Perez Yonatan, Shorer Zamir, Liani-Leibson Keren, Chabosseau Pauline, Kadir Rotem, Volodarsky Michael, Halperin Daniel, Barber-Zucker Shiran, Shalev Hanna, Schreiber Ruth, Gradstein Libe, Gurevich Evgenia, Zarivach Raz, Rutter Guy A, Landau Daniel, Birk Ohad S
The Morris Kahn Laboratory of Human Genetics, National Institute for Biotechnology in the Negev and Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
Pediatric Neurology unit, Division of Pediatrics, Soroka University Medical Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84101, Israel.
Brain. 2017 Apr 1;140(4):928-939. doi: 10.1093/brain/awx013.
A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through defective function of this novel activity of SLC30A9 rather than by a defect in its previously described role in transcriptional activation of Wnt signalling.
神经功能恶化在早年就很明显，逐渐发展为严重智力残疾、严重共济失调、脊柱前凸和眼球运动失用症。脑部磁共振成像（MRI）正常。6名受影响个体中有4人还患有早发性肾病，具有肾小管间质性肾炎、高血压和高钾血症倾向，不过没有人出现肾功能快速恶化。全基因组连锁分析在4号染色体上确定了一个约18兆碱基（Mb）的疾病相关位点（在D4S2971处最大优势对数分数为4.4；θ = 0）。全外显子组测序在该位点内的SLC30A9中发现了一个单一突变，该突变在家族中按预期分离，在300名贝都因对照中未发现纯合状态。我们发现SLC30A9（溶质载体家族30成员9；也称为锌转运体9，ZnT - 9）在全身广泛表达，在小脑、骨骼肌、胸腺和肾脏中表达水平较高。对过表达与增强型绿色荧光蛋白融合的SLC30A9的SH - SY5Y细胞进行共聚焦分析，结果显示其囊泡状胞质定位与内质网相关，不与内体或高尔基体标记物共定位。SLC30A9编码一种推测的锌转运体（通过相似性），之前与Wnt信号通路相关。然而，在SH - SY5Y细胞中使用双荧光素酶报告基因检测，我们发现Wnt信号通路不受该突变影响。基于蛋白质建模，所鉴定的突变预计会影响SLC30A9高度保守的阳离子外流结构域，推测会破坏其跨膜螺旋结构。对过表达野生型和突变型SLC30A9的HEK293细胞进行胞质锌离子测量，结果显示突变型SLC30A9细胞内的锌浓度低于野生型。这表明SLC30A9具有影响细胞内锌稳态的锌转运特性，并且该疾病的分子机制是通过SLC30A9这种新活性的功能缺陷，而非其先前描述的在Wnt信号通路转录激活中的缺陷。
