Molecular Analysis and Gene Therapy in Inherited dysmyelinating disorder

应用酶替代疗法(ERT)和骨髓移植(BMT)治疗日本高谢病患者15例(ERT 12例，BMT 3例)。表型分为1型(12例)和3b型(3例)。多数病例经治疗后实验室指标(血红蛋白、血小板、血管紧张素转换酶、酸性磷酸酶)和严重程度评分指数(SSI)均有改善。然而，体格发育，尤其是身高，在治疗后仍然严重迟缓(治疗前2.7SD，治疗后2.2SD)。BMT对体格发育迟缓的改善作用明显优于ERT，基因分型和脾切除不影响疗效。低剂量方案(60U/kg/剂量&lt；6个月)导致3例患者在治疗过程中发生骨侵犯。提示小儿高谢病1型患者在治疗中应注意体格发育，酶的初始剂量是影响…的最重要因素。我们对3例日本MLD患者(例1、例12和例13)的ASA基因序列进行了分析，在例1的婴儿晚期形式中，发现了两个新的突变：第一内含子3‘剪接点-2位置的366A*g突变(标记为366G)和外显子5的1542T*C(标记为1542C)，导致亮氨酸298被丝氨酸取代。对患者的RT-PCR扩增的cDNA片段进行分析，发现366G等位基因的转录本发生了异常剪接。在瞬时表达研究中，携带298Ser的突变的cDNA没有显示出AsA活性的增加，这证实了该突变是婴儿晚期痴呆的原因。在12例幼年型的病例中，发现了一个取消N-糖基化位点(350Asn*Ser)的假性缺乏(PD)等位基因。这是日本首例ASA基因PD等位基因携带者。例12未发现致病突变。例13成人型MLD为复合杂合子，445A为父源，2330T为母源。2330T影响剪接受体位点的选择，可能与患者的轻度表型有关。对MLD患者ASA基因的进一步分析应有助于进一步考虑该疾病的表型-基因相关性。较少

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