Cell Biology of Metabolic Disorders

代谢紊乱的细胞生物学

基本信息

批准号：
7734893
负责人：
William Allen Gahl
金额：
$ 39.67万
依托单位：
NATIONAL HUMAN GENOME RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734893
关键词：
17p11.2 Albinism Alleles Allosteric Site American Anabolism Biochemical Pathway Biological Biology Blood Blood Platelets Brain Candidate Disease Gene Case Study Cataloging Catalogs Cell Nucleus Cells Cellular biology Characteristics Chromosomal Rearrangement Clinical Clinical Treatment Cytidine Cytidine Monophosphate N-Acetylneuraminic Acid Cytoplasm DNA Defect Destinations Detection Disease Enzymes Fibroblasts Finnish Type Sialic Acid Storage Disease Fluorescence Fluorescent in Situ Hybridization Future Genes Genetic Genets Genomics Genotype Goals Hematuria Hemorrhage Hereditary Disease Hermanski-Pudlak Syndrome Holoprosencephaly Human Hybridization Array In Vitro Inclusion Bodies Infantile Form Sialuria Inherited Investigation Kidney Kidney Diseases Knock-in Mouse Lead Legal patent Life Light Lipoid nephrosis Localized Lysosomes Mannose Manuscripts Measures Mediation Melanosomes Membrane Metabolic Diseases Metabolism Methods Mitochondria Modeling Molecular Mus Muscle Mutant Strains Mice Mutate Mutation Mutation Analysis Myopathy N-Acetylglucosamine kinase N-acetylmannosamine Neuromuscular Diseases New Zealand Optic Atrophy Oral Organelles Pathway interactions Patients Pharmacologic Substance Phenotype Phosphotransferases Polymerase Chain Reaction Positioning Attribute Preparation Process Protein Isoforms Protein Sortings Proteins Proteinuria Protocols documentation RASD1 gene Rare Diseases Reporting Research Screening procedure Sialic Acids Sialuria Single-Gene Defect Small Interfering RNA Smith Magenis syndrome Societies Sorting - Cell Movement Subgroup Syndrome Technology Testing Therapeutic Therapeutic Agents Time Toxicology Tubular formation UDP-N-acetylglucosamine 2-epimerase Vacuole Vesicle Week Work Zebrafish base cellular imaging chediak-higashi syndrome comparative genomic hybridization epimerase feeding glomerular basement membrane glycosylation human RASD1 protein insight interstitial lysosomal proteins mannosamine mouse model novel research study sialylation sugar trafficking

项目摘要

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). In July 2007, we characterized a knock-in HIBM mouse model and demonstrated that N-acetylmannosamine (ManNAc) rescues the phenotype of the homozygous mutant mice and is a promising treatment option for human patients (Galeano et al. J Clin Inv (2007) 117:1585-1594). A Provisional Patent No. 60/932,451 (May 31, 2007) N-acetyl mannosamine as a therapeutic agent was acquired and a clinical treatment protocol for ManNAc in patients with HIBM was prepared. Negotiations regarding an extensive toxicology study for the use of ManNAc are ongoing, and the patent is in the process of being transferred to New Zealand Pharmaceuticals, a ManNAc manufacturor. We are currently testing other treatments on our murine HIBM model (sialic acid, mannose, mannosamine, and possibly other sugars in the future). 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 human renal disorders including nephrotic syndrome and minimal change disease. We are performing studies to investigate whether ManNAc could 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, and a plenary session on this subject, to be presented by Dr. Huizing at the American Society for Nephrologys annual Renal week is upcoming. Furthermore, 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) to measure GNE allelic expression levels in both HIBM and sialuria (manuscript submitted to Human Mutation). In addition, we performed in vitro siRNA silencing experiments of the dominant, mutated allele in sialuria cells, which proved promising a promising treatment strategy in vitro in sialuria patients cells (Ref. 1). 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 reports submitted to Brain, one case report in preparation). We investigated OPA3 function, and identified a novel OPA3 isoform with a rare dual mitochondrial and peroxisomal localization. We also created zebrafish models for Costeff syndrome using antisense morpholino technology (manuscript submitted to EMBO J). 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 2,3,4,5). 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). 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 submitted to Blood). - 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 comparative genome hybridization (CGH)- arrays performed by collaborating groups, will shed light on the variable phenotype of SMS patients and genotype-phenotype correlations (one manuscript submitted to Am J Med Genet, one 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 single 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 (manuscript in preparation).

