Cell Biology of Metabolic Disorders

该项目中的研究涉及罕见的人类遗传疾病的细胞生物学以及正常和异常的细胞内过程。这项研究的目标是深入了解导致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。这项研究集中在四组罕见疾病上： 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-表异构酶/甘露糖苷酸激酶(Gne)。Gne变构位点的显性突变会引起唾液酸尿症，其特征是唾液酸产生过多。Gne的隐性突变导致神经肌肉疾病遗传性包涵体肌病(HIBM)。我们已经鉴定了一种敲入的HIBM小鼠模型，并证明了N-乙酰甘露糖胺(ManNAc)挽救了纯合子突变小鼠的表型(参考文献1)，是一种有希望的人类患者的治疗选择。其他治疗方法(唾液酸、半乳糖)目前正在这个小鼠HIBM模型上进行测试。目前正在准备一份治疗HIBM患者ManNAc的临床治疗方案。在一项试点临床试验中，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模型表现出意外的肾脏表型(足细胞病变和肾小球膜分裂)，这是通过ManNAc喂养挽救的。类似的表型在一些不明原因的肾脏疾病中也有报道，包括肾病综合征和微小病变疾病。这意味着ManNAc可能是一种治疗因足细胞病变和/或肾小球基底膜节段性分裂而引起的蛋白尿和血尿的肾脏疾病的药物。一篇关于唾液酸化缺陷可能引起的肾脏疾病的综述正在准备中。 我们开发了一种等位基因特异的实时聚合酶链式反应方法(临时专利第60/718,321号(2005年9月))，使用实时聚合酶链式反应检测等位基因表达，以测量HIBM和唾液尿症(手稿正在准备中)中的GNE等位基因表达水平。此外，我们正在进行唾液酸细胞中主要突变等位基因的体外siRNA沉默实验(手稿正在准备中)。 2.伴有或不伴有视神经萎缩的3-甲基谷氨酸尿症(3MGA)。2001年，我们的团队分离到了OPA3基因，这是一个功能未知的基因，导致了以3MGA和视神经萎缩为特征的Costeff综合征。我们检测了患有或不患有3MGA和/或孤立性视神经萎缩的患者的DNA中OPA3的突变(病例报告正在准备中)。我们目前正在研究OPA3的功能，我们已经发现了一种新的OPA3亚型，具有罕见的线粒体和过氧化体双重定位(手稿正在准备中)。我们还使用反义吗啉技术(手稿正在准备中)创建了Costeff综合征的斑马鱼模型。 3.细胞内小泡的分选和形成障碍。这些疾病包括Hermansky-Pudlak综合征(HPS)、Chediak-Higashi综合征、Griscelli综合征和其他遗传上未分类的疾病。常见的临床特征是由于黑素小体缺陷而导致的白化和由于血小板缺陷而导致的出血。我们小组研究已知和未知的HPS致病基因，目的是更好地了解这种疾病的生物学。我们的小组还对HPS的七个不同亚型的临床和遗传特征进行了分类。我们对未分类的患者进行候选基因筛选(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(使用免疫荧光、免疫EM和活细胞成像)，以检查HPS细胞中有缺陷的细胞内运输和蛋白质和细胞器的分选。这种细胞不能将某些溶酶体蛋白运输到它们正确的目的地，而HPS基因产物参与识别产生溶酶体样细胞器的特定小泡(参考文献5、6、7)。 4.遗传性间质缺失综合征。常规突变筛查通常包括基于聚合酶链式反应的方法，然后进行直接测序。如果缺失断裂点延伸到聚合酶链式反应引物的位置之外，这些方法可能会错过较大基因组缺失的发生。在隐性疾病中，这可能会导致将纯合子误认为半合子。我们小组应用实时定量聚合酶链式反应来检测各种罕见疾病中的半合子和缺失断裂点。 -Hermansky-Pudlak综合征：我们确定了HPS1基因座上有大量基因组缺失的患者(Griffin等人。Clin Genet(2005)68，23-30)和HPS6基因座(手稿正在准备中)。 -全前脑：我们通过实时定量聚合酶链式反应对患者进行了候选基因区域亚显微缺失检测(Bendavid等人。J Med Genet(2006)43,496-500)，补充了多色鱼的结果。 -Smith-Magenis综合征(SMS)：这种疾病主要(超过95%)是由17p11.2的间质缺失引起的。我们小组目前正在进行实时定量聚合酶链式反应，以鉴定98例短信患者17p11.2上关键基因的半合性。我们的结果，结合FISH分析和未来合作小组进行的平铺阵列，将揭示SMS患者的不同表型和基因-表型相关性(手稿正在准备中)。在未来，这些定量实时聚合酶链式反应方法可以应用于其他缺失综合征(例如雅各布森综合征)。 -对20例短信患者进行FISH和qPCR分析，发现17p11.2无缺失。我们实验室正在对可能的基因缺陷(包括RAI1和RASD1)进行突变分析。我们还对这些患者的DNA进行了CGH阵列，以确定新的(微)缺失或复制。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.