人類遺傳疾病：第一部份：台灣帕金森氏症的分子遺傳研究； 第二部份：台灣杭丁頓氏症患者IT15基因CAG重複序列的單套型分析
No Thumbnail Available
Date
2005
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
第一部份：摘要
帕金森氏症(Parkinson's disease；以下簡稱為PD)為一種好發於老年的神經退化性疾病，臨床上病人會出現休息性的震顫、僵直、緩動症和步伐不穩等運動功能失常症狀。病理學上PD病患的中腦黑質緻密區多巴胺神經元細胞本體及突起處出現嗜伊紅性的蛋白質包涵體稱為Lewy body，造成多巴胺神經元大量的死亡。包涵體內蛋白組成以-synuclein為主。-synuclein為一突觸前蛋白(presynaptic protein)由染色體4q21-23的SNCA基因所編碼（enode）。先前細胞及動物模式研究顯示，A53T及A30P突變的-synuclein蛋白容易堆積在包涵體內，顯示-synuclein蛋白質的變異可能參與PD的分子機制。近來亦有報導顯示SNCA基因的triplication及duplication與家族性PD相關，顯示野生型-synuclein的過度表現亦可能引發PD。除SNCA基因的突變外，parkin、DJ-1、UCHL1、PINK1、LRRK2等基因的突變亦被報導可能參與的PD致病機轉。但上述基因突變僅見於少數家族性PD患者，大部分偶發性PD的發生，可能和多種候選基因的多型性變異相關。本研究即針對SNCA、parkin、HSP70等候選基因的多型性變異，進行病例-對照組(case-control)的研究，而統計後具有顯著差異的基因變異，再配合功能性的檢測，以確認這些基因變異參與PD的致病分子機制。在SNCA基因之NACP-Rep1微衛星序列，NACP-Rep1微衛星基因型或對偶基因頻率，在患者與正常人族群間沒有統計上的顯著差異，但RsaI T>C多型性的分析中，PD病患族群中C/C基因型和C對偶基因的頻率明顯要少於控制組(P值分別為0.0153、0.0135)。若以NACP-Rep1和RsaI T>C進行單套型分析，發現0-T及0-C單套型在PD和控制者族群間的分佈存在顯著差異性(0-T：29.6% v.s. 22.6%，P=0.0287；0-C：6.9% v.s. 12.1%，P=0.0082)。另外，0-T單套型明顯地增加罹患PD的危險(odds ratio為1.54；95% CI 1.11-2.15，P = 0.0103)；相反地，0-C單套型卻降低此種風險(odds ratio為0.57；95% CI 0.36-0.90，P = 0.0177)。利用同步定量PCR檢測在PD患者血液中白血球SNCA mRNA的表現量，發現RsaI T/T和RsaI T/C基因型者確實有差異(但不顯著)，進一步構築報導質體分析此多型性對轉錄活性的影響，結果無論是IMR32細胞或293細胞，RsaI-C對偶基因明顯驅動較低的報導基因轉錄活性。在HSP70基因家族的多型性分析顯示，HSP70-2和HSP70-hom的多型性的基因型分佈在PD患者和正常人之間並無顯著差異。然而，HSP70-1 -110 A>C、+190 G>C兩多型性的基因型分佈在PD患者和正常人之間有顯著的差異(P = 0.0004、0.012)，其中-110 C/C和+190 C/C基因型在PD患者族群的比率明顯高於正常族群(P = 0.001、0.006)，而無論是-110 C/C或+190 C/C基因型其罹患PD的相對風險明顯增高(-110 C/C者odds ratio為2.91，95% CI 1.51-5.96，P = 0.0002；+190 C/C者odds ratio為3.59，95% CI=1.53-9.88，P = 0.006)。利用報導基因分析啟動子的多型性，發現-110 A對偶基因無論在IMR32細胞或是293細胞中均能驅動較高報導基因的轉錄能力，而且在細胞經熱休克處理後也呈現相似的趨勢。在parkin基因的分析上，利用SSCP和定序篩檢126個PD患者的parkin基因的所有表現子，結果僅一個位於表現子中的異型合子的突變被發現(P437A)。而分析三個位於表現子的多型性S167N、R366W、Vl380L後，發現這三個多型性無論是在基因型的分佈或對偶基因的頻率均無顯著的差異，因而推測這三個基因多型性並不影響對台灣地區個體對PD的感受性。綜言之，本研究結果顯示SNCA基因RsaI T>C取代和HSP70-1的-110 A>C可能影響台灣地區個體對偶發性PD的感受性，而parkin基因的變異則較不相關。
第二部分：摘要
杭丁頓氏症(Huntington's disease；簡稱HD)是一種體染色體顯性的神經退化性疾病，肇因於染色體4p16.3位置的IT15基因中，CAG三核苷重複序列擴增。其臨床症狀包括中年發病、運動、認知及精神性的失調。IT15基因轉錄出huntingtin蛋白，CAG擴增會導致此蛋白的多麩醯胺片段(polyglutamine tract)增長而致病。正常人CAG的重複序列範圍在10 ~ 34個，而病患的CAG重複序列則會擴增到35個以上；範圍在36 ~ 39者則有不完全穿透(incomplete penetrance)的現象。統計顯示隨著擴增的範圍增大，病患發病的年齡也會提早。IT15基因擴增多發生於由父親傳給子代，而導致子代發病提早及症狀更為嚴重，稱為預期現象(anticipation)。鄰近CAG重複片段3'端處，另有一多型性的CCG三核苷重複序列。研究顯示西方的族群HD對偶基因和(CCG)7的多型性有高度的連鎖，而日本族群則和(CCG)10高度連鎖。本研究在分析53個HD患者及172名無親緣關係的正常個體後，發現HD對偶基因CAG重複序列的範圍從38到109，而正常族群的CAG重複序列範圍則是10 ~ 29。另外，在利用多重線性回歸分析發病年齡和CAG重複序列的範圍呈現逆相關性，但鄰近的CCG重複序列對發病年齡的影響並不顯著。利用CCG和兩個鄰近的雙核苷酸重複標記D4S127、D4S412進行單套型的分析，結果顯示三個標記中的兩個與HD基因呈現連鎖不平衡，而單套型分析24個HD家族的個體，發現HD對偶基因有三個主要的單套型佔所有HD染色體的87.5%。這些資料顯示，在台灣地區出現的多種HD單套型顯示疾病的發生可能來自於一次或更多的突變事件所導致。
PartI:Abstract Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder of the elderly. It is characterized by resting tremor, rigidity, bradykinesia, and postural instability. The characteristic pathological features of PD include juxtanuclear ubiquitinated proteinaceous inclusions (Lewy bodies) in neuronal perikarya and neuronal processes, leading to selective loss of dopaminergic neurons in the substantia nigra pars compacta. alpha-synuclein, the main component of Lewy bodies, is a presynaptic protein encoded by the SNCA gene on chromosome 4q21-23. Previously, point mutations A53T and A30P in the highly conserved N-terminal portion of the Alpha-synuclein protein were shown to be more prone to fibrillogenesis in the transgenic and cellular studies, suggesting that Alpha-synuclein may have an important role in the development of PD. Recently, triplication and duplication of the Alpha-synuclein gene were reported to cause PD in several distinct families, indicating that a mere over-expression of wild-type Alpha-synuclein