Complex Human Diseases - From Gene to Function to Therapy

对复杂人类疾病的理解和治疗方法的发展是分子医学面临的一些巨大挑战。分子进出细胞的运输是生物体最重要的功能之一。在许多情况下，化疗失败的一个重要原因是耐药性。从细胞中清除毒素的泵对肿瘤的抵抗力至关重要。此外，许多遗传疾病是由转运蛋白缺陷引起的。atp结合盒（ABC）基因家族编码转运蛋白，将各种化合物输送到细胞和组织的膜上。ABC转运蛋白的过度表达是化疗药物多药耐药的重要原因，这些基因在多种人类疾病中发生突变。补体基因与年龄相关性黄斑变性通过研究补体B和C2 （<I>CFH</I>, <I>C2</I>）基因的变异，我们已经确定了年龄相关性黄斑变性（AMD）患者的易感和保护性变异。老年性黄斑变性是导致老年人失明的主要原因，据估计有多达1000万美国人受其影响。通过检测&lt；I>BF</I&gt；和&lt；I>C2</I&gt；基因的遗传变异，鉴定出具有显著保护作用的单倍型。替代补体途径在对引起癌症和炎症的病原体的反应中很重要。对这部分先天免疫反应的进一步研究将有助于深入了解人类疾病。我们发现了一个缺失，删除了&lt；I>CFHR1</I&gt；与&lt；I>CFH</I&gt；相邻的&lt；I&gt；基因对AMD具有高度保护作用。我们从年龄相关性眼病研究的受试者中获得了3000多个dna，并对它们进行了相关标记分型，证实了主要等位基因的相关性，并证明CFH和CFB等位基因主要影响疾病进展到早期阶段（大红斑），HTRA1和CFHR1/3缺失影响疾病进展到晚期结局（新生血管、地理萎缩）。通过Affymetrix 6.0芯片数据分析，我们已经能够对杂合缺失患者进行基因分型，并鉴定出破坏或删除CFHR4基因的额外缺失。这些缺失的进一步表征可能揭示更多关于这个免疫调节基因簇的功能。最近发表的数据表明AMD与toll样受体3 （TLR3）基因有关。然而，我们在AREDS队列中输入了这种变异，并与来自其他7个中心的数据一起未能复制这种关联。癌症干细胞中的ABC基因在癌症干细胞中表达，允许对癌症治疗的先天抵抗。ABCG2</I&gt；是一个重要的耐药基因，我们已经筛选并鉴定了该基因编码蛋白的新抑制剂。其中一种是一类新的化合物，被称为botrylamides。botrylamides存在于海洋海绵中，其化学性质简单，可以合成衍生物并确定其结构/功能关系。<I>ABCG2</I&gt；是肿瘤干细胞中重要的标志物和耐药因子。这些化合物中的一些可以被证明抑制底物结合和/或atp酶活性的蛋白质。ABCB5基因已被证明是黑色素瘤干细胞的标志。我们对这个基因进行了进化分析，并证明它是作为一个全转运体进化的。此外，我们已经对该基因的几个变异进行了验证和应用基因分型分析，以证明该基因的变异广泛分布，并且在不同的人群中存在显著差异。代谢组学研究ABC基因功能为了解引起弹性假性黄瘤的ABCC6&lt；/I&gt；基因的功能，我们启动了代谢组学研究。男性患者的尿液已被用于生成代谢物谱。我们还对ABCC亚家族基因缺失的酵母细胞和ABCC亚家族基因缺失小鼠的血清和尿液进行了研究。这些研究揭示了这些样品中存在的一些小分子差异，并为进一步测试提供了重要的候选底物。此外，我们还描述了浮游甲壳类水蚤的ABC基因的补充，水蚤是一种全球分布的生物，对湖泊和池塘的生态至关重要。水蚤不仅是第一个甲壳类动物，也是第一个确定其基因组序列的非昆虫节肢动物。TRIM5和HIV感染一种调节蛋白TRIM5- α与HIV-1和其他逆转录病毒的传播有关。我们发现，喀麦隆大约4%的巴卡俾格米人在R332X基因上有一个突变，这是一个零等位基因，具有部分显性负活性。诸如此类的遗传因素，加上与黑猩猩体液的高频率接触，可能使该地区的人口易受艾滋病毒最初跨物种传播的影响。发现一种新的ERBB4基因变体，这是精神分裂症的候选基因。我们与美国国家心理健康研究所的Amanda Law博士和Daniel Weinberger博士合作，寻找人类ERBB4基因的剪接变体，该基因编码神经调节蛋白1基因的受体。我们发现一种新的ERBB4外显子3-跳跃转录物变体在人胎儿脑中表达，并且这种剪接变体优先存在于ERBB4 JM-b/CYT-2亚型中。此外，在ERBB4基因的内含子15上还发现了一个编码14个氨基酸的45-nt序列，这是JM-b异构体的特征。我们首次证明了ERBB4外显子3是一个可选外显子。相反，外显子E15a和外显子16是互斥的。因此，我们明确了目前人类ERBB4基因存在4个备选外显子（外显子3,15a， 16和26），应该由29个外显子组成，而不是28个外显子。慢性疲劳综合征（CFS）是一种病因不明的使人衰弱的疾病，据估计全世界有1700万人受到影响。研究CFS患者的外周血单核细胞（PBMC），我们的同事在101例患者中有68例（67%）鉴定出了人类γ -病毒，异嗜性小鼠白血病病毒相关病毒（XMRV）的DNA，而在218例健康对照中有8例（3.7%）。我们对这些样本中的RNASEL基因进行了基因分型，该基因先前与前列腺癌患者的XMRV感染有关。RNASEL基因型与CFS或XMRV阳性均无相关性。这些发现提高了XMRV可能是CFS发病机制的一个促成因素的可能性。施耐德角膜营养不良症（SCD）是一种由于角膜内胆固醇和脂质沉积异常而影响视力的遗传性疾病。此外，许多SCD患者表现出全身性高胆固醇血症和胆固醇和/或高密度脂蛋白代谢未知方面的明显失调。一种新基因（UBIAD1）的突变被发现导致这种疾病。继续对SCD进行遗传分析，可以更好地了解UBIAD1蛋白在维持胆固醇和HDL稳态中的作用，并可能阐明导致SCD的突变对蛋白质功能的影响。附加基因测序[摘要截短为7800个字符]

Summary The understanding of complex human disease and the development of therapeutic approaches are some of the great challenges of molecular medicine. The transport of molecules into and out of the cell is one of the most critical functions of living organisms. A large reason for the failure of chemotherapy in many cases is drug resistance. Pumps that remove toxins from cells are critically important in the resistance of tumors. In addition a number of genetic diseases are caused by defects in transport proteins. The ATP-binding cassette (ABC) gene family codes for transporter proteins pumping a variety of compounds across the membranes of cells and tissues. Overexpression of ABC transporters is an important cause of multidrug resistance to chemotherapy agents and these