【作者】 刘鼎；

【导师】 肖波； 李国良；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 目的本课题共收集了6个中国汉族PKD家系,将其与国内外已报道的PKD家系进行对比研究,深入挖掘中国汉族PKD家系自身特殊的临床特点,并选择具有代表性的家系进行基因定位与克隆研究,定位中国汉族PKD新的致病基因位点,期待克隆出PKD的致病基因。方法1)临床研究:综合已收集的6个PKD家系和中国已报道的12个PKD家系,与国外的42个PKD家系进行临床分析以及发病年龄、严重程度的配对t检验和wilcoxon符号秩和统计学分析。2)基因定位:选择家系结构好、患病人数多的家系A进行基因定位研究。首先选择24个微卫星位点对涵盖EKD1和EKD2的整条16号染色体进行排除定位;在排除了家系与16号染色体的连锁关系后,利用商业化的ABI全基因组连锁试剂盒Linkage Mapping Setv.2.5-MD10进行STR全基因组扫描,并在全扫的阳性区域内进一步增加微卫星位点进行精细定位;为了进一步验证STR扫描的结果,同时更加深入的挖掘该家系所包含的遗传信息,利用全基因组Linkage-12 SNP芯片对家系进行的再次扫描和定位,并采用Genehunter-2-locus软件对阳性区域进行2个致病位点相互作用检验。3)候选基因克隆:采用功能-候选克隆的策略,筛选4个已知基因(KCNG3、SLC8A1、PRKCE和FGF12基因)作为候选基因进行测序和突变分析。结果1)临床表型上,中国单纯型PKD家系存在发病年龄逐代提前和(或)严重程度加重的遗传早现现象:统计学上,中国单纯型PKD家系代与代之间的平均年龄差为5.2岁,有显著的统计学差异(配对t检验和Wilcoxon符号秩检验P值均＜0.0001),而复杂型PKD发病年龄并无显著差异(P＞0.01)。2)参数和非参数分析排除了该家系的致病基因与16号染色体存在连锁关系。STR全基因组扫描在D3S1580处获得了最大的两点LOD值1.75(θ=0),其最大多点LOD值和NPL值分别为1.60和4.64(P=0.004),将这个PKD家系的致病基因位点初步定位于D3S1262和D3S1311之间的区域。进一步的精确定位在D3S3669处得到最大的LOD值2.82(θ=0),NPL值9.83(P＜0.0001);单体型分析将致病基因定位于D3S1314和D3S1265之间约10.2 cM大小的区域,对应NCBI的Marshfield图谱,相当于3q28-29的区域。全基因组SNP芯片扫描结果发现除3q28-29外,还存在另一个连锁信号较强的区域2p21-22,其位置分别位于2号染色体rs1509562至rs1016607之间约7.18cM(LOD值为2.35;NPL值为5.30;P＜0.00001)和3号染色体rs10937369至rs711995之间约13.78cM(LOD值为2.39;NPL值为5.74;P＜0.00001)的区域。两位点相互作用检验结果在基于倍增模型的前提下得到最大的two-locus LOD值3.81,同时最大two-locus NPL值也升至7.81(P=0.0006),提示PKD可能同时存在两个致病基因位点2p21-22和3q28-29。3)在对4个候选基因中的测序分析中发现了8个DNA序列变异,其中有两个DNA序列的改变导致所编码的氨基酸发生变化,但未见与该PKD家系的表型发生共分离,说明这8个DNA序列变异均为单核苷酸多态,从而排除了这4个候选基因为该PKD家系致病基因的可能。结论1)发作性运动诱发性运动障碍可分为单纯型和复杂型两种亚型。中国单纯型发作性运动诱发性运动障碍家系可能存在遗传早现现象。2)通过STR和SNP全基因组扫描,获得了中国汉族单纯型PKD家系2个新的致病基因位点,分别位于2p21-22和3q28-29,且这两个位点可能存在一定的相互作用。3)基本排除KCNG3、SLC8A1、PRKCE和FGF12基因为新的PKD致病基因的可能。更多还原

【Abstract】 ObjectivesIn this study,We collected six Chinese PKD pedigrees with no other paroxysmal diseases such as epilepsy,migraine,or infantile convulsion. The aim of the study is to deeply research the clinical characteristics of Chinese PKD pedigrees and search for paroxysmal kinesigenic dyskinesia susceptibility gene(s) using a Chinese multiplex family of Pure PKD.Methods1) Clinical research:we contrasted the onset age and severity of these 6 pedigrees and another 12 reported by others in China with that of 60 PKD pedigrees in other countries on clinical and statistical analysis by using a simple generalized paired t-test and a Wilcoxon signed rank test.2) Gene mapping:After excluding the linkage related to chromosome 16 which contains the two known gene loci of PKD(EKD1 and EKD2),two genome-wide screens were performed,using ABI Linkage Mapping Set v.2.5-MD10 and Illumina Infinium Human Linkage-12 panel,in a Chinese multiplex family of Pure PKD with genetic anticipation.Linkage,Merlin and Genehunter-Two-Locus software were used to perform parametric and nonparametric linkage analyses under the modes of single-trait-locus and two-trait-locus linkage analysis.3) Candidate genes cloning:we performed a bioinformational inquiry of all known genes in both loci interval and selected 4 genes(KCNG3、SLC8A1、PRKCE and FGF12) as candidate genes for mutation analysis by polymerase chain reaction(PCR) and DNA direct sequencing.Results1) Clinically,pure PKD families in China showed the phenomenon of progressively earlier and more severe as it is passed on to the next generation.Statistically,the mean difference of disease onset in pure PKD in China group was 5.2 years in both statistical analysis(p＜0.0001) but no statistical significance(P＞0.01) between parent and offspring generation in complicated PKD.2) The STR result identified a suspective locus on chromosome 3q27-q29 with a maximum two-point LOD score of 1.85 at 0=0 and the maximum muliti-point NPL score of 4.64(P=0.004) on D3S1580.Fine mapping showed a maximum two-Lod score of 2.82 atθ=0 on D3S3669. The new pure PKD locus lies with an 10.2cM interval on 3q28-29, between D3S1314 and D3S1265.The SNP data demonstrated two loci with suggestive linkage:one is mapped to a 7.18 cM region on chromosome 2p21-22 between rs1509562 and rs1016607(LOD score of 2.35;NPL score of 5.30;P less than.00001);the other is mapped to the a 13.78 cM region on chromosome 3q28-29 between rs10937369 and rs711995(LOD score of 2.39;NPL score of 5.74;P less than.00001).In addition,an interaction was suggested with joint analyses of these two loci,which gave rise to a 2-locus LOD score of 3.81 under the assumption of a multiplicative model and a 2-locus NPL score of 7.81(P =0.0006).3) The mutation analysis result showed that only eight common single nucleotide polymorphisms(SNPs) were detected and they were not the causative variants. Conclusions1) Paroxysmal kinesigenic dyskinesia would be divided into pure and complicated subtype.2) This study provides genetic evidences of two novel loci for Pure PKD with anticipation and suggests that pure PKD is a new clinical subtype of PKD.3) Four candidate genes(KCNG3、SLC8A1、PRKCE and FGF12) were ruled out as the causative gene for pure PKD.更多还原