第一部份：第二型黏多醣儲積症IDS基因的分子遺傳研究

摘 要

第二黏多醣儲積症(又稱Hunter syndrome)為X染色體隱性遺傳性疾病，其起因為缺乏分解heparan sulphate及dermatan sulphate的溶小體水解酵素iduronate-2-sulfatase (IDS)。全世界有接近三百種和MPS II相關的突變被報導。本研究利用單股核酸構形多型性及DNA定序等技術，對臺灣地區10位來自不同家庭且無血緣關係的MPS II患者進行分子致因研究，結果發現5種新穎的和5種已報導過的突變。合計先前臺灣14位MPS II患者的IDS基因變異分析所發現的10種突變，於24位的臺灣MPS II患者共發現20種突變，顯示IDS基因突變的高度異質性變化。R468Q和R468W突變分別發現於3位無血緣關係的患者，其發生率共佔25.0%。利用IDS基因鄰近的DXS1123、DXS1113二核重複多型性標記，建構突變基因的單套型，結果發現無血緣關係的R468Q突變為不同的起源，但無血緣關係的R468W突變則可能具相同的起源。患者1150的R468Q突變及患者710的I485R突變發生於精蟲形成的減數分裂。白血球酵素活性檢測顯示，正常人族群及女性MPS II突變基因攜帶者的IDS酵素活性範圍分別為19.2 ~ 70.6、8.4 ~ 26.6 nmol/h/mg cell protein，分佈範圍雖然有顯著差異，且女性攜帶者的平均酵素活性值亦低於正常人平均值的一半，但因為兩樣品群活性範圍的小部分重疊，故單獨檢測白血球酵素活性的數值，並不能用來判斷是否為突變基因攜帶者。此外本研究亦對所發現的17種胺基酸置換、缺失及終止密碼突變做進一步記述。突變的IDS cDNA轉移入COS-7細胞後，各突變的cDNA所表現的IDS活性，僅為野生型cDNA的0% ~ 3.9%，顯示突變的有害；但僅231del6突變造成mRNA的不穩定性(降低57%)。西方吸漬分析及共軛焦顯微鏡分析顯示，所檢測的11種單一胺基酸置換突變，僅K347E突變酵素成熟蛋白的大小及生成量與野生型酵素相似，其餘10種點突變前軀蛋白表面上正常，但成熟蛋白量降低或不具，顯示突變蛋白的正常成熟但無法正常運送至溶小體。所檢測的6個缺失、終止密碼突變中，1055del12和E521X的成熟不正常，共軛焦顯微鏡分析顯示截短的W267X和1184delG滯留在早期的vacuolar隔間。231del6和1421delAG則未看到表現的IDS蛋白，顯示突變IDS蛋白的不穩定性及被分解。在增進IDS重組蛋白在酵素替換治療應用的研究上，PEIa銜接的IDS較易被COS-7細胞分泌出去，被細胞內噬後亦生成有活性的酵素，但銜接的PEIa並沒有預期的
結合LRP的功能。黏多醣儲積症的分子遺傳學研究，可清楚的確認患者的分子致因並提供出生前及家族檢測。進一步的突變記述來釐清突變對IDS酵素活性及成熟的影響，可將此疾病的症狀和基因型相關聯，提供臨床上預後及治療方法的評估。

第二部份：家族性高膽固醇血症LDL受體基因突變的記述

摘要

家族性高膽固醇血症(familial hypercholesterolemia，簡稱FH)為一體染色體顯性遺傳疾病，患者因低密度脂蛋白(LDL)受體基因突變而導致血漿中LDL膽固醇值過高，易發展出早發性的冠狀心臟病。至今有超過840種分散在LDL受體基因上的缺失、插入、點突變、裁接突變被報導。先前本實驗室檢測了170位膽固醇濃度大於240 mg/dl的高脂血症患者LDL受體基因，於患者中發現10種可能和疾病相關的變異，包括兩種缺失變異(Del e3-5 和Del e6-8)及八種點變異(W-18X、D69N、R94H、E207K、C308Y、I402T、A410T、A696G)。本研究延續上述發現，對檢測到的變異作進一步的記敘。結果發現A696G cDNA質體轉移細胞LDL受體的表現量和活性與野生型受體相近。含D69N突變的cDNA質體表現時出現異常的中間型蛋白。含R94H、E207K、C308Y、I402T及A410T突變的LDL受體活性為野生型的20 ~ 64%。相對的，Del e3-5及Del e6-8突變的LDL受體活性僅為野生型的0 ~ 13%。D69N、R94H、E207K、C308Y及I402T等突變受體大部分停留在內質網，A410T及Del e6-8突變受體則停留在endosome/lysosome。此LDL受體的分生研究能清楚的確定病人的高血脂原因，可應用於出生前、家族分析及提供臨床上治療的評估。

