人類遺傳疾病 第一部份：第八型脊髓小腦共濟失調症之分子遺傳及外遺傳研究 第二部份：台灣兩個Netherton徵候群病患家族之分子遺傳研究

Abstract

第一部份： 第八型脊髓小腦共濟失調症(SCA8)為退化性神經疾病，其特徵為小腦功能異常或亦包含其他部位的神經性異常，此疾病和染色體13q21位置的SCA8基因3'端CTG三核重複擴增相關。自1999年以來，SCA8基因的CTG擴增突變見於遺傳性及偶發性的運動失調患者，及帕金森氏症(PD)、阿茲海默氏症(AD)、Friedreich's運動失調症等退化性神經疾病及精神病患，甚至於極少數正常人。SCA8基因表現於腦的各部位，但轉錄的RNA裁接後並不具open reading frame。在人類及老鼠基因體中，SCA8基因的5'端皆和緊鄰的KLHL1基因(actin結合蛋白)的5'端互補，即SCA8轉錄物和KLHL1轉錄物互為antisense RNA，故SCA8基因可能藉此antisense RNA，來調節KLHL1基因的表現。先前本實驗室對SCA8致病基因進行遺傳分析，亦在台灣人的PD患者中發現的CTG重複擴增的現象(Wu et al., 2004)。本研究利用PCR-genotyping及DNA定序技術，擴大分析正常人族群、運動失調症患者、PD患者、AD患者及其他神經疾病族群SCA8基因CTG重複變異，結果共發現8位個體具CTG重複擴增的對偶基因，包括1位正常人、2位運動失調症患者、1位肌張力異常症患者、1位泛發性路易體患者及3位PD患者。RT-PCR分析正常人和CTG重複擴增病患的淋巴細胞株，發現SCA8基因和KLHL1基因皆有表現。進一步利用甲基化專一的PCR檢測及限制酵素的甲基化檢測，分析SCA8基因和KLHL1基因重疊序列上的CpG島的甲基化情形，發現在正常人和SCA8基因CTG重複擴增患者細胞中，均有不同程度的甲基化情形，但和SCA8基因CTG重複並無絕對相關性。在氧化壓力相關研究中，經由氧化劑t-butylhydroperoxide (TBH) 處理後，正常人及SCA8基因CTG重複擴增患者淋巴細胞株的WST-1細胞增生檢測、Trypan blue排除檢測、Superoxide dismutase assay結果，皆無明顯差異，故推論SCA8基因CTG重複擴增，並未影響細胞株的抗氧化能力。

第二部分：

Netherton Syndrome (NS)是一種嚴重的體染色體隱性遺傳皮膚疾病，特色是先天性的紅皮症(congenital erythroderma)、特殊不正常的髮根結構(bamboo hair)及伴隨著體內IgE濃度提高的過敏性表現(atopic manifestations)。NS的病因是由於體內缺少絲胺酸蛋白抑制因子(serine protease inhibitor)的活性，而造成體內絲胺酸蛋白(serine protease)的活性異常提高。到目前為止，NS還沒有非常好的治療方法。NS的致病基因在2000年時被發現，位於染色體5q31-32的SPINK5 (serine protease inhibitor Kazal-type 5)基因，大小為61 kb，含有33個外顯子，其產物LEKTI蛋白，是一種絲胺酸蛋白抑制因子，廣泛的表現在人體各組織中，含有1064個胺基酸，有十五個potential inhibitory Kazal-type domains (D1–D15)，包括典型的Kazal-type domain D2和D15。和NS相關的SPINK5基因突變類型主要是產生了前成熟終止密碼(premature termination codon)，造成mRNA的不穩定，而使產物蛋白LEKTI (lympho-epithelial Kazal-type related inhibitor)的生成遽減。本研究目的為探討台灣兩個NS病患家族之分子致因。藉著PCR、定序來檢視兩位患者包括啟動子在內的SPINK5基因，找出和患者性狀相關的基因突變，並利用鄰近之6個微衛星序列及SPINK5基因上的5個SNP，對患者270家族進行連鎖分析。結果在患者2567的外顯子25上找到兩個突變點：已報導過的R790X及新發現的T808I，分別遺傳自患者的父親及母親。另一患者270為一新發現的R267Q變異的同型合子，此變異的Q對偶基因在正常人族群中約佔31%，但患者父親並未帶有此變異。進一步利用同步定量PCR (real-time PCR)，來檢測患者270是否有缺失突變(deletion)，結果顯示患者270在外顯子10的DNA套數和其啟動子鄰近序列(CA)20及D5S2013(為異型合子)相同，即排除其發生缺失突變的可能性。對於患者270在不符合遺傳定律之R267Q位點，推論可能發生基因轉換(gene conversion)。PartⅠ:

