Mouse Models Of Inherited Metabolic Disorders

法布里病是一种致命的x连锁隐性代谢疾病，由溶酶体酶α -半乳糖苷酶a （AGA）活性不足引起。在受影响的半合子雄性中，血管内皮细胞和平滑肌细胞溶酶体中的底物逐渐沉积导致血管闭塞性疾病。到目前为止，还没有针对这种情况的特殊治疗方法。酶替代和基因治疗都在考虑之中，但在人体上进行这些试验将是困难和耗时的。我们已经建立了法布里小鼠模型，这将对开发这种治疗方案有价值。该小鼠模型是通过同源重组破坏小鼠胚胎干细胞AGA基因组位点而建立的。尽管这些小鼠表现出完全缺乏AGA活性，但在10周龄时，它们在临床上表现正常。超微结构分析研究显示肾脏内有同心片层内含物，使用荧光标记的α - d -半乳糖基残基凝集素共聚焦显微镜显示底物（Gb3）在肾脏和培养的成纤维细胞中积累。脂质分析显示底物在肝脏和肾脏中有明显的积累。衰老研究显示，在80周龄小鼠中，底物逐渐积累，伴有亚临床肾脏病理。野生型小鼠骨髓移植法布里小鼠的骨髓可纠正大部分靶组织的代谢缺陷，具有临床应用价值。我们之前已经证明，使用含有人AGA cDNA的双顺反式MDR载体，可以在AGA缺失小鼠培养的成纤维细胞中纠正AGA缺陷和α - d -半乳糖基残基的积累。许多法布里病患者的临床症状之一是唾液减少，导致口干。为了确定AGA缺乏对唾液腺的分子效应，我们分析了Fabry小鼠的腮腺和颌下唾液腺。采用组织病理学和电镜对腮腺和颌下唾液腺进行分析。用薄层色谱法测定Gb3含量。与年龄匹配的一岁野生型相比，腮腺和颌下唾液腺的AGA活性显著降低（~ 88%和91%）。脂质分析显示腮腺和颌下腺的Gb3水平显著升高。野生型小鼠唾液腺中未见或很少见Gb-3积累。电镜分析证实唾液腺溶酶体内存在典型的Gb3片层包涵体。在各种唾液腺细胞类型中大量积累的Gb-3可能损害法布里病患者的唾液腺功能。为了评估法布里病患者的口腔和颅面表现，以促进对该疾病的认识和早期治疗，我们通过标准化问卷调查、临床检查、全景和头侧x线片以及磁共振成像收集了法布里病患者队列的数据。发现了多种异常，包括上颌窦粘液潴留囊肿的患病率增加和上颌前突的存在。鉴于口腔和牙齿异常的高患病率，建议对这些患者进行彻底的口腔评估。为了进一步表征法布里小鼠的口腔缺陷，我们对这些小鼠进行了详细的唾液化学分析，以评估唾液腺功能。通过IP注射毛卡痛，采集野生型对照和Fabry小鼠唾液，用自动分析仪分析其成分。法布里小鼠唾液化学分析显示，小鼠体内镁、钙水平显著降低（WT组Mg、Ca分别为0.65+0.11和1.11+0.30 mmol/L，法布里组Mg、Ca分别为0.44+0.11和1.11+0.3 mmol/L, n=14; p <0.01和0.02），淀粉酶活性降低30%。法布里小鼠唾液中主要阳离子水平的显著改变和淀粉酶活性的降低表明这些小鼠唾液腺功能可能受损。这些发现也提示有必要对Fabry患者进行详细的唾液化学研究，以评估他们的唾液腺功能。

Fabry disease is a fatal X-linked recessive metabolic disorder resulting from the deficient activity of the lysosomal enzyme, alpha-galactosidase A (AGA). In affected hemizygous males, the progressive deposition of substrate in lysosomes of vascular endothelial and smooth muscle cells causes occlusive vascular disease. To date, there is no specific treatment for this condition. Both enzyme replacement and gene therapy are under consideration, but carrying out these trials in human will be difficult and time-consuming. We have developed Fabry mouse model which will be valuable to develop such therapeutic regimes. This mouse model was generated by disrupting AGA genomic locus in mouse embryonic stem cells by homologous recombination. Although these mice showed a complete lack of AGA activity, they appeared clinically normal at 10 weeks of age. Ultrastructural analysis studies revealed concentric lamellar inclusions in the kidneys and confocal microscopy using a fluorescent-labeled lectin specific for alpha-D-galactosyl residues showed accumulation of substrate (Gb3) in the kidneys as well as in the cultured fibroblasts. Lipid analysis revealed a marked accumulation of the substrate in the liver and the kidneys. Aging studies revealed progressive accumulation of the substrate with sub-clinical kidney pathology in 80 weeks old mice. Bone marrow transplant of the Fabry mice with bone marrow from wild type mice corrected the metabolic defects in most of the target tissues indicating its value in the clinical domain. We had earlier shown correction of AGA deficiency and accumulation of alpha-D-galactosyl residues in the cultured fibroblasts from the AGA null mice using biscistronic MDR vectors containing human AGA cDNA. One of the clinical symptoms noted in many patients of Fabry disease is reduced saliva resulting in a dry mouth-like condition. In order to identify molecular effects of AGA deficiency on salivary glands, we have analyzed parotid and submaxillary salivary glands of Fabry mice. Parotid and submaxillary salivary glands were analyzed by histopathology and electron microscopy. Gb3 level was determined by thin layer chromatography. AGA activities in parotid and submaxillary salivary glands were significantly lower (~88 and 91%) when compared to age matched one-year-old wild types. Lipid analysis revealed dramatic increases in Gb3 levels in both parotid and submaxillary glands. None or very little Gb-3 accumulation was seen in the salivary glands of wild-type mice. Electron microscopic analysis confirmed the presence of typical lammellar inclusion bodies of Gb3 within the lysosomes of the salivary glands. Large accumulations of Gb-3 in various salivary gland cell types potentially impair salivary gland function in patients with Fabry disease. To assess oral and craniofacial findings in a cohort of patients with Fabry disease in order to facilitate recognition of this condition and early treatment of its manifestations, we collected data from cohorts of Fabry poatients by means of a standardized questionnaire, clinical examination, panoramic and cephalometric radiographs, and magnetic resonance imaging. A variety of abnormalities were identified, including an increased prevalence of mucous-retention cysts of the maxillary sinuses and the presence of maxillary prognathism. Given the high prevalence of oral and dental abnormalities, a thorough stomatologic evaluation of these patients is recommended. To further characterize oral defects in Fabry mice, we carried out detailed sialochemical analysis in these mice to assess salivary gland function. Saliva was collected from the wild-type control and Fabry mice by IP injection of pilocarpain and analyzed for its composition using an autoanalyzer. Sialochemical analysis of Fabry mice revealed significant decrease in magnesium and calcium levels (Mg and Ca 0.65+0.11 and 1.11+0.30 in WT vs 0.44+0.11 and 1.11+0.3 mmol/L in Fabry mice, n=14; p, <0.01 and 0.02), and 30 % decrease in amylase activity. Significant alteration in the levels of major cations and the decrease in amylase activity in saliva of Fabry mice indicate possible impairment of salivary gland function in these mice. These findings also suggest a need for detailed sialochemistry studies on Fabry patients to assess their salivary gland function.