LABORATORY IDENTIFICATION OF INBORN ERRORS OF METABOLISM

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。有遗传性代谢性疾病征象的动物，特别是发育不良、代谢紊乱、癫痫发作、骨骼畸形、眼睛异常（视网膜病变、白内障、角膜混浊）、持续呕吐和慢性腹泻的幼龄动物，应筛查尿液中氨基酸、有机酸、碳水化合物和糖胺聚糖排泄的定性和定量变化。对有异常发现的动物进行进一步的血液和尿液检测，使用自动氨基酸分析、气相色谱、电泳和质谱分析来识别和量化异常代谢物。怀疑是酶阻滞的结果的缺陷是通过特定的酶测定来研究的。利用家族研究和育种实验建立了定义缺陷的遗传模型。组织的光镜和电镜研究利用特殊的染色和免疫组织化学来鉴定储存的底物和特定的基因产物。自上次报告以来，2004年共分析了783个样本，这些样本来自许多犬类和猫科动物品种，以及一些雪貂和马样本。这些样本主要是尿液（412）、血清/血浆（181）和EDTA全血（113）；其他样本包括肝脏、脾脏、肾脏、皮肤、血液和淋巴结涂片等组织。样品来自美国、澳大利亚、加拿大、法国、芬兰、意大利、日本、波多黎各和英国的33个州。代谢遗传筛查实验室以前确认的病例诊断包括Fanconi综合征（25例）、I型和非I型胱氨酸尿症（11例）、孤立性糖尿症（4例）、与肠道钴胺素吸收不良相关的甲基丙二酸尿症（5例）和各种积存病。与儿童基金会合作？为了证实纸色谱和现场测试中发现的异常，我们用高效液相色谱和气相色谱法对大量样品进行了氨基酸和有机酸定量分析。这些研究有助于识别受转诊中心目前正在调查的模型影响的伴侣动物（见各个子项目），包括纽芬兰、獒犬、苏格兰鹿猎犬、卡迪根威尔士柯基犬和家养短毛猫的胱氨酸尿症，边境牧羊犬、科莫多、澳大利亚牧羊犬、牛犬和比格犬的钴胺素吸收不良，红酶病，遗传性出血性疾病，包括杜宾犬的血管性血友病，德国牧羊犬和其他品种的血友病，混合犬的纤维蛋白原缺乏症，比格犬的因子VII缺乏症，克里蓝梗的因子XI缺乏症，以及溶酶体贮积病（MPS I， IIIB， VI， VII， I-细胞病，内曼-匹克C，克拉伯病和α -甘露糖病）。代谢遗传筛选实验室的一个重点是通过尿液电泳、酶测定和血液涂片分析来鉴定患有新型和先前描述的溶酶体贮积疾病的伴侣动物。(1)尿糖胺聚糖（GAGs）和血清酶检测鉴定喜马拉雅猫粘多糖病（MPS VI）。随后的DNA分析表明，这种缺陷是由于先前描述的在暹罗猫中引起严重疾病的突变造成的。(2)最初在一种杂交犬中发现MPS VII缺乏，导致在病理生理学、分子基础和基因转移研究方面进行了大量独立支持的研究，我们最近发现两个德国牧羊犬家族由于相同的突变而患有MPS VII。此外，在大鼠梗中发现了首例MPS VII病例。这些研究的细节可以在子项目中找到。最近，GAG和酶分析也在一只雄性家养短毛猫中诊断出MPS VII。(3) 1岁幼犬血清溶酶体酶活性升高，血涂片中存在白细胞包涵体，白细胞中芳基硫酸酯酶B活性低或无活性也可诊断为MPS VI。在我们的实验室里，我们确定了这种和迷你雪纳瑞品种的致病突变，这些品种的50多只犬通过DNA测试被表征为正常，携带者或受影响。(4)继续对Skipperkes进行MPS IIIB的DNA筛查，我们共检查了1300只Schipperkes，其中约10%为携带者，1%为受影响的狗。(5)发现的其他酶缺乏症包括在一只波斯猫和一只家养短毛猫身上诊断出的α -甘露甘露病，以及在3个月大的英国施普林格西班牙猎犬身上诊断出的α -聚焦病。Josephine Deublar遗传病检测实验室已经筛选了1000多只动物，包括犬和猫品种的丙酮酸激酶缺乏症（PK）和大约80只犬的磷酸果糖激酶缺乏症（PFK），以及通过实验室提供的其他DNA检测。各大学之间已经建立了合作关系，以进一步推进上述一些新发现。2. 酶研究2004:2-1。犬酶犬血清白红总血细胞α -甘露糖苷酶-甘露糖苷酶-葡萄糖醛酸酶-葡萄糖醛酸酶-己糖胺酶- 13 13 26半乳糖苷酶- 11 10 21 α - l -甘露糖醛酸酶3 12 15芳基硫酯酶B 2 18 20 α - focusidase 39 3 3 42丙酮酸激酶3 3总170 62 3 235 2-2。猫酶猫血清白红总血细胞α -甘露糖苷酶32 4 36 -甘露糖苷酶4 1 5 -葡萄糖醛酸酶33 10 43己糖氨基酶7 14 21半乳糖苷酶9 11 20 α - l -甘露糖醛酸酶5 12 17芳基硫酸酯酶B 6 21 27 α - focusidase 28 12 40丙酮酸激酶2 2 Total 124 85 2 211

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. Animals with signs suggestive of an inherited metabolic disease, particularly young animals with failure to thrive, metabolic disturbances, seizures, bone deformities, eye abnormalities (retinopathies, cataracts, corneal clouding), persistent vomiting and chronic diarrhea are screened for qualitative and quantitative changes in the urinary excretion of amino acids, organic acids, carbohydrates, and glycosaminoglycans. Animals with abnormal findings are further investigated by blood and urine determinations using automated amino acid analysis, gas chromatography, electrophoresis, and mass spectrometry to identify and quantify the abnormal metabolites. Defects suspected to be the result of enzymatic blocks are investigated by specific enzyme assays. Family studies and breeding experiments are used to establish the mode of inheritance of defined defects. Light and electron microscopic studies of tissues utilize special stains and immunohistochemistry to identify stored substrates and specific gene products. Since the last report, a total of 783 samples were analyzed in 2004 by metabolic screening from numerous canine and feline breeds as well as from a few ferrets and equine samples. The majority of these samples were urine (412), serum/plasma (181), and EDTA whole blood (113); other samples included tissues such as liver, spleen, kidney, skin, and blood and lymph node smears. Samples were received from 33 states in the United States, Australia, Canada, France, Finland, Italy, Japan, Puerto Rico and the United Kingdom. Individual case diagnoses of diseases previously recognized by the Metabolic Genetic Screening Laboratory included Fanconi Syndrome (25), type I and non-type I cystinuria (11), isolated glucosuria (4), methylmalonic aciduria associated with intestinal cobalamin malabsorption (5), and various storage diseases. In collaboration with the Children?s Hospital