is sufficient to cause the disease. Although causal mutations in the gene for SNCA, parkin, ubiquitin carboxy-terminal hydrolase, DJ-1, PINK1, and LRRK2 were reported, mutations in these genes do not explain the occurrence of disease in most sporadic patients. Other genetic predisposition, most of which is not yet known, is thought to cause PD. In this study, the genetic variation in the SNCA, parkin, and HSP70 genes were investigated in a case-control study to examine the involvement in the susceptibility of Taiwanese to PD. The potential association of the genetic variation was further examined using a functional study. For the SNCA gene, while no statistically significant difference for the Rep1 microsatellite was observed, the C/C genotype (0.0% vs. 2.3%, P=0.0153) and C allele (8.9% vs.14.2%, P=0.0135) of RsaI T>C substitution were found less frequently in PD patients than in controls. In addition, haplotype analysis using Rep1 microsatellite and RsaI T>C substitution revealed significant difference for 0-T haplotype (29.6% vs. 22.6%, P=0.0287) and 0-C haplotype (6.9% vs. 12.1%, P =0.0082). An increased risk of the 0-T haplotype (odds ratio 1.54; 95% CI 0.36-0.90, P=0.0103) or a reduced risk of the 0-C haplotype (odds ratio 0.57; 95% CI 0.36-0.90, P=0.0177) was evident. In addition, the SNCA mRNA expression was different (although not significantly) between PD patients with RsaI T/T and T/C genotypes and reporter constructs containing the RsaI C allele cloned into a luciferase reporter plasmid drove significantly lower transcriptional activity compared with the RsaI T allele in both IMR32 and 293 cells. For the HSP70 genes, there was no statistically significant difference in genotype distribution between patients and controls for the three coding region polymorphisms in the HSP70-2 and HSP70-hom genes. However, for HSP70-1 gene, the overall genotype distribution was significantly different at the -110 A>C site (P = 0.004) and tended to be different at the +190 G>C site (P = 0.012) between patients and controls. The frequencies of the -110 CC and +190 CC genotypes were significantly higher in PD patients than controls (P = 0.001 and 0.006, respectively). Both -110 CC (odds ratio: 2.91; 95% CI: 1.51-5.96; P = 0.002) and +190 CC (odds ratio: 3.59; 95% CI: 1.53-9.88; P = 0.006) genotypes were significantly associated with PD. Reporter constructs containing the -110 A allele drove marginally higher transcriptional activity of HSP70-1 compared with the -110 C allele in both control and heat shocked IMR32 and 293 cells. For the parkin gene, one missense mutations (P437A) in addition to 3 exonic single nucleotide polymorphisms (SNPs) (S167N, R366W, and V380L) were identified. The association of exonic SNPs with the risk of PD was investigated and no statistically significant difference was found between PD patients and normal controls. The results suggest that the genetic variation in the SNCA and HSP70-1 genes, but not the parkin gene, may have a functional relevance to the susceptibility to sporadic PD. PartII:Abstract Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized clinically by mid-life onset, progressive motor impairment, cognitive decline, and psychiatric symptoms. The disease is caused by the abnormal expansion of a CAG trinucleotide repeat (>35) in the first exon of the IT15 gene in chromosome 4p16.3. The normal range of CAG repeats is about 10 ~ 34, and repeats 36 ~ 39 triplets in length are incomplete penetrance. It shows a negative correlation between age of onset and the CAG repeats size. An interesting genetic feature of HD is anticipation which may be defined as worsening disease severity and early disease onset in successive generations. It is caused by CAG repeat expansions in