Cell Biology of Metabolic Disorders

该项目的研究涉及罕见的人类遗传性疾病的细胞生物学以及正常和异常的细胞内过程。研究目标是深入了解各种遗传代谢紊乱的分子功能变化，并致力于治疗这些疾病。该研究重点关注四组罕见疾病： 1.唾液酸代谢紊乱。唾液酸生物合成途径中的关键酶是UDP-GlcNAc 2-差向异构酶/ManNAc激酶（GNE）。GNE的变构位点的显性突变引起唾液酸过多，其特征在于唾液酸的过度产生。GNE的隐性突变导致神经肌肉疾病遗传性包涵体肌病（HIBM）。我们已经表征了敲入HIBM小鼠模型，并证明N-乙酰甘露糖胺（ManNAc）挽救了纯合突变小鼠的表型（参考文献1），并且是人类患者的有希望的治疗选择。其他治疗（唾液酸、半乳糖）目前正在该鼠HIBM模型上进行测试。目前正在制定HIBM患者的ManNAc临床治疗方案。在一项初步临床试验中，向4名HIBM患者提供了高度唾液酸化的糖蛋白（参考文献2）。小鼠HIBM模型显示出意外的肾脏表型（足细胞病和肾小球膜分裂），这是由ManNAc喂养拯救的。一些原因不明的肾脏疾病，包括肾病综合征和微小病变疾病，也报告了类似的表型。这暗示ManNAc可能是涉及由于足细胞病和/或肾小球基底膜节段性分裂引起的蛋白尿和血尿的肾脏疾病的治疗剂。一项关于可能由唾液酸化缺陷引起的肾脏疾病的综述正在准备中。 我们开发了等位基因特异性实时PCR方法临时专利No. 60/718，321（2005年9月）使用真实的时间PCR检测等位基因表达以测量HIBM和siberia中的GNE等位基因表达水平（手稿在准备中）。此外，我们正在进行siRNAs细胞中显性突变等位基因的体外siRNA沉默实验（手稿在准备中）。 2. 3-甲基戊烯二酸尿症（3 MGA）疾病，伴或不伴视神经萎缩。在2001年，我们的小组分离OPA 3，一个未知功能的基因，负责Costeff综合征，其特征是3 MGA和视神经萎缩。我们检测了来自患有/不患有3 MGA和/或孤立性视神经萎缩患者的OPA 3突变的DNA（病例报告正在准备中）。我们目前正在研究OPA 3的功能，我们已经确定了一种新的OPA 3亚型与罕见的双重线粒体和过氧化物酶体定位（手稿准备）。我们还使用反义吗啉技术创建了Costeff综合征的斑马鱼模型（手稿在准备中）。 3.胞内囊泡分类和形成障碍。这些疾病包括Hermansky-Pudlak综合征（HPS）、Chediak-Higashi综合征、Griscelli综合征和其他遗传学上未分类的疾病。常见的临床特征是由于黑素体缺陷引起的白化病和由于血小板缺陷引起的出血。我们的研究小组调查了已知和未知的HPS致病基因，目的是更好地了解这种疾病的生物学。我们的研究小组还对HPS的七个不同亚组的临床和遗传特征进行了分类。我们对未分类的患者进行了候选基因筛选（参考文献3，4）。为了研究HPS突变的影响，我们对患者材料进行细胞生物学研究（采用免疫荧光，免疫EM和活细胞成像），以检查HPS细胞中蛋白质和细胞器的缺陷细胞内运输和分选。这些细胞不能将某些溶酶体蛋白转运到正确的目的地，HPS基因产物参与识别产生溶酶体样细胞器的特异性囊泡（参考文献5、6、7）。 4.遗传性间质缺失综合征。常规突变筛查通常涉及基于PCR的方法，然后是直接测序。如果缺失断裂点延伸超出PCR引物的位置，则这些方法可能错过较大基因组缺失的发生。在隐性遗传疾病中，这可能导致将纯合子误认为半合子。我们的研究小组应用实时定量PCR检测了多种罕见疾病的半合子和缺失断点。 - Hermansky-Pudlak综合征：我们鉴定了在HPS 1基因座（Griffin等，Clin Genet（2005）68，23-30）和HPS 6基因座（手稿在准备中）上具有大基因组缺失的患者。 - 前脑无裂畸形：我们通过定量实时PCR测试了患者在候选基因区域中的亚显微缺失（Bendavid等人，J Med Genet（2006）43，496-500），补充了FISH结果。 - Smith-Magenis综合征（SMS）：这种疾病主要（大于95%）由17p11.2的间质缺失引起。我们的小组目前正在进行定量实时PCR，以确定在17 p11.2的关键基因在98例SMS半合子。我们的研究结果，结合FISH分析和未来的平铺阵列进行的合作小组，将揭示SMS患者的可变表型和基因型-表型相关性（手稿准备）。在未来，这些定量实时PCR方法可以应用于其他缺失综合征（例如，Jacobsen综合征）。 - 对20例SMS患者进行FISH和qPCR分析，未发现17p11.2缺失。我们实验室正在进行可能基因缺陷的突变分析（包括RAI 1和RASD 1）。我们还对这些患者的DNA进行了CGH阵列，以识别新的（微）缺失或重复。初步结果确定了4种新的染色体重排，后续实验室研究正在进行中。

Investigations within this project concern the cell biology of rare human genetic disorders and normal and abnormal intracellular processes. The research goal is to gain insight into changes in molecular function that underlie various genetic metabolic disorders and work towards treatments for these illnesses. The research focuses on four groups of rare disorders: 1. Disorders of sialic acid metabolism. The key enzyme in the sialic acid biosynthesis pathway is UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Dominant mutations in the allosteric site of GNE cause sialuria, characterized by overproduction of sialic acid. Recessive mutations in GNE cause the neuromuscular disorder hereditary inclusion body myopathy (HIBM). We have characterized a knock-in HIBM mouse model and demonstrated that N-acetylmannosamine (ManNAc) rescues the phenotype of the homozygous mutant mice (ref 1)and is a promising treatment option for human patients Provisional Patent No. 60/932,451 (May 31, 2007) N-acetyl mannosamine as a therapeutic agent. Other treatments (sialic acid, galactose) are currently being tested on this murine HIBM model. A clinical treatment protocol for ManNAc in patients with HIBM is currently prepared. Highly sialylated glycoproteins were supplied to 4 patients with HIBM in a pilot clinical trial (ref 2). The murine HIBM model showed an unexpected kidney phenotype (of podocytopathy and glomerular membrane splitting) which was rescued by ManNAc feeding. Similar phenotypes are reported for some unexplained renal disorders including nephrotic syndrome and minimal change disease. This implicates that ManNAc might be a therapeutic agent for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane. A review on renal disorders that may be caused by sialylation defects is in preparation. We developed an allele specific real-time PCR method Provisional Patent No. 60/718,321 (Sept 2005) Use of real time PCR for detection of allelic expression to measure GNE allelic expression levels in both HIBM and sialuria (manuscript in preparation). In addition, we are performing in vitro siRNA silencing experiments of the dominant, mutated allele in sialuria cells (manuscript in preparation). 