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

1.本研究通过FISH、Southern杂交和核苷酸序列分析，在6 q16/12 p12易位、癫痫和严重精神发育迟滞患者的12 p12断裂点上发现了一个新基因PLEKHA 5。在6 q16断裂点没有发现基因。为了研究该基因的作用，我们将Plekha 5特异性siRNA转染到小鼠Neuro 2a细胞中。当用20 μM视黄酸处理时，转染的细胞表达约40%的Plekha 5 mRNA，并且与经历模拟转染（仅载体）的细胞相比，显示出有限的神经突延伸。这一发现表明PLEKHA 5可能是这种疾病的致病基因。Plekha 5定位于细胞质。亲和纯化的PLEKHA 5显示130 kDa的大小。从一个严重精神发育迟滞家系的男性患者中分离出致病基因，并对该家系的11名成员进行了连锁分析。这11人中有3人是病人。最大 关于我们 在Xq 27的DXS 1205处获得lod评分，1.10。对患者DNA中Xq 27-ter的15个候选基因进行核苷酸序列分析，均未发现明显的突变.从一个表现为快速脑萎缩和严重精神发育迟滞的家系中分离致病基因，我们对该家系的10名成员进行了连锁分析。这10人中有4人是病人。在2 q35 -36的D2 S163和D2 S2344之间获得最大lod得分，4.75。对16个候选基因进行核苷酸序列分析，发现SLC 19 A3中存在E320 Q错义突变。患者为突变纯合子，其父母为杂合子。我们构建了由CMV启动子驱动的SLCJ 9A 3（E320 Q）表达载体pC 1-SLC 19 A3（E320 Q），并将其转染HEK 293细胞后检测了硫胺素和生物素的转运活性。然而，转运蛋白活性没有显著降低。我们现在正在生产表达Slc 193（E320 Q）的转基因小鼠，以进一步研究这种突变的影响。少

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