Fabry disease: results of the first UK hemodialysis screening study.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder in which deficiency of α-Galactosidase A (α-Gal A), leads to accumulation of glycosphingolipids in the vascular endothelium, kidneys and heart. Males with classical disease present in childhood, however some individuals with low levels of α-Gal A activity present atypically with adult onset renal impairment. Screening studies in patients with established end-stage renal failure (ESRF) suggest that up to 1.5% of patients have sub-normal α-Gal A levels. We used the dried blood spot (DBS) enzyme activity test to screen for undiagnosed Fabry disease in patients with ESRF. METHODS: Male hemodialysis patients treated at a single UK center (n = 155) were screened using the DBS assay. In patients with low enzyme activity on DBS, α-Gal A activity was assessed in plasma and leucocytes. RESULTS: 8 of the 155 (5%) patients screened showed low enzyme activity on the DBS assay. Confirmatory testing of plasma and leucocyte α-Gal A activity showed normal activity in all cases tested, indicating a false positive DBS result. CONCLUSIONS: This study is the first screening program in UK hemodialysis patients using the DBS test and did not identify any new cases of Fabry disease. In this cohort, the DBS enzyme assay had a false positive rate of 2.6%, emphasizing the need for validation with alternative techniques.

Autoimmune-prone mice share a promoter haplotype associated with reduced expression and function of the Fc receptor FcgammaRII.

Human autoimmune diseases thought to arise from the combined effects of multiple susceptibility genes include systemic lupus erythematosus (SLE) and autoimmune diabetes. Well-characterised polygenic mouse models closely resembling each of these diseases exist, and genetic evidence links receptors for the Fc portion of immunoglobulin G (FcR) with their pathogenesis in mice and humans [1] [2] [3]. FcRs may be activatory or inhibitory and regulate a variety of immune and inflammatory processes [4] [5]. FcgammaRII (CD32) negatively regulates activation of cells including B cells and macrophages [6]. FcgammaRII-deficient mice are prone to immune-mediated disease [7] [8] [9]. The gene encoding FcgammaRII, Fcgr2, is contained in genetic susceptibility intervals in mouse models of SLE such as the New Zealand Black (NZB) contribution to the (NZB x New Zealand White (NZW)) F1 strain [1] [10] [11] and the BXSB strain [12], and in human SLE [1] [2] [3]. We therefore sequenced Fcgr2 and identified a haplotype defined by deletions in the Fcgr2 promoter region that is present in major SLE-prone mouse strains (NZB, BXSB, SB/Le, MRL, 129 [13]) and non-obese diabetic (NOD) mice but absent in control strains (BALB/c, C57BL/6, DBA/2, C57BL/10) and NZW mice. The autoimmune haplotype was associated with reduced cell-surface expression of FcgammaRII on macrophages and activated B cells and with hyperactive macrophages resembling those of FcgammaRII-deficient mice, and is therefore likely to play an important role in the pathogenesis of SLE and possibly diabetes.

Analysis of cytological changes in hepatocytes from rats dosed with 3'-methyl-4-dimethylaminoazobenzene: initial response appears to involve cytokinesis of binucleated cells.

The reduction in the ratio of tetraploid (4N + 2 X 2N) to diploid (2N) hepatocytes in the adult rat after treatment with the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'M) has been investigated. Analysis of isolated hepatocytes 18-28 days after treatment has confirmed that initially some of the 2 X 2N hepatocytes are converted into 2N cells by cytokinesis, and that there is no DNA synthesis during this process. Shortly afterwards nonpolyploidizing growth commences by proliferation of some 2N cells.

Recovery of hyperplastic responsiveness in rat liver after dosing with the peroxisome proliferator methylclofenapate.

Methylclofenapate (MCP) was administered daily by gavage (25 mg/kg) for 7 days to groups of adult male rats. Dosing was interrupted for 28, 35, 56, 70 or 84 days and then resumed (25 mg/kg by gavage at 0 and 24 h). During the second period of dosing animals were killed in groups of three at 6, 12, 18, 24, 30, 36, 42 and 48 h after the resumption of dosing. Hepatocytes in S-phase, labelled with bromodeoxy-uridine, were analysed by flow cytometry, cell sorting and microscopy. It was observed that total S-phase activity was just significantly elevated (approximately 20% of maximum) over corn oil controls after an interval of 28 days between initial and subsequent dosing periods. After an interval of 35 days total S-phase activity was approximately 65% of maximum, and full hyperplastic responsiveness, equal to that observed in naive animals given MCP, was detected after interruptions in dosing of 56, 70 and 84 days. The recovery of S-phase responsiveness during the interruptions in dosing was accompanied by an increase in the proportion of 2 X 2N hepatocytes from approximately 10% in animals dosed continuously with MCP, to approximately 11.4% after 28 days interruption, 17% after 35 days and control levels (approximately 20%) after 56, 70 and 84 days. Irrespective of the magnitude of the hyperplasia elicited by the second period of dosing with MCP, the proportion of 2 X 2N cells was reduced to the same levels as those observed in animals dosed continuously with MCP (approximately 10%). Very low S-phase activity (0.05%) was observed in animals dosed continuously with MCP, this level of activity being similar to that in animals given corn oil continuously.

