臺灣一位Hurler型黏多醣儲積症（MPS IH）患者的IDUA基因突變分析及記述

Characterization of Novel Mutation of IDUA Gene in a Chinese Patient with Hurler Syndrome

研究的目的在檢視臺灣一位Hurler型黏多醣儲積症(MPS IH）患者的α-L-iduronidase(IDUA）基因突變。首先利用和IDUA基因序列互補的寡核甘酸引子及聚合鋂鏈反應(PCR），放大包含此基因各表現子片段；經洋菜膠體電泳檢查後，進行單股核酸構形多型性(SSCP）分析，以初步篩檢突變。再進一步將異常大小或構形的表現子片段選殖定序，以檢測患者的基因突變。結果發現患者為表現子9的異型合子突變，其對偶基因1上第1447-1473 cDNA序列發生缺失(1447del27），造成9個胺基酸的缺失及第453個胺基酸由絲胺酸(Ser）轉變為精胺酸(Arg）；對偶基因2上的第1474個cDNA序列則發生15個核甘酸的插入(1474insl5），造成5個胺基酸的插入。進一步構築僅含上述突變點的IDUA cDNA 重組質體，並經lipofection的方式轉移入COS-7細胞中表現後，含1447del27、1474insl5變異的IDUA重組質體轉移細胞所表現的酵素活性僅為野生型之0.1%、0.3%。北方吸漬(northern blot）分析顯示，含1447del27、1474ins15變異的重組質體轉移細胞所表現的IDUAmRNA量，分別為野生型之24%、49%。西方吸漬(western blot）分析顯示，重組質體轉移細胞所表現的突變的IDUA蛋白質，在IDUA多株抗體偵測的底限之下。本實驗結果顯示，此病人在表現子9的缺失及插入突變，影響IDUA酵素活性，造成患者臨床上嚴重的性狀。

DNA screening for α-L-iduronidase（IDUA） gene mutation was performed in a Chinese patient with Hurler syndrome（MPS IH）. The exon-containing genomic DNA segments were amplified by polymerase chain reaction（PCR） and subjected to agarose gel electrophoresis and single-strand conformation polymorphrphism（SSCP） analysis. Aberrant PCR products or SSCP bands were cloned and sequenced to study the molecular lesions. The 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. Expression of recombinant IDUA cDNA containing 1447del27 or 1474ins15 mutation showed traced amounts of α-L-iduronidase activity 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. Both 1447del27 and 1474ins15 mutations affect either synthesis or stability of IDUA protein by western blot analysis The data suggest that the1447del27 and 1474ins15 mutations result in the severe Hurler phenotype of the patient.