THE CYTOGENETICS LABORATORY

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。基础研究继续主要集中在有性异常动物的吉姆萨带状核型以及提示存在染色体异常的综合征性疾病。自上次进度报告以来，共对转介到遗传学诊所或后续咨询的犬和猫病例进行了19次细胞遗传学研究。犬类调查包括迪乔治综合征、阴蒂缺失、无精子症、鱼鳞病、PCH-1杂合子、雌雄同体、尿道下裂极端和共济失调。还检查了一只猫的双性病例。新的疾病表现包括犬软组织肿胀、鱼鳞病、共济失调、极度关节松弛、牙周疾病、迪乔治综合征、四肢萎缩矿化、HD和ED。猫的脊髓性肌萎缩、成骨不全、多发骨折、皮肤哮喘和其他结缔组织疾病也被检查。研究包括使用针对不同胶原蛋白的抗体的Western blotting和I型胶原蛋白功能和组装的脉冲追踪测定。Western blots一直具有挑战性，因为许多抗体对狗和猫以外的物种是特异性的。在皮肤脆弱的动物身上的研究结果将作为皮肤科住院医师项目的一部分进行总结，并将提交发表。细胞遗传学实验室的另一个主要作用是转诊中心目前正在调查的模型的组织培养。犬MPS IIIB、MPS VI、PFK、x连锁无水外胚囊发育异常、大疱性表皮解裂和致死性肢端皮炎的影响细胞系已被添加，以确定细胞病理学，为突变分析提供RNA和DNA，并用于未来的交叉校正研究。从低磷血症、拉布拉多肌病、肾功能衰竭、可能的MPS III型和病理性骨折的狗身上建立成纤维细胞培养。从猫的成纤维细胞培养中建立MPS VI，一种未确诊的溶酶体贮积病，丙酮酸激酶缺乏症，I细胞病，门静脉分流，GM2神经节脂质病，神经肌肉疾病，MPS VII， MPS I和GM1神经节脂质病。利用荧光原位杂交（FISH）确定犬染色体上基因的物理位置的方法已经发展起来。这种方法使用荧光染料标记的基因探针。将标记的探针与中期染色体杂交，并通过荧光显微镜检测，使克隆基因和其他标记物能够定位到特定的染色体位置。FISH方法提供了一种将与犬类遗传疾病相关的突变基因定位到其染色体位置的方法，进一步增加了这些重要模型的比较医学遗传学知识，并增强了它们作为人类遗传疾病动物模型的应用。此外，FISH研究增加了犬基因组图谱的发展，增加了其作为寻找人类遗传疾病新犬同源物的资源的价值。我们在全球范围内合作绘制狗的基因组图谱。作为这项工作的一部分，我们已经绘制了一系列标记，这些标记作为犬染色体连锁群的锚点。这些连锁群包含341个标记，分布在37个犬常染色体和X染色体上。这些基因标记同时作为犬类连锁图谱中微卫星标记的锚位点，建立连锁群的染色体位置，使犬类基因区域与人类和小鼠染色体的同源区域对齐。这提供了获取这些其他物种染色体上基因进化保守排列的丰富信息来源，极大地促进了通过定位克隆方法定位和分离犬类基因的尝试。

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. Basic studies continue to concentrate primarily on Giemsa banded karyotyping of animals with sexual anomalies as well as syndromic disorders that suggest the presence of a chromosomal anomaly. Since the last progress report, a total of 19 cytogentic studies were performed on canine and feline cases referred to the genetics clinic or following consultations. Canine investigations included DiGeorge Syndrome, os clitoris, azoospermia, ichthyosis, a PCH-1 heterozygote, intersex, hypospadias extreme, and ataxia. A feline case of intersex was also examined. Novel disease presentations included canine soft tissue swelling, ichthyosis, ataxia, extreme joint laxity, perodontal disease, DiGeorge syndrome, cystrophic mineralization in limbs, HD and ED. Feline cases of spinal muscular atrophy, ostogenesis imperfecta, multiple fractures, cutaneous asthemia, and other connective tissue disorders were also examined. Studies include Western blotting using antibodies specific for different collagens and pulse chase assays for collagen type I function and assembly. The Western blots have been challenging as many of the antibodies are specific to species other than the dog and cat. The findings in animals with skin fragility are being summarized as part of a dermatology residency project and will be submitted for publication. Another primary role of the cytogenetic laboratory is tissue culture of models currently under investigation in the Referral Center. Affected cell lines have been added for canine MPS IIIB, MPS VI, PFK, X-linked anhydrotic ectodemal dysplaisa, epidermolysis bullosa, and lethal acrodermatitis in an effort to define cellular pathology, provide RNA and DNA for mutation analysis, and for use in future cross-correction studies. Fibroblast cultures were established from dogs with hypophosphatemia, Labrador myopathy, renal failure, possible MPS III, and pathologic fractures. Fibroblast cultures from cats were established for MPS VI, an undiagnosed lysosomal storage disease, pyruvate kinase deficiency, I-cell disease, portocaval shunt, GM2 gangliosidosis, neuromuscular disease, MPS VII, MPS I, and GM1 gangliosidosis. Methods have been developed for the use of fluorescence in situ hybridization (FISH) to define the physical location of genes on canine chromosomes. This approach uses gene probes labeled with fluorescent dyes. The labeled probes are hybridized to metaphase chromosomes and detected by fluorescence microscopy, allowing cloned genes and other markers to be mapped to specific chromosome locations. The FISH method provides a means of mapping the mutant genes involved in canine genetic diseases to their chromosomal locations, further increasing knowledge of the comparative medical genetics of these important models and enhancing their use as animal models of human genetic disease. In addition, the FISH studies add to the development of the canine genome map, increasing its value as a resource for finding new canine homologs of human genetic diseases. We have collaborated in a worldwide initiative to map the dog genome. As a part of this work, we have FISH-mapped a series of markers that serve as anchors for linkage groups on canine chromosomes. These linkage groups contain 341 mapped markers distributed over 37 canine autosomes and the X Chromosome. These gene markers simultaneously serve as anchor loci for microsatellite markers in the canine linkage map and establish the chromosomal locations of the linkage groups, allowing the canine gene regions to be lined up with the homologous regions of human and mouse chromosomes. This provides access to the rich source of information on evolutionarily conserved arrangements of genes on chromosomes in these other species, greatly facilitating attempts to locate and isolate canine genes by positional cloning methods.