genes are mutated in diverse human diseases. Complement genes and Age-Related Macular Degeneration By studying variants in the complement B and C2 (<I>CFH</I>, <I>C2</I>) genes we have identified predisposing and protective variants in patients with age-related macular degeneration (AMD). AMD is the leading cause of blindness in the elderly and is estimated to effect as many as of 10 million Americans. By examining the genetic variants in the <I>BF</I> and <I>C2</I> genes significantly protective haplotypes were identified. The alternative complement pathway is important in the response to pathogens causative for cancer and inflammation. Further study of this arm of the innate immune response will lead to insights into human disease. we identified a deletion that removes the <I>CFHR1</I> gene adjacent to <I>CFH</I> is highly protective for AMD. We obtained over 3000 DNAs from subjects in the Age-Related Eye Disease Study, and typed them for the associated markers, confirming the associations of the major alleles and demonstrating that the CFH and CFB alleles mainly affect progression to early stages of disease (large drusen) and that the HTRA1 and CFHR1/3 deletion affect progression to late stage outcomes (neovascularization, geographic atrophy). Through the analysis of Affymetrix 6.0 chip data we have been able to genotype heterozygous deletion patients and identify an additional deletion that disrupts or deletes the CFHR4 gene. Further characterization of these deletions are likely to reveal more about the function of this immunoregulatory gene cluster. Recently published data implicated the Toll-like receptor 3 (TLR3) gene in AMD. However we have typed this variant in the AREDS cohort, and along with data from 7 other centers failed to replicate this association. ABC genes in Cancer Stem Cells Several ABC genes are expressed in cancer stem cells allowing for an innate resistance to cancer therapy. <I>ABCG2</I> is an important drug resistance gene and we have screened for and identified new inhibitors to the protein encoded by this gene. One of these is a new class of compounds known as botrylamides. The botrylamides are found in marine sponges and are simple enough chemically that derivatives can be synthesized and a structure/function relationship determined. <I>ABCG2</I> is an important marker and resistance factor in cancer stem cells. Several of these compounds can be shown to inhibit substrate binding and/or ATPase activity of the protein. The ABCB5 gene has been shown to be a marker for melanoma stem cells. We have performed evolutionary analysis of this gene and demonstrated that it evolved as a full-transporter. In addition we have validated and applied genotyping assays for several variants in the gene to demonstrate that variation in this gene is widely distributed and significantly different in distinct human populations. Metabolomics to study ABC gene Function To understand the function of the <I>ABCC6</I> gene causing pseudoxanthoma elasticum we have initiated a metabolomics study. Urine from male patients has been used to generate metabolite profiles. We have also performed studies of serum and urine from <I>Abcc6</I> -/- mice and from yeast cell deficient