A species comparison of acute hyperplasia induced by the peroxisome proliferator methylclofenapate: involvement of the binucleated hepatocyte.

The acute hyperplastic response induced by methylclofenapate (MCP) was studied in several rodent species with different responsiveness to the hypertrophic and hyperplastic effects caused by this chemical. The species, in descending order of responsiveness, were: mouse, rat, hamster and guinea-pig, the latter species being non-responsive. Animals were dosed at daily intervals with MCP (25, 12 or 5 mg/kg by gavage) and killed at intervals from 12 h to 240 h. The parameters of ploidy, nuclearity and DNA synthesis were examined in isolated hepatocytes. The hyperplastic response elicited by MCP in rodent livers as detected by the occurrence of S-phase cells, was almost exclusively confined to the 2 x 2N (binucleated) hepatocyte population. At the same time the proportion of 2 x 2N cells was reduced in a time and dose-dependent manner, while the fraction of 4N cells increased. These observations indicate that the 2 x 2N cells responding to MCP undergo S-phase followed by amitotic cytokinesis to form 4N cells. The response in rats, mice and hamsters was quantitatively different but qualitatively similar, while the guinea-pig was non-responsive. In a given responsive species the areas under the curves are similar for different doses, indicating that the size of the responsive population is limited. The data also indicate that although the response in the mouse was greater than in the rat, because the total number of responsive 2 x 2N cells is larger, the percentage of responsive 2 x 2N cells is higher in the rat than in the mouse. The ploidy analysis reveals that there is no detectable change in the ratio of (4N + 2 x 2N):2N hepatocytes, but only 1-2% change would be expected, despite the number of 4N cells produced, due to the increase in total cell number.

Effects produced by the non-genotoxic hepatocarcinogen methylclofenapate in dwarf mice: peroxisome induction uncoupled from DNA synthesis and nuclearity changes.

Both Snell dwarf mice (dw/dw) and their phenotypically normal heterozygotes (dw/+) were dosed with methylclofenapate (MCP) at daily intervals by gavage (25 mg/kg). Animals were killed at 12, 24, 36 and 72 h after the initial dose and the parameters of ploidy, nuclearity and DNA synthesis were measured in hepatocytes isolated by collagenase perfusion. The occurrence of peroxisome proliferation was assessed by electron microscopy after daily administration of 25 mg/kg MCP by gavage for 28 days. The hepatocytes from both phenotypes exhibited similar degrees of peroxisome proliferation but hyperplasia occurred only in the heterozygous animals. The incidence of binulceated hepatocytes in heterozygotes was approximately 50%, and at the end of acute hyperplasia this had reduced to approximately 20%; by contrast the livers of dwarf animals contained approximately 20% binucleated cells and this remained unchanged throughout the period of dosing. The hyperplasia in the wild-type mice, as measured by the occurrence of S-phase, occurred predominantly in binucleated hepatocytes. These observations are further confirmation that acute hyperplasia induced by MCP and similar liver growth inducers occurs predominantly in a sensitive sub-population of binucleated hepatocytes. The results also indicate that peroxisome proliferation and hyperplasia can occur as independent phenomena.

Studies on the hyperplastic responsiveness of binucleated rat hepatocytes.

Rats were dosed by gavage for 28 days with 25 mg/kg of 3'methyl-4-dimethylaminoazobenzene (3'M) followed by 4 days dosing with 0.5 ml/100 g corn oil. Livers from animals were taken at intervals during the 4 days of corn oil dosing and examined for S-phase activity and nuclearity. The treatment regime caused an increase in the proportion of diploid (2N) cells in the hepatocyte population, a decrease in the proportion of binucleated (2 x 2N) cells of approximately 50%, and induced an increase in cell replication. A second group of rats was dosed for 28 days with 0.5 ml/100 g corn oil followed by 4 days administration of 25 mg/kg methylclofenapate (MCP). Analysis of hepatocytes taken during the MCP treatment revealed that there was acute hyperplasia, involving mainly the 2 x 2N hepatocytes, resulting in a reduction of approximately 50% of the 2 x 2N cells and an accompanying increase in the proportion of 4N cells. When a third group was given 3'M for 28 days followed by 4 days administration of MCP, there was, at the outset of MCP dosing, a hepatocyte population typical of 3'M dosed animals, with an elevated proportion of 2N cells and a 2 x 2N fraction that was reduced to approximately 50% of control levels. During the 4 days of MCP dosing there was a wave of hyperplasia involving S-phase activity in the remaining 2 x 2N hepatocytes. The proportion of 2 x 2N cells decreased further during this period and there was a concomitant increase in 4N cells. The 2N fraction, already elevated, was not further affected. These results indicate that both genotoxic and non-genotoxic hepatocarcinogens induce acute changes in the rat hepatocyte population that involve 2 x 2N cells, and that the effects appear to involve separate sub-populations of 2 x 2N hepatocytes.