FELINE I-CELL DISEASE (MUCOLIPIDOSIS II)

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在动物群体中，我们有四个繁殖的I-细胞携带者雌性和两个繁殖的I-细胞携带者雄性。此外，两只年轻的携带者雌性将被保留到成熟后进行繁殖。在过去的一年里，两只患有i-cell疾病的猫被牺牲：一只8个月大，另一只1周大。此外，我们还存档了福尔马林固定和冷冻的不同条件下的组织，这些组织来自受影响的猫和携带者以及正常的猫。目前，我们有两只一个月大的受影响的i-cell小猫，以及一只正常的小猫作为对照。死后分析受影响的小猫在尸检时通常只有轻微的肉眼病变，包括心脏扩张、食道扩张、脆弱的骨骼和严重的皮肤增厚，死亡年龄反映了由于这种疾病的进行性损害的严重程度。在8个月后死亡的受i-cell影响的猫中，许多组织都出现了组织学损伤，包括但不限于舌头、会厌、肝脏、皮肤、心脏、主动脉和其他大动脉、一般的平滑肌肉、肾脏和骨骼。这些病变的范围从扭曲细胞和器官结构的细胞内的大而透明的空泡(如会厌、舌头、主动脉和其他大动脉和心脏瓣膜中可见的不同)，到似乎对细胞形态(肝脏、肾脏)几乎没有影响的小空泡，到组织成分(骨、皮肤)的正常组织的异常。我们检查了过去对人类患者组织进行的特殊染色。受影响细胞内溶酶体含量的生化鉴定。根据直接测量、组织染色质量和超微结构外观，低聚糖、脂类和糖胺多糖(GAG)被认为在人类和猫I-细胞患者的各种细胞中积聚。然而，在患病、正常和携带者的不同组织中，特定的底物及其相对数量尚不清楚。使用商业试剂盒(Blyscan)，我们试图定量检测受I-细胞影响的猫成纤维细胞中GAG的水平。目前，我们已经培养、收集和冷冻了受粘多糖病(MPS)I、III和VI影响的猫成纤维细胞(作为阳性对照)以及I细胞培养细胞。我们已经与图卢兹大学的玛丽·瓦尼尔博士建立了国际合作，以规范和量化组织神经节苷脂。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，我们能够在NCBI轨迹档案中搜索最近从猫科动物基因组计划(FGP)中获得的猫科动物序列，并发现与人类GNPTA基因同源的遗传序列。至此，21个外显子中除3个外显子(外显子4、5和6)之外的所有外显子都有一些全基因组鸟枪式序列。以正常的Fgp序列为指导，我们设计了一对引物，对一只患I-细胞的猫的gDNA和cDNAs进行了测序。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测序尚未完成，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。到目前为止，大约一半的外显子13(最长的外显子，大约1300个碱基)已经被测序。在该外显子的第5号外显子中发现了单一的碱基差异。然而，其重要性尚不为人所知。此外，对正常猫和病猫的GNPTA基因的大部分序列进行了测序，包括FGS中缺失的外显子，并确定了一些差异，这些差异可能与致病突变有关。

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.