【作者】 周军卫；

【导师】 李晓文；

【作者基本信息】 郑州大学 ， 医学遗传学， 2003， 硕士

【摘要】 目的：良性家族性婴儿惊厥（Benign familial infantile convulsion BFIC）是一类以孟德尔方式遗传的特发性癫痫综合征，适合于采用家系连锁分析的方法进行基因定位研究。本研究在已经报道的3个白种人BFIC位点处分别选择了位于染色体19q12-q13.1的D19S245和D19S250、16p12-q12的D16S3131和D16S3133、2q24的D2S399和D2S2330，共6个STR多态性标记，对5个BFIC家系进行连锁分析和遗传异质性检测，为最终发现我国黄种人BFIC致病基因并揭示其基因机制提供依据。 方法：本研究采集了5个BFIC家系，共70人。其中患者28人，非患者42人，分别来自河南省的新乡、南阳、周口、鹤璧四地区。每人采外周血5ml，用2％Na₂EDTA抗凝，酚-氯仿法提取基因组DNA，溶于去离子水中作为模板使用。 基因型判断采用扩增片段长度多态性（AFLP）分析，6个STR标记位点的引物序列来自基因组数据库（GDB），PCR反应总体积25ul。5％的变性聚丙烯酰胺凝胶（19：1）恒温恒功率电泳（48℃，20W）2～3h，银染显色，根据等位基因Ladder判断多态性片段的大小。 连锁分析采用LINKAGE软件包（Version 5.1）中的MLINK程序。在AD模式下，设定致病基因频率为0.001，分别计算在外显率为90％、80％、70％、60％、50％，重组率从0.000至0.5，男女重组率相等时的LOD值，根据LOD值判断连锁关系。异质性检测由遗传分析程序HOMOGM完成。 结果：连锁分析结果显示：在标记位点D16S3131处，家系HY-01、CY-01和CY-02在AD模式下，重组率为0.000，外显率为90％时，获得最大LOD值总和为2.151。大于1，但小于3，提示该位点与疾病基因可能存在连锁关系。家系XY-01和QX-01的LOD值在重组率从0.000到0.2之间均为负值。 在标记位点D19S250处，家系HY-01和CY-01在AD模式下，重组率为0.085，外显率为70％、60％时，获得最大LOD值总和分别为1.056、1.155；在重组率为0.080，郑州大学2000研究生毕业论文良性家族性婴儿惊厥基因定位的研究外显率为50%时，获得最大LOD值总和为1 .227。大于1，但小于3，表明该位点与疾病基因可能存在连锁关系。家系xy一01、CY一02和Qx一01的LoD值在重组率从0.000到0.1之间均为负值，反映各家系之间可能存在位点异质性。 在D25399、D252330、D16s3133、nl9s245等4个位点处，不能提供与致病基因存在连锁关系的信息。 异质性检测的结果显示:BFIC存在遗传异质性。在5个 BFIC家系中，疾病基因与D 1653131可能存在连锁关系的家系占39.8%，与D195250可能存在连锁关系的家系占41.3%，而与这两个标记位点均不存在连锁关系的家系占18.8%。提示不同的BFIC家系可能有不同的疾病基因位点。 结论:根据本研究的分析结果，可以得出以下结论。 1、部分BFIC家系的致病基因可能与STR标记位点D16s3131存在紧密连锁关系。在所考察的5个BFIC家系中，在标记位点D16S3131处，最大LOD值总和2.151是在AD模式下，重组率为0.000时获得的。重组率为O，提示D16S3131与BFIC疾病基因可能存在紧密连锁关系。 2、部分BFIC家系的疾病基因可能与STR标记位点D195250以一定的遗传学距离相连锁。由于在标记位点D19525O处的最大LOD值是在AD模式下，重组率为0.085和0.080时获得的，0.085和0.080的重组率反映了该标记位点与BFIC致病基因在遗传图上存在着一定的距离。 3、在BFIC患者群体中，至少存在着三个BFIC疾病基因位点。在本课题所研究的5个BFIC家系和6个STR标记位点中，发现了D16S3131和D195250与部分BFIC家系的疾病基因可能存在连锁关系。异质性检测的结果表明:有18.8%（约1/5）的家系与这两个STR位点不存在连锁关系，说明至少存在着第三个与BFIC疾病基因相连锁的染色体位点。该结论与Malacame等（2001）报道的对8个意大利BFIC家系的研究结果一致。更多还原

