Molecular Analysis and Gene Therapy in Inherited dysmyelinating disorder

遗传性髓鞘形成障碍的分子分析和基因治疗

• 批准号: 06454308
• 负责人: MAEKAWA Kihei
• 金额: $ 3.84万
• 依托单位: The Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454308/
• 关键词: MLD; Gaucher Disease; RT-PCR; Arylsulfatase A; PCR-SSCP; 遺伝子解析; ArylsulfataseA; PCR-SSCP法; MLD; PMD; PCR; ASO; SSCP; レトロウイルスベクター; アデノウイルスベクター; 異染性脳白質変性症; アリルスルファターゼA遺伝子; 遺伝型と臨床型; 遺伝子治療

We investigated the effects of enzyme replacement therapy (ERT) and bone marrow transplantation (BMT) for 15 Japanese patients with Gaucher disease (ERT : 12 cases, BMT : 3 cases). Their phenotypes were classified into possible type 1 (12 cases) and type 3b (3 cases). Laboratory findings (values of hemoglobin, platelet, angiotensin converting enzyme and acid phosphatase) and severity score index (SSI) were improved by treatment in most of cases. However, physical growth, particulary height, was still severely retarded after treatment (pre--2.7SD,post--2.2SD). BMT made physical growth retardation more improved than ERT.Genotype and splenectomy did not influence the responce of treatment. Low dose protocol (60U/kg/dose < 6 months) resulted in bone involvement during treatment in three patients. These data suggest that one should pay attention to physical growth in treatment for pediatric Gaucher disease type 1 patients and the initial dosage of enzyme is the most important factor to obta … More in sufficient clinical effects in ERT.We have analyzed on the nucleotide sequence of ASA genes in three Japanese patient (case 1,12, and 13) with MLD.In case 1 with late infantile form, two novel mutations were found : a 366a*g transition (designated 366g) in the position -2 of 3' splice site of first intron, and 1542T*C in exon 5 (designated 1542C) causing leucine 298 to be substituted by serine. The analysis of the patient's cDNA fragments amplified by RT-PCR revealed that transcripts of the 366g allele were spliced aberrantly. In a transient expression study, transfectant with the mutant cDNA carrying 298Ser did not show an increase of ASA activity, which confirms the mutation is a cause of late infantile MLD.In case 12 with juvenile form, a pseudodeficiency (PD) allele, which abolishes an N-glycosylation site (350Asn*Ser), was found. This is the first case with PD allele of ASA gene in Japanese origin. However, no disease causing mutation was detected in the case 12. Case 13 with adult form MLD was a compound heterozygote of mutant alleles : 445A of paternal origin and 2330T of maternal origin. The 2330T,affecting splice acceptor site selection, was suggested to be responsible for the mild phenotype in the patient. The further analysis of ASA gene in MLD patients should provide insight into the consideration on the phenotype-genotype correlation in the disorder. Less

我们调查了 15 名日本戈谢病患者（ERT：12 例，BMT：3 例）的酶替代疗法（ERT）和骨髓移植（BMT）的效果。他们的表型分为可能的1型（12例）和3b型（3例）。大多数病例的实验室检查结果（血红蛋白、血小板、血管紧张素转换酶和酸性磷酸酶的值）和严重程度评分指数（SSI）均通过治疗得到改善。然而，治疗后身体生长，特别是身高仍然严重迟缓（前--2.7SD，后--2.2SD）。 BMT比ERT更能改善体格生长迟缓。基因型和脾切除不影响治疗反应。低剂量方案（60U/kg/剂量<6个月）导致三名患者在治疗期间出现骨受累。这些数据表明，在儿童戈谢病1型患者的治疗中应注意身体生长，酶的初始剂量是获得足够临床效果的最重要因素。我们对三名日本MLD患者（病例1,12和13）的ASA基因核苷酸序列进行了分析。在晚期婴儿型病例1中，发现了两个新的突变：366a*g转变 (指定为366g)位于第一个内含子的3'剪接位点的-2位置，以及外显子5中的1542T*C(指定为1542C)导致亮氨酸298被丝氨酸取代。通过 RT-PCR 扩增的患者 cDNA 片段分析显示，366g 等位基因的转录本存在剪接异常。在一项瞬时表达研究中，携带 298Ser 的突变 cDNA 转染子并未显示 ASA 活性增加，这证实了该突变是婴儿晚期 MLD 的原因。在幼年型病例 12 中，发现了一个假缺陷 (PD) 等位基因，该等位基因消除了 N-糖基化位点 (350Asn*Ser)。这是日本首例携带ASA基因PD等位基因的病例。然而，在病例12中未检测到引起疾病的突变。具有成人型MLD的病例13是突变等位基因的复合杂合子：父系起源的445A和母系起源的2330T。 2330T 影响剪接受体位点选择，被认为是导致患者出现轻度表型的原因。对 MLD 患者 ASA 基因的进一步分析应该有助于深入思考该疾病的表型-基因型相关性。较少的

项目成果

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Ida H., Maekawa K., et al: "Indetification of three novel mutation..." Hum Mutat. 7. 65-68 (1996)

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