|
X染色體脆裂症 (Fragile X syndrome)是人類最常見的遺傳性智障的主要原因。和此疾病產生有關的基因FMR-1(Fragile X MentalRetardation-1)已被找到。在 FMR-1基因5'端不轉譯區內的CGG重複序列發生不正常重複次數的增加導致此疾病發生並會導致易脆X染色體出現。
由於X染色體脆裂症是遺傳性的疾病，因此建立一快速且有效的篩檢方法是有其必要性的。本研究即是發展以針對FMR-1基因CGG重複序列直接作PCR的方法，以此法可用肉眼觀察染色的瓊脂凝膠上的PCR產物的有無來進行篩檢。此外我們在反應中加入另一對針對X染色體上FRAXE基因另外一個CGG重複序列的區域設計的引子進行FRAXE的PCR，作為PCR反應的內部控制。利用此法對301名男性智障患者進行篩檢。結果篩選到 7名可能為X染色體脆裂症患者，其中5名經由南方墨跡分析法(Southern blot analysis)確定為 X染色體脆裂症患者，且均可利用細胞遺傳學方法誘發出易脆X染色體。
此外，由於國外針對高加索族群利用在CGG重複序列兩側的二個具有多型性區域的標誌DXS548及AC1(marker)進行分析，發現此病具有founder effect的現象。而在中國人族群中，此方面的相關研究很少，因此本研究的另一主題即是利用DXS548及AC1二個標誌對 FMR-1基因CGG重複序列位置作單套體 (haplotype)分析，探討中國人族群是否也有類似的現象。在分析了93名正常人及14名X染色體脆裂症患者後，結果顯示在此區域位置allele freguencies在中國人及高加索人有所不同，但在高加索人中所見到有連鎖不平衡的現象，我們根據結果推測中國人族群亦可能有founder effect的現象。
The fragile X syndrome is the most common form of inherited mental retardation. The gene, fragile X mental retardation-1 ( FMR-1 ) has been identified to be associated with this syndrome. Abnormal expansion of a CGG repeat in the 5' end untranslated region of the FMR-1 gene give rise to this syndrome phenotype and fragile X chromosome.
Since the fragile X syndrome is an inheritable disease. It is necessaryfor us to estabhsh a fast and effective method in order to screen this disease.One aim of this research is to develop a nonisotopic polymerase chain reaction technique for the identification of fragile X full mutation among men, with easy visualization of the PCR product on non-denaturing gels stained with ethidium bromide. The technique consists of PCR amplification with primers that flank the CGG repeats. Moreover, in order to produce an indispensable internal control we added to the reaction other primers to amplify FRAXE trinucleotide repeat (GCC) region.
Alleles of normal size are detected, leaving a small minority of samples to be tested by southern blotting and cytogenetic method. We applied this method to screen 301 mental retarded males and found 7 of them who were suspected to be fragile X syndrome patients. Five of these 7 people were confirmed to be the fragile X syndrome patients by southern blot analysis. And we also induced the fragile X expression by the cytogenetic method in these 5 patients.
Evidence of founder chromosome effects based on the presence of genetic linkage disequilibrium to nearby microsatellite markers has been presented in studies of Caucasian subjects, but there were few similar studies have yet been pubhshed dealing with Chinese background. So, the second aim of this research is to perform haplotype analysis of the FMR-1 gene of the CGG repeats region by two polymorphic marker, DXS548 and FRAXAC1. Analysis of the allele frequencies for DXS548 and FRAXAC1 showed that there are distinct differences between Chinese and Caucasians at these loci both in normal and fragile X affected individuals, and we presumed that linkage disequilibrium exist in Chinese population.
|