Cytogenetics And Molecular Genetics Test And Analysis Of The Hereditary Fragile X Syndrome

Obejective FXS is a common inherited mental retardation disease with the second highest incidence after Down's Syndrome. It is incomplete dominant X-linked genetic disease. Expansion of the (CGG)n trinucleotide repeat in the 5'-untranslated region(5'-UTR) of Fragile X Mental RetardationⅠgene(FMRⅠ) is its pathogenesis. The clinical symptom of pre-adolescent children effected Fragile X Sydrome(FXS) is non-specific, so laboratory diagnosis is key. The methods commonly were used in laboratory are faulty, so we hope to establish an accurate, rapid and inexpensive method of laboratory testing FXS.Methods Cytogenetics and molecular genetics methods were used. Cytogenetics method: peripheral lymphatic blood of 130 samples were cultured, 5-Fdu is added to induce to fragile sites expression. 100 cells of each sample were microscopic analyzed, the rate of fragile sites≥4% is considered to be positive. PCR combined with MS-PCR method:Firstly, genomic DNA were extracted from peripheral lymphatic blood of the same 130 samples above-mentioned. All male and female samples were amplified (CGG)n repeat sequence of FMRⅠby conventional PCR, the male samples can not be effectively amplified and all female samples were carried out MS-PCR. With sodium bisulfite treatment on genomic DNA, non-methylated cytosines were converted into uracil, the methylated cytosines remains unchanged. Take the modifed genomic DNA as templates, two different sets of primers-methylation specific primer pairs and non-methylation specific primer pairs-amplified (CGG)n repeat region of FMRⅠ. Mutation type of FMRⅠwere analysed by location of the PCR products after agarose gel electrophoresis.Results After cytogenetics analysis of 130 samples, we found:There were five cases of fragile sites rate≥4% (analysis of 100 cells, fragile sites≥4). Established PCR combined with MS-PCR method; sequencing result shows that the product of conventioned PCR include (CGG)n repeat; after sodium bisulfite treatment, non- methylated (CGG)n repeat were convented into (CAA)n repeat, methylated (CGG)n repeat were convented into (CGA)n repeat. Analyze 130 clinical suspect cases using this method, all the genomic DNA of the 130 cases were effectively amplified to (CGG)n repeat of FMRⅠg ene by conventional PCR, the"n"of product are all between 13-46;Conclusion This method detected the number of (CGG)n repeat and methylation of CpG island of FMRⅠat the same time. Compared with conventional cytogenetics methods, a separate PCR, southern blot, PCR combined MS-PCR method effectively make up for their shortcomings. Provides accurate, rapid and inexpensive FXS testing method, especially suitable clinical environment.