Identification Of A Novel Exon In The Disease Gene FMR1 Of Fragile X Syndrome

Fragile X syndrome is the most common inherited mental retardation disorder with an incidence less than only that of Down's syndrome. Fragile X mental retardation 1 gene(FMR1), located at chromosome Xq27.3, has a size of approximately 38kb and contains 17 exons and 16 introns. It has been reported that the gene has multiple alternative splicing forms, mainly involving four exons(exon 12, 14, 15 and 17), and different splicing forms may be present in different tissues. But the function of the alternative splice isoforms is still unknown. Because FXS has a high incidence and complicated clinical manifestation, the analysis of FMR1 gene alternative splice expression and the determination of when, where and what splicing variants are expressed constitute a new starting point to study FMRP function and FXS pathogenesis.In the past, we had found that there was a novel alternative splice exon in the intron 9 of FMR1 gene which was 46bp long in a suspected FXS patient. In order to know whether other species have this novel alternative splice exon, tools of bioinformatics were used to search the homologues of FMR1 gene. The results showed that Macaca mulatta, Pan troglodytes and Pongo abelii have similar gene sequences, but their posititon were different, and similar sequences can not be found in the genome of Mus musculus and Rattus norvegicus. We also used bioinformatics software to predict Homo sapiens FMR1 splice variants.On this basis, according to features of this novel alternative exon, we used semi-nested PCR to detect cDNA samples from 27 normal controls and 5 human cDNA libraries from Clontech. Electrophoresis and sequencing of semi-nested PCR products demonstrated that the novel exon existed in mRNA of various human tissues. At the same time, we used hybrid minigene splicing assay to analyse the novel alternative exon at cellular level. We used PCR to amplify the minigene (intron 9 of FMR1 gene and its adjacent exon 9 and exon 10) from the DNA of a suspected FXS patient and a norml control, and the PCR products were inserted into the pcDNATM3.1 / Hygro (+) vector. 48 hours after transfection of HeLa cells, total RNA was extracted, followed by reverse transcription and semi-nested PCR. Sequencing of the inserted segment from the patient and the normal control showed that there were three base changes: 21452 C→T, 21513 C→T and 23784 T→C, comparied to reference sequence of FMR1 in GenBank. The first two base changes were polymorphic sites, where 23784 T→C might lead to an ISE based on bioinformatical analysis. The first two base changes were also present in the insert of control. Nevertheless, semi-nested PCR and the sequencing of its products showed that the novel exon in the processed transcripts of HeLa cells transfected by of recombinant vectors from both the patient and the normal control was detectable.The present study verified the presence of a novel criptic alternative exon in the intron 9of human FMR1 gene, and laid foundation to study the relationship of FMR1 and FXS pathogenesis.