased significantly. In aspects of constructing CRISPR-Cas9 mice models, we have prepared RNA and DNA templates for microinjection.Conclusion:In this study, we clarify the relationship between the NMNAT1 mutations and the disease of LCA9 from multiple perspectives like bioinformatics, cell biology, molecular biology, biochemistry, tissues and organs. Up to now, everything goes well, part work has been completed, including bioinformatics analysis, part of the analysis of cell biology and prepared work of construction CRISPR - Cas9 mice models, the subsequent work will focus on exploring the pathogenic mechanism of the varied mutations in mice, and the use of drug therapy and gene therapy to treat LCA, paving way for clinical research and treatment.Part II A de novo mosaic mutation of PHEX gene causing hypophosphatemic ricketsX-linked dominant hypophosphatemic rickets (XLHR, MIM#307800) is a genetic bone disease, characterized mainly by renal phosphate wasting with hypophosphatemia, retardation, short stature, bone pain, abnormal tooth enamel, double lower limbs deformities and X-ray suggests rickets. PHEX, located at Xp22.1-p22.2, is the gene causing XLHR. We aim to characterize the pathogenesis of a Chinese boy who is apparently "heterozygous" in PHEX gene. Direct sequencing showed two peaks:one was a wild type "G" and the other was one base substitution to "A", though the patient was a male. TA clone assay clearly showed each sequence and the ratio. The mutation effect was predicted via bioinformatics and validated by exon-trapping assay. Real-time PCR was applied to determine the copy number of PHEX. TA clone assay showed the frequency of normal (G) to mutant allele (A) as 19:13. Normal karyotype and Real-time PCR results indicate the normal copy number of PHEX. This splice site mutation leads to 4 bp of exon 18 skipping out causing frame shift p.Gly590Glufs*28 that ends up with a loss of active site and Zn2+ binding site of PHEX, which probably interfere with renal phosphate reabsorption and bone mineralization. In conclusion, mutation at conserved splice acceptor site resulted in aberrant splicing, ending up with a damaged protein product. This novel mutation is de novo in mosaic fashion that may be induced during early post-zygotic period. Taking mosaic somatic mutation of PHEX into consideration is strongly suggested in genetic counseling and etiology research for XLHR.