The Relationship Between Gene Mutation Of Enamelin Or EMSP1 And Amelogenesis Imperfecta (AI), And A Basic Research On The EMSP1 Gene Knock-out.

Amelogenesis imperfecta (AI) is a group of complicated genetic diseases, any gene construction changing concerned with enamel development can led to the formation of AI that expressed genetics characteristics and clinical heterogeneity. According to the clinical situation, AI can be divided into four types: hypoplastic, hypocalicified, hypomaturation and compound type.In this study we collected 2 families of AI in school of stomotology Jilin university, and traced totally 37 members, 13 members that include 10 males and 3 females were sick. The extant family members are totally 34, 11 are sufferers, in which 8 are males and 3 females. All of above members denied consanguineous marriage, medicine history during pregnant, medical history and the premature delivery history.Under the agreements by patients all of the family members of the 2 families of AI were undertook clinical examination, partially with X-ray exam to investigated the relationship of clinical situation, gene structure analysis, geneotype and phenotype. The general physical checkup includes: skin, hair, nail and the bone, and remove any concomitant syndromes.The clinical examination shows that the two families are 4 and 3 generations respectively, the total examinees are 37, all have no systemic diseases and syndromes concerned with AI. In family 1 the total members are 23, in which 9 are suffers, 7 males and 2 female1. In family 2 the total members are 14, in which 4 are suffers, 3 males and 1 female. The proband in family 1 performs the yellowish brown teeth, the enamel defect of incisal 1/3 of anterior teeth and occlusial surface of posterior teeth, and combined with the characteristics of dentin exposure and open bite and so on. The clinical situations of patients in family 1 are similar to that of the proband, but no open bite. The X-ray results show there are no significant changing.The clinical situations of proband in family 2 show enamel color difference in lap and buccal surface of anterior and posterior teeth, and appear white, yellow and brown spot non-translucence areas, in incisal ridge and 1/4 and 1/3 occlusive surface the enamel appears white or yellow strip areas. But the configuration and thickness of crown have no significant changing; the radiological densities of enamel and dentin in X-ray are similar. The clinical situations of suffers are similar to that of the proband.Combined the pedigree, Witkop clinical classification and pedigree clinical situations, the family 1 is limited hypoplastic AI, belongs to AD heredity; The family 2 conforms to snow-cap type hypo maturation AI, belongs to A D heredity.In the contrast of the clinical symptoms in AI of our studies to the literatures, we found different clinical types respectively in AI, we supposed that it may caused by the gene structural change. The light cases showed the limited damage in tooth enamel. Horizontal ditch and trough in the surface. The serious cases showed small, thin, smooth and color yellow tooth, sensitive to coldly stimulates. Partial patient showed a open bite occlusion. Therefore the different pedigree showeds individual type, even the same pedigreeis showed difference in the individuals.At present, there are 5 kinds mutant genes releted to AI: AMELX (located chromosome Xp22.3-p22.1), ENAM (located chromosome 4q21), EMSP1 gene (located chromosome 19q13.3-19q13.4), the MMP20 gene (located chromosome 11q22.3-23) and the DLX3 gene (locatedchromosome 1 7q21-q22). AMELX mutation can cause X chromosome-link AI. But in all AI only 5% is related to X chromosome-link form, until now 14 kinds of mutations of AMELX gene in X chromosome has been discovered. There are 5 kinds of mutations in enamelin related to AD-AI or AR-AI. In the studies of ARAI pedigree genes, enamelin gene, EMSP1 gene and MMP20 gene have been found the mutatuion type. ADHHAT is associated with DLX3 gene mutation.At present in the domestic literature it has not found in the gene structural change associated with AI. According to the tooth enamel formation and the mineralization process, conbine the enamelin matrix protein type, the gene localization and the function, we use the PCR-SSCP analysis technology, to analyses the enamelin and enamel matrix serine protein gene structure in 2 families, to discuss the clinical genetics characteristic and expound molecularmechanism in AI. Because the SSCP analysis has the high differentiations to the 200bp-300bp PCR product, we set all the PCR products in this region. By PCR and agarose gels electrophoresis, all the PCR product appears in the corresponding 200bp-300bp region.Because the family 1 and the family 2 belongs to AD heredity, therefore, we eliminate the possibility in AMELX mutation of sex-chromosome the gene structure change in autosomalmay associated with AI. In our experiment we selects enamelin and EMSP1 gene to conducts the research. Therefore we choice the proband and her elder brother in family 1, and the proband in family 2 to withdraws and collect peripheral blood to get DNA.There are 9 exons and 8 introns in enamelin gene , and 6 exons and 5 introns EMSP1 gene. We designed 21 pairs oligos in enamelin genes, and 4 pairs oligos in EMSP1 genes by using Oligo 6.0 software. At the mean time we set up the control group by the nomal person in the same pedigree. Taking DNA as the template, expand the fragments after the PCR, and run a 8% SDS-PAGE to observe the SSCP electrophoresis. No unusual electrophoresis band were discovered in this experiment. It showed that AI in family 1 and the family 2 is not associated with enamelin and the EMSP1 genes tructural change. Therefore, through this studied we eliminate enamelin and the EMSP1 gene mutation possibility, in 2 families. It may existed other related genes in AI forming process.At present, besides analyzes gene structure in concerned patient, the goal gene knock-out models are often used to observe the clinical symptoms in the gene function study. It has been reported to establish genes mutation animal models on AMELX, AMBN and MMP20 gene. These studies set up foundation for the relationship study between the gene function and the geneotype or phenotype of AI. But of all the reported studies on the related genes of AI, knock-out model of EMSP1 gene has not been reported. In this research we planed to establish a knock-out mice model of EMSP1 gene. Firstly based on the EMSP1 gene sequence analysis and the mouse EMSP1 gene sequence provied on GenBank, we designed two pairs oligos using Oligo 6.0 software. Two homologous fragments of 5000bp and 1700bp by PCR as long arm or short arm were inserted in the two sides of neo gene in general gene knock-out vector pSSC-9. PCR method and the restrictive enzyme digestion identified the colonies. The result indicated we succeeds constructed the mouse EMSP1 knock-out exon 4 and exon 5 regions gene targeting vector. Next, in order to guarantee the ES cells with EMSP1 gene knock-out targeting vector can be able smoothly to transplant to the pregnant mouse embryo, it needs to modify the ES cells in vitro to maintain its proliferation ability and differentiation suppress ability. The present commercialized ES cell has the proliferation ability and multiple- differentiation ability. It is not fit to use to tranfections of knock-out target gene. LIF gene has the function of differentiation suppress ability. So we expands mouse full-lenth LIF gene by RT-PCR technology, and using the homologous reorganization method to construct a neomycin resistance mouse LIF plasmid of pcDNA3.1-LIF. We set up a MEFs expressing LIF to suppress the differentiation of ES cells. For further target gene knock-out animal model we established a period work foundation.