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).

该项目的研究涉及罕见的人类遗传疾病以及正常和异常细胞内过程的细胞生物学。研究目标是深入了解各种遗传代谢紊乱背后的分子功能变化，并致力于治疗这些疾病。该研究重点关注三组罕见疾病： 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 变构位点的显性突变导致唾液尿，其特征是唾液酸过量产生。 GNE 的隐性突变会导致神经肌肉疾病遗传性包涵体肌病 (HIBM)。去年，我们对 GNE 的各个方面进行了广泛的综述（当前生物学，正在审查中），在人类和小鼠中鉴定了新的 GNE 亚型，并对这些亚型进行了分子建模（生物化学，正在审查中），对对人类 HIBM 患者进行基因治疗试验（参考文献 5），并在我们的 HIBM 小鼠模型中定义了肾脏表型，并使用该模型开发了凝集素组，用于筛查人类肾脏疾病的低唾液酸化（美国杂志病理学，正在审查中）。我们的进一步研究集中在唾液酸合成途径中 GNE 和其他酶的亚细胞定位和表达水平（蛋白质印迹和实时定量 PCR），并鉴定可作为唾液酸化状态参数的人类血液标记物（主要通过聚糖-剖析研究）。 2007 年，我们对敲入 HIBM 小鼠模型进行了表征，并证明 N-乙酰甘露糖胺 (ManNAc) 可以挽救纯合突变小鼠的表型，并且是对人类患者有希望的治疗方法 (J Clin Inv (2007) 117:1585-1594) 。关于使用 ManNAc 的 IND（研究性新药）申请的广泛毒理学研究的谈判正在通过 NIH-TRND（罕见和被忽视疾病的治疗）计划进行，我们的 ManNAc 专利（编号 60/932,451）已获得许可至 ManNAc 制造商新西兰制药公司。去年，我们进一步表征了我们的 HIBM 敲入小鼠模型的成年发病肌肉表型，并在我们的小鼠模型上测试了替代 HIBM 治疗方法，包括用唾液酸途径中间体喂养和 GNE 基因治疗，主要是嵌入脂质体 (Lipoplex) 中静脉注射。结果正在汇编成手稿。我们描述了一种向新生小鼠眼眶后静脉注射化合物的方法（参考文献 3）。我们提交了一份员工发明报告，内容涉及使用脂质体向哺乳动物系统性输送糖类（即 ManNAc 和唾液酸）。 我们的小鼠模型显示出意想不到的肾脏表型（足细胞病和肾小球膜分裂），但通过 ManNAc 喂养可以挽救这种表型。我们开发了一个凝集素组，用于表征 HIBM 小鼠模型的肾糖基化/唾液酸化状态（美国病理学杂志，正在审查中）。我们现在正在对多种无法解释的人类肾脏疾病进行测试，包括由于足细胞病和/或肾小球基底膜节段性分裂导致的蛋白尿和血尿。涉及肾小球低唾液酸化的人类肾脏疾病可能受益于 ManNAc 作为治疗剂。 2. 溶酶体相关细胞器（LRO）生物发生障碍。此类疾病包括赫曼斯基-普德拉克综合征(HPS)、切迪亚克-东综合征、格里塞利综合征、灰血小板综合征和其他遗传上未分类的疾病。常见的临床特征是由于黑素体缺陷导致的白化病和由于血小板缺陷导致的出血。我们研究已知和未知的 LRO 疾病致病基因（通过传统和下一代测序技术），目的是更好地了解该疾病的生物学。为了研究 LRO 疾病突变的影响，我们对患者材料进行细胞生物学研究（使用免疫荧光、免疫电子显微镜和活细胞成像），以检查细胞内蛋白质和细胞器的缺陷细胞内运输和分类。这些细胞无法将某些与溶酶体相关的细胞器驻留蛋白转运到正确的目的地，并且LRO紊乱基因产物通常参与识别产生LRO的特定囊泡。我们还对 HPS 和相关 LRO 疾病不同亚型的临床和遗传特征进行了分类。 去年，我们发现了一位患有 HPS 亚型 8 (HPS-8) 的患者（世界第二位）；确定了一种新的人类 HPS 亚型，HPS-9（参考文献 4）；采用 SNP 阵列纯合性作图结合全外显子组测序来识别白化病和中性粒细胞减少症患者中两个不同基因的致病突变（参考文献 6）；通过纯合性作图和全外显子组测序将 NBEAL2 鉴定为长期以来备受追捧的灰血小板综合征基因（参考文献 1 和 7）；描述了波多黎各患者 HPS1 基因的新突变（参考文献 2）；并描述了西班牙裔非波多黎各患者的 HPS 基因突变（参考文献 8）。此外，我们还描述了 Griscelli 综合征 3 型病例（色素细胞和黑色素瘤研究，正在出版），并描述了 HPS-2 患者的肺纤维化（分子医学，正在审查中）。我们小组为代谢紊乱细胞生物学方面的其他研究小组提供建议和协助，例如协助 ACSF3 基因的细胞内定位研究，该基因在苹果酸和甲基丙二酸尿症联合亚型中发生突变（参考文献 9）。 4. 史密斯-马吉尼斯综合征 (SMS) 和相关疾病的遗传学。 SMS 是一种复杂的神经行为障碍，其特征是多种先天性异常，主要归因于 17p11.2 的从头间质缺失。 SMS 患者的分子分析可能有助于揭示可变表型和基因型-表型相关性以及可能的治疗决策。 NIH 的 SMS 患者队列包含常见 3.7 Mb 17p11.2 缺失的患者 (n=80)、非典型 17p11.2 缺失的患者 (n=24) 和非 17p11.2 缺失的患者 (n=44)。我们对非典型缺失组的全基因组 SNP 阵列分析确定了可能影响临床特征的不同 17p11.2 断点（手稿正在准备中）。对非删除队列的 SNP 阵列分析发现了几个新的（微）删除或重复。我们对非缺失亚组进行了广泛的 RAI1 基因分析，包括突变分析和表达研究，确定了 10 名患有 RAI1 突变的患者（5 名新生，5 名家族），并首次描述了 RAI1 mRNA 表达水平降低，不仅存在于常见 17p11.2 缺失，但也见于 RAI1 突变患者和一些非 17p11.2 缺失患者（参考文献 10）。