| Objectives 1. To explore and establish the Pulsed Field Gel Electrophoresis (PFGE) technology system which adopts to the characteristics of Chinese gene structure.2. To further elucidate the gene structural polymorphism of the subtelomeric domains within 4q35 and 10q26 in Chinese population, and to investigate the relationship of plasticity, translocation and somatic mosaicism in these domains with D4Z4 units' deletion.3. To establish the routine method of gene diagnosis based on triple digestion of genomic DNA with EcoRI, EcoRI/Blnl and Xapl, in order to develop and optimize the system of gene diagnosis which could adopt to the characteristics of Chinese FSHD mutation and be simple and reliable for clinical usage.4. To further investigate the characteristics of FSHD gene structure and mutation by PFGE technology system, and moreover, to discuss the molecular pathogenesis of Chinese FSHD.Methods 1. The genomic DNA was extracted from peripheral blood lymphocytes according to the specific procedure designed to minimize DNA shearing, then the very high molecular weight DNA embedded and trapped in the agarose plugs was cleaved in situ.2. The cleaved DNA was separated by PFGE to detect and differentiate the homologous EcoRI fragments from 4q35 and 10q26 regions. The size of each fragment was calculated by "curve fitting" with the PFGE marker.3. The EcoRI/P13E-11 fragments were detected based on digestion of genomic DNA with EcoRI and subsequent blotting with the probe P13E-ll.The 4-derived repeat arrays (Blnl-resistant) were recognized with EcoRI/Blnl double digestion, while the 10-derived repeat arrays (Xapl-resistant) were recognized with Xapl digestion.4. The data analysis and tables designation were performed using a commercially available statistical package, including statistics and parameter analysis, means and rates comparison.Results 1. The establishment of PFGE system is great helpful for differentiation andanalysis of the FSHD-associated configurations in Chinese.2. The standard pattern distribution of normal population: Most (74.5%, 82/110) of the unrelated healthy individuals displayed such a pattern without any translocation, hybridization and mosaicism. The mean and median of 4q35 repeat arrays are 87.9±3.3kb (Mean ± S.E.) and 78.5kb respectively. Whereas the mean and median of 10q26 homologous arrays are 90.1 ±4.1kb and 73.0kb.After correction for difference in median repeats size, no significant difference was detected between both repeat size distributions according to t-test (P=0.673>0.05). Less than 35 kb 4-type fragments were found in none of the individuals, while less than 35 kb 10-type homologous fragments were found in 12.2%(10/82) of the individuals. The shortest 10q one was 16 kb.3. The non-standard pattern distribution of normal population: 19.1% (21/110) of the individuals studied here displayed a translocation repeat array configuration on chromosome 4 and chromosome 10. 4-type repeat arrays on chromosome 10 were identified in 10 individuals (9.1%), whereas the reverse configuration was presented in 11 individuals (10.0%). These two types of comparable configuration didn't differ significantly according to chi-square test (P=0.819>0.05). Somatic mosaicism (defined as a fifth repeat array fragment on PFGE) was observed in 3.6%(4/110, 2 males and 2 females) of the individuals. Notably, one of the females carried a short 4q35 repeat array (<35 kb) of somatic mosaicism.4. Gene diagnosis based on triple DNA analysis: In all subjects, a 4q35-derived allele smaller than 35 kb was found. Among them, the shortest one was 12 kb. Owe to the complete allele information acquired by triple DNA analysis, we found that 4 familial cases carried a hybrid EcoRI fragment consisting of clusters of both 4-type and 10-type repeat arrays.5. Characterization of the FSHD-associated gene structure in Chinese using PFGE: In a same family, all cases carried a consistent causal 4q-derived EcoRI fragment as the propositus that tr...
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