Igarashi T, Günther W, Sekine T, Inatomi J, Shiraga H, Takahashi S, Suzuki J, Tsuru N, Yanagihara T, Shimazu M, Jentsch T J, Thakker R V
Department of Pediatrics, Faculty of Medicine, University of Tokyo, Japan.
Kidney Int. 1998 Dec;54(6):1850-6. doi: 10.1046/j.1523-1755.1998.00203.x.
The annual urinary screening of Japanese children above three years of age has identified a progressive renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrocalcinosis, and this represents a variant of Dent's disease. Hitherto, 12 mutations of the X-linked renal specific chloride channel, CLCN5, have been reported in the Dent'sJapan variant. To further identify such CLCN5 mutations and to define the structure-function relationships of this channel, we have investigated five unrelated, non-consanguinous Japanese families with this disorder.
Leukocyte DNA from probands was used with CLCN5 primers for PCR amplification of the coding region, and the DNA sequences of the products determined. Functional studies were performed by expressing the mutants in Xenopus oocytes.
Five CLCN5 mutations consisting of two nonsense (R648X and R704X), two missense (S270R and L278F) and one acceptor splice site mutation (ag-->cg) in intron 4 were identified. The missense and splice site mutations represent novel abnormalities. Heterologous expression in Xenopus oocytes of wild-type and the missense mutants demonstrated that the mutations, which were translated, either abolished or markedly reduced chloride conductance.
These results expand the spectrum of CLCN5 mutations associated with this renal disorder and provide insight into possible structure-function relationships. For example, both the missense mutations are located within a short putative loop between two transmembrane domains, and our results suggest that this region may have an important functional role in the regulation of channel activity.
对日本3岁以上儿童进行的年度尿液筛查发现了一种进行性肾小管疾病，其特征为低分子量蛋白尿、高钙尿症和肾钙质沉着症，这是丹特病的一种变体。迄今为止，在丹特病日本变体中已报道了X连锁肾特异性氯通道CLCN5的12种突变。为了进一步鉴定此类CLCN5突变并确定该通道的结构-功能关系，我们研究了5个患有该疾病的无关且非近亲的日本家庭。
使用先证者的白细胞DNA与CLCN5引物进行编码区的PCR扩增，并测定产物的DNA序列。通过在非洲爪蟾卵母细胞中表达突变体进行功能研究。
鉴定出5种CLCN5突变，包括2种无义突变（R648X和R704X）、2种错义突变（S270R和L278F）以及内含子4中的1种剪接受体位点突变（ag→cg）。错义突变和剪接位点突变代表新的异常情况。野生型和错义突变体在非洲爪蟾卵母细胞中的异源表达表明，这些可翻译的突变要么消除要么显著降低了氯电导。
这些结果扩展了与这种肾脏疾病相关的CLCN5突变谱，并为可能的结构-功能关系提供了见解。例如，两种错义突变均位于两个跨膜结构域之间的一个短的假定环内，我们的结果表明该区域可能在通道活性调节中具有重要的功能作用。
