Isolation and characterization of the new genes isolated from three diseases presenting with severe psychomotor retardation.

从三种表现为严重精神运动迟缓的疾病中分离出的新基因的分离和表征。

基本信息

批准号：
15390332
负责人：
WAKAMATSU Nobuaki
金额：
$ 9.34万
依托单位：
Institute for Developmental Research, Aichi Human Service Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

1. Isolation of the causal gene from the patient presenting with reciprocal 6q16/12p12 translocation, epilepsy and severe psychomotor retardation.We identified a new gene, PLEKHA5, at the breaking point of the 12p12 by FISH, Southern blot and nucleotide sequencing analyses. There was no gene located at the 6q16 breaking point. To examine the role of this gene, we transfected Plekha5-speciflc siRNA into mouse Neuro2a cells. Transfected cells express about 40% of Plekha5 mRNA and showed limited extension of the neurites compared to cells undergoing Mock transfection (vector only) when treated with 20 μM retinoic acid. This finding suggests that PLEKHA5 is likely a causal gene of this disease. Plekha5 localized at the cytoplasm. Affinity purified PLEKHA5 showed a size of 130 kDa.2. Isolation of the causal gene from male patients in a family presenting with severe psychomotor retardation.We performed linkage analysis in 11 members of the family. Three of these eleven were patients. Maximal … More lod score, 1.10, was obtained at DXS1205 of Xq27. Nucleotide sequencing analysis was performed for 15 candidate genes at Xq27-ter from the patients' DNA, but there were no apparent mutations.3. Isolation of the causal gene from patients in a family presenting with rapid brain atrophy and severe psychomotor retardation.We performed linkage analysis in 10 members of the family. Four of these ten were patients. Maximal lod score, 4.75 was obtained between D2S163 and D2S2344 at 2q35-36. Nucleotide sequence analysis was performed for 16 candidate genes and an E320Q missense mutation was found in SLC19A3. The patients were homozygous for the mutation and their parents were heterozygous. We constructed an expression vector pC1-SLC19A3 (E320Q), that expressed SLCJ9A3 (E320Q) driven by a CMV promoter and examined the transporter activity of thiamine and biotin after transfection of this vector into HEK293 cells. However, there was no remarkable reduction of transporter activity. We are now producing transgenic mouse expressing Slc193 (E320Q) to further investigate the effects of this mutation. Less

1. 从患有 6q16/12p12 相互易位、癫痫和严重精神运动迟缓的患者中分离出致病基因。我们通过 FISH、Southern 印迹和核苷酸测序分析在 12p12 断裂点鉴定了一个新基因 PLEKHA5。 6q16 断裂点处没有基因为了检查该基因的作用，我们转染了该基因。当用 20 μM 视黄酸处理时，将 Plekha5 特异性 siRNA 导入小鼠 Neuro2a 细胞中，转染的细胞表达约 40% 的 Plekha5 mRNA，并且与经历模拟转染（仅载体）的细胞相比，神经突的延伸有限。定位于细胞质的 Plekha5 的致病基因。亲和纯化的 PLEKHA5 显示大小为 130。 kDa.2. 从患有严重精神运动障碍的男性患者中分离出致病基因。我们对这 11 名家庭成员中的 3 名患者进行了连锁分析。最大 lod 评分为 1.10。对患者DNA中Xq27-ter的15个候选基因进行DXS1205测序分析，未发现明显突变。3．从患有快速脑萎缩和严重精神运动迟缓的家庭中分离出致病基因。我们对这 10 名家庭成员中的 4 名患者进行了连锁分析，在 D2S163 和 D2S2344 之间获得了最大 lod 评分 4.75。对16个候选基因进行了2q35-36的核苷酸序列分析，发现了E320Q错义突变。 SLC19A3 突变为纯合子，其父母为杂合子，我们构建了由 CMV 启动子驱动的表达载体 pC1-SLC19A3 (E320Q)，并检测了转染后硫胺素和生物素的转运蛋白活性。然而，我们现在正在生产转基因小鼠表达。 Slc193 (E320Q) 进一步研究了这种突变的影响。