人類遺傳疾病 第壹部份：第一型黏多醣儲積症：台灣二位患者的IDUA基因突變分析 第貳部份：家族性表面蛋白B-100缺陷：台灣第二型高脂血症患者的研究

Human Genetic Diseases Part I：Mucopolysaccharidosis Type I：Mutational Analysis of the IDUA Gene in Two Chinese Patients Part II：Familial Defective Apolipoprotein B-100：a Study of Hyperlipidemia Type II Patients in Taiwan

鄧燕妮(YEN-NI TENG)

指導教授 ： 李桂楨

國立臺灣師範大學/理學院/生物學系/博士(1999年)

摘要

無資料

關鍵字

艾杜醣醛酸酵素 ； 裁接接受點 ； 膽固醇 ； 低密度脂蛋白 ； 家族性表面蛋白B-100缺陷

並列摘要

The purpose of this study is to investigate two human genetic disorders, mainly autosomal recessive - mucopolysaccharidosis type I (MPS I) and autosomal dominant - familial defective apolipoprotein (apo) B-100 (FDB), at molecular level in Taiwan.
In the study of MPS I, DNA screening for α-L-iduronidase (IDUA) gene mutations for two Chinese MPS I patients was performed by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and DNA sequencing analysis. The D4 patient has 1447del27 (Ser453→Arg in addition to in frame deletion of codons 454-462) in one allele and 1474ins15 (in frame insertion of 5 unrelated amino acids at codon 463) in the other allele. Patient L5 also has heterozygous mutations; the maternal allele has L346R (T to G transversion in codon 346) and the paternal allele has 388-3c>g (C to G transversion at position -3 of the 3' splice site of intron 2). Expression of recombinant IDUA cDNA containing 1447del27 or 1474ins15 mutation showed traced amounts of α-L-iduronidase activity (0.1 %, 0.3 %) compared to that of normal cDNA upon transfection into COS-7 cells. By northern blot analysis, 24 % 1447del27 mRNA and 49 % 1474ins15 mRNA were detected. The IDUA protein produced by 1447del27 and 1474ins15 mutations was beyond the detection by IDUA polyclonal antibody. The data suggest that the 1447del27 and 1474ins15 mutations result in the severe Hurler phenotype of the D4 patient. In transfected COS-7 cells, L346R showed no appreciable α-L-iduronidase activity (0.4 % of normal activity), although it did not cause apparent reduction in IDUA mRNA or protein level. The 388-3c>g mutation caused a significant decrease in the level of IDUA mRNA. In about 4 % time, the mutation caused the cells to use a cryptic CAG sequence 58 bp upstream from the 3' splice site and lead to insertion of 58 intronic nucleotides. IDUA cDNA containing mutated acceptor splice site showed low efficiency of splicing in expression study. The results provide further support for the importance of cytosine at -3 position in RNA processing.
In the study of FDB, the apo B gene segment around codon 3500 was screened by heteroduplex analysis, single strand conformation polymorphism (SSCP) analysis and DNA sequencing analysis in a total of 373 hyperlipidemic individuals. Two single-base mutations were detected. One mutation, ACA3528→ACG change, resulted in degenerate codon with no amino acid substitution. The other mutation, CGG3500→CAG mutation, resulted in an Arg3500→Gln substitution (R3500Q). The prevalence of heterozygote in this selected population was 0.3 % (95 % confidence interval, 0.01-1.5 %) for the R3500Q mutation, and 2.4 % (95 % confidence interval, 1.1-4.5 %) for the previously described R3500W mutation. The results suggest that R3500Q mutation is not a significant factor contributing to moderate hypercholesterolemia in Chinese (P = 0.027). Family studies of the R3500Q carrier revealed an extra two individuals heterozygous for the mutation, and both of them were hypercholesterolemic. An analysis of the R3500Q allele using 6 diallelic markers and the 3'HVR marker revealed a haplotype which was the same as that reported in a Chinese American but differed from that reported in a Chinese Canadian. The data support limited multiple recurrent origins for R3500Q in Chinese population.

並列關鍵字

α-L-iduronidase ； acceptor splice site ； cholesterol ； low density lipoprotein ； familial defective apoB-100

參考文獻

秦義雯（1998）：第一型黏多醣儲積症（MPS IH/S）的分子遺傳學研究。台北市：國立臺灣師範大學碩士論文。

Anson, D. S., Bielicki, J., and Hopwood, J. J. (1992) Correction of human MPS type I fibroblasts by retroviral mediated gene transfer of the human α-L-iduronidase gene. Hum Gene Ther 3:371-379.

Bunge, S., Kleijer, W. J., Steglich, C., Beck, M., Zuther, C., Morris, C. P., Schwinger, E., Hopwood, J. J., Scott, H. S., and Gal, A. (1994) Mucopolysaccharidosis type I: identification of 8 novel mutations and determination of the frequency of the two commonα-L-iduronidase mutations (W402X and Q70X) among European patients. Hum Mol Genet 3:861-866.

Bunge, S., Kleijer, W. J., Steglich, C., Beck, M., Schwinger, E., and Gal, A. (1995) Mucopolysaccharidosis type I: identification of 13 novel mutations of theα-L-iduronidase gene. Hum Mutat 6:91-94.

Chomczynski, P., and Sacci, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 163: 156-159.