该项目中的研究涉及稀有人类遗传疾病以及正常和异常细胞内过程的细胞生物学。研究目标是洞悉分子功能的变化，这些变化是各种遗传代谢疾病的基础，并致力于治疗这些疾病。该研究侧重于四组罕见疾病： 1。唾液酸代谢的疾病。唾液酸生物合成途径中的关键酶是UDP-GLCNAC 2- epimerase/mannac激酶（GNE）。 GNE的变构位点的主要突变引起唾液酸，其特征是唾液酸生产过多。 GNE中的隐性突变导致神经肌肉疾病遗传包容体肌病（HIBM）。 2007年7月，我们表征了一种敲入HIBM小鼠模型，并证明了N-乙酰基曼诺胺（MANNAC）营救了纯合突变小鼠的表型，并且是人类患者的有前途的治疗选择（Galeano等人，J Clin Inv（2007）117：1585-1594）。获得了60/932,451号临时专利（2007年5月31日）N-乙酰甘露糖胺作为治疗剂，并制备了HIBM患者MANNAC的临床治疗方案。关于一项有关使用MANNAC的广泛毒理学研究的谈判正在进行中，并且该专利正在转移到MANNAC制造商的新西兰药品。我们目前正在对鼠HIBM模型（唾液酸，甘露糖，甘露糖胺以及可能的其他糖）进行其他处理。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），该表型被甘露纳克喂养救出。据报道，一些无法解释的人类肾脏疾病（包括肾病综合征和最小变化疾病）的表型。我们正在进行研究，以研究MANNAC是否可以是涉及肾小球肿瘤和/或分段分裂肾小球基底膜的肾脏疾病的治疗剂。正在准备对可能是由囊肿缺陷引起的肾脏疾病的综述，并且正在准备有关该主题的全体会议，由美国肾上腺素年度肾脏年度肾脏周的Huizing博士提出。此外，我们开发了一种特定的等位基因实时PCR方法临时专利60/718,321（2005年9月）使用实时PCR检测等位基因表达以测量等位基因等位基因表达），以测量HIBM和Sialuria中的GNE等位基因表达水平（手稿已列入人类突变）。此外，我们在唾液酸细胞中进行了显性等位基因的显性siRNA沉默实验，这证明了唾液酸患者细胞中有望在体外具有有希望的治疗策略（参考文献1）。 2。出现有或没有视觉萎缩的3-甲基谷氨酸（3MGA）的疾病。在2001年，我们的组孤立的OPA3是一个导致Costeff综合征的未知功能的基因，其特征是3MGA和光学萎缩。我们测试了OPA3中突变的患者/或没有3MGA和/或孤立视神经萎缩的患者的DNA（案例报告提交给大脑，一份病例报告中的一份病例报告）。我们研究了OPA3功能，并鉴定出具有罕见的双线粒体和过氧化物酶体定位的新型OPA3同工型。我们还使用反义莫诺利诺技术（提交给EMBO J）的Costeff综合征创建了斑马鱼模型。 3。细胞内囊泡分选和形成的疾病。这些疾病包括Hermansky-Pudlak综合征（HPS），Chediak-Higashi综合征，Griscelli综合征和其他遗传疾病。常见的临床特征是由于黑色素体缺陷和由于血小板缺陷而出血引起的白化病。我们的小组研究了已知和未知的引起HPS的基因，目的是更好地了解该疾病的生物学。我们的小组还将HPS七个不同亚组的临床和遗传特征分类。我们对未分类的患者进行候选基因筛查（参考2,3,4,5）。为了研究HPS突变的作用，我们对患者材料（采用免疫荧光，Immmuno-EM和Live细胞成像）进行细胞生物学研究，以检查HPS细胞中蛋白质和细胞器的细胞内运输和分类缺陷。这些细胞无法将某些溶酶体蛋白转运到其正确的目的地，而HPS基因产物参与识别引起溶酶体样细胞器的特定囊泡（参考文献5,6）。 4。遗传性质缺失综合征。常规突变筛选通常涉及基于PCR的方法，然后进行直接测序。如果缺失断点延伸到PCR引物的位置，则可能会错过这些方法的较大基因组缺失。在隐性疾病中，这可能导致误认为纯合性因半合性。我们的小组应用定量实时PCR来检测各种罕见疾病的半合理和缺失断点。 - Hermansky-Pudlak综合征：我们在HPS1基因座（Griffin等人Clin Genet（2005）68，23-30）和HPS6基因座（手稿提交给血液）上确定了大基因组缺失的患者。 - 全脑脑：我们通过定量实时PCR测试了候选基因区域的亚镜下缺失（Bendavid等人J Med Genet（2006）43，496-500），补充了多色鱼类的结果。 -Smith-Magenis综合征（SMS）：这种疾病主要是由17p11.2的间质缺失引起的（大于95％）。我们的小组目前正在执行定量实时PCR，以鉴定98例SMS患者的17p11.2关键基因中的半合子。我们的结果结合了鱼类分析和比较基因组杂交（CGH） - 由协作组执行的阵列，将阐明SMS患者的可变表型和基因型 - 表型相关性（一个手稿提交给AM J Med Genet，一份手稿，准备制备中）。将来，这些定量的实时PCR方法可以应用于其他缺失综合征（例如Jacobsen综合征）。 - 对20名SMS患者的鱼类和QPCR分析在17p11.2中尚无删除。我们实验室中的单个基因缺陷的突变分析正在进行中（包括RAI1和RASD1）。我们还对这些患者DNA进行了CGH阵列，以识别新颖（微）缺失或重复。初步结果确定了4个新型染色体重排（制备中的手稿）。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ileal Crohn's disease in a woman with Hermansky-Pudlak syndrome.

患有赫曼斯基-普德拉克综合征的女性患有回肠克罗恩病。

DOI：
10.1016/s0399-8320(06)73239-0
发表时间：
2006
期刊：
Gastroenterologie clinique et biologique
影响因子：
0
作者：
deLeusse,Antoine;Dupuy,Evelyne;Huizing,Marjan;Danel,Claire;Meyer,Guy;Jian,Raymond;Marteau,Philippe
通讯作者：
Marteau,Philippe

A new genetic isolate with a unique phenotype of syndromic oculocutaneous albinism: clinical, molecular, and cellular characteristics.