FELINE I-CELL DISEASE (MUCOLIPIDOSIS II)

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。在动物群体中，我们有四个繁殖I细胞携带者雌性和两个繁殖I细胞携带者雄性。此外，两只年轻的携带者雌性被饲养至成熟进行繁殖。在过去的一年中，两只受I细胞疾病影响的猫被处死：一只在8个月时，另一只在1周时。此外，我们还将受影响的猫和携带者以及正常猫的福尔马林固定和冷冻组织在不同条件下存档。目前，我们有两只一个月大的受影响的I细胞小猫，以及一只正常的同窝仔，将作为对照。 尸检分析死亡的受影响小猫在尸检时通常只有轻度肉眼病变，包括心脏扩张、食管扩张、脆骨和严重增厚的皮肤，死亡时的年龄反映了由于该疾病的进行性而导致的病变的严重程度。在8个月时死亡的受I细胞影响的猫中，在许多组织中观察到组织学病变，包括但不限于舌、会厌、肝脏、皮肤、心脏、主动脉和其他大动脉、一般平滑肌、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大的透明空泡（如会厌、舌、主动脉和其他大动脉和心脏瓣膜中所见），到似乎对细胞形态学影响不大的小空泡（肝、肾），到组织成分（骨、皮肤）正常组织的异常。我们已经检查了过去在人类患者组织上进行的特殊染色。 受影响细胞内溶酶体内容物的生化鉴定。基于直接测量、组织学染色质量和超微结构外观，认为寡糖、脂质和糖胺聚糖（GAG）在人类和猫I细胞患者的各种细胞内蓄积。然而，具体的底物和它们在各种组织中的相对量在受影响的，正常的，和运营商是未知的。使用商业试剂盒（Blyscan），我们试图定量培养的成纤维细胞内的GAG水平从I细胞影响的猫。目前，我们已经培养、收集和冷冻了来自粘多糖样变性（MPS）I、III和VI型感染猫的培养成纤维细胞（作为阳性对照）以及I细胞培养细胞。我们与图卢兹大学的玛丽瓦尼耶博士建立了国际合作，用于组织神经节苷脂的规格和定量。 猫N-乙酰葡糖胺-1-磷酸转移酶基因的鉴定。N-乙酰葡糖胺-1-磷酸转移酶（GNPTA）的基因序列尚未在任何物种中发表。根据我们的合作者，Genzyme公司的William坎菲尔德提供的未发表的人类序列，我们能够从NCBI TraceArchives中搜索最近可从猫基因组计划（FGP）获得的猫序列，并发现与人GNPTA基因同源的基因序列。到目前为止，除了21个外显子中的3个（外显子4、5和6）外，我们都有一些全基因组鸟枪测序。 使用正常的FGP序列作为指导，我们已经设计了引物，从I-细胞受影响的猫的gDNA和cDNA序列。四个外显子已经测序到目前为止，从gDNA（外显子2，8，12和13）。外显子2测序不完整，但似乎与正常FGP序列在这一点上同源。此外，外显子8和12是相同的FGP序列，所以没有出现携带突变，在这只受影响的猫。大约一半的外显子13（最长的外显子，在约1300 bp）已被测序在这个时候。在该外显子的5 ½部分发现了单个bp差异。然而，其重要性尚不清楚。此外，已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。

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.