Dardis Andrea, Zampieri Stefania, Filocamo Mirella, Burlina Alberto, Bembi Bruno, Pittis Maria Gabriela
Unità di Malattie Metaboliche, I.R.C.C.S. Burlo Garofolo, Trieste, Italy.
Hum Mutat. 2005 Aug;26(2):164. doi: 10.1002/humu.9353.
Niemann Pick disease (NPD) is an autosomal recessive lysosomal storage disorder caused by the deficient activity of acid sphingomyelinase due to mutations in the SMPD1 gene. We functionally characterized three novel SMPD1 mutations and 11 already reported in the Italian population. Mutant alleles were studied for enzyme activity and protein processing in transiently transfected COS-1 cells. The c.96G>A, c.100delG, c.565dupC, and c.575dupC (p.W32X, p.G34fsX42, p.P189fsX1, and p.P192fs14) alleles expressed no immunoreactive protein and consequently no enzyme activity. In contrast, cells transfected with mutants c.308T>C, c.389T>C, c.674T>C, c.732G>C, c.841G>A, c.1687G>A, c.1799G>A, and c.1799G>C (p.L103P, p.V130A, p.L225P, p.W244C, p.A281T, p.D563Y, p.R600H, p.R600P) expressed protein levels comparable to wild-type ASM expressing cells. Only three of these constructs, c.389T>C, c.1687G>A, and c.1799G>A (p.V130A, p.D563Y, p.R600H), retained residual activity while the other five expressed very low or no enzyme activity. As expected, the c.1669underscore;1670delGT (p.V557fsX18) mutant expressed a completely inactive truncated protein. Interestingly, the c.2T>G (p.M1_W32del) mutant expressed 26.9% of the wild type activity, even though no ASM protein was detected by Western blot analysis, suggesting that the amount of produced enzyme is below detection levels. The results presented in this study are consistent with the wide phenotype variability found in NP type B patients and provide valuable insights into the molecular basis of the disease.
尼曼-匹克病（NPD）是一种常染色体隐性溶酶体贮积症，由SMPD1基因突变导致酸性鞘磷脂酶活性缺乏引起。我们对意大利人群中三个新的SMPD1突变和11个已报道的突变进行了功能表征。在瞬时转染的COS-1细胞中研究了突变等位基因的酶活性和蛋白质加工情况。c.96G>A、c.100delG、c.565dupC和c.575dupC（p.W32X、p.G34fsX42、p.P189fsX1和p.P192fs14）等位基因未表达免疫反应性蛋白，因此也没有酶活性。相比之下，用突变体c.308T>C、c.389T>C、c.674T>C、c.732G>C、c.841G>A、c.1687G>A、c.1799G>A和c.1799G>C（p.L103P、p.V130A、p.L225P、p.W244C、p.A281T、p.D563Y、p.R600H、p.R600P）转染的细胞表达的蛋白水平与表达野生型酸性鞘磷脂酶（ASM）的细胞相当。这些构建体中只有三个，即c.389T>C、c.1687G>A和c.1799G>A（p.V130A、p.D563Y、p.R600H）保留了残余活性，而其他五个表达的酶活性非常低或没有酶活性。正如预期的那样，c.1669_1670delGT（p.V557fsX18）突变体表达了一种完全无活性 的截短蛋白。有趣的是，c.2T>G（p.M1_W32del）突变体表达了野生型活性的26.9%，尽管通过蛋白质印迹分析未检测到ASM蛋白，这表明产生的酶量低于检测水平。本研究中呈现的结果与B型NP患者中发现的广泛表型变异性一致，并为该疾病的分子基础提供了有价值的见解。
