The Study Of Molecular Pathogenesis Of Fragile X Syndrome

Fragile X syndrome (FXS) is a common form of inherited cognitive impairment, which have a diversity of clinical manifestation. The main is cognitive impairment, moderate to worst degree, often associated with giant testis, particular face, minimal brain dysfunction, epileptic attack, and the electroencephalograms of many patients present marked and diffused moment slow wave releasing and some other clinical pathological manifestation. Research shows that more than 99% of the FXS patients are caused by the decrease or absence of FMRP(fragile X mental retardation protein), the coding products of FMR1(fragile X mental retardation 1, located at Xq27.3), which is caused by the abnormal amplification of the unstable CGG repeat sequence in the 5' untranslated region of FMR1 and the subsequent methylation of its CpG island.FXR1/2 (fragile X related gene 1/2), located in 3q28 and 17p13.1 respectively, were found have considerable similarity with FMR1 in sequence and structure. Any two of the three genes can exist in the form of homodimers or heterodimers. So they were called by a joint name of fragile X gene family. Having both two KH1 domains and a RGG box within their products as FMR1, FXR1/2 not only associate with proteins and mRNAs, but also interact with FMR1 mRNA and FMRP. These two genes are closely relevant to the pathogenesis of FXS.Research shows FXS is correlated to the three genes, FMR1 and FXR1/2, which can interact with each other. So this article studied FMR1 and FXR1 (we have only constructed the recombinant vector of FXR2) in four parts so as to discuss the molecular mechanism of FXS. Firstly, about FMR1, a rapid and high-performance diagnostic method was established to study the differences in different races of people; Then the modulins that can interact with the FMR1 3'UTR has been screened from the protein expression library through yeast three-hybridization technique. As to FXR1, its coding product, FXR1P's interacting proteins are screened by yeast two-hybridization technique and have been validated by co-immunoprecipitation, and yeast three-hybrid system was applied to screen for FXR1P-interacting mRNAs from a RNA expression library in order to find the correlated links or network relations from the gene expression abnormality to FXS morbidity, and to construct initially the interaction network regulation map between protein and protein or mRNA, and to give new clues to reveal the function of FXR1P and the relationship between its function and the pathogenesis of FXS.The number of the (CGG)_n triplets in the 5'untranslated region of FMR1 is a major criterion for the diagnosis of FXS. Therefore, to establish a method to determine the number is very important for diagnosis of FXS in high-performance, quick screening and prenatal diagnosis. To study the polymorphism of the repeat sequence of (CGG)_n can also help to illuminate the heredity features of FMR1. Because of the high GC content in this region and the hard unwinding of double strand DNA, we adopted enzyme endurable high-temperature and basic group substitution method, added synergist of PCR and enhanced the anneal temperature to improve the condition of amplification. Then we amplified the repeat consequence of FMR1 gene with high GC content, and studied the diversity of FXS's inherited mark sequence between Han people and Zhuang people separately, a stable and quick method that fit for the screening of large-scale population has been established. We found that the maximum crest of CGG repeat number in the Han people and the Zhuang people was 28 and 29 respectively.Abnormal translational expression regulation of FMR1 may leading to FMR1 expression declination of FXS sufferers. So screening the proteins conjugating to mRNA 3'UTR may have exploratory significance for the pathogenesis of FXS. The bait plasmid of FMR1 mRNA 3'UTR was constructed and transfected into yeast strain L40-ura3/pHybLex/ Zeo-MS2. After that, FMR1 mRNA 3'UTR interacting proteins were screened from human fetal brain cDNA library; the acquired positive plasmids and bait plasmid were one by one co-transformed to yeast strain and validate the RNA-protein interactions. Through sequencing and analysis of homology of the external fractions of validated positive clones, We found two kinds of proteins, K-Alpha-1 and Homo sapiens hypothetical protein, might interact with FMR1 mRNA 3'UTR. So it is supposed that the two kinds of proteins may regulate the expression of FMR1 in expression level by combining with FMR1 mRNA 3'UTR.The screening and identification of a series of interaction proteins of FXR1P will be helpful for the clarification of the function of FXR1. We construct bait recombinant plasmid of pGBKT7-FXR1, mated the Yeast Strain AH109 transformed of bait plasmid pGBKT7-FXR1, with the Yeast Strain Y187 transformed of Matchmaker human fetal brain pGADT7-cDNA library plasmid, screened positive clones by detection the expression of a series of report genes, and identified the positive AD/library plasmids by enzymatic digestion and then validated the protein-protein interaction by retransformed the AD/library plasmids to yeast Y187 and applied one-to-one small scale mating with yeast AH109 transformed bait plasmid pGBKT7-FXR1, seven proteins which may have interaction with FXR1P were acquired, namely Btf, SAFB, CMAS, FTH1, GOLGA4, HSD17B1, CSH1; later, In the Co-IP experiment, four eukaryon recombint expression vectors, pCMV-FXR1-Myc, pCMV-Flag-FXR1, pCMV-Btf-Myc, pCMV-SAFB-Myc were constructed, and the interaction of FXR1P and Btf, SAFB, were validated by Co-IP in the mammals cells. We found FXR1P can interact with Btf, but can not interact with SAFB in vivo. Thus we concluded that Btf is a new member of the FXR1P-related regulation network and play a role in the development process of nervous and muscle system.Screening of the series of mRNA which can interact with FXR1P can conduce to the study of expression regulation function of FXR1P and the pathogenesy of FXS. We applied the bait plasmid pYESTrp3/FXR1 constructed for yeast three-hybrid technique to screen for FXR1P interacting mRNAs from a human brain gyrus hippocampi pRH3-cDNA library. 56 positive clones of His~+ and LacZ~+ were acquired, and then we transformed the acquired positive plasmid and bait plasmid anew one by one into yeast to validate the protein-RNA interactions, and got 9 positive clones. At last the positive clones were sequenced and their homology were analyzed, three of them, namely LENG4 and IQCE, DPP7, were inserted correctly. FXR1P may regulate the three genes' expression by combining with their mRNAs.