Part I: Molecular and genetic studies of the IDS gene associated with
mucopolysaccharidosis type II

Abstract

Hunter syndrome (mucopolysaccharidosis type II) is an X-linked recessive lysosomal storage disease caused by a defect of the iduronate-2-sulfatase (IDS) gene. Nearly 300 different mutations underlying mucopolysaccharidosis type II (MPS II) have been identified worldwide. To investigate the molecular lesions underlying Taiwanese MPS II, 10 probands and families were identified and screened for iduronate-2-sulfatase (IDS) mutation by single strand conformation polymorphism and DNA sequencing. Five novel and five previously reported mutations were found. Together with the previously reported 10 mutations in 14 probands and families, a total of 20 mutations were identified in 24 Taiwanese MPS II patients, supporting the mutational heterogeneity for MPS II. The identified R468Q and R468W account for 25.0% mutations found in our patients. Haplotype analysis using flanking DXS1113 and DXS1123 revealed that the unrelated R468Q alleles are independent origin whereas the unrelated R468W alleles are probably of the same origin. The R468Q mutation in patient 1150 and I485R mutation in patient 710 occurred de novo in male meioses. Leukocyte IDS measurement revealed significantly different range of IDS activity in normal controls and MPS II carriers (19.2 ~ 70.6 vs.8.4 ~ 26.6 nmol/h/mg cell protein). The mean leukocyte IDS activity in female carriers was less than a half of the normal level. However, due to a small overlapping range of normal and carriers, the level of enzyme activity can not be used alone for carrier detection. In addition, a total of 17 identified missense, small deletion, and nonsense mutations were further characterized by transient expression studies. Transfection of COS-7 cells by the mutated cDNA did not yield active enzyme, demonstrating the deleterious nature of the mutations. A 57% decrease in IDS mRNA level was seen with 231del6 mutation. Among the 11 missense mutations examined, K347E substitution showed apparent normal maturation and targeting on immunoblot and confocal fluorescence microscopy examination. The rest 10 missense mutations showed apparent normal precursor with little or reduced mature forms, indicating normal maturation but incorrect targeting of the mutant enzymes. Among the 6 deletion and nonsense mutations examined, 1055del12 and E521X showed abnormal maturation. The staining pattern of truncated W267X and 1184delG proteins suggested retention within early vacuolar compartments. The mutated 231del6 and 1421delAG proteins were unstable and largely degraded. To improve the uptake of the recombinant enzyme for enzyme replacement therapy, PEIa-fused IDS showed increased secretion profiles, leading to increased uptake compared with the wild-type enzyme. However, the expected LRP-mediated uptake was not observed. Molecular and genetic studies of the IDS gene will clearly identify the patient's cause and allow antenatal and family studies. The further characterization of gene mutations may delineate their functional consequence on IDS activity and processing and may enable future studies of genotype-phenotype correlation to estimate prognosis and to lead to possible therapeutic intervention.

Part II: Characterization of LDL Receptor Gene Mutations in
Familial Hypercholesterolemia

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by increased levels of plasma LDL cholesterol, which cause cholesterol deposition in tissues in the form of tendon xanthomas and atheroma, leading in turn to premature arteriosclerosis and coronary heart disease. FH is caused by mutations in the LDL receptor gene resulting in defective clearance of plasma LDL. To date, more than 840 mutations including gross deletions, minor deletions, insertions, point mutations, and splice-site mutations scattered over the LDL receptor gene have been reported. Previously DNA screening for LDL receptor mutations was performed in 170 unrelated hyperlipidemic Chinese patients and two clinically diagnosed familial hypercholesterolemia patients. Two deletions (Del e3-5 and Del e6-8) and eight point mutations (W-18X, D69N, R94H, E207K, C308Y, I402T, A410T, and A696G) were identified. The effects of the identified mutations on LDL receptor function were characterized in the present study. The LDL receptor level and activity were close to those of wild type in A696G transfected cells. A novel intermediate protein and reduction of LDL receptor activity were seen in D69N transfected cells. For R94H, E207K, C308Y, I402T and A410T mutations, only 20~64% of normal receptor activities were seen. Conversely, Del e3-5 and Del e6-8 lead to defective proteins with 0~13% activity. Most of the mutant receptors were localized intracellularly, with a staining pattern resembling that of ER (D69N, R94H, E207K, C308Y and I402T) or endosome/lysosome (A410T and Del e6-8). Molecular analysis of the LDL receptor gene will clearly identify the cause of the patient's hyperlipidemia and allow appropriate early treatment as well as antenatal and family studies.

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