【Abstract】 Objective: Benign familial infantile convulsion (BFIC) is a kind of idiopathic epilepsy syndrome following Mendelian inheritance. It is advisable to map the gene for BFIC pedigrees by linkage analysis. In our study, six short tandem repeat (STR) loci including D19S245 and D19S250 on chromosome 19ql2-ql3.1, D16S3131 and D16S3133 on chromosome 16pl2-ql2, D2S399 and D2S2330 on chromosome 2q24 were chosen as DNA markers for linkage analysis. This study can bring some important information to find the gene for BFIC patient in China and to discover the gene mechanism in the future.Methods: The subjects were five BFIC pedigrees, 70 members in totall, including 28 patients and 42 non-patients, from four districts including Xinxiang, Nanyang, Zhoukou and Hebi in Henan province. Genomic DNA was extracted from peripheral blood by means of phenol/chloroform followed by alcohol precipitation. 5ml peripheral blood was extracted from BFIC family member. 2%NaiEDTA was added in each sample to prevent from solidifying. DNA was dissolved in deionized water and served as template.Amplification fragment length polymorphism analysis (AFLP) was conducted at the six STR loci to judge the individal’s genetype. The primer sequences are from the Genome Database. The volume is 25ul for each PCR. PCR products were separated by constant temperature and constant power (48 , 20W) electrophoresis on 5% denaturating polyacrylamide gels for 2-3 hours and followed by silver staining. The allele ladder is used in judging the ampilified fragment lengths.Linkage analysis was performed by MLINK program from LINKAGE software package (versions. 1). Two-point LOD score were calculated at recombination rates ( 9 )from 0.000 to 0.5 assuming the male and the female has equal recombination rates. An autosomal domimant model was used with 90%, 80%, 70%, 60%, 50% penetrance assuming frequency of disease gene for BFIC to be 0.001. According to the LOD scores, the linkage relations between STR markers and the gene for BFIC was judged. The test for heterogeneity was carried out for the results of pairwise analysis with positive loci. It was preformed by the genetic analysis program HOMOGM.Results: The results produced by MLINK program are as follows: In three pedigrees including HY-01, CY-01 and CY-02, the maximum sum two-point LOD score of 2.151 for D16S3131 was obtained at recombination rates of 0.000 under autosomal dominant model with 90% penetrance. The LOD score of 2.151, which over 1 and under 3, suggested that D16S3131 maybe linked to the disease gene for BFIC. In pedigrees XY-01 and QX-01, the two-point LOD score was negative at recombination rates between 0.000 and 0.2 under autosomal dominant model.For D19S250, the maximum sum two-point LOD score of 1.056 and 1.155 were obtained at recombination rates of 0.085 under autosomal dominant model with 70% and 60% penetrance in two pedigrees including HY-01 and CY-01. Another maximum sum two-point LOD score of 1.227 was obtained at recombination rates of 0.080 under autosomal dominant model with 50% penetrance in these two pedigrees. The LOD score of 1.056, 1.155 and 1.227, which over 1 and under 3, suggested that D19S250 maybe linked to the disease gene for BFIC. In pedigrees CY-02, XY-01 and QX-01, the two-point LOD score was negative at recombination rates between 0.000 and 0.1 under autosomal dominant model. These tesults suggested there were locus heterogeneity among these BFIC pedigrees.For the other DNA markers, including D2S399, D2S2330, D16S3133 and D19S245, there were no evidence can support linkage.The results produced by HOMOGM program showed thait there were locus heterogeneity in BFIC. Among these five pedigrees, a proportion of 39.8% families was probably linked to D16S3131, a proportion of 41.3% families was probably linked to D19S250, a proportion of 18.8% families was probably unlinked to any of the two loci.Conclusion: According to these results, three conclusion can be drawn. Firstly, thedisease gene for some BFIC pedigrees maybe linked to D16S3131更多还原