2. Disorders of 3-methylglutaconic aciduria (3MGA) presenting with or without optic atrophy. In 2001, our group isolated OPA3, a gene of unknown function responsible for Costeff syndrome, which is characterized by 3MGA and optic atrophy. We tested DNA from patients with/without 3MGA and/or isolated optic atrophy for mutations in OPA3 (case report in preparation). We are currently investigating OPA3 function, and we have identified a novel OPA3 isoform with a rare dual mitochondrial and peroxisomal localization (manuscript in preparation). We also created zebrafish models for Costeff syndrome using antisense morpholino technology (manuscript in preparation). 3. Disorders of intracellular vesicle sorting and formation. These disorders include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, and other genetically unclassified disorders. Common clinical features are albinism due to defects in melanosomes and bleeding due to platelet defects. Our group investigates known and unknown HPS-causing genes, with the goal of better understanding the biology of the disease. Our group also catalogues the clinical and genetic characteristics of the seven distinct subgroups of HPS. And we perform candidate gene screening on unclassified patients (refs 3,4). To study the effects of HPS mutations, we perform cell biological studies on patients material (employing immuno-fluorescence, immmuno-EM, and live cell imaging) to examine defective intracellular trafficking and sorting of proteins and organelles in HPS cells. Such cells fail to transport certain lysosomal proteins to their correct destinations, and HPS gene products are involved in recognizing the specific vesicles that give rise to lysosome-like organelles (refs 5,6,7). 4. Genetic interstitial deletion syndromes. Routine mutation screening commonly involves PCR-based approaches followed by direct sequencing. Occurrence of larger genomic deletions may be missed by these approaches if the deletion breakpoints extend beyond the position of the PCR primers. In recessive disorders, this can lead to mistaking homozygosity for hemizygosity. Our group applied quantitative real-time PCR to detect hemizygosity and deletion breakpoints in a variety of rare disorders. - Hermansky-Pudlak syndrome: We identified patients with a large genomic deletion on the HPS1 locus (Griffin et al. Clin Genet (2005) 68, 23-30) and the HPS6 locus (manuscript in preparation). - Holoprosencephaly: We tested patients by quantitative real-time PCR for submicroscopic deletions in candidate gene regions (Bendavid et al. J Med Genet (2006) 43, 496-500), supplementing multicolor FISH results. - Smith-Magenis syndrome (SMS): This disorder is mainly (greater than 95%) caused by an interstitial deletion of 17p11.2. Our group is currently performing quantitative real-time PCR to identify hemizygosity in key genes on 17p11.2 in 98 patients with SMS. Our results, in combination with FISH analysis and future tiling arrays performed by collaborating groups, will shed light on the variable phenotype of SMS patients and genotype-phenotype correlations (manuscript in preparation). In the future, these quantitative real-time PCR methods can be applied to other deletion syndromes (e.g., Jacobsen syndrome). - FISH and qPCR analysis on 20 SMS patients identified no deletion in 17p11.2. Mutation analysis for sible gene defects are ongoing (including RAI1 and RASD1) in our lab. We also performed CGH-arrays on these patients DNA to identify novel (micro) deletions or duplications. Preliminary results identified 4 novel chromosomal rearrangements, for which follow-up laboratory research is ongoing.