in an ABCC subfamily gene. These studies reveal a number of small molecules differentially present in these samples and provide important candidate substrates for further testing. In addition we have characterized the complement of ABC genes in the planktic crustacean Daphnia a globally distributed organism of central importance for the ecology of lakes and ponds. Daphnia is not only the first crustacean, but also the first non-insect arthropod to have its genome sequence determined. TRIM5 and HIV Infection A regulatory protein, TRIM5-alpha, has been implicated in the transmission of HIV-1 and other retroviruses. We identified that approximately four percent of Baka pygmies in Cameroon have a mutation in the gene, R332X that is a null allele, with partial dominant negative activity. Genetic factors such as this, along with the high frequency of exposure to chimpanzee body fluids, may have predisposed population in this region to the initial cross-species transmission of HIV. Discovery of a new variant of ERBB4 gene, a candidate gene for schizophrenia. In collaboration with Dr. Amanda Law and Dr. Daniel Weinberger at the National Institute of Mental Health, NIH, we searched for splice variants of the human ERBB4 gene, which encodes a receptor for neuregulin 1 gene. We found that a novel ERBB4 exon 3-skipping transcript variant was expressed in the human fetal brain, and that this splice variant was present preferentially in the ERBB4 JM-b/CYT-2 isoform. In addition, a 45-nt sequence encoding 14 amino acids, characteristic of the JM-b isoform, is located in the intron 15 of the ERBB4 gene. We have for the first time shown that the ERBB4 exon 3 is an alternative exon. In contrast, both exon E15a and exon 16 are mutually exclusive. Therefore, we have clarified that there are currently four alternative exons (exons 3, 15a, 16 and 26) in human ERBB4 gene, which should consist of 29 exons instead of 28 exons. Detection of an Infectious Retrovirus, XMRV, in Patients with Chronic Fatigue Syndrome Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMC) from CFS patients, our colleagues identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), in 68 of 101 patients (67%) compared to 8 of 218 (3.7%) healthy controls. We genotyped the RNASEL gene in these samples, a gene previously implicated in XMRV infection of prostate cancer patients. There was no association with RNASEL genotype and either CFS or XMRV positivity. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS. Genetics and function of Schnyder Corneal Dystrophy Schnyder Corneal Dystrophy (SCD) is an inherited disease affecting vision due to abnormal deposition of cholesterol and lipid in the cornea. In addition, many SCD patients exhibit systemic hypercholesterolemia and apparent disregulation of an unknown aspect of cholesterol and/or HDL metabolism. Mutations in a novel gene (UBIAD1) were identified cause this disorder. Continued genetic analysis of SCD may allow a greater understanding of the role of the UBIAD1 protein in maintaining cholesterol and HDL homeostasis and may clarify the effect of mutations causing SCD on protein function. Sequencing of the gene in addition [summary truncated at 7800 characters]