CANINE MUCOPOLYSACCHARIDOSIS

犬粘多糖病

• 批准号: 7391967
• 负责人: MARK E HASKINS
• 金额: $ 0.67万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7391967
• 关键词: CANINE; MUCOPOLYSACCHARIDOSIS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. MPS IIIB in Schipperke Dogs A naturally occurring canine form of MPS IIIB has been identified through the metabolic screening laboratory. The initial description of the dogs identified with this condition has been published. We have isolated and sequenced the entire protein-coding region of the canine NAGLU gene which has been presented in abstract form. Additionally the mutation has been identified and was found to be an unusual L1 retrotransposon-mediated insertion of a polyadenylate sequence of approximately 45 A¿s. The insertion occurs in the sixth exon and is predicted to lead to the insertion of 15 lysine residues after amino acid 704 of the normal protein sequence. The insertion of so large a series of lysines would be predicted to result in the elimination of normal enzyme activity, whether or not the lysines were followed by in frame or frame shifted translations. A DNA diagnostic for this mutation has been developed and is used to diagnose animals produced in the colony as well as in the Skipperke dog population. While the DNA test was still in development, four new affetced animals were diagnosed through the metabolic screening laboratory. Urine, serum and blood samples were requested from relatives of these animals to determine carrier status and identify other affected animals. A total of 96 samples were analyzed from 28 animals. Four additional affected dogs were diagnosed and 11 carriers were detected. This study allowed preliminary accuracy testing of the newly developed DNA test in the breeding community. Preliminary evaluations of the brains of the two original affected dogs indicated striking accumulation of GM2 and GM3 gangliosides. Evalutions underway include neurologic testing, histopathologic evaluation of the CNS and biochemical analysis of the CNS, with a specific focus on ganglioside elevations. Part of the canine colony for this lysosomal storage disease in now present at Iowa State University, where it will be the focus of study by Dr. N.M. Ellinwood. A further effort associated with this model is the assessment of gene therapy for MPS IIIB. Specifically, recombinant adeno-associated vectors, of various serotypes, are being constructed, and will be evaluated in the brains of MPS IIIB affected dogs following intracerebral injection Canine MPS VI After the original discovery of mucopolysaccharidosis (MPS) VI or arylsulfatase deficiency in Siamese cats and their clinicopathologic and molecular characterization and use in gene transfer studies, we have recently identified several dogs with MPS VI. The first affected breed was the Miniature Pinscher, followed by the Welsh Corgi, Chesapeake Bay retriever, and Miniature Schnauzer. MPS VI is caused by a deficiency of the enzyme N-acetylgalactosamine-4-sulfatase, also known as 4S (EC # 3.1.6.12). 4S deficiency results in the lysosomal accumulation of glycosaminoglycans (GAG), specifically dermatan sulfate, that is found in urine. MPS VI is inherited as an autosomal recessive trait, and is clinically characterized by short stature, facial dysmorphism, corneal dystrophy, secondary neurological disturbances, and various orthopedic abnormalities. The normal canine ARSB cDNA sequence was determined using primers developed based on conserved sequences of the human and feline ARSB, and by screening clones from a canine cDNA library. When the DNA sequence from Miniature Pinschers affected with MPS VI was compared to the normal canine sequence, a single missense mutation (G to A) was identified. This mutation, occurring in exon V, replaces the tiny hydrophobic amino acid glycine with a bulky positively charged basic amino acid arginine in a highly conserved region of the protein. The same mutation (G302R) has been described in humans to cause a severe form of the disease. A DNA test based on restriction enzyme analysis of PCR products was developed. Samples were solicited for testing from AKC registered Miniature Pinschers. DNA obtained from cheek swab or EDTA blood samples from 130 Miniature Pinschers has been analyzed. One affected and 20 carrier dogs were identified. The affected dog and all identified carrier dogs were closely related. In this biased population, the frequency of the mutant allele was approximately 0.0846. To identify the mutation that is responsible for MPS VI in Miniature Schnauzers, primers were developed from intronic sequence provided by The Insititute for Genome Research (TIGR) and the Whitehead Genomic Institute as well as 5' utr sequence and 3' utr sequence generated from the normal canine cDNA. The mutation responsible for MPS VI in the Miniature Schnauzer was identified as a 56 bp deletion that eliminates the last 26 bp of the 5' utr and the first 10 codons of exon I including the initiator ATG. Since the next ATG is located 426 bp downstream in the cDNA, this truncated protein is predicted to be non-functional and unstable. Canine MPS VII A German shepherd puppy was presented to the University of Georgia because of difficulties ambulating. Laboratory diagnostic tests were pursued and a diagnosis of MPS VII was reached. The same mutation was found in this German shepherd dog as previously described in a mixed breed dog 20 years ago thus confirming that this disease originated in the German shepherd breed. No relatives have become available for further study. A juvenile female Rat terrier was presented to because of severe deformities and gait abnormalities. The puppy exhibited a dome shaped head, severe underbite and multiple limb deformities. Skeletal radiographs revealed the classical bone changes observed with many MPS disorders. White blood cells contained granular material which stained with toluidine blue. Urine contained large amounts of chondroitin and dermatin sulfate. The serum beta-glucuronidase activity was completely lacking documenting an MPS VII disorder. This dog does not have the same mutation seen in the original mixed breed and recent German Shepherd dog. Relatives of the affected dog have been examined and some were found to be carriers. At this time there are no plans to capture this model because of the close similarities of the disease in the established canine m

