Mouse Models Of Inherited Metabolic Disorders

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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.