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Cytogenetics and Molecular Genetics Test and Analysis of the Hereditary Fragile X Syndrome

Author: HaoZuoYu
Tutor: ChenYing；FanLiBin
School: Anhui Medical University,
Course: Genetics
Keywords: Fragile X Syndrome Fragile sites A Karyotype analysis Fragile X mental retardation gene Repeat sequence (CGG) n Methylation specific PCR
CLC: R749.94
Type: Master's thesis
Year: 2010
Downloads: 40
Quote: 0
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Abstract


The purpose of fragile X syndrome (Fragile X Syndrome, FXS) is the higher incidence after Down syndrome, a genetic mental retardation, for and incompletely dominant X-linked genetic disease, its pathogenesis because deoxy trinucleotide tandem repeat copy number due to the greatly increased, mainly as mental retardation. Since no specific clinical features of children before puberty, mainly based on laboratory diagnosis. Common laboratory diagnostic methods are inadequate, this study is to establish a set of accurate, rapid, and inexpensive method for laboratory testing FXS. The method using the method of combining cytogenetics and molecular genetics. Cytogenetic methods: First 130 cases samples of peripheral blood lymphocytes were cultured join the 5-Fdu induced the expression of fragile sites, each sample microscope analysis of 100 metaphase cells, count the expression of the X chromosome fragile sites fragile sites expression rate of ≥ 4% positive; molecular genetics methods: PCR combined with methylation-specific PCR (MS-PCR) of the 130 cases samples of peripheral blood genomic DNA was extracted First of all male and female samples conventional PCR amplification FMR I gene (CGG) n repeat sequence, MS-PCR, can not be effectively amplified male samples and all female samples of genomic DNA with sodium bisulfite modified unmethylated extracellular pyrimidin converted to uracil, whereas methylated cytosines remain unchanged, the modified DNA as a template, using two different sets of primer pairs (methylation-specific primer pairs and the non-methylation specific primer pair) FMR Ⅰ gene was amplified (CGG) n repeat region, according to the amplification product fragment size to determine type of mutation. The results found that of the 130 suspected cases of peripheral blood cytogenetic analysis: four cases of X chromosome fragile sites detection rate of ≥ 4% (analysis of 100 cells, fragile sites ≥ 4). PCR combined with the MS-PCR, sequencing showed that conventional PCR amplification products (CGG) n repeat sequences; after sodium bisulfite modified unmethylated (CGG) n repeat (CAA) n repeat sequence, methylation (CGG) n repeat (CGA) n repeat sequences. The use of the method and analysis of 130 clinically suspected cases, conventional PCR can be effective amplification of genomic DNA of 130 suspected cases FMR Ⅰ gene (CGG) n repeat sequence product (CGG) n repeat number between 13-46p . Conclusion explore the establishment of a method of MS-PCR in combination with PCR detection FXS. The method can also be detected FMR Ⅰ gene (CGG) n the number of repeats and CpG island methylation and FMR Ⅰ gene mutation genotyping make up for the separate application of PCR, traditional cytogenetic analysis, southern blot method shortcomings, combined with the clinical manifestations, provides an accurate, rapid, and inexpensive laboratory test method for FXS.

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