FELINE I-CELL DISEASE (MUCOLIPIDOSIS II)

猫科 I 细胞疾病（粘脂血症 II）

• 批准号: 7391957
• 负责人: URS GIGER
• 金额: $ 2.01万
• 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/7391957
• 关键词: FELINE; CELL; DISEASE; MUCOLIPIDOSIS; II

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. Within the animal colony, we have four breeding I-cell carrier females and two breeding I-cell carrier males. In addition, two young carrier females are being kept until maturity for breeding. In the past year, two cats affected with I-cell disease have been sacrificed: one at 8 months and another at 1 week. Also, we have archived formalin-fixed and frozen tissues in variable condition from affected and carrier as well as normal cats. Presently, we have two one-month old affected I-cell kittens, as well as a normal littermate which will serve as a control. Post-mortem analysis Affected kittens that die often have only mild gross lesions at necropsy, including dilated hearts, esophageal dilation, brittle bones, and severely thickened skin, with the age at death reflecting the severity of lesions due to the progressive nature of this disease. In I-cell affected cats that died at eight months, histological lesions were seen in a many tissues, including, but not limited to, the tongue, epiglottis, liver, skin, heart, aorta and other large arteries, smooth muscle in general, kidney, and bones. These lesions ranged from large clear vacuoles within cells that distorted the cellular and organ architecture (as variably seen in the epiglottis, tongue, aorta and other large arteries and cardiac valves), to small vacuoles that appear to have little effect on cellular morphology (liver, kidney), to abnormalities in the normal organization of tissue components (bone, skin). We have examined special stains that have been performed on tissue from human patients in the past. Biochemical identification of lysosomal contents within affected cells. Based on direct measurement, histological staining qualities, and ultrastructural appearance, oligosaccharides, lipids, and glycosaminoglycans (GAGs) are believed to accumulate within various cells in human and feline I-cell patients. However, specific substrates and their relative amounts in various tissues in affected, normal, and carriers are not known. Using a commercial kit (Blyscan), we have attempted to quantitate levels of GAGs within cultured fibroblasts from I-cell affected cats. Presently, we have grown, collected, and frozen cultured fibroblasts from mucopolysaccharidosis (MPS) I, III, and VI affected cats (as positive controls) as well as I-cell cultured cells. We have established an international collaboration with Dr. Marie Vanier, Toulouse University for specification and quantification of tissue gangliosides. Identification of the feline N-acetylglucosamine-1-phosphotransferase gene. The genetic sequence of N-acetylglucosamine-1-phosphotransferase (GNPTA) GNPTA has not yet been published in any species. Based on the unpublished human sequence, provided by our collaborator, William Canfield, Genzyme Corp., we were able to search the NCBI Trace Archives for recently available feline sequence from the Feline Genome Project (FGP) and found genetic sequences homologous to the human GNPTA gene. To this point, for all but three (exons 4, 5, and 6) of the 21 exons we have some whole genome shotgun sequences. Using the normal FGP sequence as a guide, we have designed primers to sequence gDNA and cDNA from an I-cell affected cat. Four exons have been sequenced so far from the gDNA (exons 2, 8, 12, and 13). Exon 2 sequencing is not complete, but appears to be homologous to the normal FGP sequence at this point. Also, exons 8 and 12 are identical to the FGP sequence, so do not appear to carry the mutation in this affected cat. Approximately half of exon 13 (the longest exon, at approximately 1300 bp) has been sequenced at this time. A single bp difference has been found in the 5¿ portion of this exon. however, its significance is not yet known. In addition, most of the GNPTA cDNA from a the normal and affected cat have been sequenced including the missing exons in the FGS and a few differences have been determined, which may relate to the disease-causing mutation.

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。在动物群体中，我们有四只繁殖携带i细胞的雌性和两只繁殖携带i细胞的雄性。此外，两只年轻的携带雌性被保留到成熟繁殖。在过去的一年里，两只患i细胞病的猫被处死：一只8个月大，另一只1周大。此外，我们还存档了受影响猫和携带者以及正常猫的不同状态的福尔马林固定和冷冻组织。目前，我们有两只一个月大的感染i细胞的小猫，以及一只正常的窝友作为对照。死亡的受影响小猫尸检时通常只有轻微的大体病变，包括心脏扩张、食管扩张、骨质脆性和皮肤严重增厚，死亡时的年龄反映了由于该病的进行性而造成的病变的严重程度。在8个月后死亡的感染i细胞的猫中，在许多组织中都可以看到组织学病变，包括但不限于舌头、会阴、肝脏、皮肤、心脏、主动脉和其他大动脉、平滑肌、肾脏和骨骼。这些病变的范围从细胞内的大而清晰的空泡扭曲细胞和器官结构（在会珠、舌、主动脉和其他大动脉和心脏瓣膜中可见），到对细胞形态（肝、肾）几乎没有影响的小空泡，到正常组织成分（骨、皮肤）的异常。我们已经检查了过去在人类患者的组织上进行的特殊染色。感染细胞内溶酶体内容物的生化鉴定。根据直接测量、组织学染色质量和超微结构外观，低聚糖、脂质和糖胺聚糖（GAGs）被认为在人类和猫i细胞患者的各种细胞内积累。然而，具体的底物及其在不同组织中的相对量，在受影响的，正常的，和携带者是未知的。使用商业试剂盒（Blyscan），我们尝试在培养的i细胞感染猫的成纤维细胞中定量测定GAGs的水平。目前，我们已经从粘多糖病（MPS） I、III和VI感染的猫（作为阳性对照）以及I细胞培养的细胞中培养、收集和冷冻培养成纤维细胞。我们与图卢兹大学的Marie Vanier博士建立了国际合作，以规范和定量组织神经节苷。猫n -乙酰氨基葡萄糖-1-磷酸转移酶基因的鉴定。n -乙酰氨基葡萄糖-1-磷酸转移酶（GNPTA）的基因序列尚未在任何物种中发表。基于我们的合作者William Canfield， Genzyme Corp.提供的未发表的人类序列，我们能够从NCBI Trace Archives中检索猫科基因组计划（FGP）中最近可用的猫科序列，并找到与人类GNPTA基因同源的基因序列。到目前为止，对于21个外显子中的3个（外显子4,5和6），我们都有一些全基因组鸟枪序列。以正常的FGP序列为指导，我们设计了引物对i细胞感染猫的gDNA和cDNA进行测序。到目前为止，已经对gDNA的四个外显子（外显子2、8、12和13）进行了测序。外显子2测序不完整，但在这一点上似乎与正常的FGP序列同源。此外，外显子8和12与FGP序列相同，因此在这只受影响的猫中似乎没有携带突变。大约一半的外显子13（最长的外显子，约1300 bp）在这个时候已经测序。在这个外显子的5¿部分发现了一个bp的差异。然而，其意义尚不清楚。此外，对正常猫和患病猫的大部分GNPTA cDNA进行了测序，包括FGS中缺失的外显子，并确定了一些差异，这可能与致病突变有关。