Spinocerebellar ataxia type 8 (SCA8) is a neurodegenerative disorder characterized by cerebellar dysfunction alone or in combination with other neurological abnormalities. The expansion of 3' CTG trinucleotide repeat on chromosome 13q21 was shown to cause dominantly inherited SCA8. Since first described in 1999, the CTG expansions of the SCA8 gene were found in various familial and sporadic ataxia patients, as well as in patients with psychiatric disorder, Friedreich's ataxia, Parkinson's disease (PD), Alzheimer's disease (AD), and in rare instances in the general population. The SCA8 transcripts are found ubiquitously expressed in various brain tissues and no extended open reading frames are present. Thus the SCA8 transcript was suggested to act as an antisense regulator of the KLHL1, a gene encoding the actin-binding protein. This antisense/sense transcriptional organization is evolutionary conserved in both human and mouse. Previously we assessed repeat sizes at the SCA8 locus and detected abnormal expansions in SCA and PD patients (Wu et al., 2004). In this study, SCA8 repeat size ranges in control subjects and in patients with ataxia, dementia, PD, and other neurological disorders were set up by polymerase chain reaction (PCR)-genotyping and DNA sequencing. A total of 8 subjects with expanded allele were found, including one normal, two ataxia, one dystonia, one parkinsonism(DLBD), and three PD. RT-PCR analysis revealed that both SCA8 and KLHL1 were expressed in lymphoblastoid cells with normal or expanded CTG repeats. Analysis of aberrant methylation by methylation specific PCR assay and restriction enzyme based-methylation assay further revealed differential methylation of the SCA8 and KLHL1 gene exon 1 region. However, CTG repeat length-dependent methylation was not observed. Finally, oxidative stress tolerance of lymphoblastoid cells carrying normal or expanded SCA8 CTG repeats was assessed by quantifying the cell viability and the amount of SOD upon t-butylhydroperoxide (TBH) treatment. The results of no significant difference suggest that cells expressed expanded SCA8 CTG repeats were not more vulnerable to TBH treatment.

Part Ⅱ:

Netherton syndrome (NS) is a severe autosomal recessive skin disorder characterized by congenital ichthyosis, hair shaft abnormalities, and atopic manifestations. NS is caused by deficiency of serine protease inhibitor, resulting in high serine protease activity. So far there is no effective treatment for NS. In year 2000, the gene for NS was mapped to 5q31-q32 and was subsequently identified as serine protease inhibitor Kazal-type 5 (SPINK5). The SPINK5 gene spans a region of 61 kb and is composed of 33 exons. It encodes LEKTI (lympho-epithelial Kazal-type related inhibitor), a predicted serine protease inhibitor highly expressed in thymus and mucous epithelia. The LEKTI protein consists of 1064 amino acids organized into 15 potential inhibitory Kazal-type domains (D1-D15). The pathogenic mutations identified in the SPINK5 gene are predominantly nonsense, predicting marked instability of mutated SPINK5 transcripts and loss of expression of LEKTI. In the study, the molecular lesions of two Taiwanese patients with NS were examined. The entire coding sequence of the SPINK5 gene, flanking intron boundaries, and the proximal promoter region from the two patients were amplified for direct sequencing. Linkage analysis using six flanking microsatellite markers and five single nucleotide polymorphisms (SNPs) in the SPINK5 gene were also performed in patient 270's family to generate haplotypes. Patient 2567 has heterozygous mutations; the maternal allele has T808I (C to T transition in codon 808) and the paternal allele has R790X (C to T transition in codon 790). Patient 270 is homozygous for a novel polymorphism R267Q (G to A transition in codon 267). The R267Q was seen frequently upon screening 200 control chromosomes, with Q allele frequency 0.31. However, the change was not detected in the patient's father. Haplotype analysis revealed that the patient was homozygous for the 5 SNP in the genomic sequence of SPINK5 as well as the flanking (GT)17 and D5S413, in addition to the discrepancy of R267Q. Quantitative real-time PCR analysis further excludes the possibility of small deletion. Thus a gene conversion event may have resulted in the homozygosity for R267Q.