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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。一种天然存在的犬类MPS IIIB已通过代谢筛选实验室确定。对患有这种疾病的狗的初步描述已经发表。我们已经分离并测序了犬NAGLU基因的整个蛋白质编码区，该基因已以抽象形式呈现。此外，该突变已被确定，并被发现是一个不寻常的L1反转录转座子介导的多腺苷酸序列插入约45 a¿s。该插入发生在第6外显子，预计将导致在正常蛋白序列的704氨基酸后插入15个赖氨酸残基。如此大的赖氨酸序列的插入预计会导致正常酶活性的消除，无论赖氨酸之后是否有帧内翻译或帧移位翻译。这种突变的DNA诊断方法已经被开发出来，并被用于诊断在该群体中生产的动物以及Skipperke犬群。虽然DNA测试仍在开发中，但通过代谢筛选实验室诊断出了四只新的受影响动物。要求这些动物的亲属提供尿液、血清和血液样本，以确定携带者状况并确定其他受影响动物。共分析了来自28只动物的96个样本。另外4只受感染的狗被诊断出来，并发现了11只携带者。这项研究为新开发的DNA检测方法在繁殖群落中的准确性提供了初步的验证。对两只最初受影响的狗的大脑的初步评估表明，GM2和GM3神经节苷脂的积累惊人。正在进行的评估包括神经学测试，中枢神经系统的组织病理学评估和中枢神经系统的生化分析，特别关注神经节苷脂升高。这种溶酶体贮积病的部分犬群现在在爱荷华州立大学，这将是N.M.埃林伍德博士研究的重点。与该模型相关的进一步工作是评估MPS IIIB的基因治疗。具体而言，各种血清型的重组腺相关载体正在构建中，并将在脑内注射犬MPS VI后，在MPS IIIB感染犬的大脑中进行评估。在最初发现暹罗猫的粘多糖病（MPS） VI或芳香基磺化酶缺乏症及其临床病理和分子表征以及在基因转移研究中的应用之后，我们最近发现了几只患有MPS VI的狗。第一个受影响的品种是迷你平切犬，其次是威尔士柯基犬，切萨皮克湾猎犬和迷你雪纳瑞。MPS VI是由n -乙酰半乳糖胺-4-硫酸酯酶（也称为4S酶）缺乏引起的（EC # 3.1.6.12）。4S缺乏导致溶酶体积累的糖胺聚糖（GAG），特别是皮肤硫酸，发现在尿中。MPS VI是一种常染色体隐性遗传性状，临床表现为身材矮小、面部畸形、角膜营养不良、继发性神经障碍和各种骨科异常。利用基于人和猫ARSB保守序列构建的引物，以及从犬cDNA文库中筛选克隆，确定正常犬ARSB cDNA序列。将感染MPS VI的迷你Pinschers的DNA序列与正常犬的DNA序列进行比较，发现了一个单一的错义突变（G到a）。这种突变发生在外显子V，在蛋白质的高度保守区域，用一个带正电的碱性氨基酸精氨酸取代了微小的疏水氨基酸甘氨酸。同样的突变（G302R）在人类中已被描述为引起严重形式的疾病。建立了一种基于PCR产物限制性内切酶分析的DNA检测方法。从AKC注册的迷你pinscher中征集样本进行测试。从130只迷你pinscher的颊拭子或EDTA血液样本中提取的DNA进行了分析。发现1只受感染犬和20只携带犬。患病犬与所有已确定的带病犬有密切关系。在这个偏倚群体中，突变等位基因的频率约为0.0846。为了确定迷你雪纳瑞MPS VI的突变，我们从基因组研究所（TIGR）和Whitehead基因组研究所提供的内含子序列以及从正常犬cDNA中产生的5' utr序列和3' utr序列中开发了引物。在迷你雪纳瑞犬中，导致MPS VI的突变被鉴定为56 bp的缺失，该缺失消除了5' utr的最后26 bp和外显子I的前10个密码子，包括启动子ATG。由于下一个ATG位于cDNA下游426bp处，因此该截断蛋白被预测为无功能且不稳定。由于行走困难，一只德国牧羊犬被送到了佐治亚大学。进行了实验室诊断检查，得出了MPS VII的诊断。在这只德国牧羊犬身上发现了与20年前在一只杂交犬身上发现的相同的突变，从而证实了这种疾病起源于德国牧羊犬品种。没有亲属可以进一步研究。一只幼年雌性大鼠梗因严重畸形和步态异常而被提出。这只小狗显示出一个圆顶状的头，严重的下咬合和多肢畸形。骨骼x线片显示许多MPS疾病的典型骨改变。白细胞含有被甲苯胺蓝染色的颗粒状物质。尿液中含有大量的软骨素和硫酸皮素。血清β -葡糖醛酸酶活性完全缺乏记录MPS VII紊乱。这只狗没有相同的突变看到在原来的混合品种和最近的德国牧羊犬。受感染狗的亲属已接受检查，其中一些被发现是携带者。目前还没有计划捕捉这个模型，因为这种疾病在已建立的犬类中非常相似