Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease.

We have identified a murine gene, metaxin, that spans the 6-kb interval separating the glucocerebrosidase gene (GC) from the thrombospondin 3 gene on chromosome 3E3-F1. Metaxin and GC are transcribed convergently; their major polyadenylylation sites are only 431 bp apart. On the other hand, metaxin and the thrombospondin 3 gene are transcribed divergently and share a common promoter sequence. The cDNA for metaxin encodes a 317-aa protein, without either a signal sequence or consensus for N-linked glycosylation. Metaxin protein is expressed ubiquitously in tissues of the young adult mouse, but no close homologues have been found in the DNA or protein data bases. A targeted mutation (A-->G in exon 9) was introduced into GC by homologous recombination in embryonic stem cells to establish a mouse model for a mild form of Gaucher disease. A phosphoglycerate kinase-neomycin gene cassette was also inserted into the 3'-flanking region of GC as a selectable marker, at a site later identified as the terminal exon of metaxin. Mice homozygous for the combined mutations die early in gestation. Since the same amino acid mutation in humans is associated with mild type 1 Gaucher disease, we suggest that metaxin protein is likely to be essential for embryonic development in mice. Clearly, the contiguous gene organization at this locus limits targeting strategies for the production of murine models of Gaucher disease.

Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the β-subunit of β-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr(-)) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (similar to 64 and 59 kDa) and secreted (63-69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditionalmethods of screening high-producing recombinant cellsmay represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

Parkinsonism in Gaucher's disease type 1: ten new cases and a review of the literature

Parkinsonism has been described in patients with Gaucher's disease (GD). We reviewed the 10 cases of patients with both parkinsonism and GD recorded in the French national GD registry, as well as 49 previously published cases. Relative to the general population, parkinsonism in GD patients (1) was more frequent, (2) occurred at an earlier age, (3) responded less well to levodopa, and (4) was more frequently associated with signs of cortical dysfunction. Enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) were ineffective on GD-associated parkinsonism, suggesting that parkinsonism itself is not an indication for ERT or SRT in this setting.

A metastatic neuroendocrine anaplastic small cell tumor in a patient with multiple endocrine neoplasia type 1 syndrome : assessment of disease status and response to doxorubicin, cyclophosphamide, etoposide chemotherapy through scintigraphic imaging with 111In-pentetreotide

Extrapulmonary small cell and small cell neuroendocrine tumors of unknown primary site are, in general, aggressive neoplasms with a short median survival. Like small cell lung cancer (SCLC), they often are responsive to chemotherapy and radiotherapy. Small cell lung cancer and well differentiated neuroendocrine carcinomas of the gastrointestinal tract and pancreas tend to express somatostatin receptors. These tumors may be localized in patients by scintigraphic imaging using radiolabeled somatostatin analogues. A patient with an anaplastic neuroendocrine small cell tumor arising on a background of multiple endocrine neoplasia type 1 syndrome is reported. The patient had a known large pancreatic gastrinoma and previously treated parathyroid adenopathy. At presentation, there was small cell cancer throughout the liver and skeleton. Imaging with a radiolabeled somatostatin analogue, 111In- pentetreotide (Mallinckrodt Medical B. V., Petten, Holland), revealed all sites of disease detected by routine biochemical and radiologic methods. After six cycles of chemotherapy with doxorubicin, cyclophosphamide, and etoposide, there was almost complete clearance of the metastatic disease. 111In-pentetreotide scintigraphy revealed uptake consistent with small areas of residual disease in the liver, the abdomen (in mesenteric lymph nodes), and posterior thorax (in a rib). The primary gastrinoma present before the onset of the anaplastic small cell cancer showed no evidence of response to the treatment. The patient remained well for 1 year and then relapsed with brain, lung, liver, and skeletal metastases. Despite an initial response to salvage radiotherapy and chemotherapy with carboplatin and dacarbazine, the patient died 6 months later.

Is childhood type 1 diabetes a wealth-related disease? An ecological analysis of European incidence rates

Aims/Hypothesis: To describe the epidemiology of childhood-onset Type 1 (insulin-dependent) diabetes in Europe, the EURODIAB collaborative group has established prospective, geographically-defined registers of children diagnosed under 15 years. A total of 16,362 cases were registered by 44 centres during the period 1989-1994. The registers cover a population of approximately 28 million children with most European countries represented. Methods In most centres a primary and a secondary source of ascertainment were used so that the completeness of registration could be assessed by the capture-recapture method. Ecological correlation and regression analyses were used to study the relationship between incidence and various environmental, health and economic indicators. Findings: The standardised average annual incidence rate during the period 1989-94 ranged from 3.2 cases per 100,000 per annum in the Former Yugoslavian Republic of Macedonia to 40.2 cases per 100,000 per annum in Finland. Indicators of national prosperity such as infant mortality (r= -0.64) and gross domestic product (r= 0.58) were most strongly and significantly correlated with incidence rate, and previously-reported associations with coffee consumption (r= 0.51), milk consumption (r= 0.58) and latitude (r= 0.40) were also observed. Conclusion/Interpretation: The wide variation in childhood type 1 diabetes incidence rates within Europe could be partially explained by indicators of national prosperity. These indicators could reflect differences in environmental risk factors such as nutrition or lifestyle that are important in determining a country's incidence rate.

Testing the possible negative association of type 1 diabetes and atopic disease by analysis of the interleukin 4 receptor gene

Variations in the interleukin 4 receptor A (IL4RA) gene have been reported to be associated with atopy, asthma, and allergy, which may occur less frequently in subjects with type 1 diabetes (T1D). Since atopy shows a humoral immune reactivity pattern, and T1D results from a cellular (T lymphocyte) response, we hypothesised that alleles predisposing to atopy could be protective for T1D and transmitted less often than the expected 50% from heterozygous parents to offspring with T1D. We genotyped seven exonic single nucleotide polymorphisms (SNPs) and the -3223 C>T SNP in the putative promoter region of IL4RA in up to 3475 T1D families, including 1244 Finnish T1D families. Only the -3223 C>T SNP showed evidence of negative association (P=0.014). There was some evidence for an interaction between -3233 C>T and the T1D locus IDDM2 in the insulin gene region (P=0.001 in the combined and P=0.02 in the Finnish data set). We, therefore, cannot rule out a genetic effect of IL4RA in T1D, but it is not a major one.

Herpes simplex virus type 1 and Alzheimer's disease: the autophagy connection.

The causes of Alzheimer's disease (AD) and of the characteristic pathological features—amyloid plaques and neurofibrillary tangles—of AD brain are unknown, despite the enormous resources provided over the years for their investigation. Indeed, the only generally accepted risk factors are age, Down syndrome, carriage of the type 4 allele of the apolipoprotein E gene (APOE-e 4), and possibly brain injury. Following the authors' previous studies implicating herpes simplex virus type 1 (HSV1) in brain of APOE-e 4 carriers as a major cause of AD, the authors propose here, on the basis of their and others' recent studies, that not only does HSV1 generate the main components of amyloid plaques and neurofibrillary tangles (NFTs)—ß -amyloid (Aß) and abnormally phosphorylated tau but also, by disrupting autophagy, it prevents degradation of these aberrant proteins, leading to their accumulation and deposition, and eventually to AD.

Secular trends, disease maps and ecological analyses of the incidence of childhood onset type 1 diabetes in Northern Ireland, 1989-2003

Aims To investigate secular trends in the incidence of Type 1 diabetes in Northern Ireland over the period 1989-2003. To highlight geographical variations in the incidence of Type 1 diabetes by producing disease maps and to compare incidence rates by relevant area characteristics.