Cell Biology of Metabolic Disorders

代谢紊乱的细胞生物学

• 批准号: 8349997
• 负责人: William Gahl
• 金额: $ 44.52万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 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/8349997
• 关键词: 17p11.2; Adult; Albinism; Allosteric Site; American; Anabolism; Biochemical; Biochemistry; Biogenesis; Biological; Biology; Blood; Blood Platelets; Candidate Disease Gene; Cataloging; Catalogs; Cell Nucleus; Cells; Cellular biology; Characteristics; Clinical; Complex; Cytidine; Cytidine Monophosphate N-Acetylneuraminic Acid; Cytoplasm; Defect; Deletion Mutation; Destinations; Disease; Drug or chemical Tissue Distribution; Employee; Enzymes; Fibroblasts; Fluorescence; Galactose; Gene Mutation; Genes; Genetic; Genome; Genotype; Goals; Gray unit of radiation dose; Griscelli Syndrome; Hematuria; Hemorrhage; Hereditary Disease; Hermanski-Pudlak Syndrome; Hispanics; Human; Human Genetics; Inclusion Bodies; Inherited; Injection of therapeutic agent; Intravenous; Investigation; Investigational New Drug Application; Journals; Kidney; Kidney Diseases; Knock-in Mouse; Lectin; Legal patent; Leukocytes; Licensing; Life; Light; Liposomes; Lysosomes; Mammals; Mannose; Manufacturer Name; Manuscripts; Maps; Mediation; Melanoma Cell; Melanosomes; Membrane; Metabolic Diseases; Metabolism; Methods; Modeling; Molecular; Molecular Analysis; Molecular Medicine; Molecular Models; Mus; Muscle; Mutant Strains Mice; Mutate; Mutation; Mutation Analysis; Myopathy; N-Acetylglucosamine kinase; N-Acetylneuraminate lyase; N-acetylmannosamine; Neuromuscular Diseases; Neutropenia; New Zealand; Newborn Infant; Oral; Organelles; Pathology; Pathway interactions; Patients; Pharmacologic Substance; Phenotype; Phosphotransferases; Pigments; Plasma; Polysaccharides; Preparation; Process; Protein Isoforms; Proteins; Proteinuria; Puerto Rican; Pulmonary Fibrosis; RNA; RNA Splicing; Rare Diseases; Renal glomerular disease; Reporting; Research; Sampling; Screening procedure; Serum; Sialic Acids; Sialuria; Smith Magenis syndrome; Sorting - Cell Movement; Subgroup; Supplementation; Syndrome; Techniques; Testing; Therapeutic; Therapeutic Agents; Time; Tissues; Toxicology; UDP-N-acetylglucosamine 2-epimerase; United States National Institutes of Health; Vesicle; Western Blotting; Work; Writing; cellular imaging; chediak-higashi syndrome; cohort; disease-causing mutation; epimerase; exome; feeding; gene therapy; glomerular basement membrane; glycosylation; insight; interstitial; mRNA Expression; mannosamine; methylmalonic aciduria; molecular modeling; mouse model; neglect; neurobehavioral disorder; next generation; novel; programs; protein expression; sialylation; sugar; trafficking

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 three 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 the last year, we wrote an extensive review on all aspects of GNE (Current Biology, under review), identified novel GNE isoforms in human and mice and performed molecular modeling on these isoforms (Biochemistry, under review), performed biochemical analysis for a gene therapy trial of a human HIBM patient (Ref. 5), and defined the kidney phenotype in our HIBM mouse model and used this model to develop a lectin panel for screening human kidney disorders for hyposialylation (American Journal of Pathology, under review). Our further studies focused on subcellular localization and expression levels of GNE and other enzymes in the sialic acid synthesis pathway (Western blotting and real-time quantitative PCR) and identifiying human blood-markers that can serve as parameters for sialylation status (mainly by glycan-profiling studies). In 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 for human patients (J Clin Inv (2007) 117:1585-1594). Negotiations regarding an extensive toxicology study for an IND (investigational New Drug) application for the use of ManNAc are ongoing through the NIH-TRND (Therapies for Rare and Neglected Diseases) program, and our ManNAc patent (No. 60/932,451) is licensed to a ManNAc manufacturer, New Zealand Pharmaceuticals. This last year we further characterized the adult onset muscle phenotype of our HIBM knock-in mouse model and tested alternative HIBM treatments on our murine model, including feeding with sialic acid pathway intermediates and GNE gene