该主题是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。 Schipperke狗中的MPS IIIB通过代谢筛查实验室发现了天然存在的MPS IIIB的犬类形式。对这种情况确定的狗的最初描述已发表。我们已经分离并测序了以抽象形式呈现的犬纳格鲁基因的整个蛋白质编码区域。另外，已经鉴定出突变，并被发现是对大约45 a？s的聚腺苷序列的不寻常的L1逆转录子介导的插入。该插入发生在第六个外显子中，预计将导致正常蛋白序列的氨基酸704后15个级别的残留物插入。插入如此大的一系列水平将被预测会导致正常酶活性，无论是在框架还是框架移动的翻译中遵循的水平。已经开发了针对该突变的DNA诊断，并用于诊断菌落和船长狗种群中产生的动物。尽管DNA测试仍在开发中，但通过代谢筛查实验室诊断出了四只新的复活动物。从这些动物的亲戚那里要求尿液，血清和血液样本确定载体状态并识别其他受影响的动物。总共分析了28只动物的96个样品。诊断出其他四只受影响的狗，并检测到11个载体。这项研究允许对新开发的DNA测试的初步准确性测试。对两只原始受影响的狗的大脑的初步评估表明GM2和GM3神经毒剂的惊人精度。正在进行的评估包括神经系统测试，中枢神经系统的组织病理学评估以及对中枢神经系统的生化分析，并特别关注神经节苷脂升高。现在在爱荷华州立大学出席的这种溶酶体储存疾病的犬殖民地的一部分，这将是N.M. Ellinwood博士的研究重点。与该模型相关的进一步努力是评估MPS IIIB基因治疗。具体而言，正在构建各种血清型的重组腺相关载体，并将在MPS IIIB受影响的狗的大脑中进行评估。脑内注射犬MPS VI MPS VI在最初发现粘多糖（MPS）VI或芳基硫酸盐症中的杂型及其临床效率及其临床效果及其siamesecoration及其siamesecortiation及其临床上的转移，并将其造成的菌丝及其临床症状转移，并将其评估。我们最近确定了几只具有MPS VI的狗。第一个受影响的品种是微型平彻（Miniature Pinscher），其次是威尔士·科吉（Welsh Corgi），切萨皮克湾（Chesapeake Bay）猎犬和微型schnauzer。 MPS VI是由酶N-乙酰乳糖胺-4-硫酸酶的缺乏引起的，也称为4S（EC＃3.1.6.12）。 4S缺乏会导致糖胺聚糖（GAG），特别是硫酸硫酸盐的溶酶体积累，在尿液中发现。 MPS VI遗传为常染色体隐性性状，在临床上以矮小的身材，面部营养不良，角膜营养不良，继发性神经系统疾病和各种骨科异常为特征。正常的犬ARSB cDNA序列是使用基于人类和猫ARSB的配置序列开发的引物，并通过从犬cDNA文库中筛选克隆来确定的。当将受到MPS VI影响的微型短数字的DNA序列与正常犬序列进行比较时，发现了单个错义突变（G至A）。这种突变发生在外显子V中，在蛋白质高度保守的区域中，用笨重的带阳性的碱性氨基酸精氨酸代替了微小的疏水氨基酸甘氨酸。在人类中已经描述了相同的突变（G302R）引起严重的疾病形式。开发了基于PCR产物限制酶分析的DNA测试。征求了来自AKC注册的微型根彻斯的样品进行测试。已经分析了从130个微型剪车的脸颊拭子或EDTA血液样本获得的DNA。一只受影响和20只载犬。受影响的狗和所有已确定的携带者狗都是密切相关的。在这种偏见的人群中，突变等位基因的频率约为0.0846。为了鉴定导致小型Schnauzers中MPS VI的突变，引物是由基因组研究（TIGR）和Whitehead Genomic Institute的内含子序列以及5'UTR序列以及由正常犬CDNA产生的3'UTR序列的。在微型Schnauzer中负责MPS VI的突变被确定为56 bp的缺失，消除了5'UTR的最后26 bp和包括引发剂ATG在内的外显子I的前10个密码子。由于下一个ATG位于cDNA下游的426 bp，因此该截断的蛋白质被预测为非功能和不稳定。犬MPS VII由于难以行动，将一只德国牧羊犬送交佐治亚大学。进行了实验室诊断测试，并进行了MPS VII的诊断。在20年前在混合品种狗中所描述的这只德国牧羊犬中发现了同样的突变，因此证实了这种疾病起源于德国牧羊犬。没有亲戚可以进一步研究。由于严重的畸形和步态异常，出现了少年雌性大鼠梗。小狗暴露了一个圆顶形的头，严重的咬伤和多个肢体畸形。骨骼X光片揭示了许多MPS疾病观察到的经典骨变化。白细胞包含用甲苯胺蓝色染色的颗粒状材料。尿液中含有大量软骨素和硫酸皮肤素。血清β-葡萄糖醛酸酶的活性完全缺乏记录MPS VII疾病的作用。这只狗在原始混合品种和最近的德国牧羊犬中没有相同的突变。已经检查了受影响的狗的亲戚，发现有些是携带者。目前，由于已建立的犬M中该疾病的紧密相似性，还没有计划捕获该模型