Establishment Of Molecular Diagnostic Methods For Fragile X Syndrome And Identification Of Novel Alternatively Spliced Transcripts Of Its Disease Gene

Fragile X syndrome is the most common inherited mental retardation disorder. The disease-determining gene, fragile X mental retardation 1 gene(FMR1) , was cloned on chromosome Xq27.3 in 1991.This gene spans 38kb in the genome and contains 17 exons and 16 introns.It had been found that the gene have multiple alternative spliced forms, involving four exons.Different spliced forms may exist in different tissues,but the function of them is still unknown.Due to the high frequency of the disease, its diverse clinical manifestations and the hereditary property of unstable transmission ,it is important to establish laboratory diagnostic methods for FXS.According to characteristics of FMR1 gene mutation in FXS,molecular diagnostic methods were established at genomic DNA and mRNA level in this study.To detect the CGG repeats and methylation of CpG islands in FMR1 gene,genomic DNA was extracted from peripheral blood leucocytes of the patients and was analysized by methylation specific PCR.And to detect the mutation in FMR1 gene, the entire coding sequence of cDNA from FMR1 gene was amplified by RT-PCR.The PCR products were purified and directly sequenced.Using this strategy,no mutation was found in a patient with FXS phenotype.Meanwhile,a cloning and sequencing strategy was used to analyze the alternative splicing of FMR1 gene in the peripheral blood leucocytes of the patient at mRNA level.The entire coding sequence of FMR1 gene was amplified by RT-PCR, and PCR products were inserted into pGEM T cloning vector,and multiple alternative spliced products of FMR1 gene were analyzed by sequencing.The results showed that complex alternative spliced products of FMR1 gene were detected in the patient, and total of 11 distinct alternative spliced products existed in 19 clones selected. A novel alternative spliced pattern was found in 6 alternative spliced transcripts, which involved the insertion of partial sequence of intron 9 of FMR1 gene. Bioinformatics was used to analyze splicing signals in intron 9 of FMR1 gene.In results,four main splicing signals were found around the insertion,which indicated that the insertion might be a cryptic exon.The insertion of this cryptic exon would lead to the introduction a premature termination codon,and would in turn give rise to non-functional or deleterious FMRP while initiating nonsense-mediated mRNA decay (NMD).The cloning and sequencing strategy was also used to analyze the alternative splicing of FMR1 gene in a whole brain tissue cDNA library and peripheral blood leucocytes from four normal controls at mRNA level. In the brain tissue, a total of 11 distinct alternative spliced products were detected in 20 clones, a novel spliced products was found, which used a novel splicing site in exon 15.In the four normal controls, the different abundance of alternative spliced products were seen and two novel spliced products were found in one control , which have never been reported.The molecular diagnostic methods established in this study would help the clinical diagnosis and prenatal diagnosis of FXS,as well as provide reliable data for genetic counseling of FXS.The finding of novel alternative spliced transcripts would shed light on the elucidation of FMRP function and the mechanism of FXS.