therapy, mostly intravenous delivered embedded in liposomes (Lipoplex). The results are being compiled for a manuscript. We described a method of retro-orbital intravenous delivery of compounds to newborn mice (Ref. 3). We filed an employee invention report for the use of liposomes to systemically deliver saccharides (i.e., ManNAc and sialic acid) to mammals. Our mouse model showed an unexpected kidney phenotype (of podocytopathy and glomerular membrane splitting) which was rescued by ManNAc feeding. We developed a lectin panel that characterized the renal glycosylation/sialylation status of our HIBM mouse model (American Journal of Pathology, under review). We are now testing this panel on a variety of unexplained human renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane. Human renal disorders involving glomerular hyposialylation may benefit from ManNAc as a therapeutic agent. 2. Disorders of lysosome-related organelles (LRO) biogenesis. Such disorders include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, Gray Platelet syndrome, and other genetically unclassified disorders. Common clinical features are albinism due to defects in melanosomes and bleeding due to platelet defects. We investigate known and unknown LRO-disorders-causing genes (by conventional and next generation sequencing techniques), with the goal of better understanding the biology of the disease. To study the effects of LRO-disorders mutations, we perform cell biological studies on patient material (using immuno-fluorescence, immmuno-EM, and live cell imaging) to examine defective intracellular trafficking and sorting of proteins and organelles in cells. Such cells fail to transport certain lysosome-related organelle resident proteins to their correct destinations, and LRO-disorder gene products are generally involved in recognizing the specific vesicles that give rise to LROs. We also catalogue the clinical and genetic characteristics of the distinct subtypes of HPS and related LRO-disorders. This last year we identified a patient (second in the world) with HPS subtype 8 (HPS-8); identified a novel human HPS subtype, HPS-9 (Ref. 4); employed SNP-array homozygosity mapping combined with whole exome sequencing to identify disease causing mutations in two different genes in a patient with albinism and neutropenia (Ref. 6); identified by homozygosity mapping and whole exome sequencing NBEAL2 as the long sought-after gene for Gray-Platelet Syndrome (Refs 1 and 7); described new mutations in the HPS1 gene among Puerto-Rican patients (Ref. 2); and described HPS gene mutations in non-Puerto-Rican patients of Hispanic descent (Ref. 8). Furthermore, we characterized Griscelli syndrome type 3 cases (Pigment Cell and Melanoma Research, in press) and describe pulmonary fibrosis in HPS-2 patients (Molecular Medicine, under review). Our group is advises and assists other research groups in the cell biology of metabolic disorders, such as assistance in intracellular localization studies on the ACSF3 gene, found mutated in a subtype of combined malinic and methylmalonic aciduria (Ref. 9). 4. Genetics of Smith-Magenis syndrome (SMS) and related disorders. SMS is a complex neurobehavioral disorder characterized by multiple congenital anomalies, primarily ascribed to a de novo interstitial deletion of 17p11.2. Molecular analysis of SMS patients may shed light on the variable phenotype and genotype-phenotype correlations and possible treatment decisions. The NIH cohort of SMS patients contains patients with the common 3.7 Mb 17p11.2 deletion (n=80), with atypical 17p11.2 deletions (n=24), and non 17p11.2 deleted patients (n= 44). Our whole genome SNP-array analysis on the atypical deletion group identified different 17p11.2 breakpoints that may influence clinical features (manuscript in preparation). SNP-array analysis of the non-deleted cohort identified several novel (micro) deletions or duplications. Our extensive RAI1 gene analysis on the non-deleted subgroup, including mutation analysis and expression studies, identified 10 patients with RAI1 mutations (5 de novo, 5 familial) and described for the first time decreased RAI1 mRNA expression levels not only in patients with the common 17p11.2 deletion but also in RAI1 mutated patients, and in some non-17p11.2 deleted patients (Ref. 10).