of Philadelphia, numerous samples were analyzed by HPLC and gas chromatography for amino acid and organic acid quantitation in order to confirm abnormalities found by paper chromatographic and spot tests. These studies assisted in the identification of companion animals affected with models currently under investigation in the Referral Center (see the various subprojects), including cystinuria in Newfoundlands, Mastiffs, Scottish Deerhounds, Cardigan Welsh Corgis, and domestic shorthair cats, cobalamin malabsorption in Border Collies, Komodor, Australian Shepherds, Cattle Dogs, and Beagles, erythroenzymopathies, hereditary bleeding disorders including von Willebrand disease in Dobermans, hemophilia in German shepherds and various other breeds, fibrinogen deficiency in a mixed breed dog, factor VII deficiency in Beagles, factor XI deficiency in Kerry Blue Terriers, and lysosomal storage diseases (MPS I, IIIB, VI, VII, I-Cell disease, Neimann-Pick C, Krabbe disease, and alpha-mannosidosis). A focus of the Metabolic Genetic Screening Laboratory is the identification of companion animals with novel and previously described lysosomal storage diseases through urine electrophoresis, enzyme assays, and blood smear analysis. (1) Examination of urinary glycosaminoglycans (GAGs) and serum enzyme assays led to the identification of a Himalyan cat with mucopolysaccharidosis VI (MPS VI). Subsequent DNA analysis indicated the deficiency is due to the mutation previously described to cause a severe form of the disease in Siamese cats. (2) After the original discovery of MPS VII deficiency in a mixed breed dog, which led to the intense independently supported studies on the pathophysiology, molecular basis and gene transfer investigations, we have recently found two families of German shepherd dogs with MPS VII due to the same mutation. Additionally, the first case of MPS VII in Rat Terriers was identified. Details of these studies can be found in the subprojects. Recently GAG and enzyme analyses also led to the diagnosis of MPS VII in a male domestic short hair cat. (3) Increased serum activity of lysosomal enzymes, the presence of white blood cell inclusion bodies in the blood smear, and little to no activity of arylsulfatase B in white blood cells also led to the diagnosis of MPS VI in a one year-old Miniature Pincher. In our laboratory we determined the disease-causing mutation in this and the Miniature schnauzer breed, over 50 canines for these breeds have been characterized as normal, carrier, or affected through DNA testing. (4) DNA screening for MPS IIIB in Skipperkes continues and we have examined a total of 1300 Schipperkes resulting in approximately 10% carriers and 1% affected dogs. (5) Other enzyme deficiencies identified included alpha-mannosidosis that was diagnosed in a Persian and one domestic short hair cat and alpha-fucosidosis diagnosed in 3 month-old English Springer Spaniel. The Josephine Deublar Genetic Disease Testing Laboratory has screened over 1000 animals, both canine and feline breeds for Pyruvate Kinase Deficiency (PK) and approximately 80 canines for Phosphofructokinase Deficiency (PFK) among other DNA test offered through the laboratory. Collaborations have been established between various universities to further some the above new findings. 2. ENZYME STUDIES 2004: 2-1. Canine Enzyme Canine Serum White Red Total Blood Cells Alpha-mannosidase 44 3 47 Beta-mannosidase 2 2 4 Beta-glucuronidase 5 1 57 Hexosaminidase 13 13 26 Galactosidase 11 10 21 Alpha-L-iduronidase 3 12 15 Arylsulfatase B 2 18 20 Alpha-fucosidase 39 3 42 Pyruvate Kinase 3 3 Total 170 62 3 235 2-2. Feline Enzyme Feline Serum White Red Total Blood Cells Alpha-mannosidase 32 4 36 Beta-mannosidase 4 1 5 Beta-glucuronidase 33 10 43 Hexosaminidase 7 14 21 Galactosidase 9 11 20 Alpha-L-iduronidase 5 12 17 Arylsulfatase B 6 21 27 Alpha-fucosidase 28 12 40 Pyruvate Kinase 2 2 Total 124 85 2 211