人類遺傳疾病　第一部份：第二型黏多醣儲積症IDS基因的分子遺傳研究第二部份：家族性高膽固醇血症LDL受體基因突變的記述

Human Genetic Diseases:Part I: Molecular and genetic studies of the IDS gene associated with mucopolysaccharidosis type II. Part II: Characterization of LDL Receptor Gene Mutations in Familial Hypercholesterolemia.

張瑞宏(Jui-Hung Chang)

指導教授 ： 李桂楨

國立臺灣師範大學/理學院/生物學系/博士(2004年)

摘要

無資料

關鍵字

黏多醣儲積症 ； 溶小體 ； 低密度脂蛋白受體 ； 突變 ； 外毒素 ； HASH(0x9d74490)

並列摘要

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.

並列關鍵字

mucopolysaccharidoses ； lysosome ； iduronate-2-sulfatase ； LDL receptor ； pseudomonas exotoxin A ； mutation

參考文獻

Lin, W. L., Tai, D. Y., Lo, E., Lee-Chen, G. J. (2002) The mutation underlying familial hypercholesterolemia in a Taiwanese family. BioFormosa 37(1):1-6.

Allured, V. S., Collier, R. J., Carroll, S. F. and McKay, D. B. (1986) Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci U S A 83(5):1320-4.

Bielicki, J., Freeman, C., Clements, P. R. and Hopwood, J. J. (1990) Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties. Biochem J 271(1):75-86.

Bondeson, M. L., Malmgren, H., Dahl, N., Carlberg, B. M. and Pettersson, U. (1995) Presence of an IDS-related locus (IDS2) in Xq28 complicates the mutational analysis of Hunter syndrome. Eur J Hum Genet 3(4):219-27.

Bonuccelli, G., Di Natale, P., Corsolini, F., Villani, G. and Filocamo, M. (2001) The effect of four mutations on the expression of iduronate-2-sulfatase in Mucopolysaccharidosis type II. Biochim Biophys Acta 1537(3):233-8.