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。该研究侧重于三组罕见疾病： 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）。在过去的一年中，我们对人和小鼠中的各个方面进行了广泛的评论（当前的生物学，正在综述中），对这些同工型进行了新型的GNE同工型，并进行了分子建模（生物化学，正在综述），对人类HibM患者的基因治疗试验进行了生物化学分析，并针对人类HIBM患者进行了模型（5），并熟悉了HIB。用于筛查人类肾脏疾病的凝集素面板（美国病理学杂志，正在综述）。我们的进一步研究集中于唾液酸合成途径中gn​​e和其他酶的亚细胞定位和表达水平（Western blotting和实时定量PCR），并确定可以用作溶酶状态参数的人类血液标记物（主要是通过Glycan-Profing-Profing Litching研究）。 在2007年，我们表征了一种敲门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，并且是人类患者的一种有希望的治疗方法（J Clin Inv（2007）117：1585-1594）。关于IND（研究新药）对使用MANNAC的广泛毒理学研究的谈判正在通过NIH-TRND（稀有和被忽视的疾病）计划进行，我们的MANNAC专利（No. 60/932,451）已获得曼尼纳克制造商，New Zealandalandicals。去年，我们进一步表征了我们的HIBM敲入小鼠模型的成年发作肌肉表型，并在我们的鼠模型上测试了替代性HIBM处理，包括用唾液酸途径中间体和局势基因疗法进食，主要是静脉内递送的嵌入在脂质体中（Lipoplex）（Lipoplex）。结果是为手稿编译的。我们描述了一种将化合物静脉内递送到新生小鼠的方法（参考文献3）。我们提交了一份员工发明报告，以便将脂质体用于系统地向哺乳动物提供糖（即甘露酸和唾液酸）。 我们的小鼠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。我们开发了一个凝集素面板，该面板表征了HIBM小鼠模型的肾脏糖基化/溶解状态（美国病理学杂志，正在综述）。现在，我们正在对由于足细胞病和/或肾小球基质膜的各种无法解释的人肾脏疾病进行测试，这些小组涉及涉及蛋白尿和血尿的各种无法解释的人肾脏疾病。涉及肾小球降低的人肾脏疾病可能受益于Mannac作为治疗剂。 2。与溶酶体相关细胞器（LRO）生物发生的疾病。这些疾病包括Hermansky-Pudlak综合征（HPS），Chediak-Higashi综合征，Griscelli综合征，灰色血小板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。我们研究了已知和未知的LRO-引起引起的基因（通过常规和下一代测序技术），目的是更好地了解该疾病的生物学。为了研究LRO-差突变的作用，我们对患者材料（使用免疫荧光，Immmuno-EM和活细胞成像）进行细胞生物学研究，以检查细胞内运输和细胞中蛋白质和细胞器的缺陷。这些细胞无法将某些与溶酶体相关的细胞器驻留蛋白运送到其正确的目的地，而LRO diSorder基因产物通常参与识别引起LROS的特定囊泡。我们还分类了HPS和相关LRO抑制剂的不同亚型的临床和遗传特征。 去年，我们确定了一名患者（世界第二），其HPS亚型8（HPS-8）；确定了一种新型的人类HPS亚型HPS-9（参考文献4）；采用SNP - 阵列纯合性映射与整个外显子组测序相结合，以鉴定白化病和中性粒细胞减少患者的两个不同基因的突变的疾病（参考文献6）；通过纯合性映射和整个外显子组测序NBEAL2鉴定为灰色植物综合征的长期基因（参考文献1和7）；描述了波多黎各患者中HPS1基因中的新突变（参考文献2）；并描述了西班牙裔血统的非普通话 - 大师患者中的HPS基因突变（参考文献8）。此外，我们表征了Griscelli综合征3型病例（印刷中的色素细胞和黑色素瘤研究），并描述了HPS-2患者的肺纤维化（分子医学，正在综述）。我们的小组的建议并协助其他研究小组参与代谢性疾病的细胞生物学，例如在ACSF3基因的细胞内定位研究中的帮助，发现在马林尼和甲基丙二酰联合和甲基丙二酰酸尿的亚型中发现了突变（参考文献9）。 4。史密斯 - 麦加尼综合征（SMS）和相关疾病的遗传学。 SMS是一种复杂的神经行为疾病，其特征是多种先天异常，主要归因于从头上质量删除17p11.2。 SMS患者的分子分析可能会揭示可变的表型和基因型 - 表型相关性以及可能的治疗决策。 NIH SMS患者队列包含患有常见3.7 MB 17P11.2缺失（n = 80）的患者，具有非典型17p11.2缺失（n = 24）和非17P11.2删除的患者（n = 44）。我们对非典型缺失组的整个基因组SNP阵列分析确定了可能影响临床特征的不同17p11.2断点（制备中的手稿）。非删除队列的SNP阵列分析确定了几种新型（微）缺失或重复。我们对非骨骼亚组的广泛RAI1基因分析（包括突变分析和表达研究）确定了10例RAI1突变患者（5个从头，5个家族性），并首次描述了RAI1 mRNA表达水平不仅在常见的17p11.2患者中降低，而且在RAI1突变患者和一些非17p11.2 delec.2 delec.2 delec.2 delec.2 delec.2 delec.2 delet。