第IIIA型黏多醣之分子遺傳學研究

Molecular Genetic Study of Mcopolysaccharidosis Type IIIA

柯明慧(Ko, Ming Hui)

指導教授 ： 李桂楨

國立臺灣師範大學/理學院/生物學系/碩士(2000年)

摘要

摘要
N-Sulfatase (NS)為一種溶小體酵素，主要代謝heparan sulfate黏多醣，當酵素活性缺失或顯著降低，將導致體染色體隱性遺傳的第三A型黏多醣儲積症(MPS IIIA)。NS基因已被分離且定序。本論文主要以PCR、SSCP及DNA定序等技術，來檢視臺灣二位MPS IIIA患者的分子致因。結果發現患者176為複異型合子，其表現子8上第377個胺基酸由Arg轉變成Cys (R377C突變，CGC→TGC)，其表現子7上第293個胺基酸由Pro轉變成Ser (P293S突變，CCC→TCC)。Bsp1286I切割的檢測試驗顯示患者的R377C突變係遺傳自父親，但因缺乏母親的DNA樣品，故無法確定P293S突變是否遺傳自母親。患者205亦為複異型合子的突變，其表現子2上第42個胺基酸由Asn轉變成Lys (N42K突變，AAC→AAA)，表現子6上第235個胺基酸由Asp轉變成Asn (D235N突變，GAC→AAC)。誤配引子PCR及TaqI切割的檢測試驗顯示D235N突變應遺傳自母親。由於缺乏父親的DNA樣品，故無法確定N42K突變突變是否遺傳自父親。含上述突變的NS cDNA重組質體，轉移至COS-7細胞中時，並未表現出NS酵素活性，但表現的NS mRNA量和野生型者相近，僅NS成熟蛋白的表現量較野生型者低，故推測上述胺基酸的改變，可能對NS蛋白質的轉譯後修飾、運送、穩定性等造成影響。

關鍵字

第IIIA型黏多醣 ； 溶小體水解校酵素 ； 轉譯後修飾 ； 蛋白穩定性 ； PCR技術 ； SSCP技術 ； HASH(0x9c884a4) ； HASH(0x9c88d90)

並列摘要

ABSTRACT
N-Sulfatase (NS) is one of the lysosomal enzymes involved in the stepwise degradation of glycosaminoglycans heparan sulfate. Loss or marked reduction of NS activity results in the autosomal recessive mucopolysaccharidosis type IIIA (MPS IIIA) disease. The coding sequences of the NS gene were isolated and sequenced. The purpose of this study was to investigate the molecular lesions of two Chinese MPS IIIA patients by polymerase chain reaction, single strand conformation polymorphism, and DNA sequence analyses. Patient 176 has heterozygous mutations; one NS allele has R377C (C→T transition in codon 377) and the other NS allele has P293S (C→T transition in codon 293). By Bsp1286I restriction analysis, mutation R377C was paternally inherited. Owing to unavailability of mother’s DNA, the origin of P293S mutation is not clear. Patient 205 also has heterozygous mutations; one NS allele has N42K (C→A transition in codon 42) and the other NS allele has D235N (G→A transition in codon 235). By PCR with mismatch primer and TaqI restriction analysis, the mutation was maternally inherited. Since father’s DNA is unavailable, the origin of N42K mutation is not certain. Transfection of COS-7 cells, by cDNA mutagenized to N42K, D235N, P293S, and R377C mutations, did not yield active enzyme. The four mutations did not cause reduction in NS mRNA level. The observed reduced mature NS protein suggests that the amino acid changes affect post-translational modification, transport, and stability of NS protein.

並列關鍵字

N-Sulfatase ； heparan sulfate ； MPSIIIA ； PCR ； SSCP ； lysosomal enzyme ； post-translational modification ； protein stability