the IT15 gene between generations, particularly when HD is inherited from the father. There is another triplet sequence, a CCG repeat, immediately 3' adjacent to the CAG repeats in the IT15 gene. It had been reported that the western HD chromosomes are strongly associated (CCG)7, and the Japanese HD chromosomes are mostly with (CCG)10. We studied the expanded CAG repeat and adjacent CCG repeat in 53 HD patients and 172 unrelated normal subjects matched to the patients for ethnic origin. The range of the CAG repeat varied from 38 to 109 in the HD patients and from 10 to 29 in the control group. A significant negative correlation was found between the age at onset and the CAG expansion, with no significant influence of the adjacent CCG repeat on the age at onset by multiple regression analysis. Allelic association using CCG repeat and 2 flanking dinucleotide repeat markers (D4S127, D4S412) within 150 kb of the HD gene revealed linkage disequilibrium for 2 of 3 markers. Haplotype analysis of 24 HD families using these markers identified 3 major haplotypes underlying 87.5% of HD chromosomes. The data suggested frequent haplotypes in the Taiwanese population on which one or more mutational events leading to the disease occurred.
PartI:Abstract Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder of the elderly. It is characterized by resting tremor, rigidity, bradykinesia, and postural instability. The characteristic pathological features of PD include juxtanuclear ubiquitinated proteinaceous inclusions (Lewy bodies) in neuronal perikarya and neuronal processes, leading to selective loss of dopaminergic neurons in the substantia nigra pars compacta. alpha-synuclein, the main component of Lewy bodies, is a presynaptic protein encoded by the SNCA gene on chromosome 4q21-23. Previously, point mutations A53T and A30P in the highly conserved N-terminal portion of the Alpha-synuclein protein were shown to be more prone to fibrillogenesis in the transgenic and cellular studies, suggesting that Alpha-synuclein may have an important role in the development of PD. Recently, triplication and duplication of the Alpha-synuclein gene were reported to cause PD in several distinct families, indicating that a mere over-expression of wild-type Alpha-synuclein is sufficient to cause the disease. Although causal mutations in the gene for SNCA, parkin, ubiquitin carboxy-terminal hydrolase, DJ-1, PINK1, and LRRK2 were reported, mutations in these genes do not explain the occurrence of disease in most sporadic patients. Other genetic predisposition, most of which is not yet known, is thought to cause PD. In this study, the genetic variation in the SNCA, parkin, and HSP70 genes were investigated in a case-control study to examine the involvement in the susceptibility of Taiwanese to PD. The potential association of the genetic variation was further examined using a functional study. For the SNCA gene, while no statistically significant difference for the Rep1 microsatellite was observed, the C/C genotype (0.0% vs. 2.3%, P=0.0153) and C allele (8.9% vs.14.2%, P=0.0135) of RsaI T>C substitution were found less frequently in PD patients than in controls. In addition, haplotype analysis using Rep1 microsatellite and RsaI T>C substitution revealed significant difference for 0-T haplotype (29.6% vs. 22.6%, P=0.0287) and 0-C haplotype (6.9% vs. 12.1%, P =0.0082). An increased risk of the 0-T haplotype (odds ratio 1.54; 95% CI 0.36-0.90, P=0.0103) or a reduced risk of the 0-C haplotype (odds ratio 0.57; 95% CI 0.36-0.90, P=0.0177) was evident. In addition, the SNCA mRNA expression was different (although not significantly) between PD patients with RsaI T/T and T/C genotypes and reporter constructs containing the RsaI C allele cloned into a luciferase reporter plasmid drove significantly lower transcriptional activity compared with the RsaI T allele in both IMR32 and 293 cells. For the HSP70 genes, there was no statistically significant difference in genotype distribution between patients and controls for the three coding region polymorphisms in the HSP70-2 and HSP70-hom genes. However, for HSP70-1 gene, the overall genotype distribution was significantly different at the -110 A>C site (P = 0.004) and tended to be different at the +190 G>C site (P = 0.012) between patients and controls. The frequencies of the -110 CC and +190 CC genotypes were significantly higher in PD patients than controls (P = 0.001 and 0.006, respectively). Both -110 CC (odds ratio: 2.91; 95% CI: 1.51-5.96; P = 0.002) and +190 CC (odds ratio: 3.59; 95% CI: 1.53-9.88; P = 0.006) genotypes were significantly associated with PD. Reporter constructs containing the -110 A allele drove marginally higher transcriptional activity of HSP70-1 compared with the -110 C allele in both control and heat shocked IMR32 and 293 cells. For the parkin gene, one missense mutations (P437A) in addition to 3 exonic single nucleotide polymorphisms (SNPs) (S167N, R366W, and V380L) were identified. The association of exonic SNPs with the risk of PD was investigated and no statistically significant difference was found between PD patients and normal controls. The results suggest that the genetic variation in the SNCA and HSP70-1 genes, but not the parkin gene, may have a functional relevance to the susceptibility to sporadic PD. PartII:Abstract Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized clinically by mid-life onset, progressive motor impairment, cognitive decline, and psychiatric symptoms. The disease is caused by the abnormal expansion of a CAG trinucleotide repeat (>35) in the first exon of the IT15 gene in chromosome 4p16.3. The normal range of CAG repeats is about 10 ~ 34, and repeats 36 ~ 39 triplets in length are incomplete penetrance. It shows a negative correlation between age of onset and the CAG repeats size. An interesting genetic feature of HD is anticipation which may be defined as worsening disease severity and early disease onset in successive generations. It is caused by CAG repeat expansions in the IT15 gene between generations, particularly when HD is inherited from the father. There is another triplet sequence, a CCG repeat, immediately 3' adjacent to the CAG repeats in the IT15 gene. It had been reported that the western HD chromosomes are strongly associated (CCG)7, and the Japanese HD chromosomes are mostly with (CCG)10. We studied the expanded CAG repeat and adjacent CCG repeat in 53 HD patients and 172 unrelated normal subjects matched to the patients for ethnic origin. The range of the CAG repeat varied from 38 to 109 in the HD patients and from 10 to 29 in the control group. A significant negative correlation was found between the age at onset and the CAG expansion, with no significant influence of the adjacent CCG repeat on the age at onset by multiple regression analysis. Allelic association using CCG repeat and 2 flanking dinucleotide repeat markers (D4S127, D4S412) within 150 kb of the HD gene revealed linkage disequilibrium for 2 of 3 markers. Haplotype analysis of 24 HD families using these markers identified 3 major haplotypes underlying 87.5% of HD chromosomes. The data suggested frequent haplotypes in the Taiwanese population on which one or more mutational events leading to the disease occurred.
Description
Keywords
帕金森氏症, 基因多型性, 熱休克蛋白, 單套型, 杭丁頓氏症, CAG三核苷重複序列, Parkinson's disease, polymorphism, alpha-synuclein, parkin, heat-shock protein 70, NACP-Rep1, haplotype analysis, polyglutamine disease, Huntington's disease